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Sample records for aldh enzymatic activity

  1. ALDH enzymatic activity and CD133 positivity and response to chemotherapy in ovarian cancer patients.

    PubMed

    Ricci, Francesca; Bernasconi, Sergio; Porcu, Luca; Erba, Eugenio; Panini, Nicolò; Fruscio, Robert; Sina, Federica; Torri, Valter; Broggini, Massimo; Damia, Giovanna

    2013-01-01

    The prognostic/predictive role of both CD133 and Aldehyde dehydrogenase (ALDH) expression in human ovarian cancer remains elusive. This is an observational study that investigated the expression of CD133 and of ALDH enzymatic activity in fresh ovarian cancer samples and their association with different clinic-pathological patient' characteristics and explored their possible predictive/prognostic role. We analyzed the expression of CD133 and ALDH enzymatic activity in 108 human ovarian cancer samples. We found that among the total patients analyzed, 13% of them was completely negative for ALDH activity and 26% was negative for CD133 staining. Both markers were variably expressed within the samples and when both studied in the same tumor sample, no statistically significant correlation between ALDH enzymatic activity and CD133 expression was found. No statistical significant correlation was found also between the percentage values of positive ALDH and CD133 cells and the number of serial passages patient's cultures underwent, suggesting that these markers do not confer by themselves a self-renewal growth advantage to the cultures. Lower levels of CD133 were associated with higher tumor grade. No correlation with response to therapy, progression free survival and overall survival was found. Our data suggest that neither ALDH enzymatic activity nor CD133 expression provide additional predictive/prognostic information in ovarian cancer patients.

  2. Regulation of Human Mitochondrial Aldehyde Dehydrogenase (ALDH-2) Activity by Electrophiles in Vitro*

    PubMed Central

    Oelze, Matthias; Knorr, Maike; Schell, Richard; Kamuf, Jens; Pautz, Andrea; Art, Julia; Wenzel, Philip; Münzel, Thomas; Kleinert, Hartmut; Daiber, Andreas

    2011-01-01

    Recently, mitochondrial aldehyde dehydrogenase (ALDH-2) was reported to reduce ischemic damage in an experimental myocardial infarction model. ALDH-2 activity is redox-sensitive. Therefore, we here compared effects of various electrophiles (organic nitrates, reactive fatty acid metabolites, or oxidants) on the activity of ALDH-2 with special emphasis on organic nitrate-induced inactivation of the enzyme, the biochemical correlate of nitrate tolerance. Recombinant human ALDH-2 was overexpressed in Escherichia coli; activity was determined with an HPLC-based assay, and reactive oxygen and nitrogen species formation was determined by chemiluminescence, fluorescence, protein tyrosine nitration, and diaminonaphthalene nitrosation. The organic nitrate glyceryl trinitrate caused a severe concentration-dependent decrease in enzyme activity, whereas incubation with pentaerythritol tetranitrate had only minor effects. 4-Hydroxynonenal, an oxidized prostaglandin J2, and 9- or 10-nitrooleate caused a significant inhibition of ALDH-2 activity, which was improved in the presence of Mg2+ and Ca2+. Hydrogen peroxide and NO generation caused only minor inhibition of ALDH-2 activity, whereas peroxynitrite generation or bolus additions lead to severe impairment of the enzymatic activity, which was prevented by the thioredoxin/thioredoxin reductase (Trx/TrxR) system. In the presence of glyceryl trinitrate and to a lesser extent pentaerythritol tetranitrate, ALDH-2 may be switched to a peroxynitrite synthase. Electrophiles of different nature potently regulate the enzymatic activity of ALDH-2 and thereby may influence the resistance to ischemic damage in response to myocardial infarction. The Trx/TrxR system may play an important role in this process because it not only prevents inhibition of ALDH-2 but is also inhibited by the ALDH-2 substrate 4-hydroxynonenal. PMID:21252222

  3. ALDH2 Mediates 5-Nitrofuran Activity in Multiple Species

    PubMed Central

    Zhou, Linna; Ishizaki, Hironori; Spitzer, Michaela; Taylor, Kerrie L.; Temperley, Nicholas D.; Johnson, Stephen L.; Brear, Paul; Gautier, Philippe; Zeng, Zhiqiang; Mitchell, Amy; Narayan, Vikram; McNeil, Ewan M.; Melton, David W.; Smith, Terry K.; Tyers, Mike; Westwood, Nicholas J.; Patton, E. Elizabeth

    2012-01-01

    Summary Understanding how drugs work in vivo is critical for drug design and for maximizing the potential of currently available drugs. 5-nitrofurans are a class of prodrugs widely used to treat bacterial and trypanosome infections, but despite relative specificity, 5-nitrofurans often cause serious toxic side effects in people. Here, we use yeast and zebrafish, as well as human in vitro systems, to assess the biological activity of 5-nitrofurans, and we identify a conserved interaction between aldehyde dehydrogenase (ALDH) 2 and 5-nitrofurans across these species. In addition, we show that the activity of nifurtimox, a 5-nitrofuran anti-trypanosome prodrug, is dependent on zebrafish Aldh2 and is a substrate for human ALDH2. This study reveals a conserved and biologically relevant ALDH2-5-nitrofuran interaction that may have important implications for managing the toxicity of 5-nitrofuran treatment. PMID:22840776

  4. The rarity of ALDH(+) cells is the key to separation of normal versus leukemia stem cells by ALDH activity in AML patients.

    PubMed

    Hoang, Van T; Buss, Eike C; Wang, Wenwen; Hoffmann, Isabel; Raffel, Simon; Zepeda-Moreno, Abraham; Baran, Natalia; Wuchter, Patrick; Eckstein, Volker; Trumpp, Andreas; Jauch, Anna; Ho, Anthony D; Lutz, Christoph

    2015-08-01

    To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH(+) cells; <1.9%; ALDH-rare AML), whereas 24 patients had relatively numerous ALDH(+) cells (≥1.9%; ALDH-numerous AML). In patients with ALDH-rare AML, normal HSC could be separated by their CD34(+) ALDH(+) phenotype, whereas LSC were exclusively detected among CD34(+) ALDH(-) cells. For patients with ALDH-numerous AML, the CD34(+) ALDH(+) subset consisted mainly of LSC and separation from HSC was not feasible. Functional analyses further showed that ALDH(+) cells from ALDH-numerous AML were quiescent, refractory to ARA-C treatment and capable of leukemic engraftment in a xenogenic mouse transplantation model. Clinically, resistance to chemotherapy and poor long-term outcome were also characteristic for patients with ALDH-numerous AML providing an additional risk-stratification tool. The difference in spectrum and relevance of ALDH activity in the putative LSC populations demonstrates, in addition to phenotypic and genetic, also functional heterogeneity of leukemic cells and suggests divergent roles for ALDH activity in normal HSC versus LSC. By acknowledging these differences our study provides a new and useful tool for prospective identification of AML cases in which separation of HSC from LSC is possible.

  5. Maternal and zygotic aldh1a2 activity is required for pancreas development in zebrafish.

    PubMed

    Alexa, Kristen; Choe, Seong-Kyu; Hirsch, Nicolas; Etheridge, Letitiah; Laver, Elizabeth; Sagerström, Charles G

    2009-12-11

    We have isolated and characterized a novel zebrafish pancreas mutant. Mutant embryos lack expression of isl1 and sst in the endocrine pancreas, but retain isl1 expression in the CNS. Non-endocrine endodermal gene expression is less affected in the mutant, with varying degrees of residual expression observed for pdx1, carbA, hhex, prox1, sid4, transferrin and ifabp. In addition, mutant embryos display a swollen pericardium and lack fin buds. Genetic mapping revealed a mutation resulting in a glycine to arginine change in the catalytic domain of the aldh1a2 gene, which is required for the production of retinoic acid from vitamin A. Comparison of our mutant (aldh1a2(um22)) to neckless (aldh1a2(i26)), a previously identified aldh1a2 mutant, revealed similarities in residual endodermal gene expression. In contrast, treatment with DEAB (diethylaminobenzaldehyde), a competitive reversible inhibitor of Aldh enzymes, produces a more severe phenotype with complete loss of endodermal gene expression, indicating that a source of Aldh activity persists in both mutants. We find that mRNA from the aldh1a2(um22) mutant allele is inactive, indicating that it represents a null allele. Instead, the residual Aldh activity is likely due to maternal aldh1a2, since we find that translation-blocking, but not splice-blocking, aldh1a2 morpholinos produce a phenotype similar to DEAB treatment. We conclude that Aldh1a2 is the primary Aldh acting during pancreas development and that maternal Aldh1a2 activity persists in aldh1a2(um22) and aldh1a2(i26) mutant embryos.

  6. Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes.

    PubMed

    Mali, Vishal R; Deshpande, Mandar; Pan, Guodong; Thandavarayan, Rajarajan A; Palaniyandi, Suresh S

    2016-02-01

    Reactive oxygen species (ROS)-mediated reactive aldehydes induce cellular stress. In cardiovascular diseases such as ischemia-reperfusion injury, lipid-peroxidation derived reactive aldehydes such as 4-hydroxy-2-nonenal (4HNE) are known to contribute to the pathogenesis. 4HNE is involved in ROS formation, abnormal calcium handling and more importantly defective mitochondrial respiration. Aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)(+)-dependent isozymes which can detoxify endogenous and exogenous aldehydes into non-toxic carboxylic acids. Therefore we hypothesize that 4HNE afflicts mitochondrial respiration and leads to cell death by impairing ALDH2 activity in cultured H9C2 cardiomyocyte cell lines. H9C2 cardiomyocytes were treated with 25, 50 and 75 μM 4HNE and its vehicle, ethanol as well as 25, 50 and 75 μM disulfiram (DSF), an inhibitor of ALDH2 and its vehicle (DMSO) for 4 h. 4HNE significantly decreased ALDH2 activity, ALDH2 protein levels, mitochondrial respiration and mitochondrial respiratory reserve capacity, and increased 4HNE adduct formation and cell death in cultured H9C2 cardiomyocytes. ALDH2 inhibition by DSF and ALDH2 siRNA attenuated ALDH2 activity besides reducing ALDH2 levels, mitochondrial respiration and mitochondrial respiratory reserve capacity and increased cell death. Our results indicate that ALDH2 impairment can lead to poor mitochondrial respiration and increased cell death in cultured H9C2 cardiomyocytes.

  7. Alkynol natural products target ALDH2 in cancer cells by irreversible binding to the active site.

    PubMed

    Heydenreuter, Wolfgang; Kunold, Elena; Sieber, Stephan A

    2015-11-11

    Falcarinol and stipudiol are natural products with potent anti-cancer activity found in several vegetables. Here, we use a chemical proteomic strategy to identify ALDH2 as a molecular target of falcarinol in cancer cells and confirm enzyme inhibition via covalent alkylation of the active site. Furthermore, the synthesis of stipudiol led to the observation that ALDH2 exhibits preference for alkynol-based binders. Inhibition of ALDH2 impairs detoxification of reactive aldehydes and limits oxidative stress response, two crucial pathways for cellular viability.

  8. Blood ALDH1 and GST activity in diabetes type 2 and its correlation with glycated hemoglobin.

    PubMed

    Giebułtowicz, J; Sołobodowska, S; Bobilewicz, D; Wroczyński, P

    2014-01-01

    There is increasing evidence that oxidative stress (OS) plays a major role in the pathogenesis of diabetes mellitus (DM) and the development of its complications. As one of the consequences of OS is increased lipid peroxidation (LP), the aim of our studies was to check, how the activity of 2 enzymes involved in the detoxification of aldehydes formed during LP, glutathione S-transferase (GST) and aldehyde dehydrogenase 1 (ALDH 1) is changed in patients suffering from DM.GST and ALDH1A1 activities were determined in whole blood samples of DM type 2 patients (n=64) and healthy controls (n=60) using spectrophotometer (for GST activity) and fluorometer (for ALDH1 activity) and they were found to be significantly increased in diabetics when compared with healthy control (p<0.05). Intriguingly, grouping the DM patients on the basis of the glucose level and HbA1c revealed unusually low ALDH activity in the group of patients (n=16) with a relatively high level of these 2 parameters.The increase of ALDH1A1 and GST activity in DM seems to be associated with the severity of the disease and might be a compensatory effect against oxidative stress. Surprisingly low ALDH activity in DM patients with relatively high glucose and HbA1c levels can be a factor predisposing to the development of diabetic complications.

  9. Vasodilatory effect of nitroglycerin in Japanese subjects with different aldehyde dehydrogenase 2 (ALDH2) genotypes.

    PubMed

    Miura, Takeshi; Nishinaka, Toru; Terada, Tomoyuki; Yonezawa, Kazuya

    2017-03-23

    The functional genetic polymorphism of aldehyde dehydrogenase 2 (ALDH2) influences the enzymatic activities of its wild type (Glu504 encoded by ALDH2*1) and mutant type (Lys504 encoded by ALDH2*2) proteins. The enzymatic activities of mutant-type ALDH2 are limited compared with those of the wild type. ALDH2 has been suggested as a critical factor for nitroglycerin-mediated vasodilation by some human studies and in vitro studies. Currently, there is no research on direct observations of the vasodilatory effect of nitroglycerin sublingual tablets, which is the generally used dosage form. In the present study, the contribution of ALDH2 to the vasodilatory effect of nitroglycerin sublingual tablets was investigated among three genotype groups (ALDH2*1/*1, ALDH2*1/*2, and ALDH2*2/*2) in Japanese. The results by direct assessments of in vivo nitroglycerin-mediated dilation showed no apparent difference in vasodilation among all genotypes of ALDH2. Furthermore, to analyze the effect of other factors (age and flow-mediated dilation), multiple regression analysis and Pearson's correlation coefficient analysis were carried out. These analyses also indicated that the genotypes of ALDH2 were not related to the degree of vasodilation. These results suggest the existence of other predominant pathway(s) for nitroglycerin biotransformation, at least with regard to clinical nitroglycerin (e.g., a sublingual tablet) in Japanese subjects.

  10. Activation of ALDH2 with Low Concentration of Ethanol Attenuates Myocardial Ischemia/Reperfusion Injury in Diabetes Rat Model.

    PubMed

    Kang, Pin-Fang; Wu, Wen-Juan; Tang, Yang; Xuan, Ling; Guan, Su-Dong; Tang, Bi; Zhang, Heng; Gao, Qin; Wang, Hong-Ju

    2016-01-01

    The aim of this paper is to observe the change of mitochondrial aldehyde dehydrogenase 2 (ALDH2) when diabetes mellitus (DM) rat heart was subjected to ischemia/reperfusion (I/R) intervention and analyze its underlying mechanisms. DM rat hearts were subjected to 30 min regional ischemia and 120 min reperfusion in vitro and pretreated with ALDH2 activator ethanol (EtOH); cardiomyocyte in high glucose (HG) condition was pretreated with ALDH2 activator Alda-1. In control I/R group, myocardial tissue structure collapse appeared. Compared with control I/R group, left ventricular parameters, SOD activity, the level of Bcl-2/Bax mRNA, ALDH2 mRNA, and protein expressions were decreased and LDH and MDA contents were increased, meanwhile the aggravation of myocardial structure injury in DM I/R group. When DM I/R rats were pretreated with EtOH, left ventricular parameters, SOD, Bcl-2/Bax, and ALDH2 expression were increased; LDH, MDA, and myocardial structure injury were attenuated. Compared with DM + EtOH I/R group, cyanamide (ALDH2 nonspecific blocker), atractyloside (mitoPTP opener), and wortmannin (PI3K inhibitor) groups all decreased left ventricular parameters, SOD, Bcl-2/Bax, and ALDH2 and increased LDH, MDA, and myocardial injury. When cardiomyocyte was under HG condition, CCK-8 activity and ALDH2 protein expression were decreased. Alda-1 increased CCK-8 and ALDH2. Our findings suggested enhanced ALDH2 expression in diabetic I/R rats played the cardioprotective role, maybe through activating PI3K and inhibiting mitoPTP opening.

  11. Activation of ALDH2 with Low Concentration of Ethanol Attenuates Myocardial Ischemia/Reperfusion Injury in Diabetes Rat Model

    PubMed Central

    Kang, Pin-Fang; Wu, Wen-Juan; Tang, Yang; Xuan, Ling; Guan, Su-Dong; Tang, Bi; Zhang, Heng

    2016-01-01

    The aim of this paper is to observe the change of mitochondrial aldehyde dehydrogenase 2 (ALDH2) when diabetes mellitus (DM) rat heart was subjected to ischemia/reperfusion (I/R) intervention and analyze its underlying mechanisms. DM rat hearts were subjected to 30 min regional ischemia and 120 min reperfusion in vitro and pretreated with ALDH2 activator ethanol (EtOH); cardiomyocyte in high glucose (HG) condition was pretreated with ALDH2 activator Alda-1. In control I/R group, myocardial tissue structure collapse appeared. Compared with control I/R group, left ventricular parameters, SOD activity, the level of Bcl-2/Bax mRNA, ALDH2 mRNA, and protein expressions were decreased and LDH and MDA contents were increased, meanwhile the aggravation of myocardial structure injury in DM I/R group. When DM I/R rats were pretreated with EtOH, left ventricular parameters, SOD, Bcl-2/Bax, and ALDH2 expression were increased; LDH, MDA, and myocardial structure injury were attenuated. Compared with DM + EtOH I/R group, cyanamide (ALDH2 nonspecific blocker), atractyloside (mitoPTP opener), and wortmannin (PI3K inhibitor) groups all decreased left ventricular parameters, SOD, Bcl-2/Bax, and ALDH2 and increased LDH, MDA, and myocardial injury. When cardiomyocyte was under HG condition, CCK-8 activity and ALDH2 protein expression were decreased. Alda-1 increased CCK-8 and ALDH2. Our findings suggested enhanced ALDH2 expression in diabetic I/R rats played the cardioprotective role, maybe through activating PI3K and inhibiting mitoPTP opening. PMID:27829984

  12. Contribution of ALDH2 polymorphism to alcoholism-associated hypertension.

    PubMed

    Hu, Nan; Zhang, Yingmei; Nair, Sreejayan; Culver, Bruce W; Ren, Jun

    2014-01-01

    Chronic alcohol intake is considered as an independent lifestyle factor that may influence the risk of a number of cardiovascular anomalies including hypertension. In healthy adults, binge drinking and chronic alcohol ingestion lead to the onset and development of hypertension although the precise mechanism(s) remains obscure. Although oxidative stress and endothelial injury have been postulated to play a major contributing role to alcoholism-induced hypertension, recent evidence depicted a rather unique role for the genotype of the acetaldehyde-metabolizing enzyme mitochondrial aldehyde dehydrogenase (ALDH2), which is mainly responsible for detoxifying ethanol consumed, in alcoholism-induced elevation of blood pressure. Genetic polymorphism of ALDH2 in human results in altered ethanol pharmacokinetic properties and ethanol metabolism, leading to accumulation of the ethanol metabolite acetaldehyde following alcohol intake. The unfavorable consequence of the ALDH2 variants is believed to be governed by the accumulation of the ethanol metabolite acetaldehyde. Presence of the mutant or inactive ALDH2*2 gene often results in an increased risk of hypertension in human. Such association between blood pressure and ALDH2 enzymatic activity may be affected by the interplay between gene and environment, such as life style and ethnicity. The aim of this mini-review is to summarize the possible contribution of ALDH2 genetic polymorphism in the onset and development of alcoholism-related development of hypertension. Furthermore, the double-edged sword of ALDH2 gene and genetic polymorphism in alcoholism and alcoholic tissue damage and relevant patents will be discussed.

  13. Aldh1 Expression and Activity Increase During Tumor Evolution in Sarcoma Cancer Stem Cell Populations

    PubMed Central

    Martinez-Cruzado, Lucia; Tornin, Juan; Santos, Laura; Rodriguez, Aida; García-Castro, Javier; Morís, Francisco; Rodriguez, Rene

    2016-01-01

    Tumors evolve from initial tumorigenic events into increasingly aggressive behaviors in a process usually driven by subpopulations of cancer stem cells (CSCs). Mesenchymal stromal/stem cells (MSCs) may act as the cell-of-origin for sarcomas, and CSCs that present MSC features have been identified in sarcomas due to their ability to grow as self-renewed floating spheres (tumorspheres). Accordingly, we previously developed sarcoma models using human MSCs transformed with relevant oncogenic events. To study the evolution/emergence of CSC subpopulations during tumor progression, we compared the tumorigenic properties of bulk adherent cultures and tumorsphere-forming subpopulations both in the sarcoma cell-of-origin models (transformed MSCs) and in their corresponding tumor xenograft-derived cells. Tumor formation assays showed that the tumorsphere cultures from xenograft-derived cells, but not from the cell-of-origin models, were enriched in CSCs, providing evidence of the emergence of bona fide CSCs subpopulations during tumor progression. Relevant CSC-related factors, such as ALDH1 and SOX2, were increasingly upregulated in CSCs during tumor progression, and importantly, the increased levels and activity of ALDH1 in these subpopulations were associated with enhanced tumorigenicity. In addition to being a CSC marker, our findings indicate that ALDH1 could also be useful for tracking the malignant potential of CSC subpopulations during sarcoma evolution. PMID:27292183

  14. Transcriptional activity of novel ALDH1L1 promoters in the rat brain following AAV vector-mediated gene transfer

    PubMed Central

    Mudannayake, Janitha M; Mouravlev, Alexandre; Fong, Dahna M; Young, Deborah

    2016-01-01

    Aldehyde dehydrogenase family 1, member L1 (ALDH1L1) is a recently characterized pan-astrocytic marker that is more homogenously expressed throughout the brain than the classic astrocytic marker, glial fibrillary acidic protein. We generated putative promoter sequence variants of the rat ALDH1L1 gene for use in adeno-associated viral vector-mediated gene transfer, with an aim to achieve selective regulation of transgene expression in astrocytes in the rat brain. Unexpectedly, ALDH1L1 promoter variants mediated transcriptional activity exclusively in neurons in the substantia nigra pars compacta as assessed by luciferase reporter expression at 3 weeks postvector infusion. This selectivity for neurons in the substantia nigra pars compacta also persisted in the context of adeno-associated viral serotype 5, 8 or 9 vector-mediated gene delivery. An in vivo promoter comparison showed the highest performing ALDH1L1 promoter variant mediated higher transgene expression than the neuronal-specific synapsin 1 and tyrosine hydroxylase promoters. The ALDH1L1 promoter was also transcriptionally active in dentate granule neurons following intrahippocampal adeno-associated viral vector infusion, whereas transgene expression was detected in both striatal neurons and astrocytes following vector infusion into the striatum. Our results demonstrate the potential suitability of the ALDH1L1 promoter as a new tool in the development of gene therapy and disease modelling applications. PMID:27990448

  15. Enzymatically active ultrathin pepsin membranes.

    PubMed

    Raaijmakers, Michiel J T; Schmidt, Thomas; Barth, Monika; Tutus, Murat; Benes, Nieck E; Wessling, Matthias

    2015-05-11

    Enzymatically active proteins enable efficient and specific cleavage reactions of peptide bonds. Covalent coupling of the enzymes permits immobilization, which in turn reduces autolysis-induced deactivation. Ultrathin pepsin membranes were prepared by facile interfacial polycondensation of pepsin and trimesoyl chloride. The pepsin membrane allows for simultaneous enzymatic conversion and selective removal of digestion products. The large water fluxes through the membrane expedite the transport of large molecules through the pepsin layers. The presented method enables the large-scale production of ultrathin, cross-linked, enzymatically active membranes.

  16. A High-Content Assay Enables the Automated Screening and Identification of Small Molecules with Specific ALDH1A1-Inhibitory Activity

    PubMed Central

    Yasgar, Adam; Titus, Steven A.; Wang, Yuhong; Danchik, Carina; Yang, Shyh-Ming; Vasiliou, Vasilis; Jadhav, Ajit; Maloney, David J.; Simeonov, Anton

    2017-01-01

    Aldehyde dehydrogenase enzymes (ALDHs) have a broad spectrum of biological activities through the oxidation of both endogenous and exogenous aldehydes. Increased expression of ALDH1A1 has been identified in a wide-range of human cancer stem cells and is associated with cancer relapse and poor prognosis, raising the potential of ALDH1A1 as a therapeutic target. To facilitate quantitative high-throughput screening (qHTS) campaigns for the discovery, characterization and structure-activity-relationship (SAR) studies of small molecule ALDH1A1 inhibitors with cellular activity, we show herein the miniaturization to 1536-well format and automation of a high-content cell-based ALDEFLUOR assay. We demonstrate the utility of this assay by generating dose-response curves on a comprehensive set of prior art inhibitors as well as hundreds of ALDH1A1 inhibitors synthesized in house. Finally, we established a screening paradigm using a pair of cell lines with low and high ALDH1A1 expression, respectively, to uncover novel cell-active ALDH1A1-specific inhibitors from a collection of over 1,000 small molecules. PMID:28129349

  17. Antioxidant function of corneal ALDH3A1 in cultured stromal fibroblasts.

    PubMed

    Lassen, Natalie; Pappa, Aglaia; Black, William J; Jester, James V; Day, Brian J; Min, Elysia; Vasiliou, Vasilis

    2006-11-01

    Aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in epithelial cells and stromal keratocytes of mammalian cornea and is believed to play an important role in cellular defense. To explore a potential protective role against oxidative damage, a rabbit corneal fibroblastic cell line (TRK43) was stably transfected with the human ALDH3A1 and subjected to oxidative stress induced by H(2)O(2), mitomycin C (MMC), or etoposide (VP-16). ALDH3A1-transfected cells were more resistant to H(2)O(2,) MMC, and VP-16 compared to the vector-transfected cells. All treatments induced apoptosis only in vector-transfected cells, which was associated with increased levels of 4-hydroxy-2-nonenal (4-HNE)-adducted proteins. Treatment with H(2)O(2) resulted in a rise in reduced glutathione (GSH) levels in all groups but was more pronounced in the ALDH3A1-expressing cells. Treatment with the DNA-damaging agents led to GSH depletion in control groups, although the depletion was significantly less in ALDH3A1-expressing cells. Increased carbonylation of ALDH3A1 but not significant decline in enzymatic activity was observed after all treatments. In conclusion, our results suggest that ALDH3A1 may act to protect corneal cells against cellular oxidative damage by metabolizing toxic lipid peroxidation products (e.g., 4-HNE), maintaining cellular GSH levels and redox balance, and operating as an antioxidant.

  18. Structural and functional modifications of corneal crystallin ALDH3A1 by UVB light.

    PubMed

    Estey, Tia; Chen, Ying; Carpenter, John F; Vasiliou, Vasilis

    2010-12-21

    As one of the most abundantly expressed proteins in the mammalian corneal epithelium, aldehyde dehydrogenase 3A1 (ALDH3A1) plays critical and multifaceted roles in protecting the cornea from oxidative stress. Recent studies have demonstrated that one protective mechanism of ALDH3A1 is the direct absorption of UV-energy, which reduces damage to other corneal proteins such as glucose-6-phosphate dehydrogenase through a competition mechanism. UV-exposure, however, leads to the inactivation of ALDH3A1 in such cases. In the current study, we demonstrate that UV-light caused soluble, non-native aggregation of ALDH3A1 due to both covalent and non-covalent interactions, and that the formation of the aggregates was responsible for the loss of ALDH3A1 enzymatic activity. Spectroscopic studies revealed that as a result of aggregation, the secondary and tertiary structure of ALDH3A1 were perturbed. LysC peptide mapping using MALDI-TOF mass spectrometry shows that UV-induced damage to ALDH3A1 also includes chemical modifications to Trp, Met, and Cys residues. Surprisingly, the conserved active site Cys of ALDH3A1 does not appear to be affected by UV-exposure; this residue remained intact after exposure to UV-light that rendered the enzyme completely inactive. Collectively, our data suggest that the UV-induced inactivation of ALDH3A1 is a result of non-native aggregation and associated structural changes rather than specific damage to the active site Cys.

  19. Expression pattern, ethanol-metabolizing activities, and cellular localization of alcohol and aldehyde dehydrogenases in human large bowel: association of the functional polymorphisms of ADH and ALDH genes with hemorrhoids and colorectal cancer.

    PubMed

    Chiang, Chien-Ping; Jao, Shu-Wen; Lee, Shiao-Pieng; Chen, Pei-Chi; Chung, Chia-Chi; Lee, Shou-Lun; Nieh, Shin; Yin, Shih-Jiun

    2012-02-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for metabolism of ethanol. Functional polymorphisms of ADH1B, ADH1C, and ALDH2 genes occur among racial populations. The goal of this study was to systematically determine the functional expressions and cellular localization of ADHs and ALDHs in human rectal mucosa, the lesions of adenocarcinoma and hemorrhoid, and the genetic association of allelic variations of ADH and ALDH with large bowel disorders. Twenty-one surgical specimens of rectal adenocarcinoma and the adjacent normal mucosa, including 16 paired tissues of rectal tumor, normal mucosae of rectum and sigmoid colon from the same individuals, and 18 surgical mixed hemorrhoid specimens and leukocyte DNA samples from 103 colorectal cancer patients, 67 hemorrhoid patients, and 545 control subjects recruited in previous study, were investigated. The isozyme/allozyme expression patterns of ADH and ALDH were identified by isoelectric focusing and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting using the corresponding purified class-specific antibodies; the cellular activity and protein localizations were detected by immunohistochemistry and histochemistry, respectively. Genotypes of ADH1B, ADH1C, and ALDH2 were determined by polymerase chain reaction-restriction fragment length polymorphisms. At 33mM ethanol, pH 7.5, the activity of ADH1C*1/1 phenotypes exhibited 87% higher than that of the ADH1C*1/*2 phenotypes in normal rectal mucosa. The activity of ALDH2-active phenotypes of rectal mucosa was 33% greater than ALDH2-inactive phenotypes at 200μM acetaldehyde. The protein contents in normal rectal mucosa were in the following order: ADH1>ALDH2>ADH3≈ALDH1A1, whereas those of ADH2, ADH4, and ALDH3A1 were fairly low. Both activity and content of ADH1 were significantly decreased in rectal tumors, whereas the ALDH activity remained

  20. Biochemical genetics of opossum aldehyde dehydrogenase 3: evidence for three ALDH3A-like genes and an ALDH3B-like gene.

    PubMed

    Holmes, Roger S

    2010-04-01

    Mammalian ALDH3 isozymes participate in peroxidic and fatty aldehyde metabolism, and in anterior eye tissue UV-filtration. BLAT analyses were undertaken of the opossum genome using rat ALDH3A1, ALDH3A2, ALDH3B1, and ALDH3B2 amino acid sequences. Two predicted opossum ALDH3A1-like genes and an ALDH3A2-like gene were observed on chromosome 2, as well as an ALDH3B-like gene, which showed similar intron-exon boundaries with other mammalian ALDH3-like genes. Opossum ALDH3 subunit sequences and structures were highly conserved, including residues previously shown to be involved in catalysis and coenzyme binding for rat ALDH3A1. Eleven glycine residues were conserved for all of the opossum ALDH3-like sequences examined, including two glycine residues previously located within the stem of the rat ALDH3A1 active site funnel. Phylogeny studies of human, rat, opossum, and chicken ALDH3-like sequences indicated that the common ancestor for ALDH3A- and ALDH3B-like genes predates the appearance of birds during vertebrate evolution.

  1. SET overexpression decreases cell detoxification efficiency: ALDH2 and GSTP1 are downregulated, DDR is impaired and DNA damage accumulates.

    PubMed

    Almeida, Luciana O; Goto, Renata N; Pestana, Cezar R; Uyemura, Sérgio A; Gutkind, Silvio; Curti, Carlos; Leopoldino, Andréia M

    2012-12-01

    Alcohol and tobacco consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Aldehyde dehydrogenase 2 (ALDH2) and glutathione S-transferase pi 1 (GSTP1) are important enzymes for cellular detoxification and low efficiencies are implicated in cancer. We assessed the potential role of SET protein overexpression, a histone acetylation modulator accumulated in HNSCC, in gene regulation and protein activity of ALDH2 and GSTP1. SET was knocked down in HN13, HN12 and Cal27, and overexpressed in HEK293 cells; ethanol and cisplatin were the chemical agents. Cells with SET overexpression (HEK293/SET, HN13 and HN12) showed lower ALDH2 and GSTP1 mRNA levels and trichostatin A increased them (real-time PCR). Ethanol upregulated GSTP1 and ALDH2 mRNAs, whereas cisplatin upregulated GSTP1 in HEK293 cells. SET-chromatin binding revealed SET interaction with ALDH2 and GSTP1 promoters, specifically via SET NAP domain; ethanol and cisplatin abolished SET binding. ALDH2 and GSTP1 efficiency was assessed by enzymatic and comet assay. A lower ALDH2 activity was associated with greater DNA damage (tail intensity) in HEK293/SET compared with HEK293 cells, whereas HN13/siSET showed ALDH2 activity higher than HN13 cells. HN13/siSET cells showed increased tail intensity. Cisplatin-induced DNA damage response showed negative relationship between SET overexpression and BRCA2 recruitment. SET downregulated repair genes ATM, BRCA1 and CHEK2 and upregulated TP53. Cisplatin-induced cell-cycle arrest occurred in G(0) /G(1) and S in HEK293 cells, whereas HEK293/SET showed G(2) /M stalling. Overall, cisplatin was more cytotoxic for HN13 than HN13/siSET cells. Our data suggest a role for SET in cellular detoxification, DNA damage response and genome integrity.

  2. Exploring the evolutionary route of the acquisition of betaine aldehyde dehydrogenase activity by plant ALDH10 enzymes: implications for the synthesis of the osmoprotectant glycine betaine

    PubMed Central

    2014-01-01

    Background Plant ALDH10 enzymes are aminoaldehyde dehydrogenases (AMADHs) that oxidize different ω-amino or trimethylammonium aldehydes, but only some of them have betaine aldehyde dehydrogenase (BADH) activity and produce the osmoprotectant glycine betaine (GB). The latter enzymes possess alanine or cysteine at position 441 (numbering of the spinach enzyme, SoBADH), while those ALDH10s that cannot oxidize betaine aldehyde (BAL) have isoleucine at this position. Only the plants that contain A441- or C441-type ALDH10 isoenzymes accumulate GB in response to osmotic stress. In this work we explored the evolutionary history of the acquisition of BAL specificity by plant ALDH10s. Results We performed extensive phylogenetic analyses and constructed and characterized, kinetically and structurally, four SoBADH variants that simulate the parsimonious intermediates in the evolutionary pathway from I441-type to A441- or C441-type enzymes. All mutants had a correct folding, average thermal stabilities and similar activity with aminopropionaldehyde, but whereas A441S and A441T exhibited significant activity with BAL, A441V and A441F did not. The kinetics of the mutants were consistent with their predicted structural features obtained by modeling, and confirmed the importance of position 441 for BAL specificity. The acquisition of BADH activity could have happened through any of these intermediates without detriment of the original function or protein stability. Phylogenetic studies showed that this event occurred independently several times during angiosperms evolution when an ALDH10 gene duplicate changed the critical Ile residue for Ala or Cys in two consecutive single mutations. ALDH10 isoenzymes frequently group in two clades within a plant family: one includes peroxisomal I441-type, the other peroxisomal and non-peroxisomal I441-, A441- or C441-type. Interestingly, high GB-accumulators plants have non-peroxisomal A441- or C441-type isoenzymes, while low-GB accumulators

  3. Assessment of the reproductive toxicity of inhalation exposure to ethyl tertiary butyl ether in male mice with normal, low active and inactive ALDH2.

    PubMed

    Weng, Zuquan; Ohtani, Katsumi; Suda, Megumi; Yanagiba, Yukie; Kawamoto, Toshihiro; Nakajima, Tamie; Wang, Rui-Sheng

    2014-04-01

    No data are available regarding aldehyde dehydrogenase 2 (ALDH2) polymorphisms related to the reproductive toxicity possibly caused by ethyl tertiary butyl ether (ETBE). In this study, two inhalation experiments were performed in Aldh2 knockout (KO), heterogeneous (HT) and wild type (WT) C57BL/6 male mice exposed to ETBE, and the data about general toxicity, testicular histopathology, sperm head numbers, sperm motility and sperm DNA damage were collected. The results showed that the 13-week exposure to 0, 500, 1,750 and 5,000 ppm ETBE significantly decreased sperm motility and increased levels of sperm DNA strand breaks and 8-hydroxy-deoxyguanosine in both WT and KO mice, the effects were found in 1,750 and 5,000 ppm groups of WT mice, and all of the three exposed groups of KO mice compared to the corresponding control; furthermore, ETBE also caused decrease in the relative weights of testes and epididymides, the slight atrophy of seminiferous tubules of testis and reduction in sperm numbers of KO mice exposed to ≥500 ppm. In the experiment of exposure to lower concentrations of ETBE (0, 50, 200 and 500 ppm) for 9 weeks, the remarkable effects of ETBE on sperm head numbers, sperm motility and sperm DNA damage were further observed in KO and HT mice exposed to 200 ppm ETBE, but not in WT mice. Our findings suggested that only exposure to high concentrations of ETBE might result in reproductive toxicity in mice with normal active ALDH2, while low active and inactive ALDH2 enzyme significantly enhanced the ETBE-induced reproductive toxicity in mice, even exposed to low concentrations of ETBE, mainly due to the accumulation of acetaldehyde as a primary metabolite of ETBE.

  4. Targeting aberrant expression of Notch-1 in ALDH(+) cancer stem cells in breast cancer.

    PubMed

    Pal, Deeksha; Kolluru, Venkatesh; Chandrasekaran, Balaji; Baby, Becca V; Aman, Masarath; Suman, Suman; Sirimulla, Suman; Sanders, Mary Ann; Alatassi, Houda; Ankem, Murali K; Damodaran, Chendil

    2017-03-01

    We have previously reported that high aldehyde dehydrogenase (ALDH) enzyme activity in breast cancer cells results in breast cancer stem cell (BCSC) properties by upregualting Notch-1 and epithelial mesenchymal markers. This results in chemoresistance in breast cancer. Here, we examined the functional and clinical significance of ALDH expression by measuring the ALDH levels in breast cancer tissues by immunohistochemistry. There was a significantly higher ALDH expression in higher grade breast cancer tumor tissues (Grade- II and III) versus normal breast tissues. Injection of BCSC (ALDH(+) and CD44(+) /CD22(-) ) cells resulted in aggressive tumor growth in athymic mice versus ALDH(-) cells. The ALDH(+) and CD44(+) /CD22(-) tumors grow rapidly and are larger than ALDH(-) tumors which were slow growing and smaller. Molecularly, ALDH(+) tumors expressed higher expression of Notch-1 and EMT markers than ALDH(-) tumors. Oral administration of the naturally occurring Psoralidin (Pso, 25 mg/kg of body weight) significantly inhibited the growth in ALDH(+) and ALDH(-) tumors as well. Psoralidin inhibited Notch-1 mediated EMT activation in ALDH(+) and ALDH(-) tumors-this confirms our in vitro findings. Our results suggest that Notch-1 could be an attractive target and inhibition of Notch-1 by Psoralidin may prevent pathogenesis of breast cancer as well as metastasis. © 2016 Wiley Periodicals, Inc.

  5. Extracellular enzymatic activity of Microsporum canis isolates.

    PubMed

    Papini, R; Mancianti, F

    The enzymatic activity of 70 feline and canine Microsporum canis isolates was determined by the Api-Zym test. The liquid phase of cultures, inoculated into Tryptic Soy Broth, was used to examine 19 enzymes. Considerable differences were observed among the extracellular enzymatic patterns. All the isolates produced alkaline phosphatase and beta-glucosidase, while lipase (C14), trypsin, chymotrypsin, beta-glucuronidase, and alpha-fucosidase activity was never revealed. Esterase (C4) activity was present in 57 samples (81%), esterase lipase (C8) in 31 (44%), leucine arylamidase in 35 (50%), valine arylamidase and cystine arylamidase in 7 (10%), acid phosphatase in 64 (91%), naphthol-AS-BI-phosphohydrolase in 60 (86%), alpha-galactosidase in 5 (7%), beta-galactosidase in 6 (8%), alpha-glucosidase in 25 (36%), N-acetyl-beta-glucosaminidase in 41 (58%), and alpha-mannosidase in 51 (73%). The beta-galactosidase activity of M. canis has not been reported previously. Remarkable variations of intensity for each enzymatic activity were also detected. It is believed that these results could provide basic data for further investigations on the pathogenic role of enzymes secreted by M. canis.

  6. Evaluation of STAT3 Signaling in ALDH+ and ALDH+/CD44+/CD24− Subpopulations of Breast Cancer Cells

    PubMed Central

    Lin, Li; Hutzen, Brian; Lee, Hsiu-Fang; Peng, Zhengang; Wang, Wenlong; Zhao, Chongqiang; Lin, Huey-Jen; Sun, Duxin; Li, Pui-Kai; Li, Chenglong; Korkaya, Hasan; Wicha, Max S.; Lin, Jiayuh

    2013-01-01

    Background STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH+), or cell surface molecule CD44-positive (CD44+) but CD24-negative (CD24−) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH+ and ALDH+/CD44+/CD24− subpopulations of breast cancer cells is unknown. Methods and Results We examined STAT3 activation in ALDH+ and ALDH+/CD44+/CD24− subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH+) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH−) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH+ subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH+ subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH+ subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH+ breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH+ cells were further selected for the stem cell markers CD44+ and CD24−. Conclusion These studies demonstrate an important role for STAT3 signaling in ALDH+ and ALDH+/CD44+/CD24− subpopulations of breast cancer cells which may have cancer stem cell properties and suggest

  7. The prognostic roles of ALDH1 isoenzymes in gastric cancer

    PubMed Central

    Li, Kai; Guo, Xiaoguang; Wang, Ziwei; Li, Xiaofeng; Bu, Youquan; Bai, Xuefeng; Zheng, Liansheng; Huang, Ying

    2016-01-01

    Increased aldehyde dehydrogenase 1 (ALDH1) activity has been determined to be present in the stem cells of several kinds of cancers including gastric cancer (GC). Nevertheless, which ones of ALDH1’s isoenzymes are leading to ALDH1 activity remains elusive. In this study, we examined the prognostic value and hazard ratio (HR) of individual ALDH1 isoenzymes in patients with GC using “The Kaplan–Meier plotter” database. mRNA high expression level of ALDH1A1 was not found to be significantly correlated with the overall survival (OS) of all patients with GC followed for 20 years, HR =0.86 (95% confidence interval [CI]: 0.7–1.05), P=0.13. mRNA high expression level of ALDH1A2 was also not significantly correlated with OS for all patients with GC, HR =1.13 (95% CI: 0.91–1.41), P=0.25. mRNA high expression level of ALDH1A3 was found to be significantly correlated with worsened OS in either intestinal-type patients, HR =2.24 (95% CI: 1.44–3.49), P=0.00026, or diffuse-type patients, HR =1.91 (95% CI: 1.02–3.59), P=0.04. Interestingly, mRNA high expression level of ALDH1B1 was found to be significantly correlated with better OS for all patients with GC, HR =0.66 (95% CI: 0.53–0.81), P=7.8e–05, and mRNA high expression level of ALDH1L1 was found to be significantly correlated with worsened OS for all patients with GC, HR =1.23 (95% CI: 1–1.51), P=0.048. Furthermore, our results also indicate that ALDH1A3 and ALDH1L1 are potential major contributors to the ALDH1 activity in GC, since mRNA high expression levels of ALDH1A3 and ALDH1L1 were found to be significantly correlated with worsened OS for all patients with GC. Based on our study, ALDH1A3 and ALDH1L1 are potential prognostic markers and therapeutic targets for patients with GC. PMID:27354812

  8. Enzymatic activities in coniferous leaf litter

    SciTech Connect

    Spalding, B.P.

    1980-07-01

    Assays for measuring the activities of cellulase, xylanase, mannase, amylase, ..beta..-glucosidase, invertase, and protease employing buffered suspensions of ground coniferous and deciduous leaf litter exhibited zero-order kinetics. Only a small percentage of the whole-litter activities of invertase, ..beta..-glucosidase, and protease were extractable into 0.05M potassium acetate, pH 5.0; however, extractable activities of cellulase and xylanase represented from 39 to 174% of the whole-litter activities indicating their soluble exocellar nature. Extractable protease and amylase activities were best correlated with the average daily rates of CO/sub 2/ evolution in a group of 90 leaf litter samples equally representing 18 coniferous species. Enzymatic activities were readily detectable in extracts of all samples but classification of the samples by species provided little differentiation in the distribution of either enzymatic activities or rates of CO/sub 2/ evolution. Mannase, cellulase, and xylanase activities were well-correlated with each other in all samples. Assays attempting to measure a pool of readily-metabolizable substances in litter by extractable reducing substances, ninhydrin-positive substances, glucose, and phenolics failed to show correlation coefficients >0.41 with rates of CO/sub 2/ evolution. Addition of D-(+)-catechin to litter extracts, up to levels equivalent to those observed in the group of samples, did not inhibit any carbohydrase thus suggesting the lack of inhibition of litter-decomposing enzymes by the concentrations of phenolics present in these coniferous leaf litters.

  9. Mutations affecting enzymatic activity in liver arginase

    SciTech Connect

    Vockley, J.G.; Tabor, D.E.; Goodman, B.K.

    1994-09-01

    The hydrolysis of arginine to ornithine and urea is catalyzed by arginase in the last step of the urea cycle. We examined a group of arginase deficient patients by PCR-SSCP analysis to characterize the molecular basis of this disorder. A heterogeneous population of nonsense mutations, microdeletions, and missense mutations has been identified in our cohort. Microdeletions which introduce premature stop codons downstream of the deletion and nonsense mutations result in no arginase activity. These mutations occur randomly along the gene. The majority of missense mutations identified appear to occur in regions of high cross-species homology. To test the effect of these missense mutations on arginase activity, site-directed mutagenesis was used to re-create the patient mutations for in vivo expression studies in a prokaryotic fusion-protein expression system. Of 4 different missense mutations identified in 6 individuals, only one was located outside of a conserved region. The three substitution mutations within the conserved regions had a significant effect on enzymatic activity (0-3.1 nmole/30min, normal is 1300-1400 nmoles/30min, as determined by in vitro arginase assay), while the fourth mutation, a T to S substitution, did not. In addition, site-directed mutagenesis was utilized to create mutations not in residues postulated to play a significant role in the enzymatic function or active site formation in manganese-binding proteins such as arginase. We have determined that the substitution of glycine for a histidine residue, located in a very highly conserved region of exon 3, and the substitution of a histidine and an aspartic acid residue within a similarly conserved region in exon 4, totally abolishes enzymatic activity. Mutations substituting glycine for an additional histidine and aspartic acid residue in exon 4 and two aspartic acid residues in exon 7 have also been created. We are currently in the process of characterizing these mutations.

  10. Plant ALDH10 Family

    PubMed Central

    Kopečný, David; Končitíková, Radka; Tylichová, Martina; Vigouroux, Armelle; Moskalíková, Hana; Soural, Miroslav; Šebela, Marek; Moréra, Solange

    2013-01-01

    Plant ALDH10 family members are aminoaldehyde dehydrogenases (AMADHs), which oxidize ω-aminoaldehydes to the corresponding acids. They have been linked to polyamine catabolism, osmoprotection, secondary metabolism (fragrance), and carnitine biosynthesis. Plants commonly contain two AMADH isoenzymes. We previously studied the substrate specificity of two AMADH isoforms from peas (PsAMADHs). Here, two isoenzymes from tomato (Solanum lycopersicum), SlAMADHs, and three AMADHs from maize (Zea mays), ZmAMADHs, were kinetically investigated to obtain further clues to the catalytic mechanism and the substrate specificity. We also solved the high resolution crystal structures of SlAMADH1 and ZmAMADH1a because these enzymes stand out from the others regarding their activity. From the structural and kinetic analysis, we can state that five residues at positions 163, 288, 289, 444, and 454 (PsAMADHs numbering) can, directly or not, significantly modulate AMADH substrate specificity. In the SlAMADH1 structure, a PEG aldehyde derived from the precipitant forms a thiohemiacetal intermediate, never observed so far. Its absence in the SlAMADH1-E260A structure suggests that Glu-260 can activate the catalytic cysteine as a nucleophile. We show that the five AMADHs studied here are capable of oxidizing 3-dimethylsulfoniopropionaldehyde to the cryo- and osmoprotectant 3-dimethylsulfoniopropionate. For the first time, we also show that 3-acetamidopropionaldehyde, the third aminoaldehyde besides 3-aminopropionaldehyde and 4-aminobutyraldehyde, is generally oxidized by AMADHs, meaning that these enzymes are unique in metabolizing and detoxifying aldehyde products of polyamine degradation to nontoxic amino acids. Finally, gene expression profiles in maize indicate that AMADHs might be important for controlling ω-aminoaldehyde levels during early stages of the seed development. PMID:23408433

  11. ALDH2 protects against stroke by clearing 4-HNE

    PubMed Central

    Guo, Jin-Min; Liu, Ai-Jun; Zang, Pu; Dong, Wen-Zhe; Ying, Li; Wang, Wei; Xu, Pu; Song, Xu-Rui; Cai, Jun; Zhang, She-Qing; Duan, Jun-Li; Mehta, Jawahar L; Su, Ding-Feng

    2013-01-01

    Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that metabolizes ethanol and toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE). Using an unbiased proteomic search, we identified ALDH2 deficiency in stroke-prone spontaneously hypertensive rats (SHR-SP) as compared with spontaneously hypertensive rats (SHR). We concluded the causative role of ALDH2 deficiency in neuronal injury as overexpression or activation of ALDH2 conferred neuroprotection by clearing 4-HNE in in vitro studies. Further, ALDH2-knockdown rats revealed the absence of neuroprotective effects of PKCε. Moderate ethanol administration that is known to exert protection against stroke was shown to enhance the detoxification of 4-HNE, and to protect against ischemic cerebral injury through the PKCε-ALDH2 pathway. In SHR-SP, serum 4-HNE level was persistently elevated and correlated inversely with the lifespan. The role of 4-HNE in stroke in humans was also suggested by persistent elevation of its plasma levels for at least 6 months after stroke. Lastly, we observed that 21 of 1 242 subjects followed for 8 years who developed stroke had higher initial plasma 4-HNE levels than those who did not develop stroke. These findings suggest that activation of the ALDH2 pathway may serve as a useful index in the identification of stroke-prone subjects, and the ALDH2 pathway may be a potential target of therapeutic intervention in stroke. PMID:23689279

  12. Importance of ALDH1A enzymes in determining human testicular retinoic acid concentrations

    PubMed Central

    Arnold, Samuel L.; Kent, Travis; Hogarth, Cathryn A.; Schlatt, Stefan; Prasad, Bhagwat; Haenisch, Michael; Walsh, Thomas; Muller, Charles H.; Griswold, Michael D.; Amory, John K.; Isoherranen, Nina

    2015-01-01

    Retinoic acid (RA), the active metabolite of vitamin A, is required for spermatogenesis and many other biological processes. RA formation requires irreversible oxidation of retinal to RA by aldehyde dehydrogenase enzymes of the 1A family (ALDH1A). While ALDH1A1, ALDH1A2, and ALDH1A3 all form RA, the expression pattern and relative contribution of these enzymes to RA formation in the testis is unknown. In this study, novel methods to measure ALDH1A protein levels and intrinsic RA formation were used to accurately predict RA formation velocities in individual human testis samples and an association between RA formation and intratesticular RA concentrations was observed. The distinct localization of ALDH1A in the testis suggests a specific role for each enzyme in controlling RA formation. ALDH1A1 was found in Sertoli cells, while only ALDH1A2 was found in spermatogonia, spermatids, and spermatocytes. In the absence of cellular retinol binding protein (CRBP)1, ALDH1A1 was predicted to be the main contributor to intratesticular RA formation, but when CRBP1 was present, ALDH1A2 was predicted to be equally important in RA formation as ALDH1A1. This study provides a comprehensive novel methodology to evaluate RA homeostasis in human tissues and provides insight to how the individual ALDH1A enzymes mediate RA concentrations in specific cell types. PMID:25502770

  13. Enzymatic activity of fungi isolated from crops

    PubMed Central

    Cholewa, Grażyna; Sobczak, Paweł; Silny, Wojciech; Nadulski, Rafał; Wojtyła-Buciora, Paulina; Zagórski, Jerzy

    2016-01-01

    Aim To detect and assess the activity of extracellular hydrolytic enzymes and to find differences in enzymograms between fungi isolated from wheat and rye samples and grown on Czapek-Dox Broth and Sabouraud Dextrose Broth enriched with cereal (wheat or rye). Isolated strains were also classified in the scale of biosafety levels (BSL). Material and methods The study used 23 strains of fungi cultured from samples of wheat and rye (grain, grain dust obtained during threshing and soil) collected in the Lublin region (eastern Poland). API ZYM test (bioMérieux) was carried out according to the manufacturer’s instructions. Classification of BSL (Biosafety levels) was based on the current literature. Results High enzymatic activity was found in strains cultured in media containing 1% of wheat grain (Bipolaris holmi, Penicillium decumbens) and with an addition of 1% of rye grain (Cladosporium herbarum, Aspergillus versicolor, Alternaria alternata). The total number of enzymes varied depending on the type of media, and in most cases it was higher in the culture where an addition of cereal grains was used. Conclusions Isolated strains of fungi reveal differences in the profiles of the enzyme assay. It can be assumed that the substrate enriched in grains stimulate the higher activity of mold enzymes. PMID:28035224

  14. Changes in enzymatic activity in composts containing chicken feathers.

    PubMed

    Bohacz, Justyna; Korniłłowicz-Kowalska, Teresa

    2009-07-01

    Enzymatic activity, i.e. respiratory activity, dehydrogenase activity, phosphatase activity, caseinian protease activity, BAA protease activity and urease activity, was determined to investigate the process of biochemical transformations and to select enzymatic indices of maturity of composts prepared from feathers and lignocellulose wastes (bark, straw). Composting was conducted for 7 months, with periodic determinations of activity of the enzymes. The study revealed significant differences in the enzymatic activity, related with the duration of composting and with the substrate composition of the composts. Generally, composts enriched with straw were characterised by higher enzymatic activity than composts without any addition of straw. It was found that the activity of such enzymes as cellulase and protease, towards the end of the period of composting decreased and stabilised. The enzymes enumerated can be taken into consideration in estimation of the maturity of composts prepared from feathers and lignocellulose wastes.

  15. ALDH1A Isozymes Are Markers of Human Melanoma Stem Cells and Potential Therapeutic Targets

    PubMed Central

    Luo, Yuchun; Dallaglio, Katiuscia; Chen, Ying; Robinson, William A; Robinson, Steven E; McCarter, Martin D; Wang, Jianbin; Gonzalez, Rene; Thompson, David C; Norris, David A; Roop, Dennis R; Vasiliou, Vasilis; Fujita, Mayumi

    2012-01-01

    Although the concept of cancer stem cells (CSCs) is well accepted for many tumors, the existence of such cells in human melanoma has been the subject of debate. In the present study, we demonstrate the existence of human melanoma cells that fulfill the criteria for CSCs (self-renewal and differentiation) by serially xenotransplanting cells into NOD/SCID mice. These cells possess high aldehyde dehydrogenase (ALDH) activity with ALDH1A1 and ALDH1A3 being the predominant ALDH isozymes. ALDH-positive melanoma cells are more tumorigenic than ALDH-negative cells in both NOD/SCID mice and NSG mice. Biological analyses of the ALDH-positive melanoma cells reveal the ALDH isozymes to be key molecules regulating the function of these cells. Silencing ALDH1A by siRNA or shRNA leads to cell cycle arrest, apoptosis and decreased cell viability in vitro and reduced tumorigenesis in vivo. ALDH-positive melanoma cells are more resistant to chemotherapeutic agents and silencing ALDH1A by siRNA sensitizes melanoma cells to drug-induced cell death. Furthermore, we, for the first time, examined the molecular signatures of ALDH-positive CSCs from patient-derived tumor specimens. The signatures of melanoma CSCs include retinoic acid (RA)-driven target genes with RA response elements and genes associated with stem cell function. These findings implicate that ALDH isozymes are not only biomarkers of CSCs but also attractive therapeutic targets for human melanoma. Further investigation of these isozymes and genes will enhance our understanding of the molecular mechanisms governing CSCs and reveal new molecular targets for therapeutic intervention of cancer. PMID:22887839

  16. Enzymatic activity of allergenic house dust and storage mite extracts.

    PubMed

    Morales, Maria; Iraola, Víctor; Leonor, Jose R; Carnés, Jerónimo

    2013-01-01

    Proteases are involved in the pathogenicity of allergy, increasing epithelial permeability and acting as adjuvants. Enzymatic activity is therefore important for the allergenicity of an extract and also affects its stability and safety. However, the enzymatic activity of extracts is not usually evaluated. The objective of this study was to evaluate the enzymatic activity of the most allergenic mite extracts and to investigate their allergenic properties. Extracts from nine allergenic mite species (Dermatophagoides pteronyssinus, Dermatophagoides farinae Hughes, Euroglyphus maynei, Lepidoglyphus destructor, Tyrophagus putrescentiae (Schrank), Glycyphagus domesticus (DeGeer), Acarus siro L., Chortoglyphus arcuatus, and Blomia tropicalis) were characterized. Protein and allergen profiles were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western-blot, respectively. Gelatinolytic activity was evaluated with a zymogram and the activity of other enzymes (cysteine, serine proteases, and esterases) was evaluated individually or with the API-ZYM system. The main differences in protease activity were found between house dust mites and storage mites. House dust mites presented higher cysteine protease activity while storage mites presented higher serine protease activity. These differences are in line with their trophic specialization. A wide range of different activities was found in all the extracts analyzed, reflecting the fact that the extracts preserve the activity of many enzymes, this being necessary for a correct diagnosis. However, enzymes may act as adjuvants and, therefore, could lead to undesirable effects in immunotherapies, making this activity not suitable for treatment products. Modified extracts with lower enzymatic activity could be more appropriate for immunotherapy.

  17. Effects of organic carbon sequestration strategies on soil enzymatic activities

    NASA Astrophysics Data System (ADS)

    Puglisi, E.; Suciu, N.; Botteri, L.; Ferrari, T.; Coppolecchia, D.; Trevisan, M.; Piccolo, A.

    2009-04-01

    Greenhouse gases emissions can be counterbalanced with proper agronomical strategies aimed at sequestering carbon in soils. These strategies must be tested not only for their ability in reducing carbon dioxide emissions, but also for their impact on soil quality: enzymatic activities are related to main soil ecological quality, and can be used as early and sensitive indicators of alteration events. Three different strategies for soil carbon sequestration were studied: minimum tillage, protection of biodegradable organic fraction by compost amendment and oxidative polimerization of soil organic matter catalyzed by biometic porfirins. All strategies were compared with a traditional agricultural management based on tillage and mineral fertilization. Experiments were carried out in three Italian soils from different pedo-climatic regions located respectively in Piacenza, Turin and Naples and cultivated with maize or wheat. Soil samples were taken for three consecutive years after harvest and analyzed for their content in phosphates, ß-glucosidase, urease and invertase. An alteration index based on these enzymatic activities levels was applied as well. The biomimetic porfirin application didn't cause changes in enzymatic activities compared to the control at any treatment or location. Enzymatic activities were generally higher in the minimum tillage and compost treatment, while differences between location and date of samplings were limited. Application of the soil alteration index based on enzymatic activities showed that soils treated with compost or subjected to minimum tillage generally have a higher biological quality. The work confirms the environmental sustainability of the carbon sequestering agronomical practices studied.

  18. Enzymatic and Non-Enzymatic Virulence Activities of Dermatophytes on Solid Media

    PubMed Central

    Elavarashi, Elangovan; Rangarajan, Sudha

    2017-01-01

    Introduction Dermatophytes are keratinophilic fungi causing superficial cutaneous infections that account 20-25% of the global population. As per literature search, there is a dearth in the study on virulence factors of dermatophytes from the Indian sub-continent and moreover the association of the virulence factors and the host tissue in vitro helps in understanding the host-pathogen interaction. Aim To analyse the enzymatic and non-enzymatic virulence activities of dermatophytes on solid media. Materials and Methods A total of 11 isolates, three standard American Type Culture Collection (ATCC) strains- Trichophyton rubrum- 28188, Trichophyton mentagrophytes- 9533, Trichophyton tonsurans- 28942, one CBS KNAW Fungal Biodiversity Centre strain- Arthroderma grubyi- 243.66, five clinical isolates- T. rubrum, T. mentagrophytes, Trichophyton rubrum var. raubitschekii, Trichophyton interdigitale, Epidermophyton floccosum, and two laboratory isolates - Microsporum gypseum and Microsporum canis were screened for the production of virulence enzymes such as phospholipase, lipase, protease, gelatinase and non-enzyme virulence factors (haemolytic activity) of dermatophytes. The clinical isolates were identified from a tertiary care hospital, Chennai. These dermatophytes were tested upon specific substrates on solid media such as egg yolk, tween 80, bovine serum albumin, gelatin powder and sheep blood respectively. Results The virulence activity of phospholipase, lipase, protease and gelatinase was observed from all the dermatophyte species. T. rubrum, T. rubrum ATCC strain, T. rubrum var. raubitschekii, T. mentagrophytes, T. mentagrophytes ATCC strain, T. interdigitale and A. grubyi CBS strain produced complete haemolysis, whereas other dermatophytes showed no haemolytic activity. Conclusion Phospholipase, lipase, protease and gelatinase act as enzymatic virulence marker and the T. rubrum complex, T. mentagrophytes complex and A. grubyi showed complete haemolysis and hence

  19. Production of Enzymatically Active Human Acetylcholinesterase in E. Coli

    DTIC Science & Technology

    1993-10-01

    AD-A282 703 lE1l1lm11I AD( CONTRACT NO: DAMD17-90-C-0107 TITLE: PRODUCTION OF ENZYMATICALLY ACTIVE HUMAN ACETYLCHOLINESTERASE IN E . COLI PRINCIPAL...FUNDING NUMBERS Production of Enzymatically Active Human Contract No. Acetylcholinesterase in E . coli DAMD17-90-C-0107 6. AUTHOR(S) M. Gorecki, Ph.D. and M...S493pMFL-52Ser - Run #1 37 Table 8: Summary of reconstitution and purification of rhAChE derived from E . coli S493pMFL-52Ser - Run #2 38 Table 9

  20. Aldehyde dehydrogenase (ALDH) 3A1 expression by the human keratocyte and its repair phenotypes.

    PubMed

    Pei, Ying; Reins, Rose Y; McDermott, Alison M

    2006-11-01

    Transparency is essential for normal corneal function. Recent studies suggest that corneal cells express high levels of so-called corneal crystallins, such as aldehyde dehydrogenase (ALDH) and transketolase (TKT) that contribute to maintaining cellular transparency. Stromal injury leads to the appearance of repair phenotype keratocytes, the corneal fibroblast and myofibroblast. Previous studies on keratocytes from species such as bovine and rabbit indicate that the transformation from the normal to repair phenotype is accompanied by a loss of corneal crystallin expression, which may be associated with loss of cellular transparency. Here we investigated if a similar loss occurs with human keratocyte repair phenotypes. Human corneal epithelial cells were collected by scraping and keratocytes were isolated by collagenase digestion from cadaveric corneas. The cells were either processed immediately (freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. RT-PCR, western blotting and immunolabeling were used to detect mRNA and protein expression of ALDH isozymes and TKT. ALDH enzyme activity was also quantitated and immunolabeling was performed to determine the expression of ALDH3A1 in human corneal tissue sections from normal and diseased corneas. Human corneal keratocytes isolated from three donors expressed ALDH1A1 and ALDH3A1 mRNA, and one donor also expressed ALDH2 and TKT. Corneal epithelial cells expressed ALDH1A1, ALDH2, ALDH3A1 and TKT. Compared to normal keratocytes, corneal fibroblast expression of ALDH3A1 mRNA was reduced by 27% (n=5). ALDH3A1 protein expression as detected by western blotting was markedly reduced in passage zero fibroblasts and undetectable in higher passages (n=3). TKT protein expression was reduced in fibroblasts compared to keratocytes (n=2). ALDH3A1 enzyme activity was not

  1. Pharmacological recruitment of aldehyde dehydrogenase 3A1 (ALDH3A1) to assist ALDH2 in acetaldehyde and ethanol metabolism in vivo

    PubMed Central

    Chen, Che-Hong; Cruz, Leslie A.; Mochly-Rosen, Daria

    2015-01-01

    Correcting a genetic mutation that leads to a loss of function has been a challenge. One such mutation is in aldehyde dehydrogenase 2 (ALDH2), denoted ALDH2*2. This mutation is present in ∼0.6 billion East Asians and results in accumulation of toxic acetaldehyde after consumption of ethanol. To temporarily increase metabolism of acetaldehyde in vivo, we describe an approach in which a pharmacologic agent recruited another ALDH to metabolize acetaldehyde. We focused on ALDH3A1, which is enriched in the upper aerodigestive track, and identified Alda-89 as a small molecule that enables ALDH3A1 to metabolize acetaldehyde. When given together with the ALDH2-specific activator, Alda-1, Alda-89 reduced acetaldehyde-induced behavioral impairment by causing a rapid reduction in blood ethanol and acetaldehyde levels after acute ethanol intoxication in both wild-type and ALDH2-deficient, ALDH2*1/*2, heterozygotic knock-in mice. The use of a pharmacologic agent to recruit an enzyme to metabolize a substrate that it usually does not metabolize may represent a novel means to temporarily increase elimination of toxic agents in vivo. PMID:25713355

  2. Enzymatic activity of rodents acclimated to cold and long scotophase

    NASA Astrophysics Data System (ADS)

    Fourie, F. Le R.; Haim, A.

    1980-09-01

    Rodents representative of a diurnal species ( Rhabdomys pumilio) as well as a nocturnal species ( Praomys natalensis) were acclimated to cold (Ta = 8°C) at a photoperiod of LD 12:12 and a long scotophase (LD 8; 16) at a temperature of 25° C(Ta). Control groups were kept for both species at Ta = 25° C and LD 12:12 and winter acclimated individuals were obtained during July and August to serve as further reference. Blood samples obtained from the tail were analysed for enzymes representative of three major biochemical pathways. The enzymatic activity of LDH (glycolytic pathway), MDH (Krebs cycle) and G6PDH (hexose monophosphate shunt, as an indicator of gonadal activity) were monitored to represent metabolic activity of the respective cycles. Cold acclimated as well as winter acclimatized mice revealed similar enzymatic patterns for both species and significant increases in LDH and MDH were recorded with a concurrent decrease in G6PDH activity. Specimens exposed to long scotophase exhibited similar enzymatic patterns for both species studied, but enzymatic activity was higher than those of cold acclimated individuals. From these results it is concluded that cold as well as long scotophase induce metabolic adaptations through biochemical activity in the experimental animals. The effect of long scotophase is assumed to be an important factor in the induction of winter acclimatization.

  3. Macrophage migration inhibitory factor (MIF) enzymatic activity and lung cancer.

    PubMed

    Mawhinney, Leona; Armstrong, Michelle E; O' Reilly, Ciaran; Bucala, Richard; Leng, Lin; Fingerle-Rowson, Gunter; Fayne, Darren; Keane, Michael P; Tynan, Aisling; Maher, Lewena; Cooke, Gordon; Lloyd, David; Conroy, Helen; Donnelly, Seamas C

    2015-04-16

    The cytokine macrophage migration inhibitory factor (MIF) possesses unique tautomerase enzymatic activity, which contributes to the biological functional activity of MIF. In this study, we investigated the effects of blocking the hydrophobic active site of the tautomerase activity of MIF in the pathogenesis of lung cancer. To address this, we initially established a Lewis lung carcinoma (LLC) murine model in Mif-KO and wild-type (WT) mice and compared tumor growth in a knock-in mouse model expressing a mutant MIF lacking enzymatic activity (Mif (P1G)). Primary tumor growth was significantly attenuated in both Mif-KO and Mif (P1G) mice compared with WT mice. We subsequently undertook a structure-based, virtual screen to identify putative small molecular weight inhibitors specific for the tautomerase enzymatic active site of MIF. From primary and secondary screens, the inhibitor SCD-19 was identified, which significantly attenuated the tautomerase enzymatic activity of MIF in vitro and in biological functional screens. In the LLC murine model, SCD-19, given intraperitoneally at the time of tumor inoculation, was found to significantly reduce primary tumor volume by 90% (p < 0.001) compared with the control treatment. To better replicate the human disease scenario, SCD-19 was given when the tumor was palpable (at d 7 after tumor inoculation) and, again, treatment was found to significantly reduce tumor volume by 81% (p < 0.001) compared with the control treatment. In this report, we identify a novel inhibitor that blocks the hydrophobic pocket of MIF, which houses its specific tautomerase enzymatic activity, and demonstrate that targeting this unique active site significantly attenuates lung cancer growth in in vitro and in vivo systems.

  4. PKC-ALDH2 Pathway Plays a Novel Role in Adipocyte Differentiation

    PubMed Central

    Yu, Yu-Hsiang; Liao, Pei-Ru; Guo, Chien-Jung; Chen, Che-Hong; Mochly-Rosen, Daria; Chuang, Lee-Ming

    2016-01-01

    The ALDH2 gene encodes the mitochondrial aldehyde dehydrogenase 2 (ALDH2), a critical enzyme involved in ethanol clearance through acetaldehyde metabolism. ALDH2 also catalyzes the metabolism of other bioreactive aldehydes, including propionaldehyde, butyraldehyde, and 4-hydroxykenals (4-HNE). Increased levels of 4-HNE in adipose tissue positively correlate with obesity and insulin resistance. However, it remains unclear whether ALDH2 is involved in regulation of adipocyte differentiation. Here, we found that ALDH2 protein levels were lower in white adipose tissue of high-fat diet-fed mice and ob/ob mice relative to lean mice. Knockdown of ALDH2 expression in 3T3-L1 preadipocytes caused an increase in intracellular 4-HNE, thereby attenuated adipocyte differentiation. By contrast, an ALDH2 activator, Alda-1, significantly accelerated adipogenesis, which was accompanied by an increase in adipogenic gene expression. Consistently, adipogenesis was reduced when protein kinase C ε (PKCε), an ALDH2 phosphorylating activator, was silenced in 3T3-L1 preadipocytes, whereas treatment with a PKCε agonist in 3T3-L1 preadipocytes enhanced adipogenesis. Whole-genome microarray profiling of Alda-1-treated cells demonstrated several upregulated transcripts encoding proteins involved in metabolism and the majority of these transcripts are for proteins involved in PPAR signaling pathways. Furthermore, PKCε-ALDH2 interaction alleviates 4-HNE induced aberrant PPARγ regulation on adipogenesis. Taken together, these results demonstrate that ALDH2 activation enhances adipogenesis and signaling pathways involving PPARγ. Thus, activation of PKCε-ALDH2 regulatory axis may be a therapeutic target for treating obesity and type 2 diabetes. PMID:27575855

  5. Distinct prognostic values and potential drug targets of ALDH1 isoenzymes in non-small-cell lung cancer

    PubMed Central

    You, Qinghua; Guo, Huanchen; Xu, Dongxiang

    2015-01-01

    Increased aldehyde dehydrogenase 1 (ALDH1) activity has been found in the stem cell populations of leukemia and some solid tumors including non-small-cell lung cancer (NSCLC). However, which ALDH1’s isoenzymes are contributing to ALDH1 activity remains elusive. In addition, the prognostic value of individual ALDH1 isoenzyme is not clear. In the current study, we investigated the prognostic value of ALDH1 isoenzymes in NSCLC patients through the Kaplan–Meier plotter database, which contains updated gene expression data and survival information from a total of 1,926 NSCLC patients. High expression of ALDH1A1 mRNA was found to be correlated to a better overall survival (OS) in all NSCLC patients followed for 20 years (hazard ratio [HR] 0.88 [0.77–0.99], P=0.039). In addition, high expression of ALDH1A1 mRNA was also found to be correlated to better OS in adenocarcinoma (Ade) patients (HR 0.71 [0.57–0.9], P=0.0044) but not in squamous cell carcinoma (SCC) patients (HR 0.92 [0.72–1.16], P=0.48). High expression of ALDH1A2 and ALDH1B1 mRNA was found to be correlated to worser OS in all NSCLC patients, as well as in Ade, but not in SCC patients. High expression of both ALDH1A3 and ALDH1L1 mRNA was not found to be correlated to OS in all NSCLC patients. These results strongly support that ALDH1A1 mRNA in NSCLC is associated with better prognosis. In addition, our current study also supports that ALDH1A2 and ALDH1B1 might be major contributors to the ALDH1 activity in NSCLC, since high expression of ALDH1A2 and ALDH1B1 mRNA was found to be significantly correlated to worser OS in all NSCLC patients. Based on our study, ALDH1A2 and ALDH1B1 might be excellent potential drug targets for NSCLC patients. PMID:26366059

  6. Biological activity of camel milk casein following enzymatic digestion.

    PubMed

    Salami, Maryam; Moosavi-Movahedi, Ali Akbar; Moosavi-Movahedi, Faezeh; Ehsani, Mohammad Reza; Yousefi, Reza; Farhadi, Mohammad; Niasari-Naslaji, Amir; Saboury, Ali Akbar; Chobert, Jean-Marc; Haertlé, Thomas

    2011-11-01

    The aim of this study was to investigate the effects of enzymatic hydrolysis with digestive enzymes of camel whole casein and beta-casein (β-CN) on their antioxidant and Angiotensin Converting Enzyme (ACE)-inhibitory properties. Peptides in each hydrolysate were fractionated with ultra-filtration membranes. The antioxidant activity was determined using a Trolox equivalent antioxidant capacity (TEAC) scale. After enzymatic hydrolysis, both antioxidant and ACE-inhibitory activities of camel whole casein and camel β-CN were enhanced. Camel whole casein and β-CN showed significant ACE-inhibitory activities after hydrolysis with pepsin alone and after pepsinolysis followed by trypsinolysis and chymotrypsinolysis. Camel β-CN showed high antioxidant activity after hydrolysis with chymotrypsin. The results of this study suggest that when camel milk is consumed and digested, the produced peptides start to act as natural antioxidants and ACE-inhibitors.

  7. Some enzymatic activities associated with purified parapoxvirions.

    PubMed Central

    Caplen, H S; Holowczak, J A

    1983-01-01

    Purified virions of milker's nodule virus, a parapoxvirus, were shown to contain an RNA polymerase, a nucleotide phosphohydrolase, and a protein kinase associated with or encapsulated within the DNA-containing core of the virus. In vitro, the activated viral RNA polymerase transcribed only 7 to 8% of the genome, in the form of 8S to 14S polyadenylated RNA molecules which were complementary to sequences present in milker's nodule virus DNA but not vaccinia virus DNA or DNA prepared from the host cells in which the virus was propagated. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that in vitro, the activated viral protein kinase phosphorylated viral polypeptides of 95, 60, 33.5, 15, and 13.8 kilodaltons. Images PMID:6188861

  8. Mechanobiocatalysis: Modulating Enzymatic Activity with Mechanical Force

    DTIC Science & Technology

    2015-09-28

    displayed by enzymes and other materials. It was demonstrated that the application of forces to enzymes properly outfitted with polymers resulted in...intrinsic activities displayed by enzymes and other materials. It was demonstrated that the application of forces to enzymes properly outfitted with polymers ...of eYFP-containing polymer composites via the application of mechanical force, as well as showing that the photophysical properties displayed by

  9. [Histidine triad protein superfamily--biological function and enzymatic activity].

    PubMed

    Krakowiak, Agnieszka; Fryc, Izabela

    2012-01-01

    The HIT superfamily consists of proteins that share the histidine triad motif, His-X-His-X-His-X-X (where X is a hydrophobic amino acid), which constitutes enzymatic catalytic center. These enzymes act as nucleotidylyl hydrolase or transferase, and the mutation of the second histidine in the triad abolishes their activity. HIT proteins were found ubiquitous in all organisms and they were classified into 5 branches, which are represented by human proteins: HINT1, FHIT, Aprataxin, GALT and DCPS. Because HINT1 orthologs, which belong to the evolutionally oldest family branch, were found from prokaryotes to eukaryotes, it is clear that HIT motif was conserved during the evolution what means that the enzymatic activity is necessary for functions of these proteins. However, in few cases, e.g. HINT1 and FHIT, the connection between the biological function and the enzymatic activity is still obscure. In this review, the relations between biology and activity for 7 HIT proteins, which were found in human, are highlighted.

  10. BACE1 and BACE2 enzymatic activities in Alzheimer's disease.

    PubMed

    Ahmed, Rachel R; Holler, Christopher J; Webb, Robin L; Li, Feng; Beckett, Tina L; Murphy, M Paul

    2010-02-01

    beta-Secretase is the rate limiting enzymatic activity in the production of the amyloid-beta peptide (Abeta) and is thought to be involved in Alzheimer's disease (AD) pathogenesis. Although BACE1 (beta-site APP Cleaving Enzyme 1, EC 3.4.23.46) has received significant attention, the related BACE2 (EC 3.4.23.45) has not. Though BACE2 is also expressed in the brain, its potential role in AD has not been resolved. In this study, we compared the activities of both BACE1 and BACE2, which were isolated from the same samples of frontal cortex from both AD-affected individuals and age-matched controls. BACE1 activity showed a significant positive correlation with the amount of extractable Abeta, and BACE1 protein and activity were significantly increased in AD cases. Unexpectedly, there were substantial total amounts of BACE2 protein and enzymatic activity in the human brain. BACE2 activity did not change significantly in the AD brain, and was not related to Abeta concentration. These data indicate that BACE1 likely accounts for most of the Abeta produced in the human brain, and that BACE2 activity is not a likely contributor. However, as both forms of BACE compete for the same substrate pool, even small changes in BACE2 activity could have consequences for human disease.

  11. Enzymatic activity preservation and protection through entrapment within degradable hydrogels.

    PubMed

    Mariani, Angela M; Natoli, Mary E; Kofinas, Peter

    2013-11-01

    This work aims to develop a repeatable enzyme entrapment method that preserves activity within an amicable environment while resisting activity reduction in the presence of environmental challenges. Advances in such methods have wide potential use in biosensor applications. In this work β-galactosidase (lactase) enzyme was entrapped within hydrogel matrices of acrylamide (ACR) crosslinked with N,N'-methylenebisacrylamide (BIS, non-degradable) or poly(ethylene glycol) diacrylate (PEGDA, degradable) to create "biogels." Diffusivity studies of control, enzyme free, hydrogel constructs showed near-Fickian swelling behavior in PBS regardless of crosslinker type or density. As expected, the swelling rate, Ks , decreased when increasing the crosslink density from 78.6 to 14.7 min⁻¹ over a range of 1-20 mol% PEGDA indicating that diffusivity into the matrix is dependent on crosslink density. Fabricated biogels were evaluated for maintained enzyme activity in the 7 and 8 pH range. PEGDA crosslinked gels consistently showed improved enzymatic activity retention as compared to BIS crosslinked gels. As PEGDA crosslink density increased from 5 to 10 mol%, enzymatic activity retention post-initial entrapment increased. Higher PEGDA crosslink densities between 15% and 40% decreased enzymatic activity due to assumed steric hindrance of the entrapped enzyme and also decreased substrate and product diffusion. Increased enzymatic stability was observed in 40 mol% PEGDA crosslinked gels. The biogels were pH challenged to 8.0 and stability, measured as retention of activity, was observed to be 91%. Free, non-entrapped, solution based enzyme conversion only retained 23% activity under the same pH challenge conditions. No significant loss of active enzyme was determined to elute out of the biogels during storage in PBS or during biogel wash and recycling. This entrapment method illustrates the potential to sterically hinder and diffusively impede enzymes from performing their

  12. Disruption of the Sjögren-Larsson Syndrome Gene Aldh3a2 in Mice Increases Keratinocyte Growth and Retards Skin Barrier Recovery.

    PubMed

    Naganuma, Tatsuro; Takagi, Shuyu; Kanetake, Tsukasa; Kitamura, Takuya; Hattori, Satoko; Miyakawa, Tsuyoshi; Sassa, Takayuki; Kihara, Akio

    2016-05-27

    The fatty aldehyde dehydrogenase (FALDH) ALDH3A2 is the causative gene of Sjögren Larsson syndrome (SLS). To date, the molecular mechanism underlying the symptoms characterizing SLS has been poorly understood. Using Aldh3a2(-/-) mice, we found here that Aldh3a2 was the major FALDH active in undifferentiated keratinocytes. Long-chain base metabolism was greatly impaired in Aldh3a2(-/-) keratinocytes. Phenotypically, the intercellular spaces were widened in the basal layer of the Aldh3a2(-/-) epidermis due to hyperproliferation of keratinocytes. Furthermore, oxidative stress-induced genes were up-regulated in Aldh3a2(-/-) keratinocytes. Upon keratinocyte differentiation, the activity of another FALDH, Aldh3b2, surpassed that of Aldh3a2 As a result, Aldh3a2(-/-) mice were indistinguishable from wild-type mice in terms of their whole epidermis FALDH activity, and their skin barrier function was uncompromised under normal conditions. However, perturbation of the stratum corneum caused increased transepidermal water loss and delayed barrier recovery in Aldh3a2(-/-) mice. In conclusion, Aldh3a2(-/-) mice replicated some aspects of SLS symptoms, especially at the basal layer of the epidermis. Our results suggest that hyperproliferation of keratinocytes via oxidative stress responses may partly contribute to the ichthyosis symptoms of SLS.

  13. First enzymatically activated Taxotere prodrugs designed for ADEPT and PMT.

    PubMed

    Bouvier, Emmanuel; Thirot, Sylvie; Schmidt, Frédéric; Monneret, Claude

    2004-03-01

    Described here are the syntheses and preliminary biological evaluations of the first two enzymatically activated prodrugs of docetaxel (Taxotere) reported to date. These prodrugs were designed as potential candidates for selective chemotherapy in ADEPT or PMT. They are constituted of a glucuronic acid moiety, a double spacer and the cytotoxic drug, differing only by the spacer substitution. The prodrugs were stable in a buffer, and the in vitro studies showed good detoxification and hydrolysis kinetics. As docetaxel was efficiently released in both cases, these compounds are very valuable candidates for further biological evaluations.

  14. Proteomic Analysis of Mitochondria-Enriched Fraction Isolated from the Frontal Cortex and Hippocampus of Apolipoprotein E Knockout Mice Treated with Alda-1, an Activator of Mitochondrial Aldehyde Dehydrogenase (ALDH2)

    PubMed Central

    Stachowicz, Aneta; Olszanecki, Rafał; Suski, Maciej; Głombik, Katarzyna; Basta-Kaim, Agnieszka; Adamek, Dariusz; Korbut, Ryszard

    2017-01-01

    The role of different genotypes of apolipoprotein E (apoE) in the etiology of Alzheimer’s disease is widely recognized. It has been shown that altered functioning of apoE may promote 4-hydroxynonenal modification of mitochondrial proteins, which may result in mitochondrial dysfunction, aggravation of oxidative stress, and neurodegeneration. Mitochondrial aldehyde dehydrogenase (ALDH2) is an enzyme considered to perform protective function in mitochondria by the detoxification of the end products of lipid peroxidation, such as 4-hydroxynonenal and other reactive aldehydes. The goal of our study was to apply a differential proteomics approach in concert with molecular and morphological techniques to elucidate the changes in the frontal cortex and hippocampus of apolipoprotein E knockout (apoE−/−) mice upon treatment with Alda-1—a small molecular weight activator of ALDH2. Despite the lack of significant morphological changes in the brain of apoE−/− mice as compared to age-matched wild type animals, the proteomic and molecular approach revealed many changes in the expression of genes and proteins, indicating the impairment of energy metabolism, neuroplasticity, and neurogenesis in brains of apoE−/− mice. Importantly, prolonged treatment of apoE−/− mice with Alda-1 led to the beneficial changes in the expression of genes and proteins related to neuroplasticity and mitochondrial function. The pattern of alterations implies mitoprotective action of Alda-1, however, the accurate functional consequences of the revealed changes require further research. PMID:28218653

  15. Efficient enzymatic acrylation through transesterification at controlled water activity.

    PubMed

    Nordblad, Mathias; Adlercreutz, Patrick

    2008-04-15

    Enzymatic acrylation is a process of potentially strong interest to the chemical industry. Direct esterification involving acrylic acid is unfortunately rather slow, with inhibition phenomena appearing at high acid concentrations. In the present study the acrylation of 1-octanol catalyzed by immobilized Candida antarctica lipase B (Novozym 435) was shown to be as much as an order of magnitude faster when ethyl acrylate served as the donor of the acrylic group. Water activity is a key parameter for optimizing the rate of ester synthesis. The optimum water activity for the esterification of octanol by acrylic acid was found to be 0.75, that for its esterification by propionic acid to be 0.45 and the transesterification involving ethyl acrylate to be fastest at a water activity of 0.3. The reasons for these differences in optimum water activity are discussed in terms of enzyme specificity, substrate solvation, and mass transfer effects.

  16. ALDH2 modulates autophagy flux to regulate acetaldehyde-mediated toxicity thresholds

    PubMed Central

    Tanaka, Koji; Whelan, Kelly A; Chandramouleeswaran, Prasanna M; Kagawa, Shingo; Rustgi, Sabrina L; Noguchi, Chiaki; Guha, Manti; Srinivasan, Satish; Amanuma, Yusuke; Ohashi, Shinya; Muto, Manabu; Klein-Szanto, Andres J; Noguchi, Eishi; Avadhani, Narayan G; Nakagawa, Hiroshi

    2016-01-01

    A polymorphic mutation in the acetaldehyde dehydrogenase 2 (ALDH2) gene has been epidemiologically linked to the high susceptibility to esophageal carcinogenesis for individuals with alcohol use disorders. Mice subjected to alcohol drinking show increased oxidative stress and DNA adduct formation in esophageal epithelia where Aldh2 loss augments alcohol-induced genotoxic effects; however, it remains elusive as to how esophageal epithelial cells with dysfunctional Aldh2 cope with oxidative stress related to alcohol metabolism. Here, we investigated the role of autophagy in murine esophageal epithelial cells (keratinocytes) exposed to ethanol and acetaldehyde. We find that ethanol and acetaldehyde trigger oxidative stress via mitochondrial superoxide in esophageal keratinocytes. Aldh2-deficient cells appeared to be highly susceptible to ethanol- or acetaldehyde-mediated toxicity. Alcohol dehydrogenase-mediated acetaldehyde production was implicated in ethanol-induced cell injury in Aldh2 deficient cells as ethanol-induced oxidative stress and cell death was partially inhibited by 4-methylpyrazole. Acetaldehyde activated autophagy flux in esophageal keratinocytes where Aldh2 deficiency increased dependence on autophagy to cope with ethanol-induced acetaldehyde-mediated oxidative stress. Pharmacological inhibition of autophagy flux by chloroquine stabilized p62/SQSTM1, and increased basal and acetaldehyde-mediate oxidative stress in Aldh2 deficient cells as documented in monolayer culture as well as single-cell derived three-dimensional esophageal organoids, recapitulating a physiological esophageal epithelial proliferation-differentiation gradient. Our innovative approach indicates, for the first time, that autophagy may provide cytoprotection to esophageal epithelial cells responding to oxidative stress that is induced by ethanol and its major metabolite acetaldehyde. Defining autophagymediated cytoprotection against alcohol-induced genotoxicity in the context of

  17. Enzymatic Activity of the Scaffold Protein Rapsyn for Synapse Formation.

    PubMed

    Li, Lei; Cao, Yu; Wu, Haitao; Ye, Xinchun; Zhu, Zhihui; Xing, Guanglin; Shen, Chengyong; Barik, Arnab; Zhang, Bin; Xie, Xiaoling; Zhi, Wenbo; Gan, Lin; Su, Huabo; Xiong, Wen-Cheng; Mei, Lin

    2016-12-07

    Neurotransmission is ensured by a high concentration of neurotransmitter receptors at the postsynaptic membrane. This is mediated by scaffold proteins that bridge the receptors with cytoskeleton. One such protein is rapsyn (receptor-associated protein at synapse), which is essential for acetylcholine receptor (AChR) clustering and NMJ (neuromuscular junction) formation. We show that the RING domain of rapsyn contains E3 ligase activity. Mutation of the RING domain that abolishes the enzyme activity inhibits rapsyn- as well as agrin-induced AChR clustering in heterologous and muscle cells. Further biological and genetic studies support a working model where rapsyn, a classic scaffold protein, serves as an E3 ligase to induce AChR clustering and NMJ formation, possibly by regulation of AChR neddylation. This study identifies a previously unappreciated enzymatic function of rapsyn and a role of neddylation in synapse formation, and reveals a potential target of therapeutic intervention for relevant neurological disorders.

  18. A designed supramolecular protein assembly with in vivo enzymatic activity.

    PubMed

    Song, Woon Ju; Tezcan, F Akif

    2014-12-19

    The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(k(cat)/K(m))/k(uncat)] for ampicillin hydrolysis of 2.3 × 10(6) and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.

  19. Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants

    SciTech Connect

    Dabulis, K.; Klibanov, A.M. )

    1993-03-05

    When seven different hydrolytic enzymes (four proteases and three lipases) were lyophilized from aqueous solution containing a ligand, N-Ac-L-Phe-NH[sub 2], their catalytic activity in anhydrous solvents was far greater (one to two orders of magnitude) than that of the enzymes lyophilized without the ligand. This ligand-induced activation was expressed regardless of whether the substrate employed in organic solvents structurally resembled the ligand. Furthermore, nonligand lyoprotectants [sorbitol, other sugars, and poly(ethylene glycol)] also dramatically enhanced enzymatic activity in anhydrous solvents when present in enzyme aqueous solution prior to lyophilization. The effects of the ligand and of the lyoprotectants were nonadditive, suggesting the same mechanism of action. Excipient-activated and nonactivated enzymes exhibited identical activities in water. Also, addition of the excipients directly to suspensions of nonactivated enzymes in organic solvents had no appreciable effect on catalytic activity. These observations indicate that the mechanism of the excipient-induced activation is based on the ability of the excipients to alleviate reversible denaturation of enzymes upon lyophilization. Activity enhancement induced by the excipients is displayed even after their removal by washing enzymes with anhydrous solvents. Subtilisin Carlsberg, lyophilized with sorbitol, was found to be a much more efficient practical catalyst than its regular' counterpart.

  20. Enzymatic activity preservation through entrapment within degradable hydrogel networks

    NASA Astrophysics Data System (ADS)

    Mariani, Angela Marie

    This dissertation aimed to design and develop a "biogel;" a reproducible, abiotic, and biocompatible polymer hydrogel matrix, that prolongs enzymatic stability allowing for rapid production of biomolecules. The researched entrapment method preserves enzyme activity within an amicable environment while resisting activity reduction in the presence of increased pH environmental challenges. These biogels can be used in a number of applications including repeated production of small molecules and in biosensors. Five main objectives were accomplished: 1) Biogels capable of maintaining enzymatic functionality post-entrapment procedures were fabricated; 2) Biogel activity dependence on crosslinker type and crosslink density was determined; 3) Biogel composition effects on sustained activity after storage were compared; 4) Biogel activity dependence on charged monomer moieties was evaluated, and 5) Combined optimization knowledge gained from the first four objectives was utilized to determine the protection of enzymes within hydrogels when challenged with an increased pH above 8. Biogels were fabricated by entrapping β-galactosidase (lactase) enzyme within acrylamide (ACR) gels crosslinked with poly(ethylene glycol) diacrylate (PEGDA, degradable through hydrolysis) or N,N'-methylenebisacrylamide (BIS, non-degradable). Initial hydrogel entrapment reduced activity to 40% in ACR/PEGDA gels, compared to a 75% reduction in initial activity of ACR/BIS biogels. Once entrapped, these enzymes resist activity reduction in the presence of environmental challenges, such as altering the pH from 7 to above 8. When biogels were challenged at a pH of 8, activity retention positively correlated to PEGDA crosslinker density; increasing from 48% to 91% retention in 30 to 40 mole % PEGDA biogels as compared to solution based control which retained only 23%. Retention of activity when perturbed from pH 7 is advantageous for biogel applications including the repeated production of desired small

  1. The enzymatic activity from the sediment of the Gilau dam reservoir - Cluj county.

    PubMed

    Curticapean, Manuela-Claudia; Dragan-Bularda, Mihail

    2007-01-10

    The enzymological studies on the sediment of the accumulation lake that has the main purpose of supplying drinking water to the city of Cluj-Napoca and the nearby villages, were aimed at the comprehensive understanding of the complex processes that happen in these habitats of special significance. In the sediment samples the following enzymatic activities have been quantitatively determined: phosphatase, actual and potential dehydrogenase, catalase, urease and protease. Non-enzymatic catalytic activity was also measured. Based on the relative values for the enzymatic activities, the enzymatic indicator of the sediment quality (EISQ) was calculated (ranging from 0.1 to 0.7). The enzymatic activities have been qualitatively determined for maltase, saccharase, lactase, cellobiase, amylase, dextranase, levanase, cellulase and inulinase. The correlation between the enzymatic and bacteriologic potential was statistically calculated.

  2. Nanoparticle-mediated remote control of enzymatic activity.

    PubMed

    Knecht, Leslie D; Ali, Nur; Wei, Yinan; Hilt, J Zach; Daunert, Sylvia

    2012-10-23

    Nanomaterials have found numerous applications as tunable, remotely controlled platforms for drug delivery, hyperthermia cancer treatment, and various other biomedical applications. The basis for the interest lies in their unique properties achieved at the nanoscale that can be accessed via remote stimuli. These properties could then be exploited to simultaneously activate secondary systems that are not remotely actuatable. In this work, iron oxide nanoparticles are encapsulated in a bisacrylamide cross-linked polyacrylamide hydrogel network along with a model dehalogenase enzyme, L-2-HAD(ST). This thermophilic enzyme is activated at elevated temperatures and has been shown to have optimal activity at 70 °C. By exposing the Fe(3)O(4) nanoparticles to a remote stimulus, an alternating magnetic field (AMF), enhanced system heating can be achieved, thus remotely activating the enzyme. The internal heating of the nanocomposite hydrogel network in the AMF results in a 2-fold increase in enzymatic activity as compared to the same hydrogel heated externally in a water bath, suggesting that the internal heating of the nanoparticles is more efficient than the diffusion-limited heating of the water bath. This system may prove useful for remote actuation of biomedical and environmentally relevant enzymes and find applications in a variety of fields.

  3. Pepper aldehyde dehydrogenase CaALDH1 interacts with Xanthomonas effector AvrBsT and promotes effector-triggered cell death and defence responses.

    PubMed

    Kim, Nak Hyun; Hwang, Byung Kook

    2015-06-01

    Xanthomonas type III effector AvrBsT induces hypersensitive cell death and defence responses in pepper (Capsicum annuum) and Nicotiana benthamiana. Little is known about the host factors that interact with AvrBsT. Here, we identified pepper aldehyde dehydrogenase 1 (CaALDH1) as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and co-immunoprecipitation assays confirmed the interaction between CaALDH1 and AvrBsT in planta. CaALDH1:smGFP fluorescence was detected in the cytoplasm. CaALDH1 expression in pepper was rapidly and strongly induced by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) Ds1 (avrBsT) infection. Transient co-expression of CaALDH1 with avrBsT significantly enhanced avrBsT-triggered cell death in N. benthamiana leaves. Aldehyde dehydrogenase activity was higher in leaves transiently expressing CaALDH1, suggesting that CaALDH1 acts as a cell death enhancer, independently of AvrBsT. CaALDH1 silencing disrupted phenolic compound accumulation, H2O2 production, defence response gene expression, and cell death during avirulent Xcv Ds1 (avrBsT) infection. Transgenic Arabidopsis thaliana overexpressing CaALDH1 exhibited enhanced defence response to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis infection. These results indicate that cytoplasmic CaALDH1 interacts with AvrBsT and promotes plant cell death and defence responses.

  4. Controlling enzymatic activity by immobilization on graphene oxide.

    PubMed

    Bolibok, Paulina; Wiśniewski, Marek; Roszek, Katarzyna; Terzyk, Artur P

    2017-04-01

    In this study, graphene oxide (GO) has been applied as a matrix for enzyme immobilization. The protein adsorption capacity of GO is much higher than of other large surface area carbonaceous materials. Its structure and physicochemical properties are reported beneficial also for enzymatic activity modifications. The experimental proof was done here that GO-based biocatalytic systems with immobilized catalase are modifiable in terms of catalyzed reaction kinetic constants. It was found that activity and stability of catalase, considered here as model enzyme, closely depend on enzyme/GO ratio. The changes in kinetic parameters can be related to secondary structure alterations. The correlation between enzyme/GO ratio and kinetic and structure parameters is reported for the first time and enables the conscious control of biocatalytic processes and their extended applications. The biological activity of obtained biocatalytic systems was confirmed in vitro by the use of functional test. The addition of immobilized catalase improved the cells' viability after they were exposed to hydrogen peroxide and tert-butyl-hydroperoxide used as source of reactive oxygen species.

  5. Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

    NASA Astrophysics Data System (ADS)

    Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

    2015-04-01

    On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

  6. Aberrant expression of aldehyde dehydrogenase 1A (ALDH1A) subfamily genes in acute lymphoblastic leukaemia is a common feature of T-lineage tumours.

    PubMed

    Longville, Brooke A C; Anderson, Denise; Welch, Mathew D; Kees, Ursula R; Greene, Wayne K

    2015-01-01

    The class 1A aldehyde dehydrogenase (ALDH1A) subfamily of genes encode enzymes that function at the apex of the retinoic acid (RA) signalling pathway. We detected aberrant expression of ALDH1A genes, particularly ALDH1A2, in a majority (72%) of primary paediatric T cell acute lymphoblastic leukaemia (T-ALL) specimens. ALDH1A expression was almost exclusive to T-lineage, but not B-lineage, ALL. To determine whether ALDH1A expression may have relevance to T-ALL cell growth and survival, the effect of inhibiting ALDH1A function was measured on a panel of human ALL cell lines. This revealed that T-ALL proliferation had a higher sensitivity to modulation of ALDH1A activity and RA signalling as compared to ALL cell lines of B-lineage. Consistent with these findings, the genes most highly correlated with ALDH1A2 expression were involved in cell proliferation and apoptosis. Evidence that such genes may be targets of regulation via RA signalling initiated by ALDH1A activity was provided by the TNFRSF10B gene, encoding the apoptotic death receptor TNFRSF10B (also termed TRAIL-R2), which negatively correlated with ALDH1A2 and showed elevated transcription following treatment of T-ALL cell lines with the ALDH1A inhibitor citral (3,7-dimethyl-2,6-octadienal). These data indicate that ALDH1A expression is a common event in T-ALL and supports a role for these enzymes in the pathobiology of this disease.

  7. Enzymatic activity of albumin shown by coelenterazine chemiluminescence.

    PubMed

    Vassel, N; Cox, C D; Naseem, R; Morse, V; Evans, R T; Power, R L; Brancale, A; Wann, K T; Campbell, A K

    2012-01-01

    Bioluminescence, the emission of light from live organisms, occurs in 18 phyla and is the major communication system in the deep sea. It has appeared independently many times during evolution but its origins remain unknown. Coelenterazine bioluminescence discovered in luminous jellyfish is the most common chemistry causing bioluminescence in the sea, occurring in seven phyla. Sequence similarities between coelenterazine luciferases and photoproteins from different phyla are poor (often < 5%). The aim of this study was to examine albumin that binds organic substances as a coelenterazine luciferase to test the hypothesis that the evolutionary origin of a bioluminescent protein was the result of the formation of a solvent cage containing just a few key amino acids. The results show for the first time that bovine and human albumin catalysed coelenterazine chemiluminescence consistent with a mono-oxygenase, whereas gelatin and haemoglobin, an oxygen carrier, had very weak activity. Insulin also catalysed coelenterazine chemiluminescence and was increased by Zn(2+). Albumin chemiluminescence was heat denaturable, exhibited saturable substrate characteristics and was inhibited by cations that bound these proteins and by drugs that bind to human albumin drug site I. Molecular modelling confirmed the coelenterazine binding site and identified four basic amino acids: lys195, arg222, his242 and arg257, potentially important in binding and catalysis similar to naturally occurring coelenterazine bioluminescent proteins. These results support the 'solvent cage' hypothesis for the evolutionary origin of enzymatic coelenterazine bioluminescent proteins. They also have important consequences in diseases such as diabetes, gut disorders and food intolerance where a mono-oxygenase could affect cell surface proteins.

  8. Enzymatic activities in limb muscles subjected to external fixation with ring-hybrid frames.

    PubMed

    Reznick, Abraham Z; Coleman, Raymond; Stein, Haim

    2007-04-01

    Enzymatic activities, which originate in the muscle envelope of tibiae with an experimental segmental bone loss, provide additional evidence for the intimate bone-muscle interrelationships in new bone formation.

  9. Enzymatic activity inside and outside of water-stable aggregates in soils under different land use

    NASA Astrophysics Data System (ADS)

    Garbuz, S. A.; Yaroslavtseva, N. V.; Kholodov, V. A.

    2016-03-01

    A method is presented for assessing the distribution of enzymatic activity inside and outside of water-stable aggregates. Two samples of water-stable aggregates >1 mm have been isolated from dry aggregates of 1-2 mm. To determine the enzymatic activity, a substrate has been added to one of the samples without disaggregation; the other sample has been preliminarily disaggregated. Enzymatic activity within waterstable aggregates has been assessed from the difference between the obtained results under the supposition that the penetration of substrate within the water-saturated aggregates is hampered, and enzymatic reactions occur only at the periphery. The levels and distributions of enzymatic (peroxidase, polyphenol oxidase, and catalase) activities in water-stable aggregates of soddy-podzolic soils under forest and plowland and typical chernozems of long-term field experiments have been studied. The peroxidase, polyphenol oxidase, and catalase activities of water-stable aggregates vary from 6 to 23, from 7 to 30, and from 5 to 7 mmol/(g h), respectively. The ratio between the enzymatic activities inside and outside of soil aggregates showed a higher dependence on soil type and land use, as well as on the input of organic matter and the structural state, than the general activity level in water-stable aggregates.

  10. Controlling enzymatic activity and kinetics in swollen mesophases by physical nano-confinement.

    PubMed

    Sun, Wenjie; Vallooran, Jijo J; Zabara, Alexandru; Mezzenga, Raffaele

    2014-06-21

    Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them into a highly confined environment. We show that the enzymatic activity of a model enzyme, horseradish peroxidase (HRP), can be accurately controlled by relaxing its confinement within the cubic phases' water channels, when the aqueous channel diameters are systematically swollen with varying amount of hydration-enhancing sugar ester. The in-meso activity and kinetics of HRP are then systematically investigated by UV-vis spectroscopy, as a function of the size of the aqueous mesophase channels. The enzymatic activity of HRP increases with the swelling of the water channels. In swollen mesophases with water channel diameter larger than the HRP size, the enzymatic activity is more than double that measured in standard mesophases, approaching again the enzymatic activity of free HRP in bulk water. We also show that the physically-entrapped enzymes in the mesophases exhibit a restricted-diffusion-induced initial lag period and report the first observation of in-meso enzymatic kinetics significantly deviating from the normal Michaelis-Menten behaviour observed in free solutions, with deviations vanishing when enzyme confinement is released by swelling the mesophase.

  11. Adsorption-induced changes in ribonuclease A structure and enzymatic activity on solid surfaces.

    PubMed

    Wei, Yang; Thyparambil, Aby A; Wu, Yonnie; Latour, Robert A

    2014-12-16

    Ribonuclease A (RNase A) is a small globular enzyme that lyses RNA. The remarkable solution stability of its structure and enzymatic activity has led to its investigation to develop a new class of drugs for cancer chemotherapeutics. However, the successful clinical application of RNase A has been reported to be limited by insufficient stability and loss of enzymatic activity when it was coupled with a biomaterial carrier for drug delivery. The objective of this study was to characterize the structural stability and enzymatic activity of RNase A when it was adsorbed on different surface chemistries (represented by fused silica glass, high-density polyethylene, and poly(methyl-methacrylate)). Changes in protein structure were measured by circular dichroism, amino acid labeling with mass spectrometry, and in vitro assays of its enzymatic activity. Our results indicated that the process of adsorption caused RNase A to undergo a substantial degree of unfolding with significant differences in its adsorbed structure on each material surface. Adsorption caused RNase A to lose about 60% of its native-state enzymatic activity independent of the material on which it was adsorbed. These results indicate that the native-state structure of RNase A is greatly altered when it is adsorbed on a wide range of surface chemistries, especially at the catalytic site. Therefore, drug delivery systems must focus on retaining the native structure of RNase A in order to maintain a high level of enzymatic activity for applications such as antitumor chemotherapy.

  12. ALDH2 attenuates Dox-induced cardiotoxicity by inhibiting cardiac apoptosis and oxidative stress.

    PubMed

    Gao, Yawen; Xu, Yan; Hua, Songwen; Zhou, Shenghua; Wang, Kangkai

    2015-01-01

    The anthracycline chemotherapy drug doxorubicin (DOX) is cardiotoxic. This study aimed to explore the effect of acetaldehyde dehydrogenase 2 (ALDH2), a detoxifying protein, on DOX-induced cardiotoxicity and unveil the underlying mechanisms. BALB/c mice were randomly divided in four groups: control group (no treatment), DOX group (DOX administration for myocardial damage induction), DOX + Daidzin group (DOX administration + Daidzin, an ALDH2 antagonist) and DOX + Alda-1 group (DOX administration + Alda-1, an ALDH2 agonist). Then, survival, haemodynamic parameters, expression of pro- and anti-apoptosis markers, reactive oxygen species (ROS) and 4-Hydroxynonenal (4-HNE) levels, expression and localization of NADPH oxidase 2 (NOX2) and its cytoplasmic subunit p47(PHOX), and ALDH2 expression and activity were assessed. Mortality rates of 0, 35, 5, and 70% were obtained in the control, DOX, DOX + Alda-1, and DOX + Daidzin groups, respectively, at the ninth weekend. Compared with control animals, DOX treatment resulted in significantly reduced left ventricular systolic pressure (LVSP) and ± dp/dt, and overtly increased left ventricular end-diastolic pressure (LVEDP); increased Bax expression and caspase-3/7 activity, and reduced Bcl-2 expression in the myocardium; increased ROS (about 2 fold) and 4-HNE adduct (3 fold) levels in the myocardium; increased NOX2 protein expression and membrane translocation of P47(PHOX). These effects were aggravated in the DOX + Daidzin group, DOX + Alda-1 treated animals showed partial or complete alleviation. Finally, Daidzin further reduced the DOX-repressed ALDH2 activity, which was partially rescued by Alda-1. These results indicated that ALDH2 attenuates DOX-induced cardiotoxicity by inhibiting oxidative stress, NOX2 expression and activity, and reducing myocardial apoptosis.

  13. Inhibition of ALDH2 by O-GlcNAcylation contributes to the hyperglycemic exacerbation of myocardial ischemia/reperfusion injury.

    PubMed

    Liu, Baoshan; Wang, Jiali; Li, Minghua; Yuan, Qiuhuan; Xue, Mengyang; Xu, Feng; Chen, Yuguo

    2016-12-27

    Although hyperglycemia is causally related to adverse outcomes after myocardial ischemia/reperfusion (I/R), the underlying mechanisms are largely unknown. Here, we investigated whether excessive O-linked-N-acetylglucosamine (O-GlcNAc) modification of acetaldehyde dehydrogenase 2 (ALDH2), an important cardioprotective enzyme, was a mechanism for the hyperglycemic exacerbation of myocardial I/R injury. Both acute hyperglycemia (AHG) and diabetes (DM)-induced chronic hyperglycemia increased cardiac dysfunction, infarct size and apoptosis index compared with normal saline (NS)+I/R rats (P<0.05). ALDH2 O-GlcNAc modification was increased whereas its activity was decreased in AHG+I/R and DM+I/R rats. High glucose (HG, 30mmol/L) markedly increased ALDH2 O-GlcNAc modification compared with Con group (5mmol/L) (P<0.05). ALDH2 O-GlcNAc modification was increased by 62.9% in Con+PUGNAc group whereas it was decreased by 44.1% in Con+DON group compared with Con group (P<0.05). Accordingly, ALDH2 activity was decreased by 18.1% in Con+PUGNAc group whereas it was increased by 17.9% in Con+DON group. Moreover, DON decreased levels of 4-hydroxy-2-nonenal (4-HNE), aldehydes, protein carbonyl accumulation and apoptosis index compared with HG+H/R group (P<0.05). Alda-1, a specific activator of ALDH2, significantly decreased ALDH2 O-GlcNAc modification and improved infarct size, apoptosis index and cardiac dysfunction induced by I/R combined with hyperglycemia. These findings demonstrate that ALDH2 O-GlcNAc modification is a key mechanism for the hyperglycemic exacerbation of myocardial I/R injury and Alda-1 has therapeutic potential for inducing cardioprotection.

  14. Development and validation of a 96-well cellular assay for the discovery of ALDH1A1 inhibitors.

    PubMed

    Ming, Wenyu; Ma, Wenzhen; Chen, Lisa H; Volk, Catherine; Michael, Mervyn Dodson; Xu, Yanping; Zhang, Fang; Wang, Xiaojun

    2013-07-01

    Retinoic acid, the active metabolite of vitamin A, plays important roles in various physiological and pathological processes. The two-step production of retinoic acid from vitamin A (retinol) is catalyzed by alcohol dehydrogenases and aldehyde dehydrogenases, which are potential therapeutic targets for numerous diseases, such as obesity, diabetes, and cancer. Currently, the lack of a suitable high-throughput cellular assay hinders efforts to identify therapeutic small molecular inhibitors of aldehyde dehydrogenase, such as ALDH1A1. In this report, we utilized high-content imaging technology and a commercially available cell permeable ALDH substrate to develop a 96-well cellular ALDH1A1 assay. This assay has a robust and sensitive readout and is amenable to automation. With this cellular assay, we identified potent selective ALDH1A1 inhibitors to explore the role of retinoic acid production in various preclinical disease models.

  15. Controlling enzymatic activity and kinetics in swollen mesophases by physical nano-confinement

    NASA Astrophysics Data System (ADS)

    Sun, Wenjie; Vallooran, Jijo J.; Zabara, Alexandru; Mezzenga, Raffaele

    2014-05-01

    Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them into a highly confined environment. We show that the enzymatic activity of a model enzyme, horseradish peroxidase (HRP), can be accurately controlled by relaxing its confinement within the cubic phases' water channels, when the aqueous channel diameters are systematically swollen with varying amount of hydration-enhancing sugar ester. The in-meso activity and kinetics of HRP are then systematically investigated by UV-vis spectroscopy, as a function of the size of the aqueous mesophase channels. The enzymatic activity of HRP increases with the swelling of the water channels. In swollen mesophases with water channel diameter larger than the HRP size, the enzymatic activity is more than double that measured in standard mesophases, approaching again the enzymatic activity of free HRP in bulk water. We also show that the physically-entrapped enzymes in the mesophases exhibit a restricted-diffusion-induced initial lag period and report the first observation of in-meso enzymatic kinetics significantly deviating from the normal Michaelis-Menten behaviour observed in free solutions, with deviations vanishing when enzyme confinement is released by swelling the mesophase.Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them

  16. Novel enhancement mechanism of tyrosine hydroxylase enzymatic activity by nitric oxide through S-nitrosylation

    PubMed Central

    Wang, Yuanyuan; Sung, Chun Chau; Chung, Kenny K. K.

    2017-01-01

    Tyrosine hydroxylase (TH) is a rate-limiting step enzyme in the synthesis of catecholamines. Catecholamines function both as hormone and neurotransmitters in the peripheral and central nervous systems, therefore TH’s expression and enzymatic activity is tightly regulated by various mechanisms. Several post-translational modifications have been shown to regulate TH’s enzymatic activity such as phosphorylation, nitration and S-glutathionylation. While phosphorylation at N-terminal of TH can activate its enzymatic activity, nitration and S-glutathionylation can inactivate TH. In this study, we found that TH can also be S-nitrosylated by nitric oxide (NO). S-nitrosylation is a reversible modification of cysteine (cys) residue in protein and is known to be an emerging signaling mechanism mediated by NO. We found that TH can be S-nitrosylated at cys 279 and TH S-nitrosylation enhances its enzymatic activity both in vitro and in vivo. These results provide a novel mechanism of how NO can modulate TH’s enzymatic activity through S-nitrosylation. PMID:28287127

  17. Soluble expression and enzymatic activity evaluation of protease from reticuloendotheliosis virus.

    PubMed

    Hu, Feng; Zhao, Yan; Qi, Xiaole; Cui, Hongyu; Gao, Yulong; Gao, Honglei; Liu, Changjun; Wang, Yongqiang; Zhang, Yanping; Li, Kai; Wang, Xiaomei; Wang, Yunfeng

    2015-10-01

    The protease (PR) encoded by most retroviruses is deeply involved in the lifecycle and infection process of retroviruses by possessing the specificity necessary to correctly cleave the viral polyproteins and host cell proteins. However, as an important representative of avian retroviruses, the enzymatic properties of PR from reticuloendotheliosis virus (REV) have not been clearly documented. The recombinant PR, its mutant fused with a His-tag, and its substrate p18-p30 fused with a GST-tag were expressed in the Escherichia coli system as soluble enzymes. The soluble PR and p18-p30 were purified using Ni-NTA His Bind Resin and Glutathione Sepharose 4B, respectively. The enzymatic activity of PR was analyzed using the substrate of p18-p30. The expressed prokaryotic protease has enzyme activity that is dependent on such conditions as temperature, pH, and ions, and its activity can be inhibited by caspase inhibitor and the divalent metal ions Ca(2+) and Ni(2+). In addition, the key role of the residue Thr (amino acids 28) for the enzymatic activity of PR was identified. Furthermore, the caspase inhibitor Z-VAD-FMK was confirmed to inhibit the PR enzymatic activity of REV. For the first time, the PR of REV was expressed in the soluble form, and the optimal enzymatic reaction system in vitro was developed and preliminarily used. This study provides essential tools and information for further understanding the infection mechanism of REV and for the development of antiviral drugs treating retroviruses.

  18. Enzymatic activity regulated by a surfactant and hydroxypropyl β-cyclodextrin.

    PubMed

    Li, Qingzhong; Zhai, Tao; Du, Kun; Li, Yanxin; Feng, Wei

    2013-12-01

    Circular dichroism spectra reveal that sodium dodecyl benzene sulfonate (SDBS) at low concentrations can effectively prevent the aggregation of lysozyme molecules, while SDBS at high concentrations can lead to conformational and structural change of the protein. SDBS is able to inhibit the enzymatic activity of lysozyme in a highly efficient dose-dependent manner. The interaction mechanism of SDBS with lysozyme has been investigated by measuring optical spectra. Based on fluorescence and UV-vis spectra, microenvironmental change in and around the active site region induced by SDBS has been revealed and explained. Two-dimensional FTIR spectra have been analyzed to identify the secondary structures and residues of lysozyme, which have a preferential interaction with SDBS. Hydroxypropyl β-cyclodextrin (HP-β-CD) was used to detach SDBS from the inactivated enzyme, and complete recovery of enzymatic activity was achieved. Thus, the enzymatic activity of lysozyme can be regulated by SDBS and HP-β-CD.

  19. Biologically Active Oxylipins from Enzymatic and Nonenzymatic Routes in Macroalgae

    PubMed Central

    Barbosa, Mariana; Valentão, Patrícia; Andrade, Paula B.

    2016-01-01

    Marine algae are rich and heterogeneous sources of great chemical diversity, among which oxylipins are a well-recognized class of natural products. Algal oxylipins comprise an assortment of oxygenated, halogenated, and unsaturated functional groups and also several carbocycles, varying in ring size and position in lipid chain. Besides the discovery of structurally diverse oxylipins in macroalgae, research has recently deciphered the role of some of these metabolites in the defense and innate immunity of photosynthetic marine organisms. This review is an attempt to comprehensively cover the available literature on the chemistry, biosynthesis, ecology, and potential bioactivity of oxylipins from marine macroalgae. For a better understanding, enzymatic and nonenzymatic routes were separated; however, both processes often occur concomitantly and may influence each other, even producing structurally related molecules. PMID:26805855

  20. Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

    PubMed

    Al Khamici, Heba; Brown, Louise J; Hossain, Khondker R; Hudson, Amanda L; Sinclair-Burton, Alxcia A; Ng, Jane Phui Mun; Daniel, Elizabeth L; Hare, Joanna E; Cornell, Bruce A; Curmi, Paul M G; Davey, Mary W; Valenzuela, Stella M

    2015-01-01

    The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

  1. Effect of length of molecular recognition moiety on enzymatic activity switching.

    PubMed

    Oshiba, Yuhei; Tamaki, Takanori; Ohashi, Hidenori; Hirakawa, Hidehiko; Yamaguchi, Satoshi; Nagamune, Teruyuki; Yamaguchi, Takeo

    2013-10-01

    We site-specifically conjugated biotin-PEG derivatives with spacer arms of different lengths to mutant P450cam (3mD) and evaluated the activity of and structural changes in the conjugates as a first step toward clarifying the mechanism whereby the activity of the 3mD conjugate is inhibited. 3mD was prepared by site-specific mutation to inhibit its enzymatic activity artificially, after which the derivative compounds were conjugated to the enzyme. 3mD has one cysteine on its surface with a reactive thiol group that can react with compounds near the active site, where a conformational change will be induced after conjugation. The activity of 3mD was retained in the biotin-PEG₂-3mD conjugate, but was dramatically reduced in the biotin-PEG₁₁-3mD conjugate. To investigate the effect of poly(ethylene glycol) (PEG) length on the enzymatic activity after conjugation, PEGs of different lengths, exceeding that in biotin-PEG₁₁, and whose termini were not biotin, were conjugated to 3mD. The activity of 3mD decreased in all these conjugates. This indicates that the activity of 3mD in these conjugates decreased after its conjugation with PEG molecules that exceeded a certain length. The biotin-PEG₂-3mD, which retains enzymatic activity after conjugation, showed avidin responsiveness; the enzymatic activity decreased after avidin binding.

  2. NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ...

  3. NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ALDH2 catalyzes oxidation of toxic aldehydes to their corresponding carboxylic acids. Magnesium ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements have monitored the blue shift of the NADH fluorescence spectrum to elucidate the extent of...

  4. A Survey of Enzymatic Activity in Commercially Available Pool and Spa Products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many pool water treatment products currently available commercially claim that they work effectively by possessing enzyme activity (specifically lipase) that degrades common oil (lipid) contaminants found in pool water. Currently, there is no standard in measuring the enzymatic activity of these enz...

  5. A Survey of Enzymatic Activity in Commercially Available Pool and Spa Products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many pool water treatment products currently available commercially claim that they work effectively by possessing enzyme activity (specifically lipase) that degrades common oil (lipid) contaminants found in pool water. Currently, there is no standard in measuring the enzymatic activity of these en...

  6. Thrombolytic efficacy and enzymatic activity of rt-PA-loaded echogenic liposomes.

    PubMed

    Bader, Kenneth B; Bouchoux, Guillaume; Peng, Tao; Klegerman, Melvin E; McPherson, David D; Holland, Christy K

    2015-08-01

    Echogenic liposomes (ELIP), that can encapsulate both recombinant tissue-type plasminogen activator (rt-PA) and microbubbles, are under development to improve the treatment of thrombo-occlusive disease. However, the enzymatic activity, thrombolytic efficacy, and stable cavitation activity generated by this agent has yet to be evaluated and compared to another established ultrasound-enhanced thrombolytic scheme. A spectrophotometric method was used to compare the enzymatic activity of the rt-PA incorporated into ELIP (t-ELIP) to that of rt-PA. An in vitro flow model was employed to measure the thrombolytic efficacy and dose of ultraharmonic emissions from stable cavitation for 120-kHz ultrasound exposure of three treatment schemes: rt-PA, rt-PA and the perfluorocarbon-filled microbubble Definity(®), and t-ELIP. The enzymatic activity of rt-PA incorporated into t-ELIP was 28 % that of rt-PA. Thrombolytic efficacy of t-ELIP or rt-PA and Definity(®) was equivalent when the dose of t-ELIP was adjusted to produce comparable enzymatic activity. Sustained bubble activity was nucleated from Definity but not from t-ELIP exposed to 120-kHz ultrasound. These results emphasize the advantages of encapsulating a thrombolytic and the importance of incorporating an insoluble gas required to promote sustained, stable cavitation activity.

  7. A new biomimetic route to engineer enzymatically active mechano-responsive materials.

    PubMed

    Rios, César; Longo, Johan; Zahouani, Sarah; Garnier, Tony; Vogt, Cédric; Reisch, Andreas; Senger, Bernard; Boulmedais, Fouzia; Hemmerlé, Joseph; Benmlih, Karim; Frisch, Benoît; Schaaf, Pierre; Jierry, Loïc; Lavalle, Philippe

    2015-04-04

    Using modified β-galactosidase covalently linked to cross-linked polyelectrolyte multilayers (PEM), catalytically active materials have been designed. Their enzymatic activity can be modulated, partially in a reversible way, simply by stretching. This strategy, based on enzyme conformational changes, constitutes a new tool for the development of biocatalytic mechano-responsive materials.

  8. Acute-phase response to benzo[a]pyrene and induction of rat ALDH3A1.

    PubMed

    Pappas, Periklis; Sotiropoulou, Marianthi; Karamanakos, Petros; Kostoula, Aggeliki; Levidiotou, Stamatia; Marselos, Marios

    2003-02-01

    The aldehyde dehydrogenase-3A1 (ALDH3A1) enzyme, encoded by a member of the [Ah]-gene family, is dramatically increased (more than 100-fold) by benzo[a]pyrene (BaP) and other polycyclic hydrocarbons. Although much is known regarding the mechanism for the drug-metabolizing enzymes up-regulated by the Ah receptor, the physiological role of that tremendously increased ALDH3A1 enzyme activity is not yet fully clarified. The aim of this study was to identify a possible acute-phase response to different classes of xenobiotics affecting the metabolic capacity of the hepatocyte, by studying possible changes of serum acute-phase proteins (APPs) of hepatic origin, before and after BaP administration. Male Wistar rats were used in different series of experiments. The effects of BaP were estimated in terms of dose-response and time-response, with regard to the serum level of several APPs such as alpha-1-acid-glycoprotein (AAG), alpha-1-antitrypsin (AAT), C-reactive protein (CRP), and haptoglobin (HPT). In parallel experiments, levels of the same proteins have been determined after a time-dependent treatment with lipopolysaccharide (LPS). The changes in serum proteins were compared with the results of BaP or LPS administration on both hepatic ALDH3A1 and total ALDH enzyme activities. The results showed that BaP induced CRP and HPT in a time-dependent way, proportional to that caused by LPS. Additionally, ALDH3A1, CRP, and HPT were induced by BaP subacute treatment, whereas another type of ALDH inducer, phenobarbital, did not affect the levels of APPs or ALDH3A1, but did increase ALDH1A3 activity. Former studies of our group have shown that the inhibitory effects of different non-steroidal anti-inflammatory drugs (NSAIDs) on the ALDH3A1 induction were most possibly due to a decreased formation of arachidonic products like prostaglandins. Considering the changes of APPs caused by BaP, this study further supports the suggestion that the induction of ALDH3A1 is related to an

  9. Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities

    SciTech Connect

    Devash, Y.; Reichman, M.; Sela, I.; Reichenbach, N.L.; Suhadolnik, R.J.

    1985-01-29

    An enzyme that converts (/sup 3/H, /sup 32/P)ATP, with a /sup 3/H:/sup 32/P ratio of 1:1, to oligoadenylates with the same /sup 3/H:/sup 32/P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of /sup 3/H:/sup 32/P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems.

  10. Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities.

    PubMed

    Devash, Y; Reichman, M; Sela, I; Reichenbach, N L; Suhadolnik, R J

    1985-01-29

    An enzyme that converts [3H, 32P]ATP, with a 3H:32P ratio of 1:1, to oligoadenylates with the same 3H:32P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of 3H:32P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Inhibitory effect of nicotinamide on enzymatic activity of selected fungal strains causing skin infection.

    PubMed

    Ciebiada-Adamiec, Anna; Małafiej, Eugeniusz; Ciebiada, Ireneusz

    2010-05-01

    Pathogenicity of fungi is connected with their ability to easily penetrate the host tissues, survive in the infected host organism and use the elements of the host tissues as nutrients. Hence, the co-occurrence of pathogenic properties with the high enzymatic activity, which is manifested through the production of various enzymes including extracellular enzymes, was observed. It can be expected that it is possible to decrease fungal pathogenicity by lowering their enzymatic activity. The aim of the study was to determine the effect of nicotinamide on enzymatic activity of the fungi, which are most frequently isolated in cases of skin infection. Enzymatic activity was analysed using 15 Candida albicans, 15 Trichophyton rubrum and 15 Trichophyton mentagrophytes strains. The strains used for the study were collected from the current diagnostic material. API ZYM tests were used in diagnostic analysis. MICs of nicotinamide were determined by the macrodilution method in liquid medium. In the case of Candida strains, the presence of nicotinamide in the broth had a significant effect on the decrease of enzymatic activity (P < 0.05) of esterase (C4), esterase lipase (C-8), valin-arylamidase, acid phosphatase and alpha-glycosydase. A considerably stronger effect of nicotinamide was observed in the case of dermatophytes (P < 0.005). Its action led to a decrease in the activity of all the enzymes under study except alpha-glucosidase produced by T. rubrum strains. Thus, nicotinamide exhibited biological activity towards C. albicans, T. rubrum and Trichophyton mentagrophytes, which resulted in a decrease in the activity of enzymes produced by the fungi.

  12. The activity of Rhizomuchor miehei lipase as a biocatalyst in enzymatic acylation of cyclic alcohol

    NASA Astrophysics Data System (ADS)

    Iftitah, Elvina Dhiaul; Srihardyastuti, Arie; Ariefin, Mokhamat

    2017-03-01

    We report the activity of Rhizomuchor miehei lipase (RML) as a biocatalyst, in particular the investigations concerning the effort of substrate-structure reactivity on the enzymatic acylation. The acylation was studied using acetic anhydride as an acyl donor and performed in n-hexane as a solvent. The selectivity of the enzymatic acylation was revealed by Gas Chromatography-Mass Spectra. We observed that, RML has shown different behavior when catalyzing the acylation of isopulegol and mixture of isopulegol and citronellal (ratio 1:1). The chemoselectivity for the O-acylation was improved when the acyl acceptor included mixture of isopulegol and citronellal

  13. Enzymatic hydrolysis of oleuropein from Olea europea (olive) leaf extract and antioxidant activities.

    PubMed

    Yuan, Jiao-Jiao; Wang, Cheng-Zhang; Ye, Jian-Zhong; Tao, Ran; Zhang, Yu-Si

    2015-02-11

    Oleuropein (OE), the main polyphenol in olive leaf extract, is likely to decompose into hydroxytyrosol (HT) and elenolic acid under the action of light, acid, base, high temperature. In the enzymatic process, the content of OE in olive leaf extract and enzyme are key factors that affect the yield of HT. A selective enzyme was screened from among 10 enzymes with a high OE degradation rate. A single factor (pH, temperature, time, enzyme quantity) optimization process and a Box-Behnken design were studied for the enzymatic hydrolysis of 81.04% OE olive leaf extract. Additionally, enzymatic hydrolysis results with different substrates (38.6% and 81.04% OE) were compared and the DPPH antioxidant properties were also evaluated. The result showed that the performance of hydrolysis treatments was best using hemicellulase as a bio-catalyst, and the high purity of OE in olive extract was beneficial to biotransform OE into HT. The optimal enzymatic conditions for achieving a maximal yield of HT content obtained by the regression were as follows: pH 5, temperature 55 °C and enzyme quantity 55 mg. The experimental result was 11.31% ± 0.15%, and the degradation rate of OE was 98.54%. From the present investigation of the antioxidant activity determined by the DPPH method, the phenol content and radical scavenging effect were both decreased after enzymatic hydrolysis by hemicellulase. However, a high antioxidant activity of the ethyl acetate extract enzymatic hydrolysate (IC50 = 41.82 μg/mL) was demonstated. The results presented in this work suggested that hemicellulase has promising and attractive properties for industrial production of HT, and indicated that HT might be a valuable biological component for use in pharmaceutical products and functional foods.

  14. Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance

    PubMed Central

    Cui, Jiandong; Zhao, Yamin; Liu, Ronglin; Zhong, Cheng; Jia, Shiru

    2016-01-01

    Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to improve catalytic performance. However, activity of the most of the enzymes was declined after immobilization. Here, we develop a surfactant-activated lipase-inorganic flowerlike hybrid nanomaterials with rational design based on interfacial activation and self-assembly. The resulting surfactant-activated lipase-inorganic hybird nanoflower (activated hNF-lipase) exhibited 460% and 200% higher activity than native lipase and conventional lipase-inorganic hybird nanoflower (hNF-lipase). Furthermore, the activated hNF-lipase displayed good reusability due to its monodispersity and mechanical properties, and had excellent long-time stability. The superior catalytic performances were attributed to both the conformational modulation of surfactants and hierarchical structure of nanoflowers, which not only anchored lipases in an active form, but also decreased the enzyme-support negative interaction and mass-transfer limitations. This new biocatalytic system is promising to find widespread use in applications related to biomedicine, biosensor, and biodiesel. PMID:27297609

  15. Effect of restricted motion in high temperature on enzymatic activity of the pancreas

    NASA Technical Reports Server (NTRS)

    Abdusattarov, A.; Smirnova, G. I.

    1980-01-01

    Effects of 30 day hypodynamia coupled with high temperature (35-36 C) on enzymatic activity of the pancreas of male adult rats were studied. The test animals were divided into four groups. Group one served as controls (freedom of movement and a temperature of 25-26 C, considered optimal). The remaining animals were divided into three additional groups: Group two freedom of movement but high temperature (35-36 C); group three hypodynamia but an optimal temperature; group four hypodynamia and 35-36 C. Considerable change in the enzymatic activity in the pancreas of the four groups is observed in three experimental groups (two, three, and four) as compared to the control (group one). The results indicate that adaption of the organism to the thermal factor and restricted movement is accompanied by a change in the enzymatic spectrum of the pancreas. With the combined effect of these two stresses under conditions of the adaption of the organism especially sharp shifts occur in the enzymatic activity.

  16. Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

    NASA Technical Reports Server (NTRS)

    Kerr, T. P.

    1985-01-01

    In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.

  17. Plasmon-Enhanced Enzymatic Reactions: A Study of Nanoparticle-Enzyme Distance- and Nanoparticle Loading-Dependent Enzymatic Activity

    PubMed Central

    Abel, Biebele; Akinsule, Alice; Andrews, Canisha; Aslan, Kadir

    2011-01-01

    A detailed investigation of the dependence of the efficiency of plasmon-enhanced enzymatic reactions on the distance between silver island films (SIFs) and horse radish peroxidase (HRP) enzyme and on the loading of SIFs on glass surfaces is presented. Three different extent of loading of SIFs on glass slides were used: 1) low, 2) medium and 3) high, which was characterized by using optical absorption spectroscopy and scanning electron microscopy. Streptavidin-linked HRP enzyme was deposited onto SIFs and glass slides by using three different strategies: strategy 1: biotin-avidin protein assay (distance between SIFs and HRP = 4–8 nm), strategy 2: self assembled monolayers (SAMs) (1–5 nm), strategy 3: polymer layer (1–5 nm). The efficiency of enzymatic conversion of O-phenylenediamine dihydrochloride (OPD) to a colored product by HRP on SIFs and glass surfaces was assessed by optical absorption spectroscopy. The distance between SIFs and HRP and the extent of loading of SIFs on the glass surfaces were shown to have significant effect on the efficiency of plasmon-enhanced enzymatic reactions. In this regard, up to an %250 increase in enzymatic conversion of OPD was observed from SIFs with high loading using strategy 1. In addition, we have studied the potential of repeated use of SIFs in plasmon-enhanced enzymatic reactions. PMID:21949594

  18. Plasmon-Enhanced Enzymatic Reactions: A Study of Nanoparticle-Enzyme Distance- and Nanoparticle Loading-Dependent Enzymatic Activity.

    PubMed

    Abel, Biebele; Akinsule, Alice; Andrews, Canisha; Aslan, Kadir

    2011-01-01

    A detailed investigation of the dependence of the efficiency of plasmon-enhanced enzymatic reactions on the distance between silver island films (SIFs) and horse radish peroxidase (HRP) enzyme and on the loading of SIFs on glass surfaces is presented. Three different extent of loading of SIFs on glass slides were used: 1) low, 2) medium and 3) high, which was characterized by using optical absorption spectroscopy and scanning electron microscopy. Streptavidin-linked HRP enzyme was deposited onto SIFs and glass slides by using three different strategies: strategy 1: biotin-avidin protein assay (distance between SIFs and HRP = 4-8 nm), strategy 2: self assembled monolayers (SAMs) (1-5 nm), strategy 3: polymer layer (1-5 nm). The efficiency of enzymatic conversion of O-phenylenediamine dihydrochloride (OPD) to a colored product by HRP on SIFs and glass surfaces was assessed by optical absorption spectroscopy. The distance between SIFs and HRP and the extent of loading of SIFs on the glass surfaces were shown to have significant effect on the efficiency of plasmon-enhanced enzymatic reactions. In this regard, up to an %250 increase in enzymatic conversion of OPD was observed from SIFs with high loading using strategy 1. In addition, we have studied the potential of repeated use of SIFs in plasmon-enhanced enzymatic reactions.

  19. Effects of ALDH2 genotype, PPI treatment and L-cysteine on carcinogenic acetaldehyde in gastric juice and saliva after intragastric alcohol administration.

    PubMed

    Maejima, Ryuhei; Iijima, Katsunori; Kaihovaara, Pertti; Hatta, Waku; Koike, Tomoyuki; Imatani, Akira; Shimosegawa, Tooru; Salaspuro, Mikko

    2015-01-01

    Acetaldehyde (ACH) associated with alcoholic beverages is Group 1 carcinogen to humans (IARC/WHO). Aldehyde dehydrogenase (ALDH2), a major ACH eliminating enzyme, is genetically deficient in 30-50% of Eastern Asians. In alcohol drinkers, ALDH2-deficiency is a well-known risk factor for upper aerodigestive tract cancers, i.e., head and neck cancer and esophageal cancer. However, there is only a limited evidence for stomach cancer. In this study we demonstrated for the first time that ALDH2 deficiency results in markedly increased exposure of the gastric mucosa to acetaldehyde after intragastric administration of alcohol. Our finding provides concrete evidence for a causal relationship between acetaldehyde and gastric carcinogenesis. A plausible explanation is the gastric first pass metabolism of ethanol. The gastric mucosa expresses alcohol dehydrogenase (ADH) enzymes catalyzing the oxidation of ethanol to acetaldehyde, especially at the high ethanol concentrations prevailing in the stomach after the consumption of alcoholic beverages. The gastric mucosa also possesses the acetaldehyde-eliminating ALDH2 enzyme. Due to decreased mucosal ALDH2 activity, the elimination of ethanol-derived acetaldehyde is decreased, which results in its accumulation in the gastric juice. We also demonstrate that ALDH2 deficiency, proton pump inhibitor (PPI) treatment, and L-cysteine cause independent changes in gastric juice and salivary acetaldehyde levels, indicating that intragastric acetaldehyde is locally regulated by gastric mucosal ADH and ALDH2 enzymes, and by oral microbes colonizing an achlorhydric stomach. Markedly elevated acetaldehyde levels were also found at low intragastric ethanol concentrations corresponding to the ethanol levels of many foodstuffs, beverages, and dairy products produced by fermentation. A capsule that slowly releases L-cysteine effectively eliminated acetaldehyde from the gastric juice of PPI-treated ALDH2-active and ALDH2-deficient subjects. These

  20. Effects of ALDH2 Genotype, PPI Treatment and L-Cysteine on Carcinogenic Acetaldehyde in Gastric Juice and Saliva after Intragastric Alcohol Administration

    PubMed Central

    Maejima, Ryuhei; Iijima, Katsunori; Kaihovaara, Pertti; Hatta, Waku; Koike, Tomoyuki; Imatani, Akira; Shimosegawa, Tooru; Salaspuro, Mikko

    2015-01-01

    Acetaldehyde (ACH) associated with alcoholic beverages is Group 1 carcinogen to humans (IARC/WHO). Aldehyde dehydrogenase (ALDH2), a major ACH eliminating enzyme, is genetically deficient in 30–50% of Eastern Asians. In alcohol drinkers, ALDH2-deficiency is a well-known risk factor for upper aerodigestive tract cancers, i.e., head and neck cancer and esophageal cancer. However, there is only a limited evidence for stomach cancer. In this study we demonstrated for the first time that ALDH2 deficiency results in markedly increased exposure of the gastric mucosa to acetaldehyde after intragastric administration of alcohol. Our finding provides concrete evidence for a causal relationship between acetaldehyde and gastric carcinogenesis. A plausible explanation is the gastric first pass metabolism of ethanol. The gastric mucosa expresses alcohol dehydrogenase (ADH) enzymes catalyzing the oxidation of ethanol to acetaldehyde, especially at the high ethanol concentrations prevailing in the stomach after the consumption of alcoholic beverages. The gastric mucosa also possesses the acetaldehyde-eliminating ALDH2 enzyme. Due to decreased mucosal ALDH2 activity, the elimination of ethanol-derived acetaldehyde is decreased, which results in its accumulation in the gastric juice. We also demonstrate that ALDH2 deficiency, proton pump inhibitor (PPI) treatment, and L-cysteine cause independent changes in gastric juice and salivary acetaldehyde levels, indicating that intragastric acetaldehyde is locally regulated by gastric mucosal ADH and ALDH2 enzymes, and by oral microbes colonizing an achlorhydric stomach. Markedly elevated acetaldehyde levels were also found at low intragastric ethanol concentrations corresponding to the ethanol levels of many foodstuffs, beverages, and dairy products produced by fermentation. A capsule that slowly releases L-cysteine effectively eliminated acetaldehyde from the gastric juice of PPI-treated ALDH2-active and ALDH2-deficient subjects. These

  1. Amphioxus allantoicase: molecular cloning, expression and enzymatic activity.

    PubMed

    Wang, Yongjun; Zhang, Shicui; Liu, Zhenhui; Li, Hongyan; Wang, Lei

    2005-06-01

    Allantoicase, one of the purine metabolism enzymes, is progressively truncated during the chordate evolution, yet it is unknown when its activity became phylogenetically extinct. In this study, a cDNA encoding allantoicase was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 2441 bp long, and contains an open reading frame encoding a protein of 392 amino acid residues. RT-PCR analysis showed that amphioxus allantoicase was strongly expressed in the hepatic caecum, and weakly expressed in other tissues including hind-gut, gill, muscle, notochord, testis and ovary. The parallel experiment was performed measuring the allantoicase activity in the same tissues revealed that its activity was high in the hepatic caecum, but low or undetectable in other tissues examined. These suggest that allantoicase remains in action in the primitive chordate amphioxus.

  2. Enzymatic assay for calmodulins based on plant NAD kinase activity

    SciTech Connect

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  3. Finding of the Low Molecular Weight Inhibitors of Resuscitation Promoting Factor Enzymatic and Resuscitation Activity

    PubMed Central

    Demina, Galina R.; Makarov, Vadim A.; Nikitushkin, Vadim D.; Ryabova, Olga B.; Vostroknutova, Galina N.; Salina, Elena G.; Shleeva, Margarita O.; Goncharenko, Anna V.; Kaprelyants, Arseny S.

    2009-01-01

    Background Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs. Methodology/Principal Findings Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC50 1–7 µg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC50. The candidate compounds suppressed resuscitation of dormant (“non-culturable”) cells of M. smegmatis at 1 µg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 µg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations. Conclusions/Significance NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins. PMID:20016836

  4. Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees.

    PubMed

    Molnárová, Jana; Vadkertiová, Renáta; Stratilová, Eva

    2014-07-01

    Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, β-glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited β-glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D-xylose the most frequently of all the yeasts tested. The majority of the fruit-inhabiting yeasts grew in the medium with higher osmotic pressure.

  5. Soil disturbance increases soil microbial enzymatic activity in arid ecoregion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Functional diversity of the soil microbial community is commonly used in the assessment of soil health as it relates to the activity of soil microflora involved in carbon cycling. Soil microbes in different microenvironments will have varying responses to different substrates, thus catabolic fingerp...

  6. Investigation of the Solubility and Enzymatic Activity of a Thioredoxin-Gelonin Fusion Protein

    DTIC Science & Technology

    1997-05-01

    tosyl-L-lysine chloromethyl ketone TPCK N -tosyl-L-phenylalanine chloromethyl ketone Tris tris( hydroxymethyl )aminomethane trxA gene for thioredoxin UV...synthesis inhibition assay and the RIP- specific N -glycosidase assay. Thioredoxin-gelonin did not exhibit any enzymatic activity in either assay, even at...Inhibition of Purified Gelonin-XOMA ........... 61 Figure 13: UV Photograph of the Polyacrylamide Gel - N -Glycosidase Activity of Purified Gelonin-XOMA

  7. A comparative study on the rectal aminopeptidase enzymatic activities of different species.

    PubMed

    Acartürk, F; Parlatan, Z I

    2003-03-01

    The aim of the present study was to compare the enzymatic activity of four different aminopeptidases (aminopeptidase N, leucine aminopeptidase, aminopeptidase A, aminopeptidase B) in rectal homogenates from different species: rabbit, rat, guinea-pig, sheep and human. Different substrates were used as the relative specific substrates for the determination of aminopeptidase enzymatic activity. For this purpose, 4-methoxy-2-naphthylamide of L-alanine for aminopeptidase N, 4-methoxy-2-naphthylamide of L-leucine for leucine aminopeptidase, 4-methoxy-2-naphthylamide of L-glutamic acid for aminopeptidase A and 4-methoxy-2-naphthylamide of L-arginine for aminopeptidase B were employed. The rectal aminopeptidase enzymatic activity was determined spectrofluorometrically. The inhibition of activity of aminopeptidase in the presence of bestatin and puromycin inhibitors was also investigated. The results showed the presence of aminopeptidase enzymatic activity in all rectal homogenates. Sheep and guinea-pig had the greatest aminopeptidase activity. The four aminopeptidase activities of rat and rabbit were not significantly different from each other. Human data was not evaluated statistically, due to insufficient sample. But the values of human data was close to those of the rabbit and rat values except for aminopeptidase A. Based on the data of the hydrolysis and inhibition of the 4-methoxy-2-naphthylamide substrates, it was rather difficult to determine the aminopeptidase type in the rectal homogenates of the species studied. It has been found that the aminopeptidase activities of rat and rabbit were not statistically different from each other and the human data were close to them.

  8. STAT3 as a potential therapeutic target in ALDH+ and CD44+/CD24+ stem cell-like pancreatic cancer cells

    PubMed Central

    Lin, Li; Jou, David; Wang, Yina; Ma, Haiyan; Liu, Tianshu; Fuchs, James; Li, Pui-Kai; Lü, Jiagao; Li, Chenglong; Lin, Jiayuh

    2016-01-01

    Persistent activation of signal transducers and activators of transcription 3 (STAT3) is commonly detected in many types of cancer including pancreatic cancer. Whether STAT3 is activated in stem cell-like pancreatic cancer cells and the effect of STAT3 inhibition, is still unknown. Flow cytometry was used to isolate pancreatic cancer stem-like cells which are identified by both aldehyde dehydrogenase (ALDH)-positive (ALDH+) as well as cluster of differentiation (CD) 44-positive/CD24-positive subpopulations (CD44+/CD24+). STAT3 activation and the effects of STAT3 inhibition by STAT3 inhibitors, LLL12, FLLL32, and Stattic in ALDH+ and CD44+/CD24+ cells were examined. Our results showed that ALDH+ and CD44+/CD24+ pancreatic cancer stem-like cells expressed higher levels of phosphorylated STAT3, an active form of STAT3, compared to ALDH-negative (ALDH−) and CD44-negative/CD24-negative (CD44−/CD24−) pancreatic cancer cells, suggesting that STAT3 is activated in pancreatic cancer stem-like cells. Small molecular STAT3 inhibitors inhibited STAT3 phosphorylation, STAT3 downstream target gene expression, cell viability, and tumorsphere formation in ALDH+ and CD44+/CD24+ cells. Our results indicate that STAT3 is a novel therapeutic target in pancreatic cancer stem-like cells and inhibition of activated STAT3 in these cells by STAT3 inhibitors may offer an effective treatment for pancreatic cancer. PMID:27748818

  9. Influence of Cr(VI) on enzymatic activity of soil

    NASA Astrophysics Data System (ADS)

    Pacha, Jerzy

    1993-03-01

    The inhibitory effect of Cr(VI) on soil hydrolases activity during two different periods (one and six months) was investigated, in order to obtain information on the change in heavy metal toxicity with time. Considering toxicity as the ecological dose-50% (EcD50) toxicity tended to increase over six months for cellulase Cx, protease and acid phosphates and to decrease for amylase. The average EcD50 value varied between 4450 and 1210 ppm for cellulase Cx, 5000 and 2320 ppm for protease, 3830 and 3295 ppm for acid phosphatase, 4030 and over 5000 ppm for amylase.

  10. Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

    PubMed Central

    Guerra, Nelson P.; Pastrana Castro, Lorenzo

    2012-01-01

    The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics, vmax decreased significantly (P < 0.05) and KM increased (although not always significantly) with the increase in t. The conformational changes produced in the starch chains as a consequence of the ageing seemed to affect negatively the diffusivity of the starch to the active site of the enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G]. PMID:22666116

  11. Immobilization of inorganic pyrophosphatase on nanodiamond particles retaining its high enzymatic activity.

    PubMed

    Rodina, Elena V; Valueva, Anastasiya V; Yakovlev, Ruslan Yu; Vorobyeva, Nataliya N; Kulakova, Inna I; Lisichkin, Georgy V; Leonidov, Nikolay B

    2015-12-21

    Nanodiamond (ND) particles are popular platforms for the immobilization of molecular species. In the present research, enzyme Escherichia coli inorganic pyrophosphatase (PPase) was immobilized on detonation ND through covalent or noncovalent bonding and its enzymatic activity was characterized. Factors affecting adsorption of PPase such as ND size and surface chemistry were studied. The obtained material is a submicron size association of ND particles and protein molecules in approximately equal amounts. Both covalently and noncovalently immobilized PPase retains a significant enzymatic activity (up to 95% of its soluble form) as well as thermostability. The obtained hybrid material has a very high enzyme loading capacity (∼1 mg mg(-1)) and may be considered as a promising delivery system of biologically active proteinaceous substances, particularly in the treatment of diseases such as calcium pyrophosphate crystal deposition disease and related pathologies. They can also be used as recoverable heterogeneous catalysts in the traditional uses of PPase.

  12. Microbial enzymatic activities in a pilot-scale MBR experimental plant under different working conditions.

    PubMed

    Molina-Muñoz, M; Poyatos, J M; Rodelas, B; Pozo, C; Manzanera, M; Hontoria, E; Gonzalez-Lopez, J

    2010-01-01

    Phosphatases, glucosidase, protease, esterase and dehydrogenase activities in a MBR (membrane bioreactor) system equipped with ultrafiltration membranes for the treatment of real urban wastewater were measured at different volatile suspended solid (VSS) concentrations, total suspended solid (TSS) concentrations, hydraulic retention times (HRT), temperatures and inflow rates. The results showed the capacity of the MBR system to remove COD and BOD(5) at TSS between 7200 and 13,300 mg/L; HRT values of 8.05 and 15.27 h; inflow rates of 14.67 and 27.81 L/h; and temperatures between 4 and 27 degrees C. The enzymatic activities are influenced by increases in VSS and TSS concentrations. These results suggest that the ability to get adapted to environmental changes of the bacterial populations and their microbial enzymatic activities is essential to understand the biological processes that occur in MBR systems and crucial for proper urban wastewater treatment when using MBR technologies.

  13. Digestive enzymatic activity during ontogenetic development in zebrafish (Danio rerio).

    PubMed

    Guerrera, Maria Cristina; De Pasquale, Francesca; Muglia, Ugo; Caruso, Gabriella

    2015-12-01

    Despite the growing importance of zebrafish (Danio rerio) as an experimental model in biomedical research, some aspect of physiological and related morphological age dependent changes in digestive system during larval development are still unknown. In this paper, a biochemical and morphological study of the digestive tract of zebrafish was undertaken to record the functional changes occurring in this species during its ontogenetic development, particularly from 24 hr to 47 days post fertilization (dpf). Endo- and exo-proteases, as well as α-amylase enzymes, were quantified in zebrafish larvae before first feeding (7 dpf). The most morphologically significant events during the ontogenesis of the gut occurred between 3 dpf (mouth opening) and 7 dpf (end of exocrine pancreas differentiation). The presence of a wide range of digestive enzymes, already active at earlier zebrafish larval stages, closely related with the omnivorous diet of this species. Increasing enzyme activities were found with increasing age, probably in relation with intestinal mucosa folding and consequent absorption surface increase. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 699-706, 2015. © 2015 Wiley Periodicals, Inc.

  14. Local modulation of steroid action: rapid control of enzymatic activity

    PubMed Central

    Charlier, Thierry D.; Cornil, Charlotte A.; Patte-Mensah, Christine; Meyer, Laurence; Mensah-Nyagan, A. Guy; Balthazart, Jacques

    2015-01-01

    Estrogens can induce rapid, short-lived physiological and behavioral responses, in addition to their slow, but long-term, effects at the transcriptional level. To be functionally relevant, these effects should be associated with rapid modulations of estrogens concentrations. 17β-estradiol is synthesized by the enzyme aromatase, using testosterone as a substrate, but can also be degraded into catechol-estrogens via hydroxylation by the same enzyme, leading to an increase or decrease in estrogens concentration, respectively. The first evidence that aromatase activity (AA) can be rapidly modulated came from experiments performed in Japanese quail hypothalamus homogenates. This rapid modulation is triggered by calcium-dependent phosphorylations and was confirmed in other tissues and species. The mechanisms controlling the phosphorylation status, the targeted amino acid residues and the reversibility seem to vary depending of the tissues and is discussed in this review. We currently do not know whether the phosphorylation of the same amino acid affects both aromatase and/or hydroxylase activities or whether these residues are different. These processes provide a new general mechanism by which local estrogen concentration can be rapidly altered in the brain and other tissues. PMID:25852459

  15. Biocatalytic virus capsid as nanovehicle for enzymatic activation of Tamoxifen in tumor cells.

    PubMed

    Tapia-Moreno, Alejandro; Juarez-Moreno, Karla; Gonzalez-Davis, Oscar; Cadena-Nava, Ruben D; Vazquez-Duhalt, Rafael

    2017-04-03

    Most of the drugs used in chemotherapy should be activated by a transformation catalyzed by cytochrome P450 (CYP) enzymes. In this work, bacteriophage P22 virus-like particles (VLPs) containing CYP activity, immunologically inert and functionalized in order to be recognized by human cervix carcinoma cells and human breast adenocarcinoma cells were designed. The CYP was encapsulated inside the virus capsid obtained from the bacteriophage P22. CYP and coat protein were both heterologously expressed in E. coli. The VLPs with enzymatic activity were covered with polyethylene glycol that was functionalized in its distal end with folic acid in order to be recognized by folate receptors exhibited on tumor cells. The capacity of biocatalytic VLPs to be recognized and internalized into tumor cells is demonstrated. The VLP-treated cells showed enhanced capacity for the transformation of the pro-drug tamoxifen, which resulted in an increase of the cell sensitivity to this oncological drug. In this work, the potential use of biocatalytic VLPs vehicles as a delivery system of medical relevant enzymes is clearly demonstrated. In addition to cancer treatment, this technology also offers an interesting platform as nano-bioreactors for intracellular delivery of enzymatic activity for other diseases originated by the lack of enzymatic activity.

  16. Enzymatic activity in the presence of surfactants commonly used in dissolution media, Part 1: Pepsin.

    PubMed

    Guzman, Maria L; Marques, Margareth R; Olivera Me, Maria E; Stippler, Erika S

    2016-01-01

    The United States Pharmacopeia (USP) General Chapters Dissolution 〈711〉 and Disintegration and Dissolution of Dietary Supplements 〈2040〉 allows the use of enzymes in dissolution media when gelatin capsules do not conform to dissolution specifications due to cross linking. Possible interactions between enzymes and surfactants when used together in dissolution media could result in loss of the enzymatic activity. Pepsin is an enzyme commonly used in dissolution media, and in this work, the activity of pepsin was determined in the presence of different surfactants as usually found in case of dissolution tests of certain gelatin capsule formulations. Pepsin enzymatic activity was determined according to the Ninth Edition of the Food Chemicals Codex (FCC) 9 method, in dissolution conditions: simulated gastric fluid, 37 °C and 50 rpm. Sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), polysorbate 80 (Tween 80) and octoxynol 9 (Triton X100) in concentrations above and below their critical micellar concentrations were selected. Results showed a significant reduction in the activity of pepsin at all the concentrations of SDS assayed. On the contrary, CTAB, Tween 80, and Triton X100 did not alter the enzymatic activity at of pepsin any of the concentration assayed. This data demonstrates a rational selection of the surfactant to be used when pepsin is required in dissolution test.

  17. Enzymatically active biomimetic micropropellers for the penetration of mucin gels

    PubMed Central

    Walker, Debora; Käsdorf, Benjamin T.; Jeong, Hyeon-Ho; Lieleg, Oliver; Fischer, Peer

    2015-01-01

    In the body, mucus provides an important defense mechanism by limiting the penetration of pathogens. It is therefore also a major obstacle for the efficient delivery of particle-based drug carriers. The acidic stomach lining in particular is difficult to overcome because mucin glycoproteins form viscoelastic gels under acidic conditions. The bacterium Helicobacter pylori has developed a strategy to overcome the mucus barrier by producing the enzyme urease, which locally raises the pH and consequently liquefies the mucus. This allows the bacteria to swim through mucus and to reach the epithelial surface. We present an artificial system of reactive magnetic micropropellers that mimic this strategy to move through gastric mucin gels by making use of surface-immobilized urease. The results demonstrate the validity of this biomimetic approach to penetrate biological gels, and show that externally propelled microstructures can actively and reversibly manipulate the physical state of their surroundings, suggesting that such particles could potentially penetrate native mucus. PMID:26824056

  18. Protein Conformational Gating of Enzymatic Activity in Xanthine Oxidoreductase

    SciTech Connect

    Ishikita, Hiroshi; Eger, Bryan T.; Okamoto, Ken; Nishino, Takeshi; Pai, Emil F.

    2012-05-24

    In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E{sub sq/hq}) is {approx}170 mV, a striking difference. The former greatly prefers NAD{sup +} as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD{sup +}), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 {angstrom} resolution crystal structures for XDH, XO, the NAD{sup +}- and NADH-complexed XDH, E{sub sq/hq} were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E{sub sq/hq} difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD{sup +} binding at XDH. Instead, the positive charge of the NAD{sup +} ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD{sup +} molecule all contribute to altering E{sub sq/hq} upon NAD{sup +} binding to XDH.

  19. Regulation of Nox and Duox Enzymatic Activity and Expression

    PubMed Central

    Lambeth, J. David; Kawahara, Tsukasa; Diebold, Becky

    2007-01-01

    Summary In recent years, it has become clear that reactive oxygen species (ROS, which include superoxide, hydrogen peroxide and other metabolites) are produced in biological systems. Rather than being simply a byproduct of aerobic metabolism, it is now recognized that specific enzymes --- the Nox (NADPH-oxidase) and Duox (Dual oxidase) enzymes ---- seem to have the sole function of generating ROS in a carefully regulated manner, and key roles in signal transduction, immune function, hormone biosynthesis and other normal biological functions are being uncovered. The prototypical Nox is the respiratory burst oxidase or phagocyte oxidase, which generates large amounts of superoxide and other reactive species in the phagosomes of neutrophils and macrophages, playing a central role in innate immunity by killing microbes. This enzyme system has been extensively studied over the past two decades, and provides a basis for comparison with the more recently described Nox and Duox enzymes, which generate ROS in a variety of cells and tissues. This review first considers the structure and regulation of the respiratory burst oxidase, and then reviews recent studies relating to the regulation of the activity of the novel Nox/Duox enzymes. The regulation of Nox and Duox expression in tissues and by specific stimuli is also considered here. An accompanying review considers biological and pathological roles of the Nox family of enzymes. PMID:17602947

  20. Changes in cathepsin gene expression and relative enzymatic activity during gilthead sea bream oogenesis.

    PubMed

    Carnevali, O; Cionna, C; Tosti, L; Cerdà, J; Gioacchini, G

    2008-01-01

    The aim of this study was to provide evidence on the modulation of lysosomal enzymes in terms of both gene expression and enzymatic activity during follicle maturation. For this purpose three lysosomal enzymes, cathepsins B, D, and L, were studied in relation to yolk formation and degradation, during the main phases of ovarian follicle growth in the pelagophil species, the sea bream Sparus aurata. Specific attention was focused on the gene expression quantification method, on the assay of enzymatic activities, and on the relationship between the proteolytic cleavage of yolk proteins (YPs), cathepsin gene expression and cathepsin activities. For the gene expression study, the cathepsins B-like and L-like mRNAs were isolated and partially or fully characterized, respectively; the sequences were used as design specific primers for the quantification of cathepsin gene expression by real-time PCR, in follicles at different stages of maturation. The enzymatic assays for cathepsins B, D, and L were optimized in terms of specificity, sensitivity and reliability, using specific substrates and inhibitors. In ovulated eggs, the lipovitellin I (LV I) was degraded and the changes in electrophoretic pattern were preceded by an increase in the activity of a cysteine proteinase, cathepsin L, and its mRNA. Cathepsin B did not appear to be involved in YP changes during the final maturation stage.

  1. MBD3L2 promotes Tet2 enzymatic activity for mediating 5-methylcytosine oxidation.

    PubMed

    Peng, Lina; Li, Yan; Xi, Yanping; Li, Wei; Li, Jin; Lv, Ruitu; Zhang, Lei; Zou, Qingping; Dong, Shihua; Luo, Huaibing; Wu, Feizhen; Yu, Wenqiang

    2016-03-01

    Ten-eleven translocation (Tet) proteins are key players involved in the dynamic regulation of cytosine methylation and demethylation. Inactivating mutations of Tet2 are frequently found in human malignancies, highlighting the essential role of Tet2 in cellular transformation. However, the factors that control Tet enzymatic activity remain largely unknown. Here, we found that methyl-CpG-binding domain protein 3 (MBD3) and its homolog MBD3-like 2 (MBD3L2) can specifically modulate the enzymatic activity of Tet2 protein, but not Tet1 and Tet3 proteins, in converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Moreover, MBD3L2 is more effective than MBD3 in promoting Tet2 enzymatic activity through strengthening the binding affinity between Tet2 and the methylated DNA target. Further analysis revealed pronounced decreases in 5mC levels at MBD3L2 and Tet2 co-occupied genomic regions, most of which are promoter elements associated with either cancer-related genes or genes involved in the regulation of cellular metabolic processes. Our data add new insights into the regulation of Tet2 activity by MBD3 and MBD3L2, and into how that affects Tet2-mediated modulation of its target genes in cancer development. Thus, they have important applications in understanding how dysregulation of Tet2 might contribute to human malignancy.

  2. Enzymatic vitreolysis with recombinant tissue plasminogen activator for vitreomacular traction

    PubMed Central

    Raczyńska, Dorota; Lipowski, Paweł; Zorena, Katarzyna; Skorek, Andrzej; Glasner, Paulina

    2015-01-01

    Aims The aim of our research was to gain data about the efficacy of intravitreal injections of a recombinant tissue plasminogen activator (rTPA) in dissolving vitreoretinal tractions (VRTs). Materials and methods The study group consisted of patients of our Ophthalmology Clinic who had received an injection of rTPA (TPA Group) for an existent vitreomacular traction confirmed by optical coherence tomography and stereoscopic examinations. The control group consisted of patients who had declined treatment despite the existence of a vitreomacular traction confirmed by the same diagnostic methods. Each group consisted of 30 people (30 eyes). The observation period was 6 months. Conclusion In both groups some of the VRTs had dissolved. In the TPA group the traction dissolved in 10 patients (33.33%) and in the control group only in 5 (16.67%). It is also important to point out that the mean baseline membrane thickness was higher in the TPA group than in the control group. Observing patients in both groups we noticed that the dissolution of vitreoretinal membrane occurred most frequently in those cases where the membrane was thin. In the TPA group, the mean membrane thickness after 6 months decreased considerably. At the same time, no significant change in the membrane thickness could be observed in the control group. Observation of the retinal thickness allows us to draw the following conclusion: in the TPA group, the retinal thickness in the macular area (edema) had decreased over the study period, whereas in the control group it had increased. In those cases where the traction had dissolved, the edema of the retina decreased by the end of the 6-month period in both groups. In the TPA group, the dissolution of the membrane occurred most often within 3 months from the primary injection. Based on statistics, we can confirm that in the control group there was a decrease in visual acuity during the 6 months of the study period. At the same time, visual acuity in the TPA

  3. Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

    PubMed Central

    Langer, Andreas; Schräml, Michael; Strasser, Ralf; Daub, Herwin; Myers, Thomas; Heindl, Dieter; Rant, Ulrich

    2015-01-01

    The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable. PMID:26174478

  4. Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

    NASA Astrophysics Data System (ADS)

    Langer, Andreas; Schräml, Michael; Strasser, Ralf; Daub, Herwin; Myers, Thomas; Heindl, Dieter; Rant, Ulrich

    2015-07-01

    The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.

  5. Interactive effect of oxytetracycline and lead on soil enzymatic activity and microbial biomass.

    PubMed

    Gao, Minling; Song, Wenhua; Zhou, Qian; Ma, Xiaojun; Chen, Xiaoying

    2013-09-01

    Interactive effect of oxytetracyline (OTC) and lead on soil enzymatic activity and population of microbes was studied in the paper. The results showed effect of pollutants on bacteria, actinomycetes and enzymatic activity increased in the order: (OTC+Pb)>Pb>OTC, (OTC+Pb)>Pb>OTC and (OTC+Pb)>OTC>Pb, respectively. However, impact of pollutants on fungi decreased in the order: (OTC+Pb)

  6. Size Dependent Platelet Subpopulations: Relationship of Platelet Volume to Ultrastructure Enzymatic Activity, and Function.

    DTIC Science & Technology

    1983-03-10

    of the present apheresis instruments to separate the larger more functional platelets from the smaller ones. The selective isolation of large... PLATELET VOLUME T. -(U) BOSTON UNIV MA SCHOOL OF I MEDICINE C B THOMPSON ET RL 10 MAR 83 BUSM-93-89 UNIIDN919CA89 /68 6ilfflfllflflflflll l...N00014-79-C-0168 TECHNICAL REPORT NO. 83-08 SIZE DEPENDENT PLATELET SUBPOPULATIONS: RELATIONSHIP OF PLATELET VOLUME TO ULTRASTRUCTURE. ENZYMATIC ACTIVITY

  7. Effect of compost temperature on oxygen uptake rate, specific growth rate and enzymatic activity of microorganisms in dairy cattle manure.

    PubMed

    Miyatake, Fumihito; Iwabuchi, Kazunori

    2006-05-01

    Investigations were carried out to find out the relationship between temperature and microbial activity in dairy cattle manure composting using oxygen uptake rate, specific growth rate and enzymatic activities during autothermal and isothermal composting experiments. In autothermal composting, oxygen uptake rate and specific growth rate were found to be most intensive in order of 43 degrees C, 60 degrees C and 54 degrees C. Isothermal composting at 54 degrees C resulted highest levels of enzymatic activity and promoted the volatile solids reduction. Based on the maximum enzymatic activity, specific growth rate appeared to be more closely linked with microbial activity in compost than with oxygen uptake rate. The enhancement of specific growth rate, enzymatic activity and volatile solids reduction were induced at 54 degrees C in cattle manure composting.

  8. Enzymatically Activated Near Infrared Nanoprobes Based on Amphiphilic Block Copolymers for Optical Detection of Cancer

    PubMed Central

    Ozel, Tugba; White, Sean; Nguyen, Elaine; Moy, Austin; Brenes, Nicholas; Choi, Bernard; Betancourt, Tania

    2015-01-01

    Background and Objective Nanotechnology offers the possibility of creating multi-functional structures that can provide solutions for biomedical problems. The nanoprobes herein described are an example of such structures, where nanoprobes have been designed to provide high specificity and contrast potential for optical detection of cancer. Specifically, enzymatically activated fluorescent nanoprobes (EANPs) were synthesized as cancer-specific contrast agents for optical imaging. Study Design/Materials and Methods EANPs were prepared by nanoprecipitation of blends of poly(lactic acid)-b-poly(ethylene glycol) and poly(lactic-co-glycolic acid)-b-poly(L-lysine). The lysine moieties were then covalently decorated with the near infrared (NIR) fluorescent molecule AlexaFluor-750 (AF750). Close proximity of the fluorescent molecules to each other resulted in fluorescence quenching, which was be reversed by enzymatically mediated cleavage of poly(L-lysine) chains. EANPs were characterized by dynamic light scattering and electron microscopy. Enzymatic development of fluorescence was studied in vitro by fluorescence spectroscopy. Biocompatibility and contrast potential of EANPs were studied in cancerous and noncancerous cells. The potential of the nanoprobes as contrast agents for NIR fluorescence imaging was studied in tissue phantoms. Results Spherical EANPs of ∼100 nm were synthesized via nanoprecipitation of polymer blends. Fluorescence activation of EANPs by treatment with a model protease was demonstrated with up to 15-fold optical signal enhancement within 120 minutes. Studies with MDA-MB-231 breast cancer cells demonstrated the cytocompatibility of EANPs, as well as enhanced fluorescence associated with enzymatic activation. Imaging studies in tissue phantoms confirmed the ability of a simple imaging system based on a laser source and CCD camera to image dilute suspensions of the nanoprobe at depths of up to 4 mm, as well as up to a 13-fold signal

  9. BACE1 and BACE2 Enzymatic Activities in Alzheimer’s Disease

    PubMed Central

    Ahmed, Rachel R.; Holler, Christopher J.; Webb, Robin L.; Li, Feng; Beckett, Tina L.; Murphy, M. Paul

    2009-01-01

    β-Secretase is the rate limiting enzymatic activity in the production of the amyloid-β peptide (Aβ) and is thought to be involved in Alzheimer’s disease (AD) pathogenesis. Although BACE1 (β-site APP Cleaving Enzyme 1, EC 3.4.23.46) has received significant attention, the related BACE2 (EC 3.4.23.45) has not. Though BACE2 is also expressed in the brain, its potential role in AD has not been resolved. In this study, we compared the activities of both BACE1 and BACE2, which were isolated from the same samples of frontal cortex from both AD-affected individuals and age-matched controls. BACE1 activity showed a significant positive correlation with the amount of extractable Aβ, and BACE1 protein and activity were significantly increased in AD cases. Unexpectedly, there were substantial total amounts of BACE2 protein and enzymatic activity in the human brain. BACE2 activity did not change significantly in the AD brain, and was not related to Aβ concentration. These data indicate that BACE1 likely accounts for most of the Aβ produced in the human brain, and that BACE2 activity is not a likely contributor. However, since both forms of BACE compete for the same substrate pool, even small changes in BACE2 activity could have consequences for human disease. PMID:19968762

  10. Characterisation of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties

    PubMed Central

    Mertsalov, Ilya B.; Novikov, Boris N.; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M.

    2016-01-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CMP-Sia synthetases that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterised its activity in vitro. Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn2+, Fe2+, Co2+ and Mn2+, while the activity with Mg2+ was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in coordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission. PMID:27114558

  11. The dual effects of Maillard reaction and enzymatic hydrolysis on the antioxidant activity of milk proteins.

    PubMed

    Oh, N S; Lee, H A; Lee, J Y; Joung, J Y; Lee, K B; Kim, Y; Lee, K W; Kim, S H

    2013-08-01

    The objective of this study was to determine the enhanced effects on the biological characteristics and antioxidant activity of milk proteins by the combination of the Maillard reaction and enzymatic hydrolysis. Maillard reaction products were obtained from milk protein preparations, such as whey protein concentrates and sodium caseinate with lactose, by heating at 55°C for 7 d in sodium phosphate buffer (pH 7.4). The Maillard reaction products, along with untreated milk proteins as controls, were hydrolyzed for 0 to 3h with commercial proteases Alcalase, Neutrase, Protamex, and Flavorzyme (Novozymes, Bagsværd, Denmark). The antioxidant activity of hydrolyzed Maillard reaction products was determined by reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, their 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and the ability to reduce ferric ions. Further characteristics were evaluated by the o-phthaldialdehyde method and sodium dodecyl sulfate-PAGE. The degree of hydrolysis gradually increased in a time-dependent manner, with the Alcalase-treated Maillard reaction products being the most highly hydrolyzed. Radical scavenging activities and reducing ability of hydrolyzed Maillard reaction products increased with increasing hydrolysis time. The combined products of enzymatic hydrolysis and Maillard reaction showed significantly greater antioxidant activity than did hydrolysates or Maillard reaction products alone. The hydrolyzed Maillard reaction products generated by Alcalase showed significantly higher antioxidant activity when compared with the other protease products and the antioxidant activity was higher for the whey protein concentrate groups than for the sodium caseinate groups. These findings indicate that Maillard reaction products, coupled with enzymatic hydrolysis, could act as potential antioxidants in the pharmaceutical, food, and dairy industries.

  12. Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.

    PubMed

    Mertsalov, Ilya B; Novikov, Boris N; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M

    2016-07-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission.

  13. Allocation of extracellular enzymatic activity in relation to litter composition, N deposition, and mass loss

    USGS Publications Warehouse

    Sinsabaugh, R. L.; Carreiro, M.M.; Repert, D.A.

    2002-01-01

    Decomposition of plant material is a complex process that requires interaction among a diversity of microorganisms whose presence and activity is subject to regulation by a wide range of environmental factors. Analysis of extracellular enzyme activity (EEA) provides a way to relate the functional organization of microdecomposer communities to environmental variables. In this study, we examined EEA in relation to litter composition and nitrogen deposition. Mesh bags containing senescent leaves of Quercus borealis (red oak), Acer rubrum (red maple) and Cornus florida (flowering dogwood) were placed on forest floor plots in southeastern New York. One-third of the plots were sprayed monthly with distilled water. The other plots were sprayed monthly with NH4NO3 solution at dose rates equivalent to 2 or 8 g N m-2 y-1. Mass loss, litter composition, fungal mass, and the activities of eight enzymes were measured on 13 dates for each litter type. Dogwood was followed for one year, maple for two, oak for three, For each litter type and treatment, enzymatic turnover activities were calculated from regressions of LN (%mass remaining) vs. cumulative activity. The decomposition of dogwood litter was more efficient than that of maple and oak. Maple litter had the lowest fungal mass and required the most enzymatic work to decompose, even though its mass loss rate was twice that of oak. Across litter types, N amendment reduced apparent enzymatic efficiencies and shifted EEA away from N acquisition and toward P acquisition, and away from polyphenol oxidation and toward polysaccharide hydrolysis. The effect of these shifts on decomposition rate varied with litter composition: dogwood was stimulated, oak was inhibited and maple showed mixed effects. The results show that relatively small shifts in the activity of one or two critical enzymes can significantly alter decomposition rates.

  14. Evaluating a cognitive model of ALDH2 and drinking behavior

    PubMed Central

    Hendershot, Christian S.; Witkiewitz, Katie; George, William H.; Wall, Tamara L.; Otto, Jacqueline M.; Liang, Tiebing; Larimer, Mary E.

    2010-01-01

    Background Despite evidence for genetic influences on alcohol use and alcohol-related cognitions, genetic factors and endophenotypes are rarely incorporated in cognitive models of drinking behavior. This study evaluated a model of ALDH2 and drinking behavior stipulating cognitive factors and alcohol sensitivity as accounting for genetic influences on drinking outcomes. Methods Participants were Asian-American young adults (n = 171) who completed measures of alcohol cognitions (drinking motives, drinking refusal self-efficacy and alcohol expectancies), alcohol sensitivity, drinking behavior and alcohol-related problems as part a prospective study. Structural equation modeling (SEM) evaluated a model of drinking behavior that stipulated indirect effects of ALDH2 on drinking outcomes through cognitive variables and alcohol sensitivity. Results The full model provided an adequate fit to the observed data, with the measurement model explaining 63% of the variance in baseline heavy drinking and 50% of the variance in alcohol-related problems at follow-up. Associations of ALDH2 with cognitive factors and alcohol sensitivity were significant, whereas the association of ALDH2 with drinking was not significant with these factors included in the model. Mediation tests indicated significant indirect effects of ALDH2 through drinking motives, drinking refusal self-efficacy and alcohol sensitivity. Conclusions Results are consistent with the perspective that genetic influences on drinking behavior can be partly explained by learning mechanisms and implicate cognitive factors as important for characterizing associations of ALDH2 and drinking. PMID:21039630

  15. Methods for determining enzymatic activity comprising heating and agitation of closed volumes

    SciTech Connect

    Thompson, David Neil; Henriksen, Emily DeCrescenzo; Reed, David William; Jensen, Jill Renee

    2016-03-15

    Methods for determining thermophilic enzymatic activity include heating a substrate solution in a plurality of closed volumes to a predetermined reaction temperature. Without opening the closed volumes, at least one enzyme is added, substantially simultaneously, to the closed volumes. At the predetermined reaction temperature, the closed volumes are agitated and then the activity of the at least one enzyme is determined. The methods are conducive for characterizing enzymes of high-temperature reactions, with insoluble substrates, with substrates and enzymes that do not readily intermix, and with low volumes of substrate and enzyme. Systems for characterizing the enzymes are also disclosed.

  16. A new method to determine optimum temperature and activation energies for enzymatic reactions.

    PubMed

    Wojcik, M; Miłek, J

    2016-08-01

    A new method for determination of the optimum temperature and activation energies based on an idea of the average rate of enzymatic reaction has been developed. A mathematical model describing the effect of temperature on a dimensionless activity for enzyme deactivation following the first-order kinetics has been derived. The necessary condition for existence of the function extreme of the optimal temperature has been applied in the model. The developed method has been verified using the experimental data for inulinase from Kluyveromyces marxianus.

  17. Structure and activity of a new low-molecular-weight heparin produced by enzymatic ultrafiltration.

    PubMed

    Fu, Li; Zhang, Fuming; Li, Guoyun; Onishi, Akihiro; Bhaskar, Ujjwal; Sun, Peilong; Linhardt, Robert J

    2014-05-01

    The standard process for preparing the low-molecular-weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield because of the formation of a high content of undesired disaccharides and tetrasaccharides. An enzymatic ultrafiltration reactor for LMWH preparation was developed to overcome this problem. The behavior, of the heparin oligosaccharides and polysaccharides using various membranes and conditions, was investigated to optimize this reactor. A novel product, LMWH-II, was produced from the controlled depolymerization of heparin using heparin lyase II in this optimized ultrafiltration reactor. Enzymatic ultrafiltration provides easy control and high yields (>80%) of LMWH-II. The molecular weight properties of LMWH-II were similar to other commercial LMWHs. The structure of LMWH-II closely matched heparin's core structural features. Most of the common process artifacts, present in many commercial LWMHs, were eliminated as demonstrated by 1D and 2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4 binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity.

  18. Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices.

    PubMed

    Matsuura, Koji; Huang, Han-Wei; Chen, Ming-Cheng; Chen, Yu; Cheng, Chao-Min

    2017-04-07

    Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P < 0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay.

  19. Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices

    PubMed Central

    Matsuura, Koji; Huang, Han-Wei; Chen, Ming-Cheng; Chen, Yu; Cheng, Chao-Min

    2017-01-01

    Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P < 0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay. PMID:28387379

  20. Blocked Enzymatic Etching of Gold Nanorods: Application to Colorimetric Detection of Acetylcholinesterase Activity and Its Inhibitors.

    PubMed

    Saa, Laura; Grinyte, Ruta; Sánchez-Iglesias, Ana; Liz-Marzán, Luis M; Pavlov, Valeri

    2016-05-04

    The anisotropic morphology of gold nanorods (AuNRs) has been shown to lead to nonuniform ligand distribution and preferential etching through their tips. We have recently demonstrated that this effect can be achieved by biocatalytic oxidation with hydrogen peroxide, catalyzed by the enzyme horseradish peroxidase (HRP). We report here that modification of AuNRs with thiol-containing organic molecules such as glutathione and thiocholine hinders enzymatic AuNR etching. Higher concentrations of thiol-containing molecules in the reaction mixture gradually decrease the rate of enzymatic etching, which can be monitored by UV-vis spectroscopy through changes in the AuNR longitudinal plasmon band. This effect can be applied to develop novel optical assays for acetylcholinesterase (AChE) activity. The biocatalytic hydrolysis of acetylthiocholine by AChE yields thiocholine, which prevents enzymatic AuNR etching in the presence of HRP. Additionally, the same bioassay can be used for the detection of nanomolar concentrations of AChE inhibitors such as paraoxon and galanthamine.

  1. Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

    NASA Astrophysics Data System (ADS)

    Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

    2014-07-01

    Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

  2. Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity

    PubMed Central

    Kalb, Suzanne R.; Boyer, Anne E.; Barr, John R.

    2015-01-01

    Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A–G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin. PMID:26404376

  3. Evolution of enzymatic activities and carbon fractions throughout composting of plant waste.

    PubMed

    Jurado, M M; Suárez-Estrella, F; Vargas-García, M C; López, M J; López-González, J A; Moreno, J

    2014-01-15

    Many alternatives for the proper disposal of horticultural plant wastes have been studied, and composting is one of the most attractive due to its insignificant environmental impact and low cost. The quality of compost for agronomical use is related to the degree of organic matter maturation and stabilization. Traditional parameters as well as temperature, ratio C/N, cationic exchange capacity, extractable carbon, or evolution of humificated substances have been successfully used to assess compost maturity and stability. However, microorganisms frequently isolated during composting release a wide range of hydrolytic enzymes, whose activity could apparently give interesting information on the rate of decomposition of organic matter and, therefore, on the product stability. The aim of this work was to study the evolution of some important enzymatic activities during composting of agricultural wastes and their comparison with other chemical parameters commonly employed as quality and maturity indexes, to establish a relationship between the degradation intensity of specific organic carbon fractions throughout the process. In this work, the chemical and biochemical parameters of plant wastes were studied along a composting process of 189 days to evaluate their importance as tools for compost characterization. Results showed an intense enzymatic activity during the first 2-3 weeks of composting (bio-oxidative phase), because of the availability of easily decomposable organic compounds. From a biological point of view, a less intense phase was observed between second and third month of composting (mesophilic or cooling phase). Finally, chemical humification parameters were more closely associated with the period between 119 and 189 days (maturation phase). Significant correlations between the enzymatic activities as well as between enzyme activities and other more traditional parameters were also highlighted, indicating that both kind of indexes can be a reliable tool to

  4. Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity.

    PubMed

    Kalb, Suzanne R; Boyer, Anne E; Barr, John R

    2015-08-31

    Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin.

  5. Ammodytoxins efficiently release arachidonic acid and induce apoptosis in a motoneuronal cell line in an enzymatic activity-dependent manner.

    PubMed

    Jenko-Pražnikar, Zala; Petan, Toni; Pungerčar, Jože

    2013-03-01

    Secreted phospholipases A2 (sPLA2s) are phospholipolytic enzymes and receptor ligands whose action affects cell death and survival. We have previously shown that ammodytoxin A (AtxA), a snake venom sPLA2, is rapidly internalized into motoneuronal NSC34 cells, inducing characteristic neurotoxic sPLA2 cell damage and apoptosis. In this study, we have analyzed the role of sPLA2 enzymatic activity, including arachidonic acid (AA) release, in the induction of motoneuronal apoptosis by AtxA and homologous recombinant sPLA2s with different enzymatic properties: an AtxA mutant (V31W) with very high enzymatic activity, enzymatically inactive S49-sPLA2 (ammodytin L, AtnL), its mutant (LW) with restored enzymatic activity, and non-toxic, enzymatically active sPLA2 (AtnI2). Addition of AA, AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, to NSC34 cells resulted in caspase-3 activation, DNA fragmentation and disruption of mitochondrial membrane potential, leading to a significant and rapid decrease in motoneuronal cell viability that was not observed in C2C12 myoblasts and HEK293 cells. AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, also liberated large amounts of AA specifically from motoneuronal cells, and this ability correlated well with the ability to induce apoptotic changes and decrease cell viability. The enzymatic activity of AtxA and similar sPLA2s is thus necessary, but not sufficient, for inducing motoneuronal apoptosis. This suggests that specific binding to the motoneuronal cell surface, followed by internalization and enzymatic activity-dependent induction of apoptosis, possibly as a consequence of extensive extra- and intracellular AA release, is necessary for Atx-induced motoneuronal cell death.

  6. Genome-Wide Identification and Functional Classification of Tomato (Solanum lycopersicum) Aldehyde Dehydrogenase (ALDH) Gene Superfamily

    PubMed Central

    Lopez-Valverde, Francisco J.; Robles-Bolivar, Paula; Lima-Cabello, Elena; Gachomo, Emma W.; Kotchoni, Simeon O.

    2016-01-01

    Aldehyde dehydrogenases (ALDHs) is a protein superfamily that catalyzes the oxidation of aldehyde molecules into their corresponding non-toxic carboxylic acids, and responding to different environmental stresses, offering promising genetic approaches for improving plant adaptation. The aim of the current study is the functional analysis for systematic identification of S. lycopersicum ALDH gene superfamily. We performed genome-based ALDH genes identification and functional classification, phylogenetic relationship, structure and catalytic domains analysis, and microarray based gene expression. Twenty nine unique tomato ALDH sequences encoding 11 ALDH families were identified, including a unique member of the family 19 ALDH. Phylogenetic analysis revealed 13 groups, with a conserved relationship among ALDH families. Functional structure analysis of ALDH2 showed a catalytic mechanism involving Cys-Glu couple. However, the analysis of ALDH3 showed no functional gene duplication or potential neo-functionalities. Gene expression analysis reveals that particular ALDH genes might respond to wounding stress increasing the expression as ALDH2B7. Overall, this study reveals the complexity of S. lycopersicum ALDH gene superfamily and offers new insights into the structure-functional features and evolution of ALDH gene families in vascular plants. The functional characterization of ALDHs is valuable and promoting molecular breeding in tomato for the improvement of stress tolerance and signaling. PMID:27755582

  7. Changes in the enzymatic activity of soil samples upon their storage

    NASA Astrophysics Data System (ADS)

    Dadenko, E. V.; Kazeev, K. Sh.; Kolesnikov, S. I.; Val'Kov, V. F.

    2009-12-01

    The influence of the duration and conditions of storage of soil samples on the activity of soil enzymes (catalase, β-fructofuranosidase, and dehydrogenase) was studied for the main soils of southern Russia (different subtypes of chernozems, chestnut soils, brown forest soils, gray forest soils, solonetzes, and solonchaks). The following soil storage conditions were tested: (1) the air-dry state at room temperature, (2) the airdry state at a low positive (in a refrigerator, +4°C) temperature, (3) naturally moist samples at a low positive temperature, and (4) naturally moist samples at a negative (in a freezer, -5°C) temperature. It was found that the sample storing caused significant changes in the enzymatic activities, which depended on the soil type, the land use, the type of enzyme, and the duration and conditions of the sample storage. In the course of the storage, the changes in the enzymatic activity had a nonlinear character. The maximum changes were observed in the initial period (up to 12 weeks). Then, a very gradual decrease in the activity of the studied enzymes was observed. Upon the long-term (>12 weeks) storage under the different conditions, the difference in the activities of the soil enzymes became less pronounced. The storage of soil samples in the air-dried state at room temperature can be recommended for mass investigations.

  8. Enzymatic improvement in the polyphenol extractability and antioxidant activity of green tea extracts.

    PubMed

    Hong, Yang-Hee; Jung, Eun Young; Park, Yooheon; Shin, Kwang-Soon; Kim, Tae Young; Yu, Kwang-Won; Chang, Un Jae; Suh, Hyung Joo

    2013-01-01

    This study describes increases in extraction efficiency and the bioconversion of catechins after treatment with several commercial enzymes. Tannase was also used to improve the anti-radical activities of green tea extracts. Enzymatic treatment with various commercial enzymes was introduced to improve the extraction efficiency of polyphenols. The total polyphenol, flavonoid, and catechin contents and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of the green tea extract treated with Viscozyme (VG) were significantly higher than those treated with other commercial enzymatic extractions (p<0.05). More than 95% of the epigallocatechingallate (EGCG) and of the epicatechingallate (ECG) was hydrolyzed to epigallocatechin (EGC) and to epicatechin (EC) in successive 20 min treatments with Viscozyme and tannase (TG). Due to its hydrolytic activity, treatment involving tannase resulted in a significant release of gallic acid (GA), EGC, and EC, leading to greater radical scavenging activities. Regarding the IC(50) values of the DPPH and 2,2-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, the green tea extract treated with TG showed values of 131.23 and 28.83 µg/mL, VG showed values of 224.70 and 32.54 µg/mL, and normal green tea extract (NG) showed values of 241.11 and 66.27 µg/mL, respectively. These results indicate that successive treatment with Viscozyme and tannase improves the extraction efficiency of polyphenols and increases radical scavenging activities.

  9. Effects of ellipticine on ALDH1A1-expressing breast cancer stem cells--an in vitro and in silico study.

    PubMed

    Pandrangi, Santhi Latha; Chikati, Rajasekhar; Chauhan, Pradeep Singh; Kumar, Chitta Suresh; Banarji, Anropa; Saxena, Sunita

    2014-01-01

    Targeting breast cancer stem cells (BCSCs) offers a promising strategy for breast cancer treatment. We examined the plant alkaloid ellipticine for its efficacy to inhibit the expression of aldehyde dehydrogenase 1 class A1 (ALDH1A1)-positive BCSCs by in vitro and in silico methods. At 3 mM concentration, ellipticine decreased the expression of ALDH1A1-positive BCSCs by 62% (p = 0.073) in the MCF7 cell line and by 53% (p = 0.024) in the SUM159 cell line compared to vehicle-treated cultures. Ellipticine significantly reduced the formation of mammospheres, whereas paclitaxel enhanced mammosphere formation in both the treated cell lines. Interestingly, when treated with a combination of ellipticine and paclitaxel, the percentage of ALDH1A1-positive BCSCs dropped by several fold in vitro. A homology model of Homo sapiens ALDH1A1 was built using the crystal structure of NAD-bound sheep liver class I aldehyde dehydrogenase [PDB ID: 1BXS] as a template. Molecular simulation and docking studies revealed that the amino acids Asn-117 and Asn-121, Glu-249, Cys-302, and Gln-350, present in the active site of human ALDH1A1, played a vital role in interacting with the drug. The present study suggests that ellipticine reduces the proliferation and self-renewal ability of ALDH1A1-positive BCSCs and can be used in combination with a cytotoxic drug like paclitaxel for potential targeting of BCSCs.

  10. Effects of Prochloraz fungicide on soil enzymatic activities and bacterial communities.

    PubMed

    Tejada, Manuel; Gómez, Isidoro; García-Martínez, Ana María; Osta, Paloma; Parrado, Juan

    2011-09-01

    We studied in the laboratory the effect of Prochloraz fungicide on the biological properties (soil enzymatic activities and soil bacterial communities) of a Plaggic Anthrosol. Five hundred grams of soil (<2mm) was mixed with three dosages of Prochloraz (1, 2, and 4 l ha(-1)) for 83 days. A non-Prochloraz polluted soil was used as control. Following commercial recommendations, fungicide was applied four times during the incubation experiment. For all treatments, the soil ergosterol and levels of dehydrogenase, urease, β-glucosidase, and phosphatase activity were measured at nine different times (0, 1, 21, 22, 41, 42, 62, 63, and 83 days). The 16S rDNA-DGGE profiles in all treatments were determined at the beginning and end of the incubation period. At the end of the experiment, a significant decrease in ergosterol by 72.3%, 80.8%, and 83.1%, compared with control soil, was observed when 1, 2, and 4 l ha(-1), respectively, was added. Soil enzymatic activities increased when the Prochloraz applied to the soil increased, possibly because the fungicide is used by bacterial communities as a source of energy and nutrients. The 16S rDNA-DGGE profiles indicated that the fungicide did not negatively affect soil bacterial biodiversity. These results suggested that the fungicide Prochloraz has a very interesting agronomic effect, possibly due to the negative effect on soil fungal population stimulating the growth of soil bacterial activity.

  11. Novel homozygous missense mutation in ALDH7A1 causes neonatal pyridoxine dependent epilepsy.

    PubMed

    Coci, Emanuele G; Codutti, Luca; Fink, Christian; Bartsch, Sophie; Grüning, Gunnar; Lücke, Thomas; Kurth, Ingo; Riedel, Joachim

    2017-04-01

    Pyridoxine dependent epilepsy (PDE) (OMIM#266100) is a neonatal form of epilepsy, caused by dysfunction of the enzyme α-aminoadipic semialdehyde dehydrogenase (ALDH7A1 or Antiquitin). This enzyme converts α-aminoadipic semialdehyde (α-AASA) into α-aminoadipate (AAA), a critical step in the lysine metabolism of the brain. ALDH7A1 dysfunction causes an accumulation of α-AASA and δ(1)-piperideine-6-carboxylic acid (P6C), which are in equilibrium with each other. P6C binds and inactivates pyridoxal 5'-phosphate (PLP), the active form of pyridoxine. Individuals affected by ALDH7A1 deficiency show pre-natal and post-natal seizures, which respond to oral pyridoxine but not to other pediatric anti-epileptic drugs. We discovered a novel missense mutation (c.566G > A, p.Gly189Glu) in homozygous state residing in the NAD+ binding domain coding region of exon 6 and affecting an highly conserved amino acid residue. The seizures stopped under post-natal pyridoxine therapy, nevertheless a longer follow-up is needed to evaluate the intellectual development of the child, who is additionally treated with oral l-arginine since the 13th month of life. Developmental delay with or without structural cortex abnormalities were reported in several patients. A brain MRI scan revealed hyperintense white matter in the right cerebellum compatible with cerebellar gliosis. Taken together, our studies enlarge the group of missense pathogenic mutations of ALDH7A1 gene and reveal a novel cerebellar finding within the PDE patients cohort.

  12. [Influence of ionizing radiation on enzymatic activity and state of nucleus-nucleolar apparatus in rat hepatocytes].

    PubMed

    Nersesova, L S; Gazariants, M G; Mkrtchian, Z S; Meliksetian, G O; Pogosian, L G; Pogosian, S A; Pogosian, L L; Karalova, E M; Avetisian, A S; Abroian, l O; Karalian, Z A; Akopian, Zh I

    2013-01-01

    The effects of a single exposure of rats to the whole-body roentgen irradiation at the doses of 3.5 Gy and 4.5 Gy on the activity of creatine kinase, purine nucleoside phosphorylase, alanine aminotransferase, aspartate aminotransferase, as well as on the state of the nuclear-nucleolar apparatus in rat hepatocytes on the 6th and 13th days after radiation exposure have been studied. Irradiation at the above doses induced changes in the levels of enzymatic activity of different values and different directions within the same time periods, as well as oscillating changes in this type of enzymatic activity over time. This demonstrates various radiosensitivity and adaptation abilities of these enzymatic activities. The changes in the enzymatic activity significantly correspond to the changes in the morphometric indices of nuclear-nucleolar apparatus of hepatocytes, as well as the distribution of hepatocytes within the ploidy classes: in particular, stabilization of the enzymatic activity on the 13th day after irradiation correlates with the increased transcriptional activity, which is detectable through the increased number of nucleoli per nucleus and the expanded space of a hepatocyte nucleus. The compensation mechanisms are likely to be targeted at the changes in the functional activity of surviving hepatocytes, rather than at the replacement of the damaged cells by the new ones.

  13. Novel, dually radiolabeled peptides for simultaneous monitoring of enzymatic activity and protein targets

    SciTech Connect

    Efrem Mebrahtu, Suzanne Lapi

    2012-12-13

    This application investigated a novel imaging approach to develop methods to incorporate multiple radionuclides into a single peptide at chemoselective sites for simultaneous monitoring of cell-bound protein targets as well as specific enzymatic activity, both of which are associated with enhanced tumor growth and metastasis. This imaging construct was synthesized in such a manner so that the PET radionuclide will remain associated with the tumor cells and the SPECT radionuclide was cleaved from the imaging agent. Measurement of the PET agent only will yield information about the tumor marker density while measurement of the amount of co-localization and mismatch of the two radionuclides will yield information about the enzymatic activity. This coincident measuring technique using both PET and SPECT agents allows us to draw correlations involving the interactions of enzymes (cathepsin, serine-protease urokinase (uPA) and matrix metalloproteases) and other cellular proteins which play a role in cancer growth and metastasis. This technique will allow for studies in xenograft or genetic models of cancer in the same animal at the same time, thus eliminating problems that may occur when trying to invoke comparisons across animals or timepoints. By using radionuclide imaging as opposed to other imaging modalities, this technique has the potential to be translatable and can exploit the high specific activity probes which can be generated with radiotracers. The proof of principle test of this system investigated simultaneous monitoring of matrix metalloprotease (MMP) activity in the extracellular matrix (ECM) as well as density of integrins on the cell surface, both of which can serve as tumor markers. The outcomes/deliverables of this project were as follows: 1. Peptides were synthesized dually labeled at chemospecific sites with PET and SPECT agents. 2. Stability (intrinsic and to radiolysis) and specific activity of these labeled compounds were determined. 3. The

  14. Effect of untreated sewage effluent irrigation on heavy metal content, microbial population and enzymatic activities of soils in Aligarh.

    PubMed

    Bansal, O P; Singh, Gajraj; Katiyar, Pragati

    2014-07-01

    The study pertains to the impact of domestic and industrial sewage water irrigation on the chemical, biological and enzymatic activities in alluvial soils of Aligarh District. Results showed that soil enzymatic [dehydogenase (DHA), acid and alkaline phosphatase, urease and catalase] activities in the soils increased up to 14 days of incubation and thereafter inhibited significantly. The enzymatic activity were in the order sewage effluent > partial sewage effluent > ground water irrigated soils. Increase in soil enzymatic activities up to 2nd week of incubation was due to decomposition of organic matter. Maximum inhibition of enzymatic activities, after 14 days of incubation were found in sewage effluent irrigated soils and minimum in ground water irrigated soils. Similar trend was also seen for microbial population. Soil enzymatic activities and microbial population were significantly and positively correlated with soil organic matter. Results also indicated that the microbial population and enzymatic activities in sewage irrigated soils decreased continually with irrigation period. The average concentration of total heavy metals in sewage irrigated soils and partial sewage irrigated soils increased and was 3 and 2 times higher for Zn; 4.5 and 1.7 times higher for Cu; 3.8 and 2.4 times higher for Cr; 5.7 and 3.5 times higher for Pb; 3.5 and 2.2 times higher for Cd and 2.7 and 2.0 times higher for Ni respectively than that of ground water irrigated soils. Results also showed that though total heavy metals concentration increased with period of sewage irrigation but the concentration of diethylene triamine pentaacetic acid (DTPA) extractable heavy metals in partial sewage irrigated and sewage irrigated soils remained almost same, which might be due to deposition of heavy metals in crops grown on the soils.

  15. Effect of ALDH1 on prognosis and chemoresistance by breast cancer subtype.

    PubMed

    Kida, Kumiko; Ishikawa, Takashi; Yamada, Akimitsu; Shimada, Kazuhiro; Narui, Kazutaka; Sugae, Sadatoshi; Shimizu, Daisuke; Tanabe, Mikiko; Sasaki, Takeshi; Ichikawa, Yasushi; Endo, Itaru

    2016-04-01

    Aldehyde dehydrogenase 1 (ALDH1) has been identified as a breast cancer stem cell marker, but its value as a predictor of prognosis and chemoresistance is controversial. This study investigated the effect of ALDH1 on prognosis and chemoresponse by breast cancer subtype. We immunohistochemically analyzed 653 invasive breast cancer specimens and evaluated correlations among clinicopathological factors, survival status, response to neoadjuvant chemotherapy, and ALDH1 expression. Of 653 specimens, 139 (21.3 %) expressed ALDH1 in tumor cells. ALDH1 expression was correlated significantly with larger tumor size, node metastasis, higher nuclear grade, and with HER2(+) and progesterone/estrogen receptor (HR)(-) subtypes. ALDH1 expression was significantly observed in HER2 type and triple-negative breast cancer (TNBC). Patients with ALDH1(+) cancers had significantly shorter disease-free survival (P < 0001) and overall survival (P = 0.044). ALDH1 expression significantly affected prognosis of luminal types, but not TNBC and HER2-enriched types. For the 234 patients treated with neoadjuvant chemotherapy, pathological complete response (pCR) rate was significantly lower in ALDH1(+) cases (13.5 vs. 30.3 %, P = 0.003). pCR and ALDH1 expression were significantly correlated in TNBC patients (P = 0.003). ALDH1(+) breast cancers tended to be aggressive, with poor prognoses. Although ALDH1(+) TNBC showed higher chemoresistance, ALDH1 had significant impact on prognosis in the luminal type but not in TNBC.

  16. Enzymatic modification of chitosan by cinnamic acids: Antibacterial activity against Ralstonia solanacearum.

    PubMed

    Yang, Caifeng; Zhou, Yu; Zheng, Yu; Li, Changlong; Sheng, Sheng; Wang, Jun; Wu, Fuan

    2016-06-01

    This study aimed to identify chitosan polymers that have antibacterial activity against the bacterial wilt pathogen. The chitosan polymers were enzymatically synthesized using chitosan and five cinnamic acids (CADs): caffeic acid (CA), ferulic acid (FA), cinnamic acid (CIA), p-coumaric acid (COA) and chlorogenic acid (CHA), using laccase from Pleurotus ostreatus as a catalyst. The reaction was performed in a phosphate buffered solution under heterogenous reaction conditions. The chitosan derivatives (CTS-g-CADs) were characterized by FT-IR, XRD, TGA and SEM. FT-IR demonstrated that the reaction products bound covalently to the free amino groups or hydroxyl groups of chitosan via band of amide I or ester band. XRD showed a reduced packing density for grafted chitosan comparing to original chitosan. TGA demonstrated that CTS-g-CADs have a higher thermostability than chitosan. Additionally, chitosan and its derivatives showed similar antibacterial activity. However, the IC50 value of the chitosan-caffeic acid derivative (CTS-g-CA) against the mulberry bacterial wilt pathogen RS-5 was 0.23mg/mL, which was two-fifths of the IC50 value of chitosan. Therefore, the enzymatically synthesized chitosan polymers can be used to control plant diseases in biotechnological domains.

  17. Non-enzymatic Glycation of Almond Cystatin Leads to Conformational Changes and Altered Activity.

    PubMed

    Siddiqui, Azad A; Sohail, Aamir; Bhat, Sheraz A; Rehman, Md T; Bano, Bilqees

    2015-01-01

    The non-enzymatic reaction between proteins and reducing sugars, known as glycation, leads to the formation of inter and intramolecular cross-links of proteins. Stable end products called as advanced Maillard products or advanced glycation end products (AGEs) have received tremendous attention since last decades. It was suggested that the formation of AGEs not only modify the conformation of proteins but also induces altered biological activity. In this study, cystatin purified from almond was incubated with three different sugars namely D-ribose, fructose and lactose to monitor the glycation process. Structural changes induced in cystatin on glycation were studied using UV-visible spectroscopy, fluorescence spectroscopy, CD and FTIR techniques. Glycated cystatin was found to migrate slower on electrophoresis as compared to control cystatin. Biological activity data of glycated cystatin showed that D-ribose was most effective in inducing conformational changes with maximum altered activity.

  18. Integrated catalysis opens new arylation pathways via regiodivergent enzymatic C–H activation

    PubMed Central

    Latham, Jonathan; Henry, Jean-Marc; Sharif, Humera H.; Menon, Binuraj R. K.; Shepherd, Sarah A.; Greaney, Michael F.; Micklefield, Jason

    2016-01-01

    Despite major recent advances in C–H activation, discrimination between two similar, unactivated C–H positions is beyond the scope of current chemocatalytic methods. Here we demonstrate that integration of regioselective halogenase enzymes with Pd-catalysed cross-coupling chemistry, in one-pot reactions, successfully addresses this problem for the indole heterocycle. The resultant ‘chemobio-transformation' delivers a range of functionally diverse arylated products that are impossible to access using separate enzymatic or chemocatalytic C–H activation, under mild, aqueous conditions. This use of different biocatalysts to select different C–H positions contrasts with the prevailing substrate-control approach to the area, and presents opportunities for new pathways in C–H activation chemistry. The issues of enzyme and transition metal compatibility are overcome through membrane compartmentalization, with the optimized process requiring no intermediate work-up or purification steps. PMID:27283121

  19. Antibacterial and enzymatic activity of microbial community during wastewater treatment by pilot scale vermifiltration system.

    PubMed

    Arora, Sudipti; Rajpal, Ankur; Bhargava, Renu; Pruthi, Vikas; Bhatia, Akansha; Kazmi, A A

    2014-08-01

    The present study investigated microbial community diversity and antibacterial and enzymatic properties of microorganisms in a pilot-scale vermifiltration system during domestic wastewater treatment. The study included isolation and identification of diverse microbial community by culture-dependent method from a vermifilter (VF) with earthworms and a conventional geofilter (GF) without earthworms. The results of the four months study revealed that presence of earthworms in VF could efficiently remove biochemical oxygen demand (BOD), chemical oxygen demand (COD), total and fecal coliforms, fecal streptococci and other pathogens. Furthermore, the burrowing activity of earthworms promoted the aeration conditions in VF which led to the predominance of the aerobic microorganisms, accounting for complex microbial community diversity. Antibacterial activity of the isolated microorganisms revealed the mechanism behind the removal of pathogens, which is reported for the first time. Specifically, cellulase, amylase and protease activity is responsible for biodegradation and stabilization of organic matter.

  20. Non-enzymatic glycation reduces heparin cofactor II anti-thrombin activity.

    PubMed

    Ceriello, A; Marchi, E; Barbanti, M; Milani, M R; Giugliano, D; Quatraro, A; Lefebvre, P

    1990-04-01

    The effects of non-enzymatic glycation on heparin cofactor II activity, at glucose concentrations which might be expected in physiological or diabetic conditions have been evaluated in this study. Radiolabelled glucose incorporation was associated with a loss of heparin cofactor anti-thrombin activity. The heparin cofactor heparin and dermatan sulfate-dependent inhibition of thrombin was significantly reduced, showing a remarkable decrease of the maximum second order rate constant. This study shows that heparin cofactor can be glycated at glucose concentrations found in the blood, and that this phenomenon produces a loss of heparin cofactor-antithrombin activity. These data suggest, furthermore, a possible link between heparin cofactor glycation and the pathogenesis of thrombosis in diabetes mellitus.

  1. A novel protective mechanism for mitochondrial aldehyde dehydrogenase (ALDH2) in type i diabetes-induced cardiac dysfunction: role of AMPK-regulated autophagy.

    PubMed

    Guo, Yuli; Yu, Wenjun; Sun, Dongdong; Wang, Jiaxing; Li, Congye; Zhang, Rongqing; Babcock, Sara A; Li, Yan; Liu, Min; Ma, Meijuan; Shen, Mingzhi; Zeng, Chao; Li, Na; He, Wei; Zou, Qian; Zhang, Yingmei; Wang, Haichang

    2015-02-01

    Mitochondrial aldehyde dehydrogenase (ALDH2) is known to offer myocardial protection against stress conditions including ischemia-reperfusion injury, alcoholism and diabetes mellitus although the precise mechanism is unclear. This study was designed to evaluate the effect of ALDH2 on diabetes-induced myocardial injury with a focus on autophagy. Wild-type FVB and ALDH2 transgenic mice were challenged with streptozotozin (STZ, 200mg/kg, i.p.) for 3months to induce experimental diabetic cardiomyopathy. Diabetes triggered cardiac remodeling and contractile dysfunction as evidenced by cardiac hypertrophy, decreased cell shortening and prolonged relengthening duration, the effects of which were mitigated by ALDH2. Lectin staining displayed that diabetes promoted cardiac hypertrophy, the effect of which was alleviated by ALDH2. Western blot analysis revealed dampened autophagy protein markers including LC3B ratio and Atg7 along with upregulated p62 following experimental diabetes, the effect of which was reconciled by ALDH2. Phosphorylation level of AMPK was decreased and its downstream signaling molecule FOXO3a was upregulated in both diabetic cardiac tissue and in H9C2 cells with high glucose exposure. All these effect were partly abolished by ALDH2 overexpression and ALDH2 agonist Alda1. High glucose challenge dampened autophagy in H9C2 cells as evidenced by enhanced p62 levels and decreased levels of Atg7 and LC3B, the effect of which was alleviated by the ALDH2 activator Alda-1. High glucose-induced cell death and apoptosis were reversed by Alda-1. The autophagy inhibitor 3-MA and the AMPK inhibitor compound C mitigated Alda-1-offered beneficial effect whereas the autophagy inducer rapamycin mimicked or exacerbated high glucose-induced cell injury. Moreover, compound C nullified Alda-1-induced protection against STZ-induced changes in autophagy and function. Our results suggested that ALDH2 protects against diabetes-induced myocardial dysfunction possibly through an

  2. Assessment of the human fecal microbiota: I. Measurement and reproducibility of selected enzymatic activities

    PubMed Central

    Flores, Roberto; Shi, Jianxin; Gail, Mitchell H.; Ravel, Jacques; Goedert, James J.

    2012-01-01

    Background The intestinal microbial community has major effects on human health, but optimal research methods are unsettled. To facilitate epidemiologic and clinical research, we sought to optimize conditions and to assess reproducibility of selected core functions of the distal gut microbiota, β-glucuronidase and β-glucosidase bioactivities. Methods and results A colorimetric kinetic method was optimized and used to quantify activities of β-glucuronidase and β-glucosidase in human feces. Enzyme detection was optimal with neutral pH, snap freezing in liquid nitrogen, and rapid thawing to 37°C before protein extraction. Enzymatic stability was assessed by delayed freezing for 2–48 hours to mimic field settings. Activities decayed approximately 20% within 2 hours and 40% within 4 hours at room temperature. To formally assess reproducibility, 51 volunteers (25 male; mean age 39) used two devices to self-collect and rapidly chill four replicates of a stool. Devices were compared for mean enzymatic activities and intraclass correlation coefficients (ICC) in paired replicates of the self-collected specimens. Reproducibility was excellent with both devices for β-glucuronidase (ICC 0.92). The larger collection device had significantly higher reproducibility for β-glucosidase (ICC 0.92 vs. 0.76, P<0.0001) and higher mean activities for both enzymes (P<0.0001). Conclusions Optimal measurement of these core activities of the microbiota required a sufficient quantity of rapidly chilled or frozen specimens collected in PBS at pH7.0. Application of these methods to clinical and epidemiologic research could provide insights on how the intestinal microbiota affects human health. PMID:22409163

  3. Enzymatic activity of lactic acid bacteria (with antimicrobial properties) isolated from a traditional Spanish cheese.

    PubMed

    González, Leticia; Sacristán, Noelia; Arenas, Ricardo; Fresno, José M; Eugenia Tornadijo, M

    2010-08-01

    Twenty-four strains of lactic acid bacteria (LAB) isolated from a traditional Spanish cheese (Genestoso cheese) were evaluated for their enzymatic activities (acidifying and proteolytic abilities and carboxypeptidase, aminopeptidase, dipeptidase, caseinolytic and esterase activities), in order to select indigenous strains of technical interest for the manufacture of cheese. These strains were selected on the basis of their antimicrobial activity relative to five reference strains and were identified as Lactococcus lactis subsp. lactis (thirteen strains), Leuconostoc mesenteroides (two strains), Leuconostoc pseudomesenteroides (one strain), Lactobacillus paracasei (two strains), Lactobacillus plantarum (one strain) and Enterococcus faecalis (five strains). Lactococcus strains were those that showed the greatest degree of acidifying and proteolytic activity. The cell-free extracts (CFE) of L. paracasei exhibited the highest level of aminopeptidase activity. The highest level of caseinolytic activity was shown by the CFE of one strain of L. lactis. High values were also obtained with the CFE of Lactobacillus and of several Leuconostoc. The highest level of dipeptidase activity was found amongst the strains of L. lactis. Carboxypeptidase activity was generally very low or undetectable for the majority of strains. The greatest degree of esterolytic activity was detected for Enterococcus.

  4. A water activity control system for enzymatic reactions in organic media.

    PubMed

    Petersson, Anna E V; Adlercreutz, Patrick; Mattiasson, Bo

    2007-06-01

    A water activity control system for enzymatic synthesis in organic media, for litre-scale reactors has been constructed. Water activity, a(w), is a key factor when using enzymes in non-conventional media and the optimum value varies for different enzymes. The control system consists of a water activity sensor in the headspace of a jacketed glass reactor (equipped with narrow steel tubes to introduce air), gas-washing bottles containing blue silica gel (a(w)=0) and water (a(w)=1), a PC to monitor water activity and a programmable logic controller (PLC) to control the water activity. The system was evaluated by adjusting water activity in the medium, with a deviation from the set point of less than +/-0.05. Synthesis of cetyl palmitate, under controlled water activity and catalysed by two different lipase preparations, namely, Novozym 435 (immobilised Candida antarctica lipase B) and immobilised Candida rugosa lipase, were also performed. Novozym 435 catalyses reactions very well at extremely low water activity while C. rugosa lipase shows low activity for a(w)<0.5.

  5. AID enzymatic activity is inversely proportional to the size of cytosine C5 orbital cloud.

    PubMed

    Rangam, Gopinath; Schmitz, Kerstin-Maike; Cobb, Alexander J A; Petersen-Mahrt, Svend K

    2012-01-01

    Activation induced deaminase (AID) deaminates cytosine to uracil, which is required for a functional humoral immune system. Previous work demonstrated, that AID also deaminates 5-methylcytosine (5 mC). Recently, a novel vertebrate modification (5-hydroxymethylcytosine - 5 hmC) has been implicated in functioning in epigenetic reprogramming, yet no molecular pathway explaining the removal of 5 hmC has been identified. AID has been suggested to deaminate 5 hmC, with the 5 hmU product being repaired by base excision repair pathways back to cytosine. Here we demonstrate that AID's enzymatic activity is inversely proportional to the electron cloud size of C5-cytosine - H > F > methyl > hydroxymethyl. This makes AID an unlikely candidate to be part of 5 hmC removal.

  6. Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases

    PubMed Central

    Tarhonskaya, Hanna; Rydzik, Anna M.; Leung, Ivanhoe K. H.; Loik, Nikita D.; Chan, Mun Chiang; Kawamura, Akane; McCullagh, James S. O.; Claridge, Timothy D. W.; Flashman, Emily; Schofield, Christopher J.

    2014-01-01

    Accumulation of (R)-2-hydroxyglutarate in cells results from mutations to isocitrate dehydrogenase that correlate with cancer. A recent study reports that (R)-, but not (S)-2-hydroxyglutarate, acts as a co-substrate for the hypoxia-inducible factor prolyl hydroxylases via enzyme-catalysed oxidation to 2-oxoglutarate. Here we investigate the mechanism of 2-hydroxyglutarate-enabled activation of 2-oxoglutarate oxygenases, including prolyl hydroxylase domain 2, the most important human prolyl hydroxylase isoform. We observe that 2-hydroxyglutarate-enabled catalysis by prolyl hydroxylase domain 2 is not enantiomer-specific and is stimulated by ferrous/ferric ion and reducing agents including L-ascorbate. The results reveal that 2-hydroxyglutarate is oxidized to 2-oxoglutarate non-enzymatically, likely via iron-mediated Fenton-chemistry, at levels supporting in vitro catalysis by 2-oxoglutarate oxygenases. Succinic semialdehyde and succinate are also identified as products of 2-hydroxyglutarate oxidation. Overall, the results rationalize the reported effects of 2-hydroxyglutarate on catalysis by prolyl hydroxylases in vitro and suggest that non-enzymatic 2-hydroxyglutarate oxidation may be of biological interest. PMID:24594748

  7. Enzymatic heme oxygenase activity in soluble extracts of the unicellular red alga, Cyanidium caldarium.

    PubMed

    Beale, S I; Cornejo, J

    1984-12-01

    Extracts of the phycocyanin-containing unicellular red alga, Cyanidium caldarium, catalyzed enzymatic cleavage of the heme macrocycle to form the linear tetrapyrrole bilin structure. This is the key first step in the branch of the tetrapyrrole biosynthetic pathway leading to phycobilin photosynthetic accessory pigments. A mixed-function oxidase mechanism, similar to the biliverdin-forming reaction catalyzed by animal cell-derived microsomal heme oxygenase, was indicated by requirements for O2 and a reduced pyridine nucleotide. To avoid enzymatic conversion of the bilin product to phycocyanobilins and subsequent degradation during incubation, mesoheme IX was substituted for the normal physiological substrate, protoheme IX. Mesobiliverdin IX alpha was identified as the primary incubation product by comparative reverse-phase high-pressure liquid chromatography and absorption spectrophotometry. The enzymatic nature of the reaction was indicated by the requirement for cell extract, absence of activity in boiled cell extract, high specificity for NADPH as cosubstrate, formation of the physiologically relevant IX alpha bilin isomer, and over 75% inhibition by 1 microM Sn-protoporphyrin, which has been reported to be a competitive inhibitor of animal microsomal heme oxygenase. On the other hand, coupled oxidation of mesoheme, catalyzed by ascorbate plus pyridine or myoglobin, yielded a mixture of ring-opening mesobiliverdin IX isomers, was not inhibited by Sn-protoporphyrin, and could not use NADPH as the reductant. Unlike the animal microsomal heme oxygenase, the algal reaction appeared to be catalyzed by a soluble enzyme that was not sedimentable by centrifugation for 1 h at 200,000g. Although NADPH was the preferred reductant, small amounts of activity were obtained with NADH or ascorbate. A portion of the activity was retained after gel filtration of the cell extract to remove low-molecular-weight components. Considerable stimulation of activity, particularly in

  8. A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity

    PubMed Central

    Góra, Monika; Buckle, Ashley M.; Porebski, Benjamin T.; Kemp, E. Helen; Sutton, Brian J.; Czarnocka, Barbara; Banga, J. Paul

    2014-01-01

    Background: Thyroid peroxidase (TPO) is a dimeric membrane-bound enzyme of thyroid follicular cells, responsible for thyroid hormone biosynthesis. TPO is also a common target antigen in autoimmune thyroid disease (AITD). With two active sites, TPO is an unusual enzyme, and thus there is much interest in understanding its structure and role in AITD. Homology modeling has shown TPO to be composed of different structural modules, as well as a propeptide sequence. During the course of studies to obtain homogeneous preparations of recombinant TPO for structural studies, we investigated the role of the large propeptide sequence in TPO. Methods: An engineered recombinant human TPO preparation expressed in Chinese hamster ovary (CHO) cells lacking the propeptide (TPOΔpro; amino acid residues 21–108) was characterized and its properties compared to wild-type TPO. Plasma membrane localization was determined by cell surface protein biotinylation, and biochemical studies were performed to evaluate enzymatic activity and the effect of deglycosylation. Immunological investigations using autoantibodies from AITD patients and other epitope-specific antibodies that recognize conformational determinants on TPO were evaluated for binding to TPOΔpro by flow cytometry, immunocytochemistry, and capture enzyme-linked immunosorbent assay. Molecular modeling and dynamics simulation of TPOΔpro comprising a dimer of myeloperoxidase-like domains was performed in order to investigate the impact of propeptide removal and the role of glycosylation. Results: The TPOΔpro was expressed on the cell surface at comparable levels to wild-type TPO. The TPOΔpro was enzymatically active and recognized by patients' autoantibodies and a panel of epitope-specific antibodies, confirming structural integrity of the two major conformational determinants recognized by autoantibodies. Faithful intracellular trafficking and N-glycosylation of TPOΔpro was also maintained. Molecular modeling and dynamics

  9. Vertical and horizontal distributions of microbial abundances and enzymatic activities in propylene-glycol-affected soils.

    PubMed

    Biró, Borbála; Toscano, Giuseppe; Horváth, Nikoletta; Matics, Heléna; Domonkos, Mónika; Scotti, Riccardo; Rao, Maria A; Wejden, Bente; French, Helen K

    2014-01-01

    The natural microbial activity in the unsaturated soil is vital for protecting groundwater in areas where high loads of biodegradable contaminants are supplied to the surface, which usually is the case for airports using aircraft de-icing fluids (ADF) in the cold season. Horizontal and vertical distributions of microbial abundance were assessed along the western runway of Oslo Airport (Gardermoen, Norway) to monitor the effect of ADF dispersion with special reference to the component with the highest chemical oxygen demand (COD), propylene glycol (PG). Microbial abundance was evaluated by several biondicators: colony-forming units (CFU) of some physiological groups (aerobic and anaerobic heterotrophs and microscopic fungi), most probable numbers (MPN) of PG degraders, selected catabolic enzymatic activities (fluorescein diacetate (FDA) hydrolase, dehydrogenase, and β-glucosidase). High correlations were found between the enzymatic activities and microbial counts in vertical soil profiles. All microbial abundance indicators showed a steep drop in the first meter of soil depth. The vertical distribution of microbial abundance can be correlated by a decreasing exponential function of depth. The horizontal trend of microbial abundance (evaluated as total aerobic CFU, MPN of PG-degraders, and FDA hydrolase activity) assessed in the surface soil at an increasing distance from the runway is correlated negatively with the PG and COD loads, suggesting the relevance of other chemicals in the modulation of microbial growth. The possible role of potassium formate, component of runway de-icers, has been tested in the laboratory by using mixed cultures of Pseudomonas spp., obtained by enrichment with a selective PG medium from soil samples taken at the most contaminated area near the runway. The inhibitory effect of formate on the growth of PG degraders is proven by the reduction of biomass yield on PG in the presence of formate.

  10. ALDH3A1 — EDRN Public Portal

    Cancer.gov

    ALDH3A1 is a member of the aldehyde dehydrogenase family. They are involved in the metabolism of corticosteroids, biogenic amines, neurotransmitters, and lipid peroxidation. They are also involved in the detoxification of alcohol-derived acetaldehyde. The ALDH3A1 protein is an enzyme that forms a cytoplasmic homodimer that oxidizes aromatic and medium-chain (6 carbons or more) saturated and unsaturated aldehyde substrates. It is thought to promote resistance to UV and 4-hydroxy-2-nonenal-induced oxidative damage in the cornea. There are several splice variants that encode the same protein.

  11. Enzymatic and metabolic activities of four anaerobic sludges and their impact on methane production from ensiled sorghum forage.

    PubMed

    Sambusiti, C; Rollini, M; Ficara, E; Musatti, A; Manzoni, M; Malpei, F

    2014-03-01

    Biochemical methane potential (BMP) tests were run on ensiled sorghum forage using four inocula (urban, agricultural, mixture of agricultural and urban, granular) and differences on their metabolic and enzymatic activities were also discussed. Results indicate that no significant differences were observed in terms of BMP values (258±14NmLCH4g(-1)VS) with a slightly higher value when agricultural sludge was used as inoculum. Significant differences can be observed among different inocula, in terms of methane production rate. In particular the fastest biomethanization occurred when using the urban sludge (hydrolytic kinetic constant kh=0.146d(-1)) while the slowest one was obtained from the agricultural sludge (kh=0.049d(-1)). Interestingly, positive correlations between the overall enzymatic activities and methane production rates were observed for all sludges, showing that a high enzymatic activity may favour the hydrolysis of complex substrate and accelerate the methanization process of sorghum.

  12. Mitochondrial Respiration Chain Enzymatic Activities in the Human Brain: Methodological Implications for Tissue Sampling and Storage.

    PubMed

    Ronsoni, Marcelo Fernando; Remor, Aline Pertile; Lopes, Mark William; Hohl, Alexandre; Troncoso, Iris H Z; Leal, Rodrigo Bainy; Boos, Gustavo Luchi; Kondageski, Charles; Nunes, Jean Costa; Linhares, Marcelo Neves; Lin, Kátia; Latini, Alexandra Susana; Walz, Roger

    2016-04-01

    Mitochondrial respiratory chain complexes enzymatic (MRCCE) activities were successfully evaluated in frozen brain samples. Epilepsy surgery offers an ethical opportunity to study human brain tissue surgically removed to treat drug resistant epilepsies. Epilepsy surgeries are done with hemodynamic and laboratory parameters to maintain physiology, but there are no studies analyzing the association among these parameters and MRCCE activities in the human brain tissue. We determined the intra-operative parameters independently associated with MRCCE activities in middle temporal neocortex (Cx), amygdala (AMY) and head of hippocampus (HIP) samples of patients (n = 23) who underwent temporal lobectomy using multiple linear regressions. MRCCE activities in Cx, AMY and HIP are differentially associated to trans-operative mean arterial blood pressure, O2 saturation, hemoglobin, and anesthesia duration to time of tissue sampling. The time-course between the last seizure occurrence and tissue sampling as well as the sample storage to biochemical assessments were also associated with enzyme activities. Linear regression models including these variables explain 13-17 % of MRCCE activities and show a moderate to strong effect (r = 0.37-0.82). Intraoperative hemodynamic and laboratory parameters as well as the time from last seizure to tissue sampling and storage time are associated with MRCCE activities in human samples from the Cx, AMYG and HIP. Careful control of these parameters is required to minimize confounding biases in studies using human brain samples collected from elective neurosurgery.

  13. Correlation between quantified promoter methylation and enzymatic activity of O6-methylguanine-DNA methyltransferase in glioblastomas.

    PubMed

    Kishida, Yugo; Natsume, Atsushi; Toda, Hiroshi; Toi, Yuki; Motomura, Kazuya; Koyama, Hiroko; Matsuda, Keiji; Nakayama, Osamu; Sato, Makoto; Suzuki, Masaaki; Kondo, Yutaka; Wakabayashi, Toshihiko

    2012-04-01

    The DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT, AGT) is a determinant of the resistance of tumor cells to alkylating anticancer agents that target the O(6) position of guanine. MGMT promoter methylation in tumors is regarded as the most common predictor of the responsiveness of glioblastoma to alkylating agents. However, MGMT promoter methylation status has been investigated mainly by methylation-specific PCR, which is a qualitative and subjective assay. In addition, the actual enzymatic activities associated with the methylation status of MGMT have not been explored. In the present study, MGMT promoter methylation in glioblastomas was quantified by bisulfite pyrosequencing, and its correlation with enzymatic activity was determined using a novel quantitative assay for studying the functional activity of MGMT. MGMT enzymatic activity was assessed using fluorometrically labeled oligonucleotide substrates containing MGMT-specific DNA lesions and capillary electrophoresis to detect and quantify these lesions. In comparison with existing traditional assays, this assay was equally sensitive but less time consuming and easier to perform. MGMT promoter methylation was assessed in 41 glioblastomas by bisulfite pyrosequencing, and five samples with different values were chosen for comparison with enzymatic assays. Bisulfite pyrosequencing using primers designed to work in the upstream promoter regions of MGMT demonstrated high quantitative capability and reproducibility in triplicate measurements. In comparative studies, MGMT promoter methylation values obtained by bisulfite pyrosequencing were inversely proportional to the measured enzymatic activity. The present results indicate that the quantification of MGMT methylation by bisulfite pyrosequencing represents its enzymatic activity and thus, its therapeutic responsiveness to alkylating agents.

  14. The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica

    PubMed Central

    Vaz, Aline B. M.; Rosa, Luiz H.; Vieira, Mariana L. A.; de Garcia, Virginia; Brandão, Luciana R.; Teixeira, Lia C. R. S.; Moliné, Martin; Libkind, Diego; van Broock, Maria; Rosa, Carlos A.

    2011-01-01

    The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4°C and 20°C, indicating that they could be metabolically active in the sampled substrates. PMID:24031709

  15. Enzymatic crosslinking and degradation of gelatin as a switch for bone morphogenetic protein-2 activity.

    PubMed

    Kuwahara, Kenrick; Fang, Josephine Y; Yang, Zhi; Han, Bo

    2011-12-01

    Current therapies for tissue regeneration rely on the presence or direct delivery of growth factors to sites of repair. Bone morphogenetic protein-2 (BMP-2), combined with a carrier (usually collagen), is clinically proven to induce new bone formation during spinal fusion and nonunion repair. However, due to BMP-2's short half-life and its diffusive properties, orders of magnitude above physiological levels are required to ensure effectiveness. In addition, a high dose of this multifunctional growth factor is known to induce adverse effects in patients. To circumvent these challenges, we proposed and tested a new approach for BMP-2 delivery, by controlling BMP activity via carrier binding and localized proteolysis. BMP-2 was covalently bound to gelatin through site-specific enzymatic crosslinking using a microbial transglutaminase. Binding of BMP-2 to gelatin can completely switch off BMP-2 activity, as evidenced by loss of its transdifferentiating ability toward C2C12 promyoblasts. When gelatin sequestered BMP-2 is incubated with either microbial collagenase or tissue-derived matrix metalloproteinases, BMP-2 activity is fully restored. The activity of released BMP-2 correlates with the protease activity in a dose- and time-dependent manner. This observation suggests a novel way of delivering BMP-2 and controlling its activity. This improved delivery method, which relies on a physiological feedback, should enhance the known potential of this and other growth factors for tissue repair and regeneration.

  16. Many disease-associated variants of hTERT retain high telomerase enzymatic activity

    PubMed Central

    Zaug, Arthur J.; Crary, Sharon M.; Jesse Fioravanti, Matthew; Campbell, Kristina; Cech, Thomas R.

    2013-01-01

    Mutations in the gene for telomerase reverse transcriptase (hTERT) are associated with diseases including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis and cancer. Understanding the molecular basis of these telomerase-associated diseases requires dependable quantitative measurements of telomerase enzyme activity. Furthermore, recent findings that the human POT1-TPP1 chromosome end-binding protein complex stimulates telomerase activity and processivity provide incentive for testing variant telomerases in the presence of these factors. In the present work, we compare multiple disease-associated hTERT variants reconstituted with the RNA subunit hTR in two systems (rabbit reticulocyte lysates and human cell lines) with respect to telomerase enzymatic activity, processivity and activation by telomere proteins. Surprisingly, many of the previously reported disease-associated hTERT alleles give near-normal telomerase enzyme activity. It is possible that a small deficit in telomerase activity is sufficient to cause telomere shortening over many years. Alternatively, mutations may perturb functions such as the recruitment of telomerase to telomeres, which are essential in vivo but not revealed by simple enzyme assays. PMID:23901009

  17. Differential enzymatic activity of common haplotypic versions of the human acidic Mammalian chitinase protein.

    PubMed

    Seibold, Max A; Reese, Tiffany A; Choudhry, Shweta; Salam, Muhammad T; Beckman, Kenny; Eng, Celeste; Atakilit, Amha; Meade, Kelley; Lenoir, Michael; Watson, H Geoffrey; Thyne, Shannon; Kumar, Rajesh; Weiss, Kevin B; Grammer, Leslie C; Avila, Pedro; Schleimer, Robert P; Fahy, John V; Rodriguez-Santana, Jose; Rodriguez-Cintron, William; Boot, Rolf G; Sheppard, Dean; Gilliland, Frank D; Locksley, Richard M; Burchard, Esteban G

    2009-07-17

    Mouse models have shown the importance of acidic mammalian chitinase activity in settings of chitin exposure and allergic inflammation. However, little is known regarding genetic regulation of AMCase enzymatic activity in human allergic diseases. Resequencing the AMCase gene exons we identified 8 non-synonymous single nucleotide polymorphisms including three novel variants (A290G, G296A, G339T) near the gene area coding for the enzyme active site, all in linkage disequilibrium. AMCase protein isoforms, encoded by two gene-wide haplotypes, and differentiated by these three single nucleotide polymorphisms, were recombinantly expressed and purified. Biochemical analysis revealed the isoform encoded by the variant haplotype displayed a distinct pH profile exhibiting greater retention of chitinase activity at acidic and basic pH values. Determination of absolute kinetic activity found the variant isoform encoded by the variant haplotype was 4-, 2.5-, and 10-fold more active than the wild type AMCase isoform at pH 2.2, 4.6, and 7.0, respectively. Modeling of the AMCase isoforms revealed positional changes in amino acids critical for both pH specificity and substrate binding. Genetic association analyses of AMCase haplotypes for asthma revealed significant protective associations between the variant haplotype in several asthma cohorts. The structural, kinetic, and genetic data regarding the AMCase isoforms are consistent with the Th2-priming effects of environmental chitin and a role for AMCase in negatively regulating this stimulus.

  18. A Selective Glutathione Probe based on AIE Fluorogen and its Application in Enzymatic Activity Assay

    NASA Astrophysics Data System (ADS)

    Lou, Xiaoding; Hong, Yuning; Chen, Sijie; Leung, Chris Wai Tung; Zhao, Na; Situ, Bo; Lam, Jacky Wing Yip; Tang, Ben Zhong

    2014-03-01

    In this work, we design and synthesize a malonitrile-functionalized TPE derivative (TPE-DCV), which can react with thiol group through thiol-ene click reaction, leading to the fluorescence change of the system. Combined with the unique AIE property, TPE-DCV can selectively detect glutathione (GSH) but not cysteine or homocysteine. As the cleavage of GSSG with the aid of glutathione reductase produces GSH, which turns on the fluorescence of TPE-DCV, the ensemble of TPE-DCV and GSSG can thus serve as a label-free sensor for enzymatic activity assay of glutathione reductase. We also apply TPE-DCV for the detection of intracellular GSH in living cells.

  19. Chemical, enzymatic and cellular antioxidant activity studies of Agaricus blazei Murrill.

    PubMed

    Hakime-Silva, Ricardo A; Vellosa, José C R; Khalil, Najeh M; Khalil, Omar A K; Brunetti, Iguatemy L; Oliveira, Olga M M F

    2013-09-01

    Mushrooms possess nutritional and medicinal properties that have long been used for human health preservation and that have been considered by researchers as possible sources of free radical scavengers. In this work, the antioxidant properties of water extracts from Agaricus blazei Murill, produced by maceration and decoction, are demonstrated in vitro. Resistance to oxidation is demonstrated through three mechanisms: i) inhibition of enzymatic oxidative process, with 100% inhibition of HRP (horseradish peroxidase) and MPO (myeloperoxidase); ii) inhibition of cellular oxidative stress, with 80% inhibition of the oxidative burst of polymorphonuclear neutrophils (PMNs); and iii) direct action over reactive species, with 62% and 87% suppression of HOCl and superoxide anion radical (O2• -), respectively. From the data, it was concluded that the aqueous extract of A. blazei has significant antioxidant activity, indicating its possible application for nutraceutical and medicinal purposes.

  20. Specific inflammatory response of Anemonia sulcata (Cnidaria) after bacterial injection causes tissue reaction and enzymatic activity alteration.

    PubMed

    Trapani, M R; Parisi, M G; Parrinello, D; Sanfratello, M A; Benenati, G; Palla, F; Cammarata, M

    2016-03-01

    The evolution of multicellular organisms was marked by adaptations to protect against pathogens. The mechanisms for discriminating the ''self'' from ''non-self" have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of substances that varied in type and dimension, and observed clear, strong and specific reactions, especially after injection of Escherichia coli and Vibrio alginolyticus. Moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different bacterial strains alters the expression of these enzymes and suggesting a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. Our study shows for the first time, a specific reaction and enzymatic responses following injection of bacteria in a cnidarian.

  1. Linking Microbial Enzymatic Activities and Functional Diversity of Soil around Earthworm Burrows and Casts.

    PubMed

    Lipiec, Jerzy; Frąc, Magdalena; Brzezińska, Małgorzata; Turski, Marcin; Oszust, Karolina

    2016-01-01

    The aim of this work was to evaluate the effect of earthworms (Lumbricidae) on the enzymatic activity and microbial functional diversity in the burrow system [burrow wall (BW) 0-3 mm, transitional zone (TZ) 3-7 mm, bulk soil (BS) > 20 mm from the BW] and cast aggregates of a loess soil under a pear orchard. The dehydrogenase, β-glucosidase, protease, alkaline phosphomonoesterase, and acid phosphomonoesterase enzymes were assessed using standard methods. The functional diversity (catabolic potential) was assessed using the Average Well Color Development and Richness Index following the community level physiological profiling from Biolog Eco Plates. All measurements were done using soil from each compartment immediately after in situ sampling in spring. The enzymatic activites including dehydrogenase, protease, β-glucosidase and alkaline phosphomonoesterase were appreciably greater in the BW or casts than in BS and TZ. Conversely, acid phosphomonoesterase had the largest value in the BS. Average Well Color Development in both the TZ and the BS (0.98-0.94 A590 nm) were more than eight times higher than in the BWs and casts. The lowest richness index in the BS (15 utilized substrates) increased by 86-113% in all the other compartments. The PC1 in principal component analysis mainly differentiated the BWs and the TZ. Utilization of all substrate categories was the lowest in the BS. The PC2 differentiated the casts from the other compartments. The enhanced activity of a majority of the enzymes and increased microbial functional diversity in most earthworm-influenced compartments make the soils less vulnerable to degradation and thus increases the stability of ecologically relevant processes in the orchard ecosystem.

  2. Linking Microbial Enzymatic Activities and Functional Diversity of Soil around Earthworm Burrows and Casts

    PubMed Central

    Lipiec, Jerzy; Frąc, Magdalena; Brzezińska, Małgorzata; Turski, Marcin; Oszust, Karolina

    2016-01-01

    The aim of this work was to evaluate the effect of earthworms (Lumbricidae) on the enzymatic activity and microbial functional diversity in the burrow system [burrow wall (BW) 0–3 mm, transitional zone (TZ) 3–7 mm, bulk soil (BS) > 20 mm from the BW] and cast aggregates of a loess soil under a pear orchard. The dehydrogenase, β-glucosidase, protease, alkaline phosphomonoesterase, and acid phosphomonoesterase enzymes were assessed using standard methods. The functional diversity (catabolic potential) was assessed using the Average Well Color Development and Richness Index following the community level physiological profiling from Biolog Eco Plates. All measurements were done using soil from each compartment immediately after in situ sampling in spring. The enzymatic activites including dehydrogenase, protease, β-glucosidase and alkaline phosphomonoesterase were appreciably greater in the BW or casts than in BS and TZ. Conversely, acid phosphomonoesterase had the largest value in the BS. Average Well Color Development in both the TZ and the BS (0.98–0.94 A590 nm) were more than eight times higher than in the BWs and casts. The lowest richness index in the BS (15 utilized substrates) increased by 86–113% in all the other compartments. The PC1 in principal component analysis mainly differentiated the BWs and the TZ. Utilization of all substrate categories was the lowest in the BS. The PC2 differentiated the casts from the other compartments. The enhanced activity of a majority of the enzymes and increased microbial functional diversity in most earthworm-influenced compartments make the soils less vulnerable to degradation and thus increases the stability of ecologically relevant processes in the orchard ecosystem. PMID:27625645

  3. Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway.

    PubMed

    Keller, Markus A; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V; Griffin, Julian L; Ralser, Markus

    2016-01-01

    Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks.

  4. Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway

    PubMed Central

    Keller, Markus A.; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V.; Griffin, Julian L.; Ralser, Markus

    2016-01-01

    Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks. PMID:26824074

  5. A novel technical approach for the measurement of individual ACAT-1 and ACAT-2 enzymatic activity in the testis.

    PubMed

    Chen, Li; Lafond, Julie; Pelletier, R-Marc

    2009-01-01

    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is implicated in the esterification of cholesterol when the latter is present at concentrations exceeding metabolic demands. Thus, ACAT contributes to the maintenance of cholesterol homeostasis which in testis is essential for the production of fertile gametes. However, the role of individual isoform of the enzyme in the maintenance of cholesterol homeostasis in the gonads has not been addressed yet because approaches to measure the enzymatic activity of each isoform were lacking. Here, we used the selective ACAT-1 inhibitor, K-604, to measure the individual enzymatic activity of ACAT-1 and ACAT-2 in enriched fractions of mouse seminiferous tubules. K-604 inhibited adult mouse ACAT-1 much more than ACAT-2 with IC(50) values of 100 and 1,000 microM, respectively, in the tubules. Next, the inhibitor concentration (100 microM) that inhibits the activity of ACAT-1 but not the activity of ACAT-2 was determined and applied to measure ACAT-1 and ACAT-2 enzymatic activities in mouse seminiferous tubule-enriched fractions. ACAT-2 activity reached 2173 CPMB/200 microg protein, while ACAT-1 enzymatic activity was 713 CPMB/200 microg proteins in the tubules. We also compared the effect of another inhibitor Manassantin B with K-604. Increasing the concentration (0-1,000 microM) of Manassantin B resulted in the inhibition of the activity of both ACAT-1 and ACAT-2. The results show that only K-604 is a useful tool to determine the individual ACAT-1 and ACAT-2 enzymatic activities in the seminiferous tubules.

  6. Glutamic acid-149 is important for enzymatic activity of yeast inorganic pyrophosphatase.

    PubMed

    Gonzalez, M A; Cooperman, B S

    1986-11-04

    Modification of Saccharomyces cerevisiae inorganic pyrophosphatase (PPase) with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide is known to lead to a loss of enzymatic activity, the rate of which is decreased in the presence of ligands binding to the active site [Cooperman, B. S., & Chiu, N. Y. (1973) Biochemistry 12, 1676-1682; Heitman, P., & Uhlig, H. J. (1974) Acta Biol. Med. Ger. 32, 565-594]. In this work we show that, when such inactivation is carried out in the presence of [14C]glycine ethyl ester (GEE), GEE is covalently incorporated into PPase, incorporation into the most highly labeled tryptic peptide is site-specific, as evidenced by the reduction of such incorporation in the presence of the active site ligands Zn2+ and Pi, the extent of formation of this specifically labeled peptide correlates with the fractional loss of PPase activity, and the specifically labeled peptide corresponds to residues 145-153 and the position of incorporation within this peptide is Glu-149. The significance of our findings for the location of the active site and for the catalytic mechanism of PPase is briefly considered in the light of the 3-A X-ray crystallographic structure of Arutyunyun and his colleagues [Arutyunyun, E. G., et al. (1981) Dokl. Akad. Nauk SSSR 258, 1481-1485; Kuranova, I. P., et al. (1983) Bioorg. Khim. 9, 1611-1919; Terzyan, S. S., et al. (1984) Bioorg. Khim. 10, 1469-1482].

  7. Urease immobilized polymer hydrogel: Long-term stability and enhancement of enzymatic activity.

    PubMed

    Kutcherlapati, S N Raju; Yeole, Niranjan; Jana, Tushar

    2016-02-01

    A method has been developed in which an enzyme namely urease was immobilized inside hydrogel matrix to study the stability and enzymatic activity in room temperature (∼27-30°C). This urease coupled hydrogel (UCG) was obtained by amine-acid coupling reaction and this procedure is such that it ensured the wider opening of mobile flap of enzyme active site. A systematic comparison of urea-urease assay and the detailed kinetic data clearly revealed that the urease shows activity for more than a month when stored at ∼27-30°C in case of UCG whereas it becomes inactive in case of free urease (enzyme in buffer solution). The aqueous microenvironment inside the hydrogel, unusual morphological features and thermal behaviour were believed to be the reasons for unexpected behaviour. UCG displayed enzyme activity at basic pH and up to 60°C. UCG showed significant enhancement in activity against thermal degradation compared to free urease. In summary, this method is a suitable process to stabilize the biomacromolecules in standard room temperature for many practical uses.

  8. Effect of Direct-Current Electric Field on Enzymatic Activity and the Concentration of Laccase.

    PubMed

    Wang, Chunxing; Zhang, Huiling; Ren, Dajun; Li, Qian; Zhang, Shuqin; Feng, Tao

    2015-09-01

    This work investigates the effect of direct-current electric field on the extracellular enzymatic activity, concentration and other experimental parameters of laccase from Trametes versicolor. The results showed that laccase could significantly contribute to the change of pH at the end of graphite electrode. In addition, it increased the electrical conductivity of the water. In the experiment, the optimum pH and catalytic pH range for laccase activity were 3.0 and pH 2.5-4.0. The application of 6 V direct current showed significant effects on the laccase enzyme activity. The activity of laccase was enhanced in the anodic region, but at the same time was strongly inhibited at the cathode. The electric charge characteristics of laccase were changed when exposed to electric field, and some laccases molecules moved to the anode, which produced a slight migration phenomenon. This study is the basis of combination of laccase and electrical technology, at the same time, providing a new direction of enhancing laccase activity. Compared to immobilization, using electric field is simple, no chemical additives, and great potential.

  9. Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels.

    PubMed

    Peng, Huan; Rübsam, Kristin; Jakob, Felix; Schwaneberg, Ulrich; Pich, Andrij

    2016-11-14

    the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion, or energy utilization fields.

  10. An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity.

    PubMed

    Zanin, Mark; Duan, Susu; Wong, Sook-San; Kumar, Gyanendra; Baviskar, Pradyumna; Collin, Emily; Russell, Charles; Barman, Subrata; Hause, Benjamin; Webby, Richard

    2017-01-15

    Neuraminidase (NA) is a sialidase expressed on the surface of influenza A viruses that releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. It is a homotetramer, with each monomer consisting of a transmembrane region, a stalk, and a globular head with sialidase activity. We recently characterized two swine viruses of the pandemic H1N1 lineage, A/swine/Virginia/1814-1/2012 (pH1N1low-1) and A/swine/Virginia/1814-2/2012 (pH1N1low-2), with almost undetectable NA enzymatic activity compared to that of the highly homologous A/swine/Pennsylvania/2436/2012 (pH1N1-1) and A/swine/Minnesota/2499/2012 (pH1N1-2) viruses. pH1N1-1 transmitted to aerosol contact ferrets, but pH1N1low-1 did not. The aim of this study was to identify the molecular determinants associated with low NA activity as potential markers of aerosol transmission. We identified the shared unique substitutions M19V, A232V, D248N, and I436V (N1 numbering) in pH1N1low-1 and pH1N1low-2. pH1N1low-1 also had the unique Y66D substitution in the stalk domain, where 66Y was highly conserved in N1 NAs. Restoration of 66Y was critical for the NA activity of pH1N1low-1 NA, although 19M or 248D in conjunction with 66Y was required to recover the level of activity to that of pH1N1 viruses. Studies of NA stability and molecular modeling revealed that 66Y likely stabilized the NA homotetramer. Therefore, 66Y in the stalk domain of N1 NA was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.

  11. Aldehyde dehydrogenase activity in cancer stem cells from canine mammary carcinoma cell lines.

    PubMed

    Michishita, M; Akiyoshi, R; Suemizu, H; Nakagawa, T; Sasaki, N; Takemitsu, H; Arai, T; Takahashi, K

    2012-08-01

    Increasing evidence suggests that diverse solid tumours arise from a small population of cells known as cancer stem cells or tumour-initiating cells. Cancer stem cells in several solid tumours are enriched for aldehyde dehydrogenase (ALDH) activity. High levels of ALDH activity (ALDH(high)) were detected in four cell lines derived from canine mammary carcinomas. ALDH(high) cells were enriched in a CD44(+)CD24(-) population having self-renewal capacity. Xenotransplantation into immunodeficient mice demonstrated that 1×10(4) ALDH(high) cells were sufficient for tumour formation in all injected mice, whereas 1×10(4) ALDH(low) cells failed to initiate any tumours. ALDH(high)-derived tumours contained both ALDH(+) and ALDH(-) cells, indicating that these cells had cancer stem cell-like properties.

  12. Seasonal and spatial distribution of extracellular enzymatic activities and microbial incorporation of dissolved organic substrates in marine sediments

    SciTech Connect

    Meyer-Reil, L.

    1987-08-01

    Seasonal and spatial distributions of extracellular enzymatic activities and microbial incorporations of dissolved organic substrates were followed in sediments of the brackish water Kiel Bight (Baltic Sea). Enzymatic hydrolysis of polymeric organic compounds was determined by means of fluorogenic substrates; incorporation of dissolved organic substrates into microbial biomass was measured by using tritiated substances (acetate, leucine, and thymidine). Based on a recently developed core injection technique, substrates were injected in microliter portions into undisturbed sediment cores. Enzymatic and incorporation activities underwent strong seasonal variations related to the enrichment of organic material in the sediment surface following sedimentation events. The input of the phytoplankton bloom during autumn caused stimulation of both enzymatic hydrolysis of polymeric organic compounds and microbial incorporation of dissolved organic substrates. Following input by spring phytoplankton bloom, mainly incorporation activities were stimulated. In late spring the development of the benthic fauna obviously greatly influenced microbial activities. During summer individual periods of high microbial activities were observed which might be traced back to short-term sedimentation events.

  13. Effect of enzymatic mash treatment and storage on phenolic composition, antioxidant activity, and turbidity of cloudy apple juice.

    PubMed

    Oszmiański, Jan; Wojdylo, Aneta; Kolniak, Joanna

    2009-08-12

    The effects of different commercial enzymatic mash treatments on yield, turbidity, color, and polyphenolic and sediment of procyanidins content of cloudy apple juice were studied. Addition of pectolytic enzymes to mash treatment had positive effect on the production of cloud apple juices by improving polyphenolic contents, especially procyanidins and juice yields (68.3% in control samples to 77% after Pectinex Yield Mash). As summary of the effect of enzymatic mash treatment, polyphenol contents in cloudy apple juices significantly increased after Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL maceration were applied but no effect was observed after Pectinex Ultra-SPL I Panzym XXL use, compared to the control samples. The content of polymeric procyanidins represented 50-70% of total polyphenols, but in the present study, polymeric procyanidins were significantly lower in juices than in fruits and also affected by enzymatic treatment (Pectinex AFP L-4 and Panzym Yield Mash) compared to the control samples. The enzymatic treatment decreased procyanidin content in most sediment with the exception of Pectinex Smash XXL and Pectinex AFP L-4. Generally in samples that were treated by pectinase, radical scavenging activity of cloudy apple juices was increased compared to the untreated reference samples. The highest radical scavenging activity was associated with Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL enzyme and the lowest activity with Pectinex Ultra SP-L and Pectinex APFL-4. However, in the case of enzymatic mash treatment cloudy apple juices showed instability of turbidity and low viscosity. These results must be ascribed to the much higher hydrolysis of pectin by enzymatic preparation which is responsible for viscosity. During 6 months of storage at 4 degrees C small changes in analyzed parameters of apple juices were observed.

  14. Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

    PubMed Central

    Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

    2008-01-01

    The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

  15. An invertebrate coagulation system activated by endotoxin: evidence for enzymatic mediation

    PubMed Central

    Young, Neal S.; Levin, Jack; Prendergast, Robert A.

    1972-01-01

    Lysates prepared from the amebocytes of Limulus polyphemus, the horseshoe crab, are gelled by endotoxin. Studies were carried out to characterize the components of amebocyte lysate and to examine the kinetics of their reaction with endotoxin. Analysis of amebocyte lysate using sucrose density gradients showed two peaks at 46% and 86% gradient volumes. G50 and G75 Sephadex column chromatography resulted in three protein peaks. One fraction contained a clottable protein, which had a molecular weight of approximately 27,000, and was heat stable. Another fraction contained a high molecular weight, heat labile material, which was activated by endotoxin and reacted with the clottable protein to form a gel. The rate of the reaction between endotoxin and amebocyte lysate was dependent upon the concentration of endotoxin and the concentration of the fraction containing the high molecular weight material. The activity of this fraction was inhibited by diisopropyl fluorophosphate, parachloromercuribenzoate, and para-chloromercuriphenyl sulfonate, suggesting that enzymatic activity depended upon serine hydroxyl and sulfhydryl groups. The reaction between endotoxin and the fractions of lysate was temperature and pH dependent. The data suggest that endotoxin activates an enzyme which then gels the clottable protein contained in amebocyte lysate. Images PMID:4624351

  16. Toxicological effects of dimethomorph on soil enzymatic activity and soil earthworm (Eisenia fetida).

    PubMed

    Wang, Caixia; Zhang, Qingming; Wang, Feifei; Liang, Wenxing

    2017-02-01

    The objective of this study was to evaluate the toxicity of the fungicide dimethomorph to soil microbial activity and the earthworm Eisenia fetida. Multiple biomarkers, namely, four soil enzymes (urease, dehydrogenase, invertase, and acid phosphatase), four earthworm biochemical indices (dismutase, catalase, cellulase, and malondialdehyde), and the transcriptional levels of both target genes (dismutase and catalase) were measured at 1, 10, and 100 mg kg(-1) after 1, 7, 21, and 28 days. The degradation rate of dimethomorph in soil was also determined, and the results indicated that most parameters did not differ from the controls at 1 and 10 mg kg(-1) dimethomorph by the last exposure time (28 d). However, high concentrations (100 mg kg(-1)) of dimethomorph had varying effects on soil enzymatic activity and earthworms. These effects gradually decreased with prolonged exposure times. Positive correlations (R(2) > 0.57) between the target gene expression levels and antioxidant enzyme activities were observed in this study. We also found that earthworms have improved soil microbial activity and accelerated the degradation of dimethomorph. Overall, higher concentrations of dimethomorph might pose an ecological hazard to soil environments in the short term.

  17. Protective role of ALDH2 against acetaldehyde-derived DNA damage in oesophageal squamous epithelium.

    PubMed

    Amanuma, Yusuke; Ohashi, Shinya; Itatani, Yoshiro; Tsurumaki, Mihoko; Matsuda, Shun; Kikuchi, Osamu; Nakai, Yukie; Miyamoto, Shin'ichi; Oyama, Tsunehiro; Kawamoto, Toshihiro; Whelan, Kelly A; Nakagawa, Hiroshi; Chiba, Tsutomu; Matsuda, Tomonari; Muto, Manabu

    2015-09-16

    Acetaldehyde is an ethanol-derived definite carcinogen that causes oesophageal squamous cell carcinoma (ESCC). Aldehyde dehydrogenase 2 (ALDH2) is a key enzyme that eliminates acetaldehyde, and impairment of ALDH2 increases the risk of ESCC. ALDH2 is produced in various tissues including the liver, heart, and kidney, but the generation and functional roles of ALDH2 in the oesophagus remain elusive. Here, we report that ethanol drinking increased ALDH2 production in the oesophagus of wild-type mice. Notably, levels of acetaldehyde-derived DNA damage represented by N(2)-ethylidene-2'-deoxyguanosine were higher in the oesophagus of Aldh2-knockout mice than in wild-type mice upon ethanol consumption. In vitro experiments revealed that acetaldehyde induced ALDH2 production in both mouse and human oesophageal keratinocytes. Furthermore, the N(2)-ethylidene-2'-deoxyguanosine levels increased in both Aldh2-knockout mouse keratinocytes and ALDH2-knockdown human keratinocytes treated with acetaldehyde. Conversely, forced production of ALDH2 sharply diminished the N(2)-ethylidene-2'-deoxyguanosine levels. Our findings provide new insight into the preventive role of oesophageal ALDH2 against acetaldehyde-derived DNA damage.

  18. Induced Expression of Cancer Stem Cell Markers ALDH1A3 and Sox-2 in Hierarchical Reconstitution of Apoptosis-resistant Human Breast Cancer Cells

    PubMed Central

    Kashii-Magaribuchi, Karin; Takeuchi, Rie; Haisa, Yuko; Sakamoto, Akemi; Itoh, Aimi; Izawa, Yuki; Isa, Miyuki; Fukuzawa, Mayu; Murakami, Motonobu; Takahashi, Rei

    2016-01-01

    We established an experimental system that can induce p53-dependent apoptosis by doxycycline treatment to analyze characteristics of the apoptosis-resistant cancer cell subpopulation in the human breast cancer cell line HCC1937. Expression patterns of the stem cell markers, ALDH1A3 and Sox-2, the luminal differentiation marker, GATA3 and the proliferation index marker, Ki-67 were analyzed using immunostaining and fluorescence-activated cell sorting (FACS). After doxycycline treatment, the number of viable cells was gradually decreased over seven days in a time-dependent manner due to p53-induced apoptosis; however, the number of smaller-sized ALDH1A3+ cells assessed by immunostaining increased sharply after 1 day of doxycycline treatment, suggesting their apoptosis-resistant nature. The expression of ALDH1A3 was also detected in 78% of small-sized Ki-67+ proliferating progenitor cells, followed by the transient expression of GATA3, which presumably indicated the ability to differentiate into luminal progenitor cells. Although 42.2–58.5% of residual cells were positive for both ALDH1A3 and GATA3, their expression patterns exhibited an inverse correlation. The expression pattern of another stem cell marker, Sox-2, was similar, but more drastically altered after p53 induction compared with ALDH1A3. These findings may aid in understanding the hierarchical responses of cancer stem cells to therapeutic stresses. PMID:27917009

  19. ALDH1 might influence the metastatic capability of HeLa cells.

    PubMed

    Yao, Tingting; Lu, Rongbiao; Li, Yiqing; Peng, Yongpai; Ding, Miao; Xie, Xiaofei; Lin, Zhongqiu

    2015-09-01

    Recent data suggest that tumor persistence and recurrence could be caused by the presence of cancer stem cells (CSCs). Aldehyde dehydrogenase 1 (ALDH1) has been implicated in cancer pathogenesis and used as a CSC marker. We previously reported that cervical carcinoma contains a small subpopulation of cells expressing ALDH1 [1]. In this study, we used small interfering RNA to suppress ALDH1 expression and introduced an ALDH1 reporting vector into HeLa cells followed by various in vitro assays. We showed that knockdown of ALDH1 expression reduced the cell migration ability of HeLa cells, whereas augmented expression of ALDH1 increased cell migration. However, there was no difference in the cellular proliferation, apoptosis, cell cycle, and invasion. These results indicate that ALDH1 is directly involved in HeLa migration.

  20. Effect of compatible and noncompatible osmolytes on the enzymatic activity and thermal stability of bovine liver catalase.

    PubMed

    Sepasi Tehrani, H; Moosavi-Movahedi, A A; Ghourchian, H; Ahmad, F; Kiany, A; Atri, M S; Ariaeenejad, Sh; Kavousi, K; Saboury, A A

    2013-12-01

    Catalase is an important antioxidant enzyme that catalyzes the disproportionation of H2O2 into harmless water and molecular oxygen. Due to various applications of the enzyme in different sectors of industry as well as medicine, the enhancement of stability of the enzyme is important. Effect of various classes of compatible as well as noncompatible osmolytes on the enzymatic activity, disaggregation, and thermal stability of bovine liver catalase have been investigated. Compatible osmolytes, proline, xylitol, and valine destabilize the denatured form of the enzyme and, therefore, increase its disaggregation and thermal stability. The increase in the thermal stability is accompanied with a slight increase of activity in comparison to the native enzyme at 25 °C. On the other hand, histidine, a noncompatible osmolyte stabilizes the denatured form of the protein and hence causes an overall decrease in the thermal stability and enzymatic activity of the enzyme. Chemometric results have confirmed the experimental results and have provided insight into the distribution and number of mole fraction components for the intermediates. The increase in melting temperature (Tm) and enzymatic rate could be further amplified by the intrinsic effect of temperature enhancement on the enzymatic activity for the industrial purposes.

  1. Efficient enzymatic degradation process for hydrolysis activity of the Carrageenan from red algae in marine biomass.

    PubMed

    Kang, Dae Hee; Hyeon, Jeong Eun; You, Seung Kyou; Kim, Seung Wook; Han, Sung Ok

    2014-12-20

    Carrageenan is a generic name for a family of polysaccharides obtained from certain species of red algae. New methods to produce useful cost-efficiently materials from red algae are needed to convert enzymatic processes into fermentable sugars. In this study, we constructed chimeric genes cCgkA and cCglA containing the catalytic domain of κ-carrageenase CgkA and λ-carrageenase CglA from Pseudoalteromonas carrageenovora fused with a dockerin domain. Recombinant strains expressing the chimeric carrageenase resulted in a halo formation on the carrageenan plate by alcian blue staining. The recombinant cCgkA and cCglA were assembled with scaffoldin miniCbpA via cohesin and dockerin interaction. Carbohydrate binding module (CBM) in scaffoldin was used as a tag for cellulose affinity purification using cellulose as a support. The hydrolysis process was monitored by the amount of reducing sugar released from carrageenan. Interestingly, these results indicated that miniCbpA, cCgkA and cCglA assembled into a complex and that the dockerin-fused enzymes on the scaffoldin had synergistic activity in the degradation of carrageenan. The observed enhancement of activity by carrageenolytic complex was 3.1-fold-higher compared with the corresponding enzymes alone. Thus, the assemblies of advancement of active enzyme complexes will facilitate the commercial production of useful products from red algae biomass which represents inexpensive and sustainable feed-stocks.

  2. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

    PubMed

    Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

    2015-06-05

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

  3. Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

    PubMed Central

    Barnes, H J; Arlotto, M P; Waterman, M R

    1991-01-01

    When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system. Images PMID:1829523

  4. Enzymatic properties of immobilized Alcaligenes faecalis cells with cell-associated beta-glucosidase activity

    SciTech Connect

    Wheatly, M.A.; Phillips, C.R.

    1984-06-01

    Enzymatic properties of Alcaligenes faecalis cells immobilized in polyacrylamide were characterized and compared with those reported for the extracted enzyme, and with those measured for free cells. Many of the properties reflected those of the extracted enzyme rather than those measured in the free whole cells prior to immobilization, suggesting cell disruption during immobilization. These properties included the pH activity profile, a slightly broader pH stability profile, and the activation energy. Electron micrographs showed evidence of cell debris among the polymer matrix. The immobilized cells were not viable, and did not consume glucose. Thermal stability was less after immobilization with a half-line of 16 h at 45 degrees C, and 3.5 h at 50 degrees C. The immobilized preparation was more stable when stored lyophilized rather than in buffer, losing 23 and 52% activity, respectively, after six months. The enzyme was irreversibly inhibited by both acetate and citrate buffers. If the immobilized enzyme is to be used in conjunction with cellulases from Trichoderma reesei for cellulase saccharification, the optimal conditions would be pH 5.5 and 45 degrees C in a buffer containing no carboxylic acid groups.

  5. Enzymatic activity and proteomic profile of class III peroxidases during sugarcane stem development.

    PubMed

    Cesarino, Igor; Araújo, Pedro; Sampaio Mayer, Juliana Lischka; Paes Leme, Adriana Franco; Mazzafera, Paulo

    2012-06-01

    Class III peroxidases are present as large multigene families in all land plants. This large number of genes together with the diversity of processes catalyzed by peroxidases suggests possible functional specialization of each isoform. However, assigning a precise role for each individual peroxidase gene has continued to be a major bottleneck. Here we investigated the enzyme activity and translational profile of class III peroxidases during stem development of sugarcane as a first step in the estimation of physiological functions of individual isoenzymes. Internodes at three different developmental stages (young, developing and mature) were divided into pith (inner tissue) and rind (outer tissue) fractions. The rind of mature internodes presented the highest enzymatic activity and thus could be considered the ideal tissue for the discovery of peroxidase gene function. In addition, activity staining of 2DE gels revealed different isoperoxidase profiles and protein expression regulation among different tissue fractions. In-gel tryptic digestion of excised spots followed by peptide sequencing by LC-MS/MS positively matched uncharacterized peroxidases in the sugarcane database SUCEST. Multiple spots matching the same peroxidase gene were found, which reflects the generation of more than one isoform from a particular gene by post-translational modifications. The identified sugarcane peroxidases appear to be monocot-specific sequences with no clear ortholog in dicot model plant Arabidopsis thaliana.

  6. Ultrasound-assisted enzymatic extraction and antioxidant activity of polysaccharides from pumpkin (Cucurbita moschata).

    PubMed

    Wu, Hao; Zhu, Junxiang; Diao, Wenchao; Wang, Chengrong

    2014-11-26

    An efficient ultrasound-assisted enzymatic extraction (UAEE) of Cucurbita moschata polysaccharides (CMCP) was established and the CMCP antioxidant activities were studied. The UAEE operating parameters (extraction temperature, ultrasonic power, pH, and liquid-to-material ratio) were optimized using the central composite design (CCD) and the mass transfer kinetic study in UAEE procedure was used to select the optimal extraction time. Enzymolysis and ultrasonication that were simultaneously conducted was selected as the UAEE synergistic model and the optimum extraction conditions with a maximum polysaccharide yield of 4.33 ± 0.15% were as follows: extraction temperature, 51.5 °C; ultrasonic power, 440 W; pH, 5.0; liquid-to-material ratio, 5.70:1 mL/g; and extraction time, 20 min. Evaluation of the antioxidant activity in vitro suggested that CMCP has good potential as a natural antioxidant used in the food or medicine industry because of their high reducing power and positive radical scavenging activity for DPPH radical.

  7. Characterization of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus: enzymatic activity and active site structure.

    PubMed

    Terasaka, Erina; Okada, Norihiro; Sato, Nozomi; Sako, Yoshihiko; Shiro, Yoshitsugu; Tosha, Takehiko

    2014-07-01

    Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating a more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

  8. The diagnostic value of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) measurement in the sera of gastric cancer patients.

    PubMed

    Jelski, Wojciech; Orywal, Karolina; Laniewska, Magdalena; Szmitkowski, Maciej

    2010-12-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are present in gastric cancer cells (GC). Moreover, the activity of total ADH and class IV isoenzymes is significantly higher in cancer tissue than in healthy mucosa. The activity of these enzymes in cancer cells is probably reflected in the sera and could thus be helpful for diagnostics of gastric cancer. The aim of this study was to investigate a potential role of ADH and ALDH as tumor markers for gastric cancer. We defined diagnostic sensitivity, specificity, predictive value for positive and negative results, and receiver-operating characteristics (ROC) curve for tested enzymes. Serum samples were taken from 168 patients with gastric cancer before treatment and from 168 control subjects. Total ADH activity and class III and IV isoenzymes were measured by photometric but ALDH activity and ADH I and II by the fluorometric method, with class-specific fluorogenic substrates. There was significant increase in the activity of ADH IV isoenzyme and ADH total in the sera of gastric cancer patients compared to the control. The diagnostic sensitivity for ADH IV was 73%, specificity 79%, positive and negative predictive values were 81 and 72% respectively. Area under ROC curve for ADH IV was 0.67. The results suggest a potential role for ADH IV as marker of gastric cancer.

  9. Synergistic upregulation of NONO and PSPC1 regulates Sertoli cell response to MEHP via modulation of ALDH1A1 signaling.

    PubMed

    Dong, Bing-Wei; Jin, Xiao-Hang; Yan, Chang-You; Yang, Tian; Cai, Guo-Qing; Lu, Jian

    2017-03-01

    Members of the Drosophila behavior/human splicing protein family, including splicing factor proline/glutamine rich (SFPQ), non-POU domain-containing octamer-binding protein (NONO), and paraspeckle protein component 1 (PSPC1), are abundantly expressed in testicular Sertoli cells (SCs), but their roles remain obscure. Here, we show that treatment with mono-(2-ethylhexyl) phthalate (MEHP), a well-known SC toxicant, selectively stimulates the expression levels of NONO and PSPC1. Simultaneous inhibition of NONO and PSPC1 expression in SCs enhances MEHP-induced oxidative stress and potentiates SC death. Mechanistically, NONO and PSPC1 transcriptionally activate aldehyde dehydrogenase 1 (Aldh1a1), by synergistically binding to the distinct CCGGAGTC sequence in the Aldh1a1 promoter. Together, the NONO/PSPC1-ALDH1A1 cascade may serve as an indispensable defense mechanism against MEHP insult in SCs.

  10. Formation of marine snow and enhanced enzymatic activities in oil-contaminated seawater

    NASA Astrophysics Data System (ADS)

    Ziervogel, K.; McKay, L.; Yang, T.; Rhodes, B.; Nigro, L.; Gutierrez, T.; Teske, A.; Arnosti, C.

    2010-12-01

    The fate of oil spilled into the ocean depends on its composition, as well as on biological, chemical, and physical characteristics of the spill site. We investigated the effects of oil addition from the Deepwater Horizon (DH) spill on otherwise uncontaminated water collected close to the spill site. Incubation on a roller table mimicked the physical dynamics of natural seawater, leading to the formation of marine snow-oil aggregates. We measured the enzymatic activities of heterotrophic microbes associated with the aggregates and in the surrounding water, and assessed microbial population and community composition as oil-marine snow aggregates formed and aged in the water. Surface seawater taken near the spill site in May 2010 that had no visible crude oil was incubated in 1-l glass bottles with (oil-bottles) and without (no-oil bottles) a seawater-oil mixture collected from the same site. In the oil-bottles formation of brownish, densely packed marine snow (2-3 cm diameter) was observed within the first hour of the roller table incubation. In contrast no-oil bottles showed aggregate formation only after 3 days, and aggregates were almost transparent, less abundant, and smaller in size (< 1cm diameter). Subsamples of the water surrounding the aggregates were taken throughout 21 days of the roller table incubation, and analyzed for bacterial abundance and community structure as well as the activities of hydrolytic enzymes that are used by heterotrophic bacteria to degrade organic matter. We monitored oil-degrading activities with MUF-stearate and -butyrate, and also measured b-glucosidase, alkaline phosphatase, aminopeptidase, and six different polysaccharide hydrolase activities. Enzymatic activities were up to one order of magnitude higher in the oil-bottles compared with the no-oil bottles throughout the entire incubation time. Butyrate hydrolysis was elevated throughout the time course of the incubation, and stearate hydrolysis was particularly high over the

  11. Single-molecule kinetics under force: probing protein folding and enzymatic activity with optical tweezers

    NASA Astrophysics Data System (ADS)

    Wong, Wesley

    2010-03-01

    Weak non-covalent bonds between and within single molecules govern many aspects of biological structure and function (e.g. DNA base-paring, receptor-ligand binding, protein folding, etc.) In living systems, these interactions are often subject to mechanical forces, which can greatly alter their kinetics and activity. My group develops and applies novel single-molecule manipulation techniques to explore and quantify these force-dependent kinetics. Using optical tweezers, we have quantified the force-dependent unfolding and refolding kinetics of different proteins, including the cytoskeletal protein spectrin in collaboration with E. Evans's group [1], and the A2 domain of the von Willebrand factor blood clotting protein in collaboration with T. Springer's group [2]. Furthermore, we have studied the kinetics of the ADAMTS13 enzyme acting on a single A2 domain, and have shown that physiolgical forces in the circulation can act as a cofactor for enzymatic cleavage, regulating hemostatic activity [2]. References: 1. E. Evans, K. Halvorsen, K. Kinoshita, and W.P. Wong, Handbook of Single Molecule Biophysics, P. Hinterdorfer, ed., Springer (2009). 2. X. Zhang, K. Halvorsen, C.-Z. Zhang, W.P. Wong, and T.A. Springer, Science 324 (5932), 1330-1334 (2009).

  12. Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance.

    PubMed

    Xia, Xi; Wu, Congming; Cui, Yaowen; Kang, Mengjiao; Li, Xiaowei; Ding, Shuangyang; Shen, Jianzhong

    2015-11-20

    Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities.

  13. Three in one: Identification, expression and enzymatic activity of lysozymes in amphioxus.

    PubMed

    Xu, Na; Pan, Junli; Liu, Shousheng; Xue, Qinggang; Zhang, Shicui

    2014-10-01

    The lysozymes identified so far in animals belong to the g-type, c-type, and i-type. Vertebrate animals possess only the former two types, i.e., g- and c-types, while all the three types have been reported in invertebrates. Here we demonstrate that (1) three cDNAs that encode g-, c-, and i-type lysozymes, respectively, were identified in a single species of the amphioxus Branchiostoma japonicum; (2) all the 3-type genes displayed distinct tissue-specific expression pattern; (3) recombinant g-, c-, and i-type lysozymes all exhibited enzymatic activities; and (4) native g-, c-, and i-type lysozymes were identified in the different tissues of amphioxus. Collectively, these results suggest the presence of all the 3-type lysozymes in a single animal species, first such data ever reported. The presence of biologically active i-type lysozyme in amphioxus also suggests that i-type lysozyme gene is retained at least in Protochordata, contrasting to the previous proposal that i-type lysozyme gene has been lost in a common ancestor of all chordates.

  14. Green Tea and Bone Marrow Transplantation: From Antioxidant Activity to Enzymatic and Multidrug-resistance Modulation.

    PubMed

    Peluso, Ilaria; Palmery, Maura; Vitalone, Annabella

    2016-10-25

    Epigallocatechin-3-gallate (EGCG), the main flavonoid of green tea (GT), could play an active role in the prevention of oxidative-stress-related diseases, such as hematologic malignancies. Some effects of EGCG are not imputable to antioxidant activity, but involve modulation of antioxidant enzymes and uric acid (UA) levels. The latter is the major factor responsible of the plasma non-enzymatic antioxidant capacity (NEAC). However, hyperuricemia is a frequent clinical feature caused by tumor lysis syndrome or cyclosporine side effects, both before and after bone marrow transplantation (BMT). Besides this, food-drug interactions could be associated with GT consumption and could have clinical implications. The molecular mechanisms involved in the redox and drug metabolizing/transporting pathways were discussed, with particular reference to the potential role of GT and EGCG in BMT. Moreover, on reviewing data on NEAC, isoprostanes, uric acid, and various enzymes from human studies on GT, its extract, or EGCG, an increase in NEAC, without effect on isoprostanes, and contrasting results on UA and enzymes were observed. Currently, few and contrasting available evidences suggest caution for GT consumption in BMT patients and more studies are needed to better understand the potential impact of EGCG on oxidative stress and metabolizing/transporting systems.

  15. Phenylpropanoid Glycoside Analogues: Enzymatic Synthesis, Antioxidant Activity and Theoretical Study of Their Free Radical Scavenger Mechanism

    PubMed Central

    López-Munguía, Agustín; Hernández-Romero, Yanet; Pedraza-Chaverri, José; Miranda-Molina, Alfonso; Regla, Ignacio; Martínez, Ana; Castillo, Edmundo

    2011-01-01

    Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols. PMID:21674039

  16. Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine.

    PubMed

    Wang, Xiaohong; Schröder, Heinz C; Müller, Werner E G

    2014-01-01

    In recent years a paradigm shift in understanding of human bone formation has occurred that starts to change current concepts in tissue engineering of bone and cartilage. New discoveries revealed that fundamental steps in biomineralization are enzyme driven, not only during hydroxyapatite deposition, but also during initial bioseed formation, involving the transient deposition and subsequent transformation of calcium carbonate to calcium phosphate mineral. The principal enzymes mediating these reactions, carbonic anhydrase and alkaline phosphatase, open novel targets for pharmacological intervention of bone diseases like osteoporosis, by applying compounds acting as potential activators of these enzymes. It is expected that these new findings will give an innovation boost for the development of scaffolds for bone repair and reconstruction, which began with the use of bioinert materials, followed by bioactive materials and now leading to functional regenerative tissue units. These new developments have become possible with the discovery of the morphogenic activity of bioinorganic polymers, biocalcit, bio-polyphosphate and biosilica that are formed by a biogenic, enzymatic mechanism, a driving force along with the development of novel rapid-prototyping three-dimensional (3D) printing methods and bioprinting (3D cell printing) techniques that may allow a fabrication of customized implants for patients suffering in bone diseases in the future.

  17. Inhibition of the enzymatic activity of heme oxygenases by azole-based antifungal drugs.

    PubMed

    Kinobe, Robert T; Dercho, Ryan A; Vlahakis, Jason Z; Brien, James F; Szarek, Walter A; Nakatsu, Kanji

    2006-10-01

    Ketoconazole (KTZ) and other azole antifungal agents are known to have a variety of actions beyond the inhibition of sterol synthesis in fungi. These drugs share structural features with a series of novel heme oxygenase (HO) inhibitors designed in our laboratory. Accordingly, we hypothesized that therapeutically used azole-based antifungal drugs are effective HO inhibitors. Using gas chromatography to quantify carbon monoxide formation in vitro and in vivo, we have shown that azole-containing antifungal drugs are potent HO inhibitors. Terconazole, sulconazole, and KTZ were the most potent drugs with IC(50) values of 0.41 +/- 0.01, 1.1 +/- 0.4, and 0.3 +/- 0.1 microM for rat spleen microsomal HO activity, respectively. Kinetic characterization revealed that KTZ was a noncompetitive HO inhibitor. In the presence of KTZ (2.5 and 10 microM), K(m) values for both rat spleen and brain microsomal HO were not altered; however, a significant decrease in the catalytic capacity (V(max)) was observed (P < 0.005). KTZ was also found to weakly inhibit nitric-oxide synthase with an IC(50) of 177 +/- 2 microM but had no effect on the enzymatic activity of NADPH cytochrome P450 reductase. Because these drugs were effective within the concentration range observed in humans, it is possible that inhibition of HO may play a role in some of the pharmacological actions of these antimycotic drugs.

  18. Determination of myoglobin based on its enzymatic activity by stopped-flow spectrophotometry

    NASA Astrophysics Data System (ADS)

    Zheng, Qi; Liu, Zhihong; Cai, Ruxiu

    2005-04-01

    A new method has been developed for the determination of myoglobin (Mb) based on its enzymatic activity for the oxidation of o-phenylenediamine (OPDA) with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation reaction. The catalytic activity of Mb was compared to other three kinds of catalyst. The time dependent absorbance of the reaction product, 2,3-diamimophenazine (DAPN), at a wavelength of 426 nm was recorded. The initial reaction rate obtained at 40 °C was found to be proportional to the concentration of Mb in the range of 1.0 × 10 -6 to 4.0 × 10 -9 mol L -1. The detection limit of Mb was found to be 9.93 × 10 -10 mol L -1. The relative standard deviations were within 5% for the determination of different concentrations of Mb. Excess of bovine serum albumin (BSA), Ca(II), Mg(II), Cu(II), glucose, caffeine, lactose and uric acid did not interfere.

  19. Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance

    PubMed Central

    Xia, Xi; Wu, Congming; Cui, Yaowen; Kang, Mengjiao; Li, Xiaowei; Ding, Shuangyang; Shen, Jianzhong

    2015-01-01

    Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities. PMID:26584633

  20. Immobilization of α-amylase onto a calix[4]arene derivative: Evaluation of its enzymatic activity.

    PubMed

    Veesar, Irshad Ali; Solangi, Imam Bakhsh; Memon, Shahabuddin

    2015-06-01

    In order to enhance the cost-effectiveness practicability of enzymes in many industries such as pharmaceutical, food, medical and some other technological processes, there is great need to immobilize them onto a solid supports. In this study, a new and efficient immobilization of α-amylase from Saccharomyces cerevisiae has been developed by using the surface functionalization of calix[4]arene as support. A glutaraldehyde-containing amino group functionalized calix[4]arene was used to immobilize α-amylase covalently. In this procedure, imide bonds are formed between amino groups on the protein and aldehyde groups on the calix[4]arene surface. The surface modified support was characterized using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM). The effect of various preparation conditions on the immobilized α-amylase process such as immobilization time, enzyme concentration, temperature and pH were investigated. The influence of pH and temperature on the activity of free and immobilized α-amylase was also studied using starch as substrate. The optimum reaction temperature and pH value for the enzymatic conversion catalyzed by the immobilized α-amylase were 25°C and 7, respectively. Compared to the free enzyme, the immobilized α-amylase retained 85% of its original activity and exhibited significant thermal stability than the free one and excellent durability.

  1. Post-Effort Changes in Activity of Traditional Diagnostic Enzymatic Markers in Football Players’ Blood

    PubMed Central

    Chamera, Tomasz; Spieszny, Michał; Klocek, Tomasz; Kostrzewa-Nowak, Dorota; Nowak, Robert; Lachowicz, Milena; Buryta, Rafał; Ficek, Krzysztof; Eider, Jerzy; Moska, Waldemar; Cięszczyk, Paweł

    2015-01-01

    Summary Background Long-term and intensive physical effort causes metabolic and biochemical adaptations for both athletic and non-athletic objectives. Knowing the importance of aerobic training in football players, the aim of this study was to evaluate changes in the activity of: creatinine kinase (CK), creatine kinase MB (CKMB), lactate dehydrogenase (LDH), α-hydroxybutyrate dehydrogenase (HBDH), cholinesterase (ChE) and alkaline phosphatase (ALP) in response to a semi-long distance outdoor run under aerobic conditions among both female and male football players. Methods Sixteen participants aged 21.9±2 years (women) and 18.4±0.5 years (men), all of them voluntarily recruited football players, took part in an outdoor run, the women covering a distance of 7.4±0.3 km while men covered a distance of 10.7±1.0 km. Plasma activities of the studied enzymes were determined using an appropriate diagnostic assay kit. Results Our results indicate that total LDH activity could be a useful tool in evaluating physical fitness among athletes. We simultaneously established that ChE could not be a marker useful in assessing metabolic response to physical effort in athletes. Moreover, our results suggest that post-effort changes in ALP activity might be used to estimate early symptoms of certain vitamin deficiencies in an athlete’s diet. Conclusions We confirmed that the assessment of activity of selected traditional diagnostic enzymatic markers provides information about muscle state after physical effort. PMID:28356830

  2. Subchronic exposure to ethyl tertiary butyl ether resulting in genetic damage in Aldh2 knockout mice.

    PubMed

    Weng, Zuquan; Suda, Megumi; Ohtani, Katsumi; Mei, Nan; Kawamoto, Toshihiro; Nakajima, Tamie; Wang, Rui-Sheng

    2013-09-15

    Ethyl tertiary butyl ether (ETBE) is biofuel additive recently used in Japan and some other countries. Limited evidence shows that ETBE has low toxicity. Acetaldehyde (AA), however, as one primary metabolite of ETBE, is clearly genotoxic and has been considered to be a potential carcinogen. The aim of this study was to evaluate the effects of ALDH2 gene on ETBE-induced genotoxicity and metabolism of its metabolites after inhalation exposure to ETBE. A group of wild-type (WT) and Aldh2 knockout (KO) C57BL/6 mice were exposed to 500ppm ETBE for 1-6h, and the blood concentrations of ETBE metabolites, including AA, tert-butyl alcohol and 2-methyl-1,2-propanediol, were measured. Another group of mice of WT and KO were exposed to 0, 500, 1750, or 5000ppm ETBE for 6h/day with 5 days per weeks for 13 weeks. Genotoxic effects of ETBE in these mice were measured by the alkaline comet assay, 8-hydroxyguanine DNA-glycosylase modified comet assay and micronucleus test. With short-term exposure to ETBE, the blood concentrations of all the three metabolites in KO mice were significantly higher than the corresponding concentrations of those in WT mice of both sexes. After subchronic exposure to ETBE, there was significant increase in DNA damage in a dose-dependent manner in KO male mice, while only 5000ppm exposure significantly increased DNA damage in male WT mice. Overall, there was a significant sex difference in genetic damage in both genetic types of mice. These results showed that ALDH2 is involved in the detoxification of ETBE and lack of enzyme activity may greatly increase the sensitivity to the genotoxic effects of ETBE, and male mice were more sensitive than females.

  3. Molecular docking studies and anti-enzymatic activities of Thai mango seed kernel extract against snake venoms.

    PubMed

    Leanpolchareanchai, Jiraporn; Pithayanukul, Pimolpan; Bavovada, Rapepol; Saparpakorn, Patchreenart

    2009-03-31

    The ethanolic extract from seed kernels of Thai mango (MSKE) (Mangifera indica L. cv. 'Fahlun') (Anacardiaceae) and its major phenolic principle (pentagalloyl glucopyranose) exhibited dose-dependent inhibitory effects on enzymatic activities of phospholipase A(2) (PLA(2)), hyaluronidase and L-amino acid oxidase (LAAO) of Calloselasma rhodostoma (CR) and Naja naja kaouthia (NK)venoms by in vitro tests. The anti-hemorrhagic and anti-dermonecrotic activities of MSKE against both venoms were clearly supported by in vivo tests. Molecular docking studies indicated that the phenolic molecules of the MSKE could selectively bind to the active sites or their proximity, or modify conserved residues that are critical for the catalysis of PLA(2), and selectively bind to the LAAO binding pocket of both CR and NK venoms and thereby inhibit their enzymatic activities. The results imply a potential use of MSKE against snake venoms.

  4. Fibulin-3 negatively regulates ALDH1 via c-MET suppression and increases γ-radiation-induced sensitivity in some pancreatic cancer cell lines

    SciTech Connect

    Kim, In-Gyu; Lee, Jae-Ha; Kim, Seo-Yoen; Kim, Jeong-Yul; Cho, Eun-Wie

    2014-11-21

    Highlights: • FBLN-3 gene was poorly expressed in some pancreatic cancer lines. • FBLN-3 promoter region was highly methylated in some pancreatic cancer cell lines. • FBLN-3 inhibited c-MET activation and expression and reduced cellular level of ALDH1. • FBLN-3/c-Met/ALDH1 axis modulates stemness and EMT in pancreatic cancer cells. - Abstract: Fibulin-3 (FBLN-3) has been postulated to be either a tumor suppressor or promoter depending on the cell type, and hypermethylation of the FBLN-3 promoter is often associated with human disease, especially cancer. We report that the promoter region of the FBLN-3 was significantly methylated (>95%) in some pancreatic cancer cell lines and thus FBLN-3 was poorly expressed in pancreatic cancer cell lines such as AsPC-1 and MiaPaCa-2. FBLN-3 overexpression significantly down-regulated the cellular level of c-MET and inhibited hepatocyte growth factor-induced c-MET activation, which were closely associated with γ-radiation resistance of cancer cells. Moreover, we also showed that c-MET suppression or inactivation decreased the cellular level of ALDH1 isozymes (ALDH1A1 or ALDH1A3), which serve as cancer stem cell markers, and subsequently induced inhibition of cell growth in pancreatic cancer cells. Therefore, forced overexpression of FBLN-3 sensitized cells to cytotoxic agents such as γ-radiation and strongly inhibited the stemness and epithelial to mesenchymal transition (EMT) property of pancreatic cancer cells. On the other hand, if FBLN3 was suppressed in FBLN-3-expressing BxPC3 cells, the results were opposite. This study provides the first demonstration that the FBLN-3/c-MET/ALDH1 axis in pancreatic cancer cells partially modulates stemness and EMT as well as sensitization of cells to the detrimental effects of γ-radiation.

  5. Solution structure, enzymatic, and non-enzymatic reactivity of 3-isoadenosylcobalamin, a structural isomer of coenzyme B12 with surprising coenzymic activity.

    PubMed

    Brown, Kenneth L; Zou, Xiang; Chen, Guodong; Xia, Zuping; Marques, Helder M

    2004-02-01

    The coenzymic activity of eight analogs of coenzyme B(12) (5'-deoxyadenosyl-cobalamin, AdoCbl) with structural alterations in the Ado ligand has been investigated with the AdoCbl-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii. Six of the analogs were partially active coenzymes, and one, 3-iso-5'-deoxyadenosylcobalamin (3-IsoAdoCbl) was nearly as active as AdoCbl itself. NMR-restrained molecular modeling of 3-IsoAdoCbl revealed a highly conformationally mobile structure which required a four state model to be consistent with the NMR data. Thus, two conformations, one with the IsoAdo ligand over the eastern quadrant of the corrin, and one with the IsoAdo ligand over the northern quadrant, each undergo a facile syn/anti conformational equilibrium in the IsoAdo ligand. Spectrophotometric measurement of the kinetics of RTPR-induced cleavage of the carbon-cobalt bond of 3-IsoAdoCbl showed that it binds to the enzyme with the same affinity as AdoCbl, but its homolysis is only 20% as rapid. Investigation of the non-enzymatic thermolysis of 3-IsoAdoCbl showed that like AdoCbl, 3-IsoAdoCbl decomposes by competing homolytic and heterolytic pathways. A complete temperature-dependent kinetic and product analysis, followed by correction for the base-off species permitted deconvolution of the specific rate constant for both pathways. Eyring plots for the homolysis and heterolysis rate constant cross at 93 degrees C, so that homolysis is the predominant pathway at high temperature, but heterolysis is the predominant pathway at low temperature. At 37 degrees C, the homolysis of 3-IsoAdoCbl is 5.5-fold faster than that of AdoCbl, and the enzyme catalyzes carbon-cobalt bond homolysis in 3-IsoAdoCbl by a factor of 5.9 x 10(7), only 3.9% of the catalytic efficiency with AdoCbl itself. It seems likely that the conformational flexibility of 3-IsoAdoCbl allows it to adopt a coformation in which the hydrogen bonding patterns of the adenine moiety are

  6. Purification, enzymatic properties, and active site environment of a novel manganese(III)-containing acid phosphatase.

    PubMed

    Sugiura, Y; Kawabe, H; Tanaka, H; Fujimoto, S; Ohara, A

    1981-10-25

    A new manganese-containing acid phosphatase has been isolated and crystallized from sweet potato tubers. The pure enzyme contains one atom of manganese per Mr = 110,000 polypeptide and shows phosphatase activity toward various phosphate substrates. The pH optimum of the enzyme was 5.8 and the enzyme activity was inhibited by Cu2+, Zn2+, Hg2+, AsO43-, and MoO42-. This stable metalloenzyme is red-violet in color with an intense absorption band at 515 nm (epsilon - 2460). Our electronic, circular dichroism, and electron spin resonance findings strongly indicate that the Mn-valence state of the native enzyme is trivalent. When the Mn-enzyme is excited by the 5145 A line of Ar+ laser, prominent Raman lines at 1230, 1298, 1508, and 1620 cm-1 were detected. This Raman spectrum can probably be interpreted in terms of internal vibration of a coordinated tyrosine phenolate anion. The tryptophan-modified enzyme showed a positive Raman band at 370 cm-1, which is preferentially assigned to a Mn(III)-S streching mode. The modification of the Mn-enzyme by N-bromosuccinimide led to a large decrease in the fluorescence intensity of 335 nm which was dominated by its tryptophan residues within a considerable hydrophobic environment. The acid phosphatase activity was significantly decreased by the tryptophan modification. With respect to the active site donor sets, the Mn(III)-containing acid phosphatase is distinctly different from the Zn(II)-containing alkaline phosphatase. Of interest is also the appreciable similarity of some enzymatic and spectroscopic properties between the present enzyme and uteroferrin.

  7. Mercury Reduces the Enzymatic Activity of Neprilysin in Differentiated SH-SY5Y Cells

    PubMed Central

    Chin-Chan, Miguel; Segovia, José; Quintanar, Liliana; Arcos-López, Trinidad; Hersh, Louis B.; Chow, K. Martin; Rodgers, David W.; Quintanilla-Vega, Betzabet

    2015-01-01

    Levels of amyloid beta (Aβ) in the central nervous system are regulated by the balance between its synthesis and degradation. Neprilysin (NEP) is associated with Alzheimer’s disease (AD) by its ability to degrade Aβ. Some studies have involved the exposure to mercury (Hg) in AD pathogenesis; therefore, our aim was to investigate the effects on the anabolism and catabolism of Aβ in differentiated SH-SY5Y cells incubated with 1–20 μM of Hg. Exposure to 20 µM of Hg induced an increase in Aβ-42 secretion, but did not increase the expression of the amyloid precursor protein (APP). Hg incubation (10 and 20 µM) increased NEP protein levels; however, it did not change NEP mRNA levels nor the levels of the amyloid intracellular domain peptide, a protein fragment with transcriptional activity. Interestingly, Hg reduced NEP activity at 10 and 20 µM, and circular dichroism analysis using human recombinant NEP showed conformational changes after incubation with molar equivalents of Hg. This suggests that the Hg-induced inhibition of NEP activity may be mediated by a conformational change resulting in reduced Aβ-42 degradation. Finally, the comparative effects of lead (Pb, 50 μM) were evaluated. We found a significant increase in Aβ-42 levels and a dramatic increase in APP protein levels; however, no alteration in NEP levels was observed nor in the enzymatic activity of this metalloprotease, despite the fact that Pb slightly modified the rhNEP conformation. Overall, our data suggest that Hg and Pb increase Aβ levels by different mechanisms. PMID:25673500

  8. Mercury Reduces the Enzymatic Activity of Neprilysin in Differentiated SH-SY5Y Cells.

    PubMed

    Chin-Chan, Miguel; Segovia, José; Quintanar, Liliana; Arcos-López, Trinidad; Hersh, Louis B; Chow, K Martin; Rodgers, David W; Quintanilla-Vega, Betzabet

    2015-05-01

    Levels of amyloid beta (Aβ) in the central nervous system are regulated by the balance between its synthesis and degradation. Neprilysin (NEP) is associated with Alzheimer's disease (AD) by its ability to degrade Aβ. Some studies have involved the exposure to mercury (Hg) in AD pathogenesis; therefore, our aim was to investigate the effects on the anabolism and catabolism of Aβ in differentiated SH-SY5Y cells incubated with 1-20 μM of Hg. Exposure to 20 µM of Hg induced an increase in Aβ-42 secretion, but did not increase the expression of the amyloid precursor protein (APP). Hg incubation (10 and 20 µM) increased NEP protein levels; however, it did not change NEP mRNA levels nor the levels of the amyloid intracellular domain peptide, a protein fragment with transcriptional activity. Interestingly, Hg reduced NEP activity at 10 and 20 µM, and circular dichroism analysis using human recombinant NEP showed conformational changes after incubation with molar equivalents of Hg. This suggests that the Hg-induced inhibition of NEP activity may be mediated by a conformational change resulting in reduced Aβ-42 degradation. Finally, the comparative effects of lead (Pb, 50 μM) were evaluated. We found a significant increase in Aβ-42 levels and a dramatic increase in APP protein levels; however, no alteration in NEP levels was observed nor in the enzymatic activity of this metalloprotease, despite the fact that Pb slightly modified the rhNEP conformation. Overall, our data suggest that Hg and Pb increase Aβ levels by different mechanisms.

  9. The diagnostic value of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) measurement in the sera of patients with brain tumor

    PubMed Central

    Laniewska-Dunaj, Magdalena; Orywal, Karolina; Kochanowicz, Jan; Rutkowski, Robert; Szmitkowski, Maciej

    2017-01-01

    Introduction Alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) exist in the brain. Alcohol dehydrogenase and ALDH are also present in brain tumor cells. Moreover, the activity of class I isoenzymes was significantly higher in cancer than healthy brain cells. The activity of these enzymes in tumor tissue is reflected in the serum and could thus be helpful for diagnostics of brain neoplasms. The aim of this study was to investigate the potential role of ADH and ALDH as markers for brain tumors. Material and methods Serum samples were taken for routine biochemical investigation from 115 patients suffering from brain tumors (65 glioblastomas, 50 meningiomas). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. Results There was a significant increase in the activity of ADH I isoenzyme and ADH total in the sera of brain tumor patients compared to the controls. The diagnostic sensitivity for ADH I was 78%, specificity 85%, and positive and negative predictive values were 86% and 76% respectively. The sensitivity and specificity of ADH I increased with the stage of the carcinoma. Area under receiver-operating characteristic curve for ADH I was 0.71. Conclusions The results suggest a potential role for ADH I as a marker for brain tumor. PMID:28261287

  10. MiR-187 Targets the Androgen-Regulated Gene ALDH1A3 in Prostate Cancer.

    PubMed

    Casanova-Salas, Irene; Masiá, Esther; Armiñán, Ana; Calatrava, Ana; Mancarella, Caterina; Rubio-Briones, José; Scotlandi, Katia; Vicent, Maria Jesús; López-Guerrero, José Antonio

    2015-01-01

    miRNAs are predicted to control the activity of approximately 60% of all protein-coding genes participating in the regulation of several cellular processes and diseases, including cancer. Recently, we have demonstrated that miR-187 is significantly downregulated in prostate cancer (PCa) and here we propose a proteomic approach to identify its potential targets. For this purpose, PC-3 cells were transiently transfected with miR-187 precursor and miRNA mimic negative control. Proteins were analyzed by a two-dimensional difference gel electrophoresis (2D-DIGE) and defined as differentially regulated if the observed fold change was ±1.06. Then, MALDI-TOF MS analysis was performed after protein digestion and low abundance proteins were identified by LC-MS/MS. Peptides were identified by searching against the Expasy SWISS PROT database, and target validation was performed both in vitro by western blot and qRT-PCR and in clinical samples by qRT-PCR, immunohistochemistry and ELISA. DIGE analysis showed 9 differentially expressed spots (p<0.05) and 7 showed a down-regulated expression upon miR-187 re-introduction. Among these targets we identified aldehyde dehydrogenase 1A3 (ALDH1A3). ALDH1A3 expression was significantly downregulated in PC3, LNCaP and DU-145 cells after miR-187 re-introduction. Supporting these data, the expression of ALDH1A3 was found significantly (p<0.0001) up-regulated in PCa samples and inversely correlated (p<0.0001) with miR-187 expression, its expression being directly associated with Gleason score (p = 0.05). The expression of ALDH1A3 was measured in urine samples to evaluate the predictive capability of this biomarker for the presence of PCa and, at a signification level of 10%, PSA and also ALDH1A3 were significantly associated with a positive biopsy of PCa. In conclusion, our data illustrate for the first time the role of ALDH1A3 as a miR-187 target in PCa and provide insights in the utility of using this protein as a new biomarker for PCa.

  11. MiR-187 Targets the Androgen-Regulated Gene ALDH1A3 in Prostate Cancer

    PubMed Central

    Casanova-Salas, Irene; Masiá, Esther; Armiñán, Ana; Calatrava, Ana; Mancarella, Caterina; Rubio-Briones, José; Scotlandi, Katia; Vicent, Maria Jesús; López-Guerrero, José Antonio

    2015-01-01

    miRNAs are predicted to control the activity of approximately 60% of all protein-coding genes participating in the regulation of several cellular processes and diseases, including cancer. Recently, we have demonstrated that miR-187 is significantly downregulated in prostate cancer (PCa) and here we propose a proteomic approach to identify its potential targets. For this purpose, PC-3 cells were transiently transfected with miR-187 precursor and miRNA mimic negative control. Proteins were analyzed by a two-dimensional difference gel electrophoresis (2D-DIGE) and defined as differentially regulated if the observed fold change was ±1.06. Then, MALDI-TOF MS analysis was performed after protein digestion and low abundance proteins were identified by LC–MS/MS. Peptides were identified by searching against the Expasy SWISS PROT database, and target validation was performed both in vitro by western blot and qRT-PCR and in clinical samples by qRT-PCR, immunohistochemistry and ELISA. DIGE analysis showed 9 differentially expressed spots (p<0.05) and 7 showed a down-regulated expression upon miR-187 re-introduction. Among these targets we identified aldehyde dehydrogenase 1A3 (ALDH1A3). ALDH1A3 expression was significantly downregulated in PC3, LNCaP and DU-145 cells after miR-187 re-introduction. Supporting these data, the expression of ALDH1A3 was found significantly (p<0.0001) up-regulated in PCa samples and inversely correlated (p<0.0001) with miR-187 expression, its expression being directly associated with Gleason score (p = 0.05). The expression of ALDH1A3 was measured in urine samples to evaluate the predictive capability of this biomarker for the presence of PCa and, at a signification level of 10%, PSA and also ALDH1A3 were significantly associated with a positive biopsy of PCa. In conclusion, our data illustrate for the first time the role of ALDH1A3 as a miR-187 target in PCa and provide insights in the utility of using this protein as a new biomarker for PCa

  12. Enzymatic Activity of the Mycelium Compared with Oospore Development During Infection of Pea Roots by Aphanomyces euteiches.

    PubMed

    Kjøller, R; Rosendahl, S

    1998-09-01

    ABSTRACT To describe the disease cycle of the root pathogen Aphanomyces euteiches, enzymatic activity in the mycelium was compared with the development of oospores in pea roots. Plants were inoculated with two zoospore concentrations to achieve different disease levels. Hyphae were stained for fungal alkaline phosphatase activity in the roots. Additionally, enzyme activity was measured after electrophoresis of an A. euteiches-specific glucose-6-phosphate isozyme. Development of oospores in the roots was measured after staining the oospores with trypan blue. In plants inoculated with the higher zoospore concentration, the enzymatic activity of the pathogen mycelium peaked 10 to 14 days after inoculation, when oospore formation was initiated. Oospore formation was associated with a gradual increase in disease symptoms. At the last harvest, plants inoculated with the higher zoospore concentration had died. In these plants, oospores were found in 90% of the root length, while the enzymatic activity of the mycelium was low. This suggests that the pathogen mycelium is only active on living plants and does not grow saprophytically on dead plant material.

  13. Enzymatic activity of alkaline phosphatase inside protein and polymer structures fabricated via multiphoton excitation.

    PubMed

    Basu, Swarna; Campagnola, Paul J

    2004-01-01

    We demonstrate micron scale control of bioactivity through the use of multiphoton excited photochemistry, where this technique has been used to cross-link three-dimensional matrixes of alkaline phosphatase, bovine serum albumin, and polyacrylamide and combinations therein. Using a fluorescence-based assay (ELF-97), the enzymatic activity has been studied using a Michaelis-Menten analysis, and we have measured the specificity constants kcat/KM for alkaline phosphatase in both the protein and polymer matrixes to be on the order of 10(5)-10(6) M(-1) s(-1)and are comparable to known literature values in other environments. It is found that the enzyme is simply entrapped in the polymer matrix, whereas it is completely covalently bound in the protein structures. The relative reaction rate of alkaline phosphatase bound to BSA with the ELF substrate was measured as a function of cross-link density and was found to decrease in the more tightly formed matrixes, indicating a decrease in the diffusion in the matrix.

  14. The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell

    PubMed Central

    Sumarheni; Gallet, Benoit; Fender, Pascal

    2016-01-01

    Penton-dodecahedron (Pt-Dd) derived from adenovirus type 3 is a symmetric complex of pentameric penton base plus fiber which can be produced in the baculovirus system at a high concentration. The size of Pt-Dd is smaller than the virus, but this virus-like particle (VLP) has the major proteins recognized by specific receptors on the surface of almost all types of cell. In this study, by direct observation with fluorescence microscopy on a fixed and living cell, the intracellular trafficking and localization of Pt-Dd labeled with fluorescence dyes in the cytoplasm of HeLa Tub-GFP showed a rapid internalization characteristic. Subsequently, the linkage of horseradish peroxidase (HRP) with Pt-Dd as the vector demonstrated an efficient system to deliver this enzyme into the cell without interfering its enzymatic activity as shown by biochemical and cellular experiments. These results were supported by additional studies using Bs-Dd or free form of the HRP used as the control. Overall, this study strengthens the potential role of Pt-Dd as an alternative vector for delivering therapeutic agents. PMID:27242929

  15. The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell.

    PubMed

    Sumarheni; Gallet, Benoit; Fender, Pascal

    2016-01-01

    Penton-dodecahedron (Pt-Dd) derived from adenovirus type 3 is a symmetric complex of pentameric penton base plus fiber which can be produced in the baculovirus system at a high concentration. The size of Pt-Dd is smaller than the virus, but this virus-like particle (VLP) has the major proteins recognized by specific receptors on the surface of almost all types of cell. In this study, by direct observation with fluorescence microscopy on a fixed and living cell, the intracellular trafficking and localization of Pt-Dd labeled with fluorescence dyes in the cytoplasm of HeLa Tub-GFP showed a rapid internalization characteristic. Subsequently, the linkage of horseradish peroxidase (HRP) with Pt-Dd as the vector demonstrated an efficient system to deliver this enzyme into the cell without interfering its enzymatic activity as shown by biochemical and cellular experiments. These results were supported by additional studies using Bs-Dd or free form of the HRP used as the control. Overall, this study strengthens the potential role of Pt-Dd as an alternative vector for delivering therapeutic agents.

  16. Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites*

    PubMed Central

    Honaker, Matthew T.; Acchione, Mauro; Zhang, Wei; Mannervik, Bengt; Atkins, William M.

    2013-01-01

    The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes. PMID:23649628

  17. Mutation of Asn28 Disrupts the Dimerization and Enzymatic Activity of SARS 3CL

    SciTech Connect

    Barrila, J.; Gabelli, S; Bacha, U; Amzel, M; Freire, E

    2010-01-01

    Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL{sup pro}, is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL{sup pro} and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 {angstrom} from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CLpro. Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K{sub d}. The crystallographic structure of the N28A mutant determined at 2.35 {angstrom} resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL{sup pro}.

  18. Changes in antioxidant and antiinflammatory activity of black bean (Phaseolus vulgaris L.) protein isolates due to germination and enzymatic digestion.

    PubMed

    López-Barrios, Lidia; Antunes-Ricardo, Marilena; Gutiérrez-Uribe, Janet A

    2016-07-15

    Germination is an inexpensive process to improve the nutritional properties of legumes. The effect of germinating black bean seeds on the production of cotyledon protein hydrolysates (CPH) with antioxidant and antiinflammatory activities was analyzed in this research. After simulated enzymatic digestion, the oxygen radical absorbance capacity (ORAC) of CPH obtained from germinated black beans was lower than that observed for raw cotyledons. There were no significant differences among CPH cellular antioxidant activities (CAA), except for the high CAA of the 120 min hydrolysate obtained from one day germinated black bean cotyledons. The most significant changes due to germination and enzymatic hydrolysis were observed for the inhibition of nitric oxide (NO) production in macrophages. The NO synthesis inhibition observed for raw CPH was reduced after simulated gastrointestinal digestion but for germinated samples the inhibition was doubled. Peptides derived from cell wall proteins produced during germination could be responsible of antiinflammatory activity.

  19. ALDH1A3 Mutations Cause Recessive Anophthalmia and Microphthalmia

    PubMed Central

    Fares-Taie, Lucas; Gerber, Sylvie; Chassaing, Nicolas; Clayton-Smith, Jill; Hanein, Sylvain; Silva, Eduardo; Serey, Margaux; Serre, Valérie; Gérard, Xavier; Baumann, Clarisse; Plessis, Ghislaine; Demeer, Bénédicte; Brétillon, Lionel; Bole, Christine; Nitschke, Patrick; Munnich, Arnold; Lyonnet, Stanislas; Calvas, Patrick; Kaplan, Josseline; Ragge, Nicola; Rozet, Jean-Michel

    2013-01-01

    Anophthalmia and microphthalmia (A/M) are early-eye-development anomalies resulting in absent or small ocular globes, respectively. A/M anomalies occur in syndromic or nonsyndromic forms. They are genetically heterogeneous, some mutations in some genes being responsible for both anophthalmia and microphthalmia. Using a combination of homozygosity mapping, exome sequencing, and Sanger sequencing, we identified homozygosity for one splice-site and two missense mutations in the gene encoding the A3 isoform of the aldehyde dehydrogenase 1 (ALDH1A3) in three consanguineous families segregating A/M with occasional orbital cystic, neurological, and cardiac anomalies. ALDH1A3 is a key enzyme in the formation of a retinoic acid gradient along the dorso-ventral axis during early eye development. Transitory expression of mutant ALDH1A3 open reading frames showed that both missense mutations reduce the accumulation of the enzyme, potentially leading to altered retinoic acid synthesis. Although the role of retinoic acid signaling in eye development is well established, our findings provide genetic evidence of a direct link between retinoic-acid-synthesis dysfunction and early-eye-development anomalies in humans. PMID:23312594

  20. Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures.

    PubMed

    Souza, Débora Guerini; Bellaver, Bruna; Hansel, Gisele; Arús, Bernardo Assein; Bellaver, Gabriela; Longoni, Aline; Kolling, Janaina; Wyse, Angela T S; Souza, Diogo Onofre; Quincozes-Santos, André

    2016-07-01

    Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na(+)/K(+)-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate-glutamine cycle, as well as glutamate and D-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems.

  1. Activation and stabilization of the hydroperoxide lyase enzymatic extract from mint leaves (Mentha spicata) using selected chemical additives.

    PubMed

    Akacha, Najla B; Karboune, Salwa; Gargouri, Mohamed; Kermasha, Selim

    2010-03-01

    The effects of selected lyoprotecting excipients and chemical additives on the specific activity and the thermal stability of the hydroperoxide lyase (HPL) enzymatic extract from mint leaves were investigated. The addition of KCl (5%, w/w) and dextran (2.5%, w/w) to the enzymatic extract, prior to lyophilization, increased the HPL specific activity by 2.0- and 1.2-fold, respectively, compared to the control lyophilized extract. From half-life time (t (1/2)), it can be seen that KCl has enhanced the HPL stability by 1.3- to 2.3-fold, during long-period storage at -20 degrees Celsius and 4 degrees Celsius. Among the selected additives used throughout this study, glycine appeared to be the most effective one. In addition to the activation effect conferred by glycine, it also enhanced the HPL thermal stability. In contrast, polyhydroxyl-containing additives were not effective for stabilizing the HPL enzymatic extract. On the other hand, there was no signification increase in HPL activity and its thermal stability with the presence of Triton X-100. The results also showed that in the presence of glycine (10%), the catalytic efficiency of HPL was increased by 2.45-fold than that without additive.

  2. Conformational change results in loss of enzymatic activity of jack bean urease on its interaction with silver nanoparticle.

    PubMed

    Ponnuvel, Shobana; Subramanian, Balakumar; Ponnuraj, Karthe

    2015-10-01

    Urease is an enzyme produced by microbes such as bacteria, yeast and fungi. Plants also produce this enzyme. Urease action splits urea into ammonia and carbamate. This action is having important implications in agro-chemical, medicinal and environment. Therefore there is always a constant search for new and novel compounds which could inhibit this enzyme. Here we have studied the interaction of jack bean urease (JBU) with silver nanoparticle to analyze the influence of the resultant protein corona formation on the catalytic property of JBU. Several techniques like UV-Vis, gel shift assay and CD spectroscopy have been used to characterize this interaction. Urease activity assay suggests that the protein corona formation inhibits the enzymatic action of JBU. The loss of enzymatic action could be either due to the nanoparticle blocking the active site of JBU or a conformational change in the protein. The CD spectra of JBU-AgNP complexes clearly revealed significant changes in the secondary structural composition of the JBU and this could be the reason for the loss of enzymatic activity of JBU. This study revealed an interesting observation, where the interaction of AgNP with JBU resulted destabilization of hexameric nature of JBU which is otherwise highly stable. The results of the present study could be useful in the development of nanoparticle based material for inhibiting the ureolytic activity of ureases in different fields.

  3. [Effect of Siwu decoction and its combined administration on hepatic P450 enzymatic activity and mRNA expression in rats].

    PubMed

    Liang, Miao; Ma, Zeng-Chun; Yi, Jian-Feng; Wang, Yu-Guang; Tan, Hong-Ling; Xiao, Cheng-Rong; Liang, Qian-De; Tang, Xiang-Lin; Li, Hua; Shen, Guo-Lin; Gao, Yue

    2013-11-01

    To study the effect of Siwu decoction (SWD) compound and its combined administration on hepatic P450 enzymatic activity and mRNA expression in rats. Rats were orally administered with SWD and water decoction combined with other medicines for two weeks, and then sacrificed. Their livers were perfused with normal saline to prepare liver micrisomes. Mixed probe and liver microsome in vitro incubation method were adopted to detect the effect of SWD on hepatic cytochrome P450. The real-time quantitative polymerase chain reaction (Q-PCR) was used to detect the effect of SWD on the expression of hepatic cytochrome P450. Compared with the control group, the SWD compound group showed higher CYP1A2 enzymatic activity (P < 0.05); Rehmanniae-paeoniae, angelicae-paeoniae, angelicae-rhizome, paeoniae-rhizome groups had lower CYP1A2 and CYP2C19 enzymatic activities (P < 0.05); And the compound group, the single component group and the combination group showed lower CYP2B6 enzymatic activities (P < 0.05). The compound could up-regulated the mRNA expression of CYP2B1 (P < 0.05); And the four single components could down-regulated the mRNA expression of CYP2B1 (P < 0.05). SWD compound had the effect in inducing CYP1A2 enzymatic activity. The rehmanniae-paeoniae group and the angelicae-paeoniae group had identical enzymatic activity with the control group, but significant down-regulation in CYP1A2 enzymatic activity after being combined with paeoniae. The compound and its combined administration showed the inhibitory effect on CYP2B6 enzymatic activity, particularly being combined with angelicae. The compound showed identical effect with the four single components in terms of CYP1A2 mRNA expression and enzymatic activity.

  4. Proteins and enzymatic activities in Erbaluce grape berries with different response to the withering process.

    PubMed

    Vincenzi, Simone; Tolin, Serena; Cocolin, Luca; Rantsiou, Kalliopi; Curioni, Andrea; Rolle, Luca

    2012-06-30

    During the off-vine natural withering process of Erbaluce (white) grapes to obtain "Erbaluce Caluso" Passito wine, some berries change in color from green-yellow to blue. This phenomenon appears at different extents in different years and might be related to several parameters, such as temperature and humidity during withering, grape composition and Botrytis cinerea loading. To better understand the mechanism involved in color variation, the metabolic changes corresponding to this event were studied. At the end of the withering process berries with different colors were separated using a reflectance spectrophotometer, obtaining three color classes identified as "green" (L*=40.3, a*=-0.56, b*=15.20), "gold" (L*=37.7, a*=5.01, b*=14.12) and "blue" (L*=28.6, a*=0.89, b*=-0.67). The three groups of berries had different water contents, the blue berries containing about 30% less water than the green ones. Samples were crushed and the juices were analyzed. The juice yield for blue berries was less than 50% of that of the other two classes, confirming their higher dehydration level. Protein extraction from de-seeded berries was carried out using two different protocols, the first involving a treatment with phenol (to remove polyphenolic substances) and the second based on an extraction with a mild detergent (to recover the proteins to be used for enzymatic analyses). No trace of laccase activity was found in any of the samples, although DNA analysis, by quantitative PCR, suggested the presence of B. cinerea infection in the blue grapes. Chitinase activity of the blue berries was only 30% of that of the other two samples, as confirmed also by zymographic analysis on electrophoretic gels. The same was found also for esterase activity, which was lower (of about 85%) in the blue berries, which, in contrast, showed the highest beta-glucosidase activity. The electrophoretic analysis of the protein extracts revealed strong differences among the samples. Compared to the green and

  5. Positive regulation of the enzymatic activity of gastric H(+),K(+)-ATPase by sialylation of its β-subunit.

    PubMed

    Fujii, Takuto; Watanabe, Midori; Shimizu, Takahiro; Takeshima, Hiroshi; Kushiro, Keiichiro; Takai, Madoka; Sakai, Hideki

    2016-06-01

    The gastric proton pump (H(+),K(+)-ATPase) consists of a catalytic α-subunit (αHK) and a glycosylated β-subunit (βHK). βHK glycosylation is essential for the apical trafficking and stability of αHK in gastric parietal cells. Here, we report the properties of sialic acids at the termini of the oligosaccharide chains of βHK. Sialylation of βHK was found in LLC-PK1 cells stably expressing αHK and βHK by staining of the cells with lectin-tagged fluorescent polymeric nanoparticles. This sialylation was also confirmed by biochemical studies using sialic acid-binding lectin beads and an anti-βHK antibody. The sialic acids of βHK are cleaved enzymatically by neuraminidase (sialidase) and nonenzymatically by an acidic solution (pH5). Interestingly, the enzymatic activity of H(+),K(+)-ATPase was significantly decreased by cleavage of the sialic acids of βHK. In contrast, βHK was not sialylated in the gastric tubulovesicles prepared from the stomach of fed hogs. The H(+),K(+)-ATPase activity in these tubulovesicles was not significantly altered by neuraminidase. Importantly, the sialylation of βHK was observed in the gastric samples prepared from the stomach of famotidine (a histamine H2 receptor antagonist)-treated rats, but not histamine (an acid secretagogue)-treated rats. The enzymatic activity of H(+),K(+)-ATPase in the samples of the famotidine-treated rats was significantly higher than in the histamine-treated rats. The effects of famotidine were weakened by neuraminidase. These results indicate that βHK is sialylated at neutral or weakly acidic pH, but not at acidic pH, suggesting that the sialic acids of βHK positively regulate the enzymatic activity of αHK.

  6. Epigenetic Therapy of Hematopoietic Malignancies: Novel Approaches for Tissue-Specific and Global Inhibition of EZH2 Enzymatic Activities

    DTIC Science & Technology

    2015-08-01

    AWARD NUMBER: W81XWH-14-1-0232 TITLE: Epigenetic Therapy of Hematopoietic Malignancies: Novel Approaches for Tissue - Specific and Global...Therapy of Hematopoietic Malignancies: Novel Approaches for Tissue - Specific and Global Inhibition of EZH2 Enzymatic Activities 5b. GRANT NUMBER...binding of EZH2 in B- versus T- cell lineages, and to identify the responsible tissue -specific recruiters. 2. KEYWORDS: Hematopoietic cancer, PRC2

  7. KIF4 motor regulates activity-dependent neuronal survival by suppressing PARP-1 enzymatic activity.

    PubMed

    Midorikawa, Ryosuke; Takei, Yosuke; Hirokawa, Nobutaka

    2006-04-21

    In brain development, apoptosis is a physiological process that controls the final numbers of neurons. Here, we report that the activity-dependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein 4 (KIF4), a microtubule-based molecular motor. The C-terminal domain of KIF4 is a module that suppresses the activity of poly (ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme known to maintain cell homeostasis by repairing DNA and serving as a transcriptional regulator. When neurons are stimulated by membrane depolarization, calcium signaling mediated by CaMKII induces dissociation of KIF4 from PARP-1, resulting in upregulation of PARP-1 activity, which supports neuron survival. After dissociation from PARP-1, KIF4 enters into the cytoplasm from the nucleus and moves to the distal part of neurites in a microtubule-dependent manner. We suggested that KIF4 controls the activity-dependent survival of postmitotic neurons by regulating PARP-1 activity in brain development.

  8. Impaired enzymatic defensive activity, mitochondrial dysfunction and proteasome activation are involved in RTT cell oxidative damage.

    PubMed

    Cervellati, Carlo; Sticozzi, Claudia; Romani, Arianna; Belmonte, Giuseppe; De Rasmo, Domenico; Signorile, Anna; Cervellati, Franco; Milanese, Chiara; Mastroberardino, Pier Giorgio; Pecorelli, Alessandra; Savelli, Vinno; Forman, Henry J; Hayek, Joussef; Valacchi, Giuseppe

    2015-10-01

    A strong correlation between oxidative stress (OS) and Rett syndrome (RTT), a rare neurodevelopmental disorder affecting females in the 95% of the cases, has been well documented although the source of OS and the effect of a redox imbalance in this pathology has not been yet investigated. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have demonstrated in RTT cells high levels of H2O2 and HNE protein adducts. These findings correlated with the constitutive activation of NADPH-oxidase (NOX) and that was prevented by a NOX inhibitor and iron chelator pre-treatment, showing its direct involvement. In parallel, we demonstrated an increase in mitochondrial oxidant production, altered mitochondrial biogenesis and impaired proteasome activity in RTT samples. Further, we found that the key cellular defensive enzymes: glutathione peroxidase, superoxide dismutase and thioredoxin reductases activities were also significantly lower in RTT. Taken all together, our findings suggest that the systemic OS levels in RTT can be a consequence of both: increased endogenous oxidants as well as altered mitochondrial biogenesis with a decreased activity of defensive enzymes that leads to posttranslational oxidant protein modification and a proteasome activity impairment.

  9. Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: The N-terminal region is more important than enzymatic activity

    PubMed Central

    Rouault, Morgane; Rash, Lachlan D.; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H.; Lambeau, Gérard

    2009-01-01

    Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, an homologous but non toxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1–22) of OS2, but not the central one (residues 58–89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102–119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity. PMID:16669624

  10. Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity.

    PubMed

    Rouault, Morgane; Rash, Lachlan D; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H; Lambeau, Gérard

    2006-05-09

    Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, a homologous but nontoxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1-22) of OS2, but not the central one (residues 58-89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102-119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera, which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity.

  11. Effects of partial decerebration and hypophyseal allograft in the thymus of chicken embryos: thymostimulin localization and enzymatic activities.

    PubMed

    Aita, M; Romano, N

    2006-01-01

    Changes in chicken embryo thymus after partial decerebration (including the hypophysis) and hypophyseal allograft were investigated. Chicken embryos were partially decerebrated at 36-40 hr of incubation and on day 12 received a hypophyseal allograft from 18-day-old donor embryos. The embryonic thymuses were collected on day 18 and examined with histological methods, tested for the anti-thymostimulin-like immune-reaction, and for histoenzymatic activities and compared with normal and sham-operated embryos at the same age. After partial decerebration, the thymic cortical and medullary compartments diminished markedly in size. Anti-thymostimulin, succinic dehydrogenase and ATPase enzymatic activities tested, yielded negative reactions. In partially decerebrated hypophyseal allografted embryos, the same thymic compartments improved and anti-thymostimulin-like immune-reaction and enzymatic activities partially recovered. These findings confirmed the key role of hypophysis in thymic ontogenic development and provided new information in metabolic enzymatic pathways and synthesis of a thymostimulin-like substance in the thymus.

  12. Expression of the Saccharomyces cerevisiae glycoprotein invertase in mouse fibroblasts: glycosylation, secretion, and enzymatic activity

    SciTech Connect

    Bergh, M.L.E.; Cepko, C.L.; Wolf, D.; Robbins, P.W.

    1987-06-01

    Oligosaccharide processing is controlled by host- and protein-dependent factors. To increase our understanding of the relative contribution of those factors the authors studied the glycosylation of yeast invertase expressed in a heterologous system. Invertase synthesized in psi-2 cells (an NIH 3T3-derived packaging line) is secreted efficiently, enzymatically active, and heavily glycosylated. It was estimated that the protein contains 8 or 9 carbohydrate chains. Two classes can be observed, of an approximate size of 100-110 kDa and 115-130 kDa, respectively. The size differences are due to differences in glycosylation. The smaller class contains two high-mannose carbohydrate chains; the remainder is of the complex type, sialylated and most likely tri- or tetraantennary. This profile parallels the situation observed with invertase glycosylation in yeast, where 2 of 9 or 10 chains remain unprocessed. The larger size class of invertase expressed in mouse fibroblasts has a different profile, since it contains probably only complex-type glycans. There are no apparent differences, however, in the size of the protein backbone between the two size classes. When invertase is synthesized in the presence of the mannosidase inhibitor 1-deoxymannojirimycin, processing is blocked completely. The glucosidase inhibitor 1-deoxynojirimycin does not inhibit processing completely. The glycosylation inhibitor tunicamycin prevents secretion of invertase completely when cells are cultured at 37/sup 0/C. At 26/sup 0/C, however, nonglycosylated invertase can be detected in the medium. These data suggest that glycosylation of invertase seems to be essential for the early steps of the secretory pathway but is less critical for later events.

  13. Enhanced enzymatic activity of glycerol-3-phosphate dehydrogenase from the cryophilic Saccharomyces kudriavzevii.

    PubMed

    Oliveira, Bruno M; Barrio, Eladio; Querol, Amparo; Pérez-Torrado, Roberto

    2014-01-01

    During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied the expression of glycerol biosynthetic pathway genes and we observed a higher expression of GPD1 gene in S. kudriavzevii compared to S. cerevisiae in micro-vinification conditions. We observed higher enzymatic activity of Gpd1p in S. kudriavzevii in response to osmotic and cold stress. Also, we determined that S. kudriavzevii Gpd1p enzyme presents increased catalytic properties that will contribute to increase glycerol production. Finally, we evaluated the glycerol production with S. cerevisiae, S. kudriavzevii or a recombinant Gpd1p variant in the same background and observed that the S. kudriavzevii enzyme produced increased glycerol levels at 12 or 28°C. This suggests that glycerol is increased in S. kudriavzevii mainly due to increased V max of the Gpd1p enzyme. All these differences indicate that S. kudriavzevii has changed the metabolism to promote the branch of the glycolytic pathway involved in glycerol production to adapt to low temperature environments and maintain the NAD(+)/NADH ratio in alcoholic fermentations. This knowledge is industrially relevant due to the potential use, for example, of S. cerevisiae-S. kudriavzevii hybrids in the wine industry where glycerol content is an important quality parameter.

  14. Effects of 3-methylcholanthrene and aspirin co-administration on ALDH3A1 in HepG2 cells.

    PubMed

    Sotiropoulou, M; Pappas, P; Marselos, M

    2001-01-30

    The effects of two different protocols of 3-methylcholanthrene (3MC) and aspirin co-administration were studied in a well-established human hepatoma cell line (HepG2). During this work, we have performed toxicity tests for cell viability/cell proliferation as well as studies on the expression of ALDH3A1 after exposure of HepG2 cells to 3MC or/and aspirin. For the evaluation of toxic concentrations of 3MC and aspirin, the WST-1 test was used. WST-1 is a reliable cytotoxicity test which is based on the cleavage of the tetrazolium salt WST-1 to formazan by mitochondrial enzymes of living cells. A broad range of drug concentrations for either 3MC (0.25-50.0 microM) or aspirin (0.05-10.0 mM) were used for cell exposure, in several periods of time. The expression of ALDH3A1 in HepG2 cells showed typical time- and dose-response curves of induction after application of 3MC (1-5 days, 1.5-5.0 microM, respectively). When cells were firstly exposed to 3MC (2.5 and 5.0 microM) and then to aspirin (0.25 mM), the induced ALDH3A1 activity was further enhanced in a statistically significant way (P<0.05). On the contrary, when aspirin application was preceded 3MC exposuring a statistically significant decrease in ALDH3A1 inducibility was observed, as compared with the application of 3MC alone.

  15. Enzymatic activities of allergen extracts from three species of dust mites and cockroaches commonly found in Korean home.

    PubMed

    Jeong, Kyoung Yong; Kim, Chungryul; Yong, Tai-Soon

    2010-06-01

    Allergen extracts from dust mites and cockroaches commonly found in Korean homes were used to evaluate their enzymatic activity as they are believed to influence allergenicity. Allergen extracts were prepared from 3 dust mite species (Dermatophagoides farinae, D. pteronyssinus, and Tyrophagus putrescentiae) and 3 cockroach species (Blattella germanica, Periplaneta americana, and P. fuliginosa) maintained in the Korea National Arthropods of Medical Importance Resource Bank. Proteins were extracted in PBS after homogenization using liquid nitrogen. The activities of various enzymes were investigated using the API Zym system. No significant difference in phosphatase, lipase, or glycosidase activity was observed among the 6 allergen extracts, but much difference was observed in protease activity. Protease activity was assessed in more detail by gelatin zymography and the EnzChek assay. Extract from T. putrescentiae showed the highest protease activity, followed by those of the cockroach extracts. Extracts from D. farinae and D. pteronyssinus showed only weak protease activity. Gelatinolytic activity was detected mainly in a 30-kDa protein in D. farinae, a 28-kDa protein in D. pteronyssinus, a > 26-kDa protein in T. putrescentiae, a > 20-kDa protein in B. germanica, and a > 23-kDa protein in P. americana and P. fuliginosa. The information on various enzymatic activities obtained in this study may be useful for future studies. In particular, the strong protease activity found in cockroach extracts could contribute to sensitization to cockroach allergens, which is known to be associated with the development of asthma.

  16. Influence of short-time imidacloprid and acetamiprid application on soil microbial metabolic activity and enzymatic activity.

    PubMed

    Wang, Fei; Yao, Jun; Chen, Huilun; Yi, Zhengji; Choi, Martin M F

    2014-09-01

    The influence of two neonicotinoids, i.e., imidacloprid (IMI) and acetamiprid (ACE), on soil microbial activities was investigated in a short period of time using a combination of the microcalorimetric approach and enzyme tests. Thermodynamic parameters such as Q T (J g(-1) soil), ∆H met (kJ mol(-1)), J Q/S (J g(-1) h(-1)), k (h(-1)), and soil enzymatic activities, dehydrogenase, phosphomonoesterase, arginine deaminase, and urease, were used to evaluate whole metabolic activity changes and acute toxicity following IMI and ACE treatment. Various profiles of thermogenic curves reflect different soil microbial activities. The microbial growth rate constant k, total heat evolution Q T (expect for IMI), and inhibitory ratio I show linear relationship with the doses of IMI and ACE. Q T for IMI increases at 0.0-20 μg g(-1) and then decreases at 20-80 μg g(-1), possibly attributing to the presence of tolerant microorganisms. The 50 % inhibitory ratios (IC50) of IMI and ACE are 95.7 and 77.2 μg g(-1), respectively. ACE displays slightly higher toxicity than IMI. Plots of k and Q T against microbial biomass-C indicate that the k and Q T are growth yield-dependent. IMI and ACE show 29.6; 40.4 and 23.0; and 23.3, 21.7, and 30.5 % inhibition of dehydrogenase, phosphomonoesterase, and urease activity, respectively. By contrast, the arginine deaminase activity is enhanced by 15.2 and 13.2 % with IMI and ACE, respectively. The parametric indices selected give a quantitative dose-response relationship of both insecticides and indicate that ACE is more toxic than IMI due to their difference in molecular structures.

  17. A carbamate-based approach to primaquine prodrugs: antimalarial activity, chemical stability and enzymatic activation.

    PubMed

    Mata, Graça; do Rosário, Virgílio E; Iley, Jim; Constantino, Luís; Moreira, Rui

    2012-01-15

    O-Alkyl and O-aryl carbamate derivatives of the antimalarial drug primaquine were synthesised as potential prodrugs that prevent oxidative deamination to the inactive metabolite carboxyprimaquine. Both O-alkyl and O-aryl carbamates undergo hydrolysis in alkaline and pH 7.4 phosphate buffers to the parent drug, with O-aryl carbamates being ca. 10(6)-10(10) more reactive than their O-alkyl counterparts. In human plasma O-alkyl carbamates were stable, whereas in contrast their O-aryl counterparts rapidly released the corresponding phenol product, with primaquine being released only slowly over longer incubation periods. Activation of the O-aryl carbamates in human plasma appears to be catalysed by butyrylcholinesterase (BuChE), which leads to carbamoylation of the catalytic serine of the enzyme followed by subsequent slow enzyme reactivation and release of parent drug. Most of the O-aryl and O-alkyl carbamates are activated in rat liver homogenates with half-lives ranging from 9 to 15 h, while the 4-nitrophenyl carbamate was hydrolysed too rapidly to determine an accurate rate constant. Antimalarial activity was studied using a model consisting of Plasmodium berghei, Balb C mice and Anopheles stephensi mosquitoes. When compared to controls, ethyl and n-hexyl carbamates were able to significantly reduce the percentage of infected mosquitos as well as the mean number of oocysts per infected mosquito, thus indicating that O-alkyl carbamates of primaquine have the potential to be developed as transmission-blocking antimalarial agents.

  18. Ultrahigh pressure-assisted enzymatic extraction maximizes the yield of longan pulp polysaccharides and their acetylcholinesterase inhibitory activity in vitro.

    PubMed

    Bai, Yajuan; Liu, Lei; Zhang, Ruifen; Huang, Fei; Deng, Yuanyuan; Zhang, Mingwei

    2017-03-01

    An extraction method employing ultrahigh pressure-assisted enzymatic treatment was developed and optimized by response surface methodology to increase the yield of longan pulp polysaccharides (LP-UE). A maximum polysaccharides yield of 8.55% was obtained under the optimal conditions of 407MPa ultrahigh pressure maintained for 6min with an enzyme to pretreated material ratio of 1:100, an enzymolysis time of 1.7h and a water to pretreated material ratio of 42ml/g. Subsequently, the physicochemical properties and acetylcholinesterase (AChE) inhibitory activity of LP-UE were compared to those of longan pulp polysaccharides (LP) extracted by hot water (LP-H), ultrahigh pressure (LP-U) or enzymatic treatment (LP-E). Results demonstrated that the extraction yield, hexuronic acid content and AChE inhibitory activity of LP-UE was the highest among the four LP samples. LP-UE was primarily made up of arabinose, glucose, and galactose and was linked mainly by β-type glycosidic linkage. The FTIR spectrum of LP-UE was very similar to those of LP-H, LP-U, and LP-E. In summary, ultrahigh pressure-assisted enzymatic treatment is a more efficient technique for extracting LP with considerable improvement of both yield and memory enhancement function.

  19. Pretreatment and Enzymatic Hydrolysis

    SciTech Connect

    2006-06-01

    Activities in this project are aimed at overcoming barriers associated with high capital and operating costs and sub-optimal sugar yields resulting from pretreatment and subsequent enzymatic hydrolysis of biomass.

  20. Priming effects and enzymatic activity in Israeli soils under treated wastewater and freshwater irrigation

    NASA Astrophysics Data System (ADS)

    Anissimova, Marina; Heinze, Stefanie; Chen, Yona; Tarchitzky, Jorge; Marschner, Bernd

    2014-05-01

    Irrigation of soils with treated wastewater (TWW) directly influences microbial processes of soil. TWW contains easily decomposable organic material, which can stimulate the activity of soil microorganisms and, as a result, lead to the excessive consumption of soil organic carbon pool. We investigated the effects of irrigation with TWW relative to those of irrigation with freshwater (FW) on the microbial parameters in soils with low (7%) and medium (13%) clay content in a lysimeter experiment. The objectives of our study were to (i) determine the impact of water quality on soil respiration and enzymatic activity influenced by clay content and depth, and (ii) work out the changes in the turnover of soil organic matter (PE, priming effects). Samples were taken from three soil depths (0-10, 10-20, and 40-60 cm). Soil respiration and PE were determined in a 21-days incubation experiment after addition of uniformly 14C-labeled fructose. Activity of 10 extracellular enzymes (EEA, from C-, N-, P-, and S-cycle), phenol oxidase and peroxidase activity (PO+PE), and dehydrogenase activity (DHA) were assayed. Microbial Community-Level Physiological Profiles (CLPP) using four substrates, and microbial biomass were determined. The results showed that the clay content acted as the main determinative factor. In the soil with low clay content the water quality had a greater impact: the highest PE (56%) was observed in the upper layer (0-10cm) under FW irrigation; EEA of C-, P-, and S-cycles was significantly higher in the upper soil layer under TWW irrigation. Microbial biomass was higher in the soil under TWW irrigation and decreased with increasing of depth (50 μg/g soil in the upper layer, 15 μg/g soil in the lowest layer). This tendency was also observed for DHA. Contrary to the low clay content, in the soil with medium clay content both irrigation types caused the highest PE in the lowest layer (65% under FW irrigation, 48% under TWW irrigation); the higher substrate

  1. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    SciTech Connect

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  2. Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies

    PubMed Central

    Koch, Claudia; Eber, Fabian J; Azucena, Carlos; Förste, Alexander; Walheim, Stefan; Schimmel, Thomas; Bittner, Alexander M; Jeske, Holger; Gliemann, Hartmut; Eiben, Sabine; Geiger, Fania C

    2016-01-01

    monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing 'systems-on-a-chip'. PMID:27335751

  3. Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies.

    PubMed

    Koch, Claudia; Eber, Fabian J; Azucena, Carlos; Förste, Alexander; Walheim, Stefan; Schimmel, Thomas; Bittner, Alexander M; Jeske, Holger; Gliemann, Hartmut; Eiben, Sabine; Geiger, Fania C; Wege, Christina

    2016-01-01

    monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing 'systems-on-a-chip'.

  4. A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity.

    PubMed

    Guo, Shihui; Skala, Wolfgang; Magdolen, Viktor; Briza, Peter; Biniossek, Martin L; Schilling, Oliver; Kellermann, Josef; Brandstetter, Hans; Goettig, Peter

    2016-01-08

    Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology.

  5. Expanded Hematopoietic Progenitor Cells Reselected for High Aldehyde Dehydrogenase Activity Demonstrate Islet Regenerative Functions.

    PubMed

    Seneviratne, Ayesh K; Bell, Gillian I; Sherman, Stephen E; Cooper, Tyler T; Putman, David M; Hess, David A

    2016-04-01

    Human umbilical cord blood (UCB) hematopoietic progenitor cells (HPC) purified for high aldehyde dehydrogenase activity (ALDH(hi) ) stimulate islet regeneration after transplantation into mice with streptozotocin-induced β cell deletion. However, ALDH(hi) cells represent a rare progenitor subset and widespread use of UCB ALDH(hi) cells to stimulate islet regeneration will require progenitor cell expansion without loss of islet regenerative functions. Here we demonstrate that prospectively purified UCB ALDH(hi) cells expand efficiently under serum-free, xeno-free conditions with minimal growth factor supplementation. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, kinetic analyses over 9 days revealed the frequency of ALDH(hi) cells diminished as culture time progressed such that total ALDH(hi) cell number was maximal (increased 3-fold) at day 6. Subsequently, day 6 expanded cells (bulk cells) were sorted after culture to reselect differentiated progeny with low ALDH-activity (ALDH(lo) subset) from less differentiated progeny with high ALDH-activity (ALDH(hi) subset). The ALDH(hi) subset retained primitive cell surface marker coexpression (32.0% ± 7.0% CD34(+) /CD38(-) cells, 37.0% ± 6.9% CD34(+) /CD133(+) cells), and demonstrated increased hematopoietic colony forming cell function compared with the ALDH(lo) subset. Notably, bulk cells or ALDH(lo) cells did not possess the functional capacity to lower hyperglycemia after transplantation into streptozotocin-treated NOD/SCID mice. However, transplantation of the repurified ALDH(hi) subset significantly reduced hyperglycemia, improved glucose tolerance, and increased islet-associated cell proliferation and capillary formation. Thus, expansion and delivery of reselected UCB cells that retain high ALDH-activity after short-term culture represents an improved strategy for the development of cellular therapies to enhance islet regeneration in situ.

  6. Amino Acid Residues Critical for the Specificity for Betaine Aldehyde of the Plant ALDH10 Isoenzyme Involved in the Synthesis of Glycine Betaine1[W][OA

    PubMed Central

    Díaz-Sánchez, Ángel G.; González-Segura, Lilian; Mújica-Jiménez, Carlos; Rudiño-Piñera, Enrique; Montiel, Carmina; Martínez-Castilla, León P.; Muñoz-Clares, Rosario A.

    2012-01-01

    Plant Aldehyde Dehydrogenase10 (ALDH10) enzymes catalyze the oxidation of ω-primary or ω-quaternary aminoaldehydes, but, intriguingly, only some of them, such as the spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (SoBADH), efficiently oxidize betaine aldehyde (BAL) forming the osmoprotectant glycine betaine (GB), which confers tolerance to osmotic stress. The crystal structure of SoBADH reported here shows tyrosine (Tyr)-160, tryptophan (Trp)-167, Trp-285, and Trp-456 in an arrangement suitable for cation-π interactions with the trimethylammonium group of BAL. Mutation of these residues to alanine (Ala) resulted in significant Km(BAL) increases and Vmax/Km(BAL) decreases, particularly in the Y160A mutant. Tyr-160 and Trp-456, strictly conserved in plant ALDH10s, form a pocket where the bulky trimethylammonium group binds. This space is reduced in ALDH10s with low BADH activity, because an isoleucine (Ile) pushes the Trp against the Tyr. Those with high BADH activity instead have Ala (Ala-441 in SoBADH) or cysteine, which allow enough room for binding of BAL. Accordingly, the mutation A441I decreased the Vmax/Km(BAL) of SoBADH approximately 200 times, while the mutation A441C had no effect. The kinetics with other ω-aminoaldehydes were not affected in the A441I or A441C mutant, demonstrating that the existence of an Ile in the second sphere of interaction of the aldehyde is critical for discriminating against BAL in some plant ALDH10s. A survey of the known sequences indicates that plants have two ALDH10 isoenzymes: those known to be GB accumulators have a high-BAL-affinity isoenzyme with Ala or cysteine in this critical position, while non GB accumulators have low-BAL-affinity isoenzymes containing Ile. Therefore, BADH activity appears to restrict GB synthesis in non-GB-accumulator plants. PMID:22345508

  7. Effects of different bulking agents on the maturity, enzymatic activity, and microbial community functional diversity of kitchen waste compost.

    PubMed

    Wang, Xiaojuan; Zhang, Wenwei; Gu, Jie; Gao, Hua; Qin, Qingjun

    2016-10-01

    Aerobic composting is an effective method for the disposal and utilization of kitchen waste. However, the addition of a bulking agent is necessary during kitchen waste composting because of its high moisture content and low C/N ratio. In order to select a suitable bulking agent, we investigated the influence of leaf litter (LL), sawdust (SD), and wheat straw (WS) on the enzymatic activity, microbial community functional diversity, and maturity indices during the kitchen waste composting process. The results showed that the addition of WS yielded the highest maturity (the C/N ratio decreased from 25 to 13, T value = 0.5, and germination index (GI) = 114.7%), whereas the compost containing SD as a bulking agent had the lowest maturity (GI = 32.4%). The maximum cellulase and urease activities were observed with the WS treatment on day 8, whereas the SD treatment had the lowest cellulase activity and the LL treatment had the lowest urease activity. The compost temperature and microbial activity (as the average well color development) showed that bulking the composts with SD prolonged the composting process. The diversity index based on the community-level physiological profile showed that the composts bulked with LL and WS had greater microbial community functional diversity compared with those bulked with SD. Thus, the maturity indexes and enzymatic activities suggest that WS is a suitable bulking agent for use in kitchen waste composting systems.

  8. Effect of R119G Mutation on Human P5CR1 Dynamic Property and Enzymatic Activity

    PubMed Central

    Li, Linhua; Ye, Yujia; Sang, Peng; Yin, Yirui; Hu, Wei; Wang, Jing; Zhang, Chao; Li, Deyun; Wan, Wen; Li, Rui; Li, Longjun; Ma, Linling; Xie, Yuehui

    2017-01-01

    Pyrroline-5-carboxylate reductase (P5CR1) is a universal housekeeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)+. The enzymatic cycle between P5C and proline is important for function in amino acid metabolism, apoptosis, and intracellular redox potential balance in mitochondria. Autosomal recessive cutis laxa (ARCL) results from a mutation in P5CR1 encoded by PYCR1. Specifically, the R119G mutation is reported to be linked to ARCL although it has not yet been characterized. We synthesized R119G P5CR1 and compared it to WT P5CR1. Foldx prediction of WT and R119G mutant P5CR1 protein stability suggests that the R119G mutation could significantly reduce protein stability. We also performed enzymatic activity assays to determine how the mutation impacts P5CR1 enzymatic function. The results of these experiments show that mutagenesis of R119 to G decreases P5CR1 catalytic efficiency for 3,4-dehydro-L-proline relative to WT. Mutagenesis and kinetic studies reveal that the activity of the mutant decreases as temperature increases from 5°C to 37°C, with almost no activity at 37°C, indicating that this mutation impairs P5CR1 function in vivo. Conversely, WT P5CR1 retains its activity after incubation at 37°C and has essentially no remaining activity at 75°C. Taken together, our experimental results indicate the R119G mutation could be an involving pathomechanism for ARCL. PMID:28194412

  9. Effect of R119G Mutation on Human P5CR1 Dynamic Property and Enzymatic Activity.

    PubMed

    Li, Linhua; Ye, Yujia; Sang, Peng; Yin, Yirui; Hu, Wei; Wang, Jing; Zhang, Chao; Li, Deyun; Wan, Wen; Li, Rui; Li, Longjun; Ma, Linling; Xie, Yuehui; Meng, Zhaohui

    2017-01-01

    Pyrroline-5-carboxylate reductase (P5CR1) is a universal housekeeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)(+). The enzymatic cycle between P5C and proline is important for function in amino acid metabolism, apoptosis, and intracellular redox potential balance in mitochondria. Autosomal recessive cutis laxa (ARCL) results from a mutation in P5CR1 encoded by PYCR1. Specifically, the R119G mutation is reported to be linked to ARCL although it has not yet been characterized. We synthesized R119G P5CR1 and compared it to WT P5CR1. Foldx prediction of WT and R119G mutant P5CR1 protein stability suggests that the R119G mutation could significantly reduce protein stability. We also performed enzymatic activity assays to determine how the mutation impacts P5CR1 enzymatic function. The results of these experiments show that mutagenesis of R119 to G decreases P5CR1 catalytic efficiency for 3,4-dehydro-L-proline relative to WT. Mutagenesis and kinetic studies reveal that the activity of the mutant decreases as temperature increases from 5°C to 37°C, with almost no activity at 37°C, indicating that this mutation impairs P5CR1 function in vivo. Conversely, WT P5CR1 retains its activity after incubation at 37°C and has essentially no remaining activity at 75°C. Taken together, our experimental results indicate the R119G mutation could be an involving pathomechanism for ARCL.

  10. The Proportion of ALDEFLUOR-Positive Cancer Stem Cells Changes with Cell Culture Density Due to the Expression of Different ALDH Isoforms

    PubMed Central

    Opdenaker, Lynn M.; Modarai, Shirin R.; Boman, Bruce M.

    2017-01-01

    A significant number of discrepancies exist within the literature regarding ALDEFLUOR-positive stem cell populations in cell lines. We hypothesized that these inconsistencies resulted from differences in culture conditions, particularly cell density. We cultured several colon cancer cell lines (N=8) at high and low densities and found a significant decrease in ALDEFLUOR-positive cell populations at high density. However, we found no changes in the CD166-positive stem cell population, self-renewal, or cell cycle distribution of cells cultured at different densities. Interestingly, when we sorted both ALDEFLUOR positive and negative populations from the different density cultures, we identified a significant number of Aldehyde dehydrogenase (ALDH) isoforms whose expression was decreased in ALDEFLUOR-positive stem cells cultured at high density. This novel finding suggests that multiple ALDH isoforms contribute to ALDEFLUOR activity in colon cancer stem cells and decreases in ALDEFLUOR-positive stem cells at high cell density are due to decreased expression of multiple ALDH isoforms. Thus, designing therapeutics to target ALDEFLUOR-positive cancer stem cells may require inhibition of multiple ALDH isoforms.

  11. Comparative study of the aldehyde dehydrogenase (ALDH) gene superfamily in the glycophyte Arabidopsis thaliana and Eutrema halophytes

    PubMed Central

    Hou, Quancan; Bartels, Dorothea

    2015-01-01

    Background and Aims Stresses such as drought or salinity induce the generation of reactive oxygen species, which subsequently cause excessive accumulation of aldehydes in plant cells. Aldehyde dehydrogenases (ALDHs) are considered as ‘aldehyde scavengers’ to eliminate toxic aldehydes caused by oxidative stress. The completion of the genome sequencing projects of the halophytes Eutrema parvulum and E. salsugineum has paved the way to explore the relationships and the roles of ALDH genes in the glycophyte Arabidopsis thaliana and halophyte model plants. Methods Protein sequences of all plant ALDH families were used as queries to search E. parvulum and E. salsugineum genome databases. Evolutionary analyses compared the phylogenetic relationships of ALDHs from A. thaliana and Eutrema. Expression patterns of several stress-associated ALDH genes were investigated under different salt conditions using reverse transcription–PCR. Putative cis-elements in the promoters of ALDH10A8 from A. thaliana and E. salsugineum were compared in silico. Key Results Sixteen and 17 members of ten ALDH families were identified from E. parvulum and E. salsugineum genomes, respectively. Phylogenetic analysis of ALDH protein sequences indicated that Eutrema ALDHs are closely related to those of Arabidopsis, and members within these species possess nearly identical exon–intron structures. Gene expression analysis under different salt conditions showed that most of the ALDH genes have similar expression profiles in Arabidopsis and E. salsugineum, except for ALDH7B4 and ALDH10A8. In silico analysis of promoter regions of ALDH10A8 revealed different distributions of cis-elements in E. salsugineum and Arabidopsis. Conclusions Genomic organization, copy number, sub-cellular localization and expression profiles of ALDH genes are conserved in Arabidopsis, E. parvulum and E. salsugineum. The different expression patterns of ALDH7B4 and ALDH10A8 in Arabidopsis and E. salsugineum suggest that E

  12. Long-term effects of fertilizer on soil enzymatic activity of wheat field soil in Loess Plateau, China.

    PubMed

    Hu, Weigang; Jiao, Zhifang; Wu, Fasi; Liu, Yongjun; Dong, Maoxing; Ma, Xiaojun; Fan, Tinglu; An, Lizhe; Feng, Huyuan

    2014-12-01

    The effects of long-term (29 years) fertilization on local agro-ecosystems in the Loess Plateau of northwest China, containing a single or combinations of inorganic (Nitrogen, N; Phosphate, P) and organic (Mature, M Straw, S) fertilizer, including N, NP, SNP, M, MNP, and a control. The soil enzymes, including dehydrogenase, urease, alkaline phosphatase, invertase and glomalin, were investigated in three physiological stages (Jointing, Dough, and Maturity) of wheat growth at three depths of the soil profile (0-15, 16-30, 31-45 cm). We found that the application of farmyard manure and straw produced the highest values of soil enzymatic activity, especially a balanced applied treatment of MNP. Enzymatic activity was lowest in the control. Values were generally highest at dough, followed by the jointing and maturity stages, and declined with soil profile depth. The activities of the enzymes investigated here are significantly correlated with each other and are correlated with soil nutrients, in particular with soil organic carbon. Our results suggest that a balanced application of fertilizer nutrients and organic manure (especially those containing P) has positive effects on multiple soil chemical parameters, which in turn enhances enzyme activity. We emphasize the role of organic manure in maintaining soil organic matter and promoting biological activity, as its application can result in a substantial increase in agricultural production and can be sustainable for many years.

  13. Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii.

    PubMed

    Pedri, Z C; Lozano, L M S; Hermann, K L; Helm, C V; Peralta, R M; Tavares, L B B

    2015-11-10

    AbstractLignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L-1) at 20 days of cultivation.

  14. Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii.

    PubMed

    Pedri, Z C; Lozano, L M S; Hermann, K L; Helm, C V; Peralta, R M; Tavares, L B B

    2015-11-01

    Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L-1) at 20 days of cultivation.

  15. Enzymatic Activity of the Soybean Ecto-Apyrase GS52 Is Essential for Stimulation of Nodulation1[W][OA

    PubMed Central

    Tanaka, Kiwamu; Nguyen, Cuong T.; Libault, Marc; Cheng, Jianlin; Stacey, Gary

    2011-01-01

    Nitrogen is an essential nutrient for plant growth. In the Rhizobium-legume symbiosis, root nodules are the sites of bacterial nitrogen fixation, in which atmospheric nitrogen is converted into a form that plants can utilize. While recent studies suggested an important role for the soybean (Glycine max) ecto-apyrase GS52 in rhizobial root hair infection and root nodule formation, precisely how this protein impacts the nodulation process remains undetermined. In this study, the biochemical characteristics of the GS52 enzyme were investigated. Computer modeling of the GS52 apyrase structure identified key amino acid residues important for catalytic activity, which were subsequently mutagenized. Although the GS52 enzyme exhibited broad substrate specificity, its activity on pyrimidine nucleotides and diphosphate nucleotides was significantly higher than on ATP. This result was corroborated by structural modeling of GS52, which predicted a low specificity for the adenine base within the substrate-binding pocket of the enzyme. The wild-type enzyme and its inactive mutant forms were expressed in soybean roots in order to evaluate the importance of GS52 enzymatic activity for nodulation. The results indicated a clear correlation between GS52 enzymatic activity and nodule number. Altogether, our study indicates that the catalytic activity of the GS52 apyrase, likely acting on extracellular nucleotides, is critical for rhizobial infection and nodulation. PMID:21346172

  16. Metabolic regulation analysis of an ethanologenic Escherichia coli strain based on RT-PCR and enzymatic activities

    PubMed Central

    Orencio-Trejo, Montserrat; Flores, Noemí; Escalante, Adelfo; Hernández-Chávez, Georgina; Bolívar, Francisco; Gosset, Guillermo; Martinez, Alfredo

    2008-01-01

    Background A metabolic regulation study was performed, based upon measurements of enzymatic activities, fermentation performance, and RT-PCR analysis of pathways related to central carbon metabolism, in an ethanologenic Escherichia coli strain (CCE14) derived from lineage C. In comparison with previous engineered strains, this E coli derivative has a higher ethanol production rate in mineral medium, as a result of the elevated heterologous expression of the chromosomally integrated genes encoding PDCZm and ADHZm (pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis). It is suggested that this behavior might be due to lineage differences between E. coli W and C. Results This study demonstrated that the glycolytic flux is controlled, in this case, by reactions outside glycolysis, i.e., the fermentative pathways. Changes in ethanol production rate in this ethanologenic strain result in low organic acid production rates, and high glycolytic and ethanologenic fluxes, that correlate with enhanced transcription and enzymatic activity levels of PDCZm and ADHZm. Furthermore, a higher ethanol yield (90% of the theoretical) in glucose-mineral media was obtained with CCE14 in comparison with previous engineered E. coli strains, such as KO11, that produces a 70% yield under the same conditions. Conclusion Results suggest that a higher ethanol formation rate, caused by ahigher PDCZm and ADHZm activities induces a metabolic state that cells compensate through enhanced glucose transport, ATP synthesis, and NAD-NADH+H turnover rates. These results show that glycolytic enzymatic activities, present in E. coli W and C under fermentative conditions, are sufficient to contend with increases in glucose consumption and product formation rates. PMID:18471274

  17. Discovery of Cell-Type-Specific and Disease-Related Enzymatic Activity Changes via Global Evaluation of Peptide Metabolism.

    PubMed

    Onagi, Jun; Komatsu, Toru; Ichihashi, Yuki; Kuriki, Yugo; Kamiya, Mako; Terai, Takuya; Ueno, Tasuku; Hanaoka, Kenjiro; Matsuzaki, Hiroyuki; Hata, Keisuke; Watanabe, Toshiaki; Nagano, Tetsuo; Urano, Yasuteru

    2017-03-08

    Cellular homeostasis is maintained by a complex network of reactions catalyzed by enormous numbers of enzymatic activities (the enzymome), which serve to determine the phenotypes of cells. Here, we focused on the enzymomics of proteases and peptidases because these enzymes are an important class of disease-related proteins. We describe a system that (A) simultaneously evaluates metabolic activities of peptides using a series of exogenous peptide substrates and (B) identifies the enzymes that metabolize the specified peptide substrate with high throughput. We confirmed that the developed system was able to discover cell-type-specific and disease-related exo- and endopeptidase activities and identify the responsible enzymes. For example, we found that the activity of the endopeptidase neurolysin is highly elevated in human colorectal tumor tissue samples. This simple but powerful enzymomics platform should be widely applicable to uncover cell-type-specific reactions and altered enzymatic functions with potential value as biomarkers or drug targets in various disease states and to investigate the mechanisms of the underlying pathologies.

  18. Quantitative Collection and Enzymatic Activity of Glucose Oxidase Nanotubes Fabricated by Templated Layer-by-Layer Assembly.

    PubMed

    Zhang, Shouwei; Demoustier-Champagne, Sophie; Jonas, Alain M

    2015-08-10

    We report on the fabrication of enzyme nanotubes in nanoporous polycarbonate membranes via the layer-by-layer (LbL) alternate assembly of polyethylenimine (PEI) and glucose oxidase (GOX), followed by dissolution of the sacrificial template in CH2Cl2, collection, and final dispersion in water. An adjuvant-assisted filtration methodology is exploited to extract quantitatively the nanotubes without loss of activity and morphology. Different water-soluble CH2Cl2-insoluble adjuvants are tested for maximal enzyme activity and nanotube stability; whereas NaCl disrupts the tubes by screening electrostatic interactions, the high osmotic pressure created by fructose also contributes to loosening the nanotubular structures. These issues are solved when using neutral, high molar mass dextran. The enzymatic activity of intact free nanotubes in water is then quantitatively compared to membrane-embedded nanotubes, showing that the liberated nanotubes have a higher catalytic activity in proportion to their larger exposed surface. Our study thus discloses a robust and general methodology for the fabrication and quantitative collection of enzymatic nanotubes and shows that LbL assembly provides access to efficient enzyme carriers for use as catalytic swarming agents.

  19. Modulation of enzymatic activity of Src-family kinases in bovine T cells transformed by Theileria parva.

    PubMed

    Fich, C; Klauenberg, U; Fleischer, B; Bröker, B M

    1998-08-01

    After infection with sporozoites of the protozoon Theileria parva (Tp) bovine T cells are readily transformed to permanent growth in vivo and in vitro. Their transformed state depends on the constant presence of the parasite but membrane signals remain important. Non-receptor tyrosine kinases play a critical role in the transduction of membrane signals in haematopoietic cells. We have investigated Src-family kinases in bovine T cells transformed by Tp. The T cell receptor-associated tyrosine kinase p60fyn had high activity in all cell lines tested. In addition, weak phosphorylation of 2 novel bands was observed associated with Fyn. In contrast to Fyn, enzymatic activity of p56lck, which in T cells has an essential role in signalling, was low. Furthermore, 1 of 3 Tp transformed cell lines was completely devoid of p56lck indicating that the enzyme is not necessary for the Tp dependent growth of the T cells. In addition to p60fyn and p56lck weak enzymatic activity of 1 splice variant of p53/56lyn was observed after infection of T cells with Tp. These data show that growth transformation by Tp influences kinase activity in bovine T cells. However, they also prove that p56lck does not play an essential role in the transformation mechanism.

  20. Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

    PubMed

    Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

    2015-10-07

    Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

  1. Serum biochemical profile, enzymatic activity and lipid peroxidation in organs of laying hens fed diets containing cashew nut shell liquid.

    PubMed

    Braz, N M; Freitas, E R; Trevisan, M T S; do Nascimento, G A J; Salles, R P R; Cruz, C E B; Farias, N N P; da Silva, I N G; Watanabe, P H

    2017-03-16

    The objective of this study was to evaluate the effect of feeding laying hens diets containing cashew nut shell liquid (CNSL) as a source of anacardic acid on the blood biochemical parameters as well as the enzymatic activity and lipid peroxidation of liver and tissues of the reproductive system (ovary, magnum, and uterus). A total of 216 Hisex White commercial laying hens were distributed randomly into six treatments, with six replicates of six birds. Treatments consisted of a diet without growth promoter (GP); a diet with GP; and diets without GP, with addition of increasing levels of CNSL (0.25, 0.50, 0.75 and 1.0%). Addition of CNSL to the diet did not affect the blood biochemical parameters (uric acid, creatinine, alanine aminotransferase, aspartate aminotransferase, total cholesterol, high density lipoproteins, low-density lipoproteins and triglycerides), the enzymatic activity (superoxide dismutase and nonprotein sulphydryl groups) in the organs (liver, ovary, magnum and uterus) or the peroxidation of lipids from the blood serum, liver, magnum and uterus (p > 0.05). However, the addition of 0.75% and 1.00% CNSL provided a lower thiobarbituric acid reactive substances content in the birds' ovary (p < 0.001) compared to birds of other treatments, whereas the treatment without the GP provided a higher value. Addition of up to 1% of the CNSL as a source of anacardic acid in the laying hens' diets does not influence blood biochemical parameters or the endogenous enzymatic activity in the liver, ovary, magnum and uterus, but affects the lipid peroxidation in the ovary, although the problem is reduced from the inclusion of 0.75% CNSL.

  2. Diiron centre mutations in Ciona intestinalis alternative oxidase abolish enzymatic activity and prevent rescue of cytochrome oxidase deficiency in flies.

    PubMed

    Andjelković, Ana; Oliveira, Marcos T; Cannino, Giuseppe; Yalgin, Cagri; Dhandapani, Praveen K; Dufour, Eric; Rustin, Pierre; Szibor, Marten; Jacobs, Howard T

    2015-12-17

    The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression.

  3. Enzyme-immobilized SiO2-Si electrode: Fast interfacial electron transfer with preserved enzymatic activity

    NASA Astrophysics Data System (ADS)

    Wang, Gang; Yau, Siu-Tung

    2005-12-01

    The enzyme, glucose oxidase (GOx), is immobilized using electrostatic interaction on the native oxide of heavily doped n-type silicon. Voltammetric measurement shows that the immobilized GOx gives rise to a very fast enzyme-silicon interfacial electron transfer rate constant of 7.9s-1. The measurement also suggests that the enzyme retains its native conformation when immobilized on the silicon surface. The preserved native conformation of GOx is further confirmed by testing the enzymatic activity of the immobilized GOx using glucose. The GOx-immobilized silicon is shown to behave as a glucose sensor that detects glucose with concentrations as low as 50μM.

  4. Beta 1-integrin ligation and TLR ligation enhance GM-CSF–induced ALDH1A2 expression in dendritic cells, but differentially regulate their anti-inflammatory properties

    PubMed Central

    Yokota-Nakatsuma, Aya; Ohoka, Yoshiharu; Takeuchi, Hajime; Song, Si-Young; Iwata, Makoto

    2016-01-01

    Retinoic acid (RA)–producing CD103+ mature dendritic cells (DCs) in mesenteric lymph nodes (MLNs) play crucial roles in gut immunity. GM-CSF and RA contribute to the expression of the RA-producing enzyme ALDH1A2. However, additional signals appeared to be required for inducing ALDH1A2high mature DCs from immature DCs. We found here that TLR ligands (Ls) and immobilized E-cadherin could provide such signals in FLT3-L–generated bone marrow (BM)–derived DCs after treatment with GM-CSF and the RA receptor agonist Am80. The TLR-L-treated DCs produced proinflammatory cytokines unlike normal ALDH1A2high MLN-DCs, whereas the E-cadherin-treated DCs did not. Immobilized VCAM-1 and semaphorin 7 A exerted effects similar to those of E-cadherin. Soluble anti-integrin β1 antibodies or inhibitors of integrin signaling molecules suppressed the effects of these immobilized proteins, whereas immobilized anti-integrin β1 antibodies enhanced the GM-CSF/Am80-induced ALDH1A2 expression without inducing proinflammatory cytokines. Sequential stimulation of splenic pre-DCs with GM-CSF/Am80 and immobilized E-cadherin or anti-integrin β1 antibody also induced differentiation to mature DCs with high ALDH activity. The E-cadherin-treated BM-DCs induced gut-tropic Foxp3+ T cells and alleviated DSS–induced colitis, whereas the TLR-L-treated DCs aggravated DSS–induced colitis. The results suggest that integrin β1-mediated signals contribute to the differentiation and maturation of RA-producing anti-inflammatory DCs. PMID:27897208

  5. Zebrafish foxc1a plays a crucial role in early somitogenesis by restricting the expression of aldh1a2 directly.

    PubMed

    Li, Jingyun; Yue, Yunyun; Dong, Xiaohua; Jia, Wenshuang; Li, Kui; Liang, Dong; Dong, Zhangji; Wang, Xiaoxiao; Nan, Xiaoxi; Zhang, Qinxin; Zhao, Qingshun

    2015-04-17

    Foxc1a is a member of the forkhead transcription factors. It plays an essential role in zebrafish somitogenesis. However, little is known about the molecular mechanisms underlying its controlling somitogenesis. To uncover how foxc1a regulates zebrafish somitogenesis, we generated foxc1a knock-out zebrafish using TALEN (transcription activator-like effector nuclease) technology. The foxc1a null embryos exhibited defective somites at early development. Analyses on the expressions of the key genes that control processes of somitogenesis revealed that foxc1a controlled early somitogenesis by regulating the expression of myod1. In the somites of foxc1a knock-out embryos, expressions of fgf8a and deltaC were abolished, whereas the expression of aldh1a2 (responsible for providing retinoic acid signaling) was significantly increased. Once the increased retinoic acid level in the foxc1a null embryos was reduced by knocking down aldh1a2, the reduced expression of myod1 was partially rescued by resuming expressions of fgf8a and deltaC in the somites of the mutant embryos. Moreover, a chromatin immunoprecipitation assay on zebrafish embryos revealed that Foxc1a bound aldh1a2 promoter directly. On the other hand, neither knocking down fgf8a nor inhibiting Notch signaling affected the expression of aldh1a2, although knocking down fgf8a reduced expression of deltaC in the somites of zebrafish embryos at early somitogenesis and vice versa. Taken together, our results demonstrate that foxc1a plays an essential role in early somitogenesis by controlling Fgf and Notch signaling through restricting the expression of aldh1a2 in paraxial mesoderm directly.

  6. Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity*

    PubMed Central

    Hughes, Siobhan; Jenkins, Victoria; Dar, Mohd Jamal; Engelman, Alan; Cherepanov, Peter

    2010-01-01

    Lens epithelium-derived growth factor (LEDGF) is an important co-factor of human immunodeficiency virus DNA integration; however, its cellular functions are poorly characterized. We now report identification of the Cdc7-activator of S-phase kinase (ASK) heterodimer as a novel interactor of LEDGF. Both kinase subunits co-immunoprecipitated with endogenous LEDGF from human cell extracts. Truncation analyses identified the integrase-binding domain of LEDGF as essential and minimally sufficient for the interaction with Cdc7-ASK. Reciprocally, the interaction required autophosphorylation of the kinase and the presence of 50 C-terminal residues of ASK. The kinase phosphorylated LEDGF in vitro, with Ser-206 being the major target, and LEDGF phosphorylated at this residue could be detected during S phase of the cell cycle. LEDGF potently stimulated the enzymatic activity of Cdc7-ASK, increasing phosphorylation of MCM2 in vitro by more than 10-fold. This enzymatic stimulation as well as phosphorylation of LEDGF depended on the protein-protein interaction. Intriguingly, removing the C-terminal region of ASK, involved in the interaction with LEDGF, resulted in a hyperactive kinase. Our results indicate that the interaction with LEDGF relieves autoinhibition of Cdc7-ASK kinase, imposed by the C terminus of ASK. PMID:19864417

  7. Interference of PR3-ANCA with the enzymatic activity of PR3: differences in patients during active disease or remission of Wegener's granulomatosis.

    PubMed

    van der Geld, Y M; Tool, A T J; Videler, J; de Haas, M; Tervaert, J W Cohen; Stegeman, C A; Limburg, P C; Kallenberg, C G M; Roos, D

    2002-09-01

    Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are strongly associated with Wegener's granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3-ANCA do not always correspond to clinical disease activity. To investigate the relationship between functional effects of PR3-ANCA and disease activity, we tested the effect of IgG samples from sera of 43 WG patients, taken during active disease or remission, for their capacity to interfere with the proteolytic activity of PR3. Furthermore, longitudinal sera of seven WG patients were included. The enzymatic activity of PR3 was determined (1) with casein or with a small synthetic substrate and (2) by complexation of PR3 with alpha1-antitrypsin (alpha1-AT). With a fixed concentration (100 microg/ml) of IgG, PR3-ANCA from patients during an active phase of WG had a higher inhibitory capacity towards the proteolytic activity of PR3 and complexation of PR3 with alpha1-AT than did PR3-ANCA from WG patients during remission. However, the number of PR3-ANCA units that gave 50% inhibition of the PR3 enzymatic activity and its complexation with alpha1-AT was lower for patients during remission than for patients during an active phase of WG, indicating a stronger inhibitory capacity at a molar base. In conclusion, PR3-ANCA from patients during remission had a relatively higher inhibitory capacity towards the enzymatic activity of PR3 than PR3-ANCA from patients during an active phase. This may indicate that during active disease the ANCA titre is increased, but the number of active ANCA molecules that recognize the enzyme-inhibiting epitopes is not increased.

  8. Effect of high compost temperature on enzymatic activity and species diversity of culturable bacteria in cattle manure compost.

    PubMed

    Miyatake, Fumihito; Iwabuchi, Kazunori

    2005-11-01

    To clarify the characteristics of thermophilic bacteria in cattle manure compost, enzymatic activity and species diversity of cultivated bacteria were investigated at 54, 60, 63, 66 and 70 degrees C, which were dependent on composting temperature. The highest level of thermophilic bacterial activity was observed at 54 degrees C. Following an increase in temperature to 63 degrees C, a reduction in bacterial diversity was observed. At 66 degrees C, bacterial diversity increased again, and diverse bacteria including Thermus spp. and thermophilic Bacillus spp. appeared to adapt to the higher temperature. At 70 degrees C, bacterial activity measured as superoxide dismutase and catalase activity was significantly higher than at 66 degrees C. However, the decomposition rate of protein in the compost was lower than the rate at 66 degrees C due to the higher compost temperature.

  9. Enhancement of antibiotic-activity through complexation with metal ions - Combined ITC, NMR, enzymatic and biological studies.

    PubMed

    Möhler, Jasper S; Kolmar, Theresa; Synnatschke, Kevin; Hergert, Marcel; Wilson, Liam A; Ramu, Soumya; Elliott, Alysha G; Blaskovich, Mark A T; Sidjabat, Hanna E; Paterson, David L; Schenk, Gerhard; Cooper, Matthew A; Ziora, Zyta M

    2017-02-01

    Alternative solutions need to be developed to overcome the growing problem of multi-drug resistant bacteria. This study explored the possibility of creating complexes of antibiotics with metal ions, thereby increasing their activity. Analytical techniques such as isothermal titration calorimetry and nuclear magnetic resonance were used to examine the structure and interactions between Cu(II), Ag(I) or Zn(II) and β-lactam antibiotics. The metal-β-lactam complexes were also tested for antimicrobial activity, by micro-broth dilution and disk diffusion methods, showing a synergistic increase in the activity of the drugs, and enzymatic inhibition assays confirming inhibition of β-lactamases responsible for resistance. The metal-antibiotic complex concept was proven to be successful with the activity of the drugs enhanced against β-lactamase-producing bacteria. The highest synergistic effects were observed for complexes formed with Ag(I).

  10. Oxidant and enzymatic antioxidant status (gene expression and activity) in the brain of chickens with cold-induced pulmonary hypertension

    NASA Astrophysics Data System (ADS)

    Hassanpour, Hossein; Khalaji-Pirbalouty, Valiallah; Nasiri, Leila; Mohebbi, Abdonnaser; Bahadoran, Shahab

    2015-11-01

    To evaluate oxidant and antioxidant status of the brain (hindbrain, midbrain, and forebrain) in chickens with cold-induced pulmonary hypertension, the measurements of lipid peroxidation, protein oxidation, antioxidant capacity, enzymatic activity, and gene expression (for catalase, glutathione peroxidase, and superoxide dismutases) were done. There were high lipid peroxidation/protein oxidation and low antioxidant capacity in the hindbrain of cold-induced pulmonary hypertensive chickens compared to control ( P < 0.05). In the hypertensive chickens, superoxide dismutase activity was decreased (forebrain, midbrain, and hindbrain), while catalase activity was increased (forebrain and midbrain) ( P < 0.05). Glutathione peroxidase activity did not change. Relative gene expression of catalase and superoxide dismutases (1 and 2) was downregulated, while glutathione peroxidase was upregulated in the brain of the cold-induced pulmonary hypertensive chickens. Probably, these situations in the oxidant and antioxidant status of the brain especially hindbrain may change its function at cardiovascular center and sympathetic nervous system to exacerbate pulmonary hypertension.

  11. Evaluation of oil removal efficiency and enzymatic activity in some fungal strains for bioremediation of petroleum-polluted soils

    PubMed Central

    2012-01-01

    Background Petroleum pollution is a global disaster and there are several soil cleaning methods including bioremediation. Methods In a field study, fugal strains were isolated from oil-contaminated sites of Arak refinery (Iran) and their growth ability was checked in potato dextrose agar (PDA) media containing 0-10% v/v crude oil, the activity of three enzymes (Catalase, Peroxidase and Phenol Oxidase) was evaluated in the fungal colonies and bioremediation ability of the fungi was checked in the experimental pots containing 3 kg sterilized soil and different concentrations of petroleum (0-10% w/w). Results Four fungal strains, Acromonium sp., Alternaria sp., Aspergillus terreus and Penicillium sp., were selected as the most resistant ones. They were able to growth in the subjected concentrations and Alternaria sp. showed the highest growth ability in the petroleum containing media. The enzyme assay showed that the enzymatic activity was increased in the oil-contaminated media. Bioremediation results showed that the studied fungi were able to decrease petroleum pollution. The highest petroleum removing efficiency of Aspergillus terreus, Penicillium sp., Alternaria sp. and Acromonium sp. was evaluated in the 10%, 8%, 8% and 2% petroleum pollution respectively. Conclusions Fungi are important microorganisms in decreasing of petroleum pollution. They have bioremediation potency that is related to their enzymatic activities. PMID:23369665

  12. Enzymatically-Processed Wheat Bran Enhances Macrophage Activity and Has in Vivo Anti-Inflammatory Effects in Mice

    PubMed Central

    Kang, Hee; Lee, Mi-Gi; Lee, Jae-Kang; Choi, Yong-Hyun; Choi, Yong-Seok

    2016-01-01

    Wheat bran is a rich source of dietary fiber, of which arabinoxylan is the most abundant non-starch polysaccharide. Arabinoxylan has been known to exert in vivo immunological activities. Based on prior findings, we pretreated wheat bran with enzymatic hydrolysis to increase the release of soluble arabinoxylan and investigated whether oral administration of wheat bran altered macrophage activity in a mouse model. After four weeks of treatment, we isolated peritoneal macrophages for phagocytic receptor analysis and lipopolysaccharide (LPS)-induced inflammatory changes. In the second experiment, mice given wheat bran were intraperitoneally stimulated with LPS and serum levels of pro- and anti-inflammatory cytokines were determined. The expression of SRA and CD36, and phagocytic activity increased (p < 0.05, respectively). Ex vivo stimulation of macrophages by LPS resulted in reduced surface expression of CD40 (p < 0.05) and decreased production of nitric oxide (p < 0.005), tumor necrosis factor (TNF)-α (p < 0.005), interleukin (IL)-6 (p < 0.01), and IL-12 (p < 0.05). Mice treated with wheat bran showed decreased levels of serum TNF-α and IL-6 (p < 0.05, respectively) and an increased level of serum anti-inflammatory IL-10 (p < 0.05) in response to intraperitoneal LPS. Enzymatically-processed wheat bran boosts macrophage phagocytic capacity possibly through up-regulation of scavenger receptors and confers anti-inflammatory effects, indicating its potential as an immuno-enhancing functional food. PMID:27043618

  13. Functionalized Graphene Oxide with Chitosan for Protein Nanocarriers to Protect against Enzymatic Cleavage and Retain Collagenase Activity

    PubMed Central

    Emadi, Fatemeh; Amini, Abbas; Gholami, Ahmad; Ghasemi, Younes

    2017-01-01

    Proteins have short half-life because of enzymatic cleavage. Here, a new protein nanocarrier made of graphene oxide (GO) + Chitosan (CS) is proposed to successfully prevent proteolysis in protein and simultaneously retain its activity. Bovine serum albumin (BSA) and collagenase were loaded on GO and GO-CS to explore the stability and activity of proteins. SEM, AFM, TEM, DSC, UV-Vis, FT-IR, RBS, Raman, SDS-PAGE and zymography were utilized as characterization techniques. The protecting role of GO and GO-CS against enzymatic cleavage was probed by protease digestion analysis on BSA, where the protease solution was introduced to GO-BSA and GO-CS-BSA at 37 °C for 0.5-1-3-6 hours. Characterizations showed the successful synthesis of few layers of GO and the coverage by CS. According to gelatin zymographic analysis, the loaded collagenase on GO and GO-CS lysed the gelatin and created non-staining bands which confirmed the activity of loaded collagenase. SDS-PAGE analysis revealed no significant change in the intact protein in the GO-BSA and GO-CS-BSA solution after 30-minute and 1-hour exposure to protease; however, free BSA was completely digested after 1 hour. After 6 hours, intact proteins were detected in GO-BSA and GO-CS-BSA solutions, while no intact protein was detected in the free BSA solution. PMID:28186169

  14. Functionalized Graphene Oxide with Chitosan for Protein Nanocarriers to Protect against Enzymatic Cleavage and Retain Collagenase Activity.

    PubMed

    Emadi, Fatemeh; Amini, Abbas; Gholami, Ahmad; Ghasemi, Younes

    2017-02-10

    Proteins have short half-life because of enzymatic cleavage. Here, a new protein nanocarrier made of graphene oxide (GO) + Chitosan (CS) is proposed to successfully prevent proteolysis in protein and simultaneously retain its activity. Bovine serum albumin (BSA) and collagenase were loaded on GO and GO-CS to explore the stability and activity of proteins. SEM, AFM, TEM, DSC, UV-Vis, FT-IR, RBS, Raman, SDS-PAGE and zymography were utilized as characterization techniques. The protecting role of GO and GO-CS against enzymatic cleavage was probed by protease digestion analysis on BSA, where the protease solution was introduced to GO-BSA and GO-CS-BSA at 37 °C for 0.5-1-3-6 hours. Characterizations showed the successful synthesis of few layers of GO and the coverage by CS. According to gelatin zymographic analysis, the loaded collagenase on GO and GO-CS lysed the gelatin and created non-staining bands which confirmed the activity of loaded collagenase. SDS-PAGE analysis revealed no significant change in the intact protein in the GO-BSA and GO-CS-BSA solution after 30-minute and 1-hour exposure to protease; however, free BSA was completely digested after 1 hour. After 6 hours, intact proteins were detected in GO-BSA and GO-CS-BSA solutions, while no intact protein was detected in the free BSA solution.

  15. Contrasting effects of untreated textile wastewater onto the soil available nitrogen-phosphorus and enzymatic activities in aridisol.

    PubMed

    Arif, Muhammad Saleem; Riaz, Muhammad; Shahzad, Sher Muhammad; Yasmeen, Tahira; Buttler, Alexandre; Garcıa-Gil, Juan Carlos; Roohi, Mahnaz; Rasool, Akhtar

    2016-02-01

    Water shortage and soil qualitative degradation are significant environmental problems in arid and semi-arid regions of the world. The increasing demand for water in agriculture and industry has resulted in the emergence of wastewater use as an alternative in these areas. Textile wastewater is produced in surplus amounts which poses threat to the environment as well as associated flora and fauna. A 60-day incubation study was performed to assess the effects of untreated textile wastewater at 0, 25, 50, 75, and 100% dilution levels on the physico-chemical and some microbial and enzymatic properties of an aridisol soil. The addition of textile wastewater provoked a significant change in soil pH and electrical conductivity and soil dehydrogenase and urease activities compared to the distilled-water treated control soil. Moreover, compared to the control treatment, soil phosphomonoesterase activity was significantly increased from 25 to 75% application rates, but decreased at 100% textile wastewater application rate. Total and available soil N contents increased significantly in response to application of textile wastewater. Despite significant increases in the soil total P contents after the addition of textile wastewater, soil available P content decreased with increasing concentration of wastewater. Changes in soil nutrient contents and related enzymatic activities suggested a dynamic match between substrate availability and soil N and P contents. Aridisols have high fixation and low P availability, application of textile wastewater to such soils should be considered only after careful assessment.

  16. Ferredoxin:NADP+ oxidoreductase in junction with CdSe/ZnS quantum dots: characteristics of an enzymatically active nanohybrid.

    PubMed

    Szczepaniak, Krzysztof; Worch, Remigiusz; Grzyb, Joanna

    2013-05-15

    Ferredoxin:NADP(+) oxidoreductase (FNR) is a plant and cyanobacterial photosynthetic enzyme, also found in non-photosynthetic tissues, where it is involved in redox reactions of biosynthetic pathways. In vivo it transfers electrons to nicotinamide adenine dinucleotide phosphate (NADP(+)), forming its reduced version, NADPH, while in vitro it can also use NADPH to reduce several substrates, such as ferricyanide, various quinones and nitriles. As an oxidoreductase catalyzing reaction of a broad range of substrates, FNR may be used in biotechnological processes. Quantum dots are semiconductor nanocrystals of a few to several nanometers diameter, having very useful luminescent properties. We present the spectroscopic and functional characteristics of a covalent conjugation of FNR and CdSe/ZnS quantum dots. Two types of quantum dots, of different diameter and emission maximum (550 and 650 nm), were used for comparison. Steady-state fluorescence and gel electrophoresis confirmed efficient conjugation, while fluorescence correlation spectroscopy (FCS) allowed for determination of the conjugates' radii. The nanohybrids sustained enzymatic activity; however, changes in maximal reaction rates and Michaelis constant were found. Detailed analysis of the kinetic parameters showed that the changes in the enzyme activity depend on the substrate used for activity measurement but also on the size of the quantum dots. The presented nanohybrids, as the first example using plant and photosynthetic enzyme as a protein partner, may became a tool to study photosynthesis as well as other biosynthetic and biotechnological processes, involving enzymatically catalyzed electron transfer.

  17. In Vitro Enzymatic Activities of Bacteriochlorophyll a Synthase Derived from the Green Sulfur Photosynthetic Bacterium Chlorobaculum tepidum.

    PubMed

    Saga, Yoshitaka; Hirota, Keiya; Harada, Jiro; Tamiaki, Hitoshi

    2015-08-18

    The activity of an enzyme encoded by the CT1610 gene in the green sulfur photosynthetic bacterium Chlorobaculum tepidum, which was annotated as bacteriochlorophyll (BChl) a synthase, BchG (denoted as tepBchG), was examined in vitro using the lysates of Escherichia coli containing the heterologously expressed enzyme. BChl a possessing a geranylgeranyl group at the 17-propionate residue (BChl aGG) was produced from bacteriochlorophyllide (BChlide) a and geranylgeranyl pyrophosphate in the presence of tepBchG. Surprisingly, tepBchG catalyzed the formation of BChl a bearing a farnesyl group (BChl aF) as in the enzymatic production of BChl aGG, indicating loose recognition of isoprenoid pyrophosphates in tepBchG. In contrast to such loose recognition of isoprenoid substrates, BChlide c and chlorophyllide a gave no esterifying product upon being incubated with geranylgeranyl or farnesyl pyrophosphate in the presence of tepBchG. These results confirm that tepBchG undoubtedly acts as the BChl a synthase in Cba. tepidum. The enzymatic activity of tepBchG was higher than that of BchG of Rhodobacter sphaeroides at 45 °C, although the former activity was lower than the latter below 35 °C.

  18. Activity-Dependent Enzymatic Assay for the Detection of Toluene-Oxidizing Bacteria Capable of Trichloroethylene Degradation

    NASA Astrophysics Data System (ADS)

    Kauffman, M. E.; Kauffman, M. E.; Keener, W. K.; Watwood, M. E.; Lehman, R. M.

    2001-12-01

    Toluene-oxidizing bacteria produce enzymes that cometabolically degrade trichloroethylene (TCE). These inducible enzymes are produced only in the presence of certain aromatic substrates such as toluene or phenol. Recent laboratory studies have utilized analog chemical substrates to identify production of bacterial enzymes capable of degrading trichloroethylene. These analog substrates produce chromogenic and/or fluorescent products when biotransformed by the enzymes of interest. In this study, 3-hydroxyphenylacetylene (3-HPA) was identified as an activity-dependent enzymatic probe for the detection of three of the four known toluene oxygenase enzymes capable of TCE degradation. Laboratory studies were conducted using pure cultures of Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas putida F1. Cell cultures grown on lactate (non-enzyme inducing) or lactate and toluene (inducing) were trapped trapped on black polycarbonate filters, exposed to 3-HPA, and examined for fluorescence using an epifluorescent microscope. Additionally, B. cepacia G4 cells were grown under the same conditions, but in the presence of mineral and basalt specimens to allow for bacterial attachment. The specimens were then exposed to 3-HPA and examined under an epifluorescent microscope. Our results demonstrate that cells induced for the production of oxygenase enzymes, both unattached and attached, are able to transform 3-HPA to a fluorescent product, although cells attached to geologic materials, such as basalt, take substantially longer to transform the probe. Cells grown under non-inducing conditions do not transform the probe, regardless of their attachment status. Additionally, well water samples taken from a TCE-contaminated aquifer were successfully assayed using the 3-HPA enzymatic probe. The development of this enzyme activity-dependent enzymatic assay provides a fast and reliable method to assess the potential for TCE and aromatic contaminant bioremediation.

  19. Human pancreas-specific protein disulfide-isomerase (PDIp) can function as a chaperone independently of its enzymatic activity by forming stable complexes with denatured substrate proteins.

    PubMed

    Fu, Xin-Miao; Zhu, Bao Ting

    2010-07-01

    Members of the PDI (protein disulfide-isomerase) family are critical for the correct folding of secretory proteins by catalysing disulfide bond formation as well as by serving as molecular chaperones to prevent protein aggregation. In the present paper, we report that the chaperone activity of the human pancreas-specific PDI homologue (PDIp) is independent of its enzymatic activity on the basis of the following lines of evidence. First, alkylation of PDIp by iodoacetamide fully abolishes its enzymatic activity, whereas it still retains most of its chaperone activity in preventing the aggregation of reduced insulin B chain and denatured GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Secondly, mutation of the cysteine residues in PDIp's active sites completely abolishes its enzymatic activity, but does not affect its chaperone activity. Thirdly, the b-b' fragment of PDIp, which does not contain the active sites and is devoid of enzymatic activity, still has chaperone activity. Mechanistically, we found that both the recombinant PDIp expressed in Escherichia coli and the natural PDIp present in human or monkey pancreas can form stable complexes with thermal-denatured substrate proteins independently of their enzymatic activity. The high-molecular-mass soluble complexes between PDIp and GAPDH are formed in a stoichiometric manner (subunit ratio of 1:3.5-4.5), and can dissociate after storage for a certain time. As a proof-of-concept for the biological significance of PDIp in intact cells, we demonstrated that its selective expression in E. coli confers strong protection of these cells against heat shock and oxidative-stress-induced death independently of its enzymatic activity.

  20. Development of a cancer-marker activated enzymatic switch from the herpes simplex virus thymidine kinase.

    PubMed

    Shelat, Nirav Y; Parhi, Sidhartha; Ostermeier, Marc

    2017-02-01

    Discovery of new cancer biomarkers and advances in targeted gene delivery mechanisms have made gene-directed enzyme prodrug therapy (GDEPT) an attractive method for treating cancer. Recent focus has been placed on increasing target specificity of gene delivery systems and reducing toxicity in non-cancer cells in order to make GDEPT viable. To help address this challenge, we have developed an enzymatic switch that confers higher prodrug toxicity in the presence of a cancer marker. The enzymatic switch was derived from the herpes simplex virus thymidine kinase (HSV-TK) fused to the CH1 domain of the p300 protein. The CH1 domain binds to the C-terminal transactivation domain (C-TAD) of the cancer marker hypoxia inducible factor 1α. The switch was developed using a directed evolution approach that evaluated a large library of HSV-TK/CH1 fusions using a negative selection for azidothymidine (AZT) toxicity and a positive selection for dT phosphorylation. The identified switch, dubbed TICKLE (Trigger-Induced Cell-Killing Lethal-Enzyme), confers a 4-fold increase in AZT toxicity in the presence of C-TAD. The broad substrate specificity exhibited by HSV-TK makes TICKLE an appealing prospect for testing in medical imaging and cancer therapy, while establishing a foundation for further engineering of nucleoside kinase protein switches.

  1. Localization and enzymatic activity profiles of the proteases responsible for tachykinin-directed oocyte growth in the protochordate, Ciona intestinalis.

    PubMed

    Aoyama, Masato; Kawada, Tsuyoshi; Satake, Honoo

    2012-03-01

    We previously substantiated that Ci-TK, a tachykinin of the protochordate, Ciona intestinalis (Ci), triggered oocyte growth from the vitellogenic stage (stage II) to the post-vitellogenic stage (stage III) via up-regulation of the gene expression and enzymatic activity of the proteases: cathepsin D, carboxypeptidase B1, and chymotrypsin. In the present study, we have elucidated the localization, gene expression and activation profile of these proteases. In situ hybridization showed that the Ci-cathepsin D mRNA was present exclusively in test cells of the stage II oocytes, whereas the Ci-carboxypeptidase B1 and Ci-chymotrypsin mRNAs were detected in follicular cells of the stage II and stage III oocytes. Double-immunostaining demonstrated that the immunoreactivity of Ci-cathepsin D was largely colocalized with that of the receptor of Ci-TK, Ci-TK-R, in test cells of the stage II oocytes. Ci-cathepsin D gene expression was detected at 2h after treatment with Ci-TK, and elevated for up to 5h, and then slightly decreased. Gene expression of Ci-carboxypeptidase B1 and Ci-chymotrypsin was observed at 5h after treatment with Ci-TK, and then decreased. The enzymatic activities of Ci-cathepsin D, Ci-carboxypeptidase B1, and Ci-chymotrypsin showed similar alterations with 1-h lags. These gene expression and protease activity profiles verified that Ci-cathepsin D is initially activated, which is followed by the activation of Ci-carboxypeptidase B1 and Ci-chymotrypsin. Collectively, the present data suggest that Ci-TK directly induces Ci-cahtepsin D in test cells expressing Ci-TK receptor, leading to the secondary activation of Ci-chymotrypsin and Ci-carboxypeptidase B1 in the follicle in the tachykininergic oocyte growth pathway.

  2. Amifostine Induces Antioxidant Enzymatic Activities in Normal Tissues and a Transplantable Tumor That Can Affect Radiation Response

    SciTech Connect

    Grdina, David J. Murley, Jeffrey S.; Kataoka, Yasushi; Baker, Kenneth L.; Kunnavakkam, Rangesh; Coleman, Mitchell C.; Spitz, Douglas R.

    2009-03-01

    Purpose: To determine whether amifostine can induce elevated manganese superoxide dismutase (SOD2) in murine tissues and a transplantable SA-NH tumor, resulting in a delayed tumor cell radioprotective effect. Methods and Materials: SA-NH tumor-bearing C3H mice were treated with a single 400 mg/kg or three daily 50 mg/kg doses of amifostine administered intraperitoneally. At selected time intervals after the last injection, the heart, liver, lung, pancreas, small intestine, spleen, and SA-NH tumor were removed and analyzed for SOD2, catalase, and glutathione peroxidase (GPx) enzymatic activity. The effect of elevated SOD2 enzymatic activity on the radiation response of SA-NH cells was determined. Results: SOD2 activity was significantly elevated in selected tissues and a tumor 24 h after amifostine treatment. Catalase and GPx activities remained unchanged except for significant elevations in the spleen. GPx was also elevated in the pancreas. SA-NH tumor cells exhibited a twofold elevation in SOD2 activity and a 27% elevation in radiation resistance. Amifostine administered in three daily fractions of 50 mg/kg each also resulted in significant elevations of these antioxidant enzymes. Conclusions: Amifostine can induce a delayed radioprotective effect that correlates with elevated levels of SOD2 activity in SA-NH tumor. If limited to normal tissues, this delayed radioprotective effect offers an additional potential for overall radiation protection. However, amifostine-induced elevation of SOD2 activity in tumors could have an unanticipated deleterious effect on tumor responses to fractionated radiation therapy, given that the radioprotector is administered daily just before each 2-Gy fractionated dose.

  3. Aerobic and anaerobic enzymatic activity of orange roughy (Hoplostethus atlanticus) and alfonsino (Beryx splendens) from the Juan Fernandez seamounts area.

    PubMed

    Saavedra, L M; Quiñones, R A; Gonzalez-Saldía, R R; Niklitschek, E J

    2016-06-01

    The aerobic and anaerobic enzymatic activity of two important commercial bathypelagic species living in the Juan Fernández seamounts was analyzed: alfonsino (Beryx splendens) and orange roughy (Hoplostethus atlanticus). These seamounts are influenced by the presence of an oxygen minimum zone (OMZ) located between 160 and 250 m depth. Both species have vertical segregation; alfonsino is able to stay in the OMZ, while orange roughy remains at greater depths. In this study, we compare the aerobic and anaerobic capacity of these species, measuring the activity of key metabolic enzymes in different body tissues (muscle, heart, brain and liver). Alfonsino has higher anaerobic potential in its white muscle due to greater lactate dehydrogenase (LDH) activity (190.2 μmol NADH min(-1) g ww(-1)), which is related to its smaller body size, but it is also a feature shared with species that migrate through OMZs. This potential and the higher muscle citrate synthase and electron transport system activities indicate that alfonsino has greater swimming activity level than orange roughy. This species has also a high MDH/LDH ratio in its heart, brain and liver, revealing a potential capacity to conduct aerobic metabolism in these organs under prolonged periods of environmental low oxygen conditions, preventing lactic acid accumulation. With these metabolic characteristics, alfonsino may have increased swimming activity to migrate and also could stay for a period of time in the OMZ. The observed differences between alfonsino and orange roughy with respect to their aerobic and anaerobic enzymatic activity are consistent with their characteristic vertical distributions and feeding behaviors.

  4. Chromophoric dissolved organic matter and microbial enzymatic activity. A biophysical approach to understand the marine carbon cycle.

    PubMed

    Gonnelli, Margherita; Vestri, Stefano; Santinelli, Chiara

    2013-12-01

    This study reports the first information on extracellular enzymatic activity (EEA) combined with a study of DOM dynamics at the Arno River mouth. DOM dynamics was investigated from both a quantitative (dissolved organic carbon, DOC) and a qualitative (absorption and fluorescence of chromophoric DOM, CDOM) perspective. The data here reported highlight that the Arno River was an important source of both DOC and CDOM for this coastal area. CDOM optical properties suggested that terrestrial DOM did not undergo simple dilution at the river mouth but, other physical-chemical and biological processes were probably at work to change its molecular characteristics. This observation was further supported by the "potential" enzymatic activity of β-glucosidase (BG) and leucine aminopeptidase (LAP). Their Vmax values were markedly higher in the river water than in the seawater and their ratio suggested that most of the DOM used by microbes in the Arno River was polysaccharide-like, while in the seawater it was mainly protein-like.

  5. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    SciTech Connect

    Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.; Juliano, Maria A.; Hayashi, Mirian A.F.; Oliveira, Vitor

    2010-01-01

    In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

  6. Glutathione peroxidase 3 of Saccharomyces cerevisiae suppresses non-enzymatic proteolysis of glutamine synthetase in an activity-independent manner.

    PubMed

    Lee, Phil Young; Kho, Chang Won; Lee, Do Hee; Kang, Sunghyun; Kang, Seongman; Lee, Sang Chul; Park, Byoung Chul; Cho, Sayeon; Bae, Kwang-Hee; Park, Sung Goo

    2007-10-19

    Glutathione peroxidase 3 (Gpx3) is ubiquitously expressed and is important antioxidant enzyme in yeast. It modulates the activities of redox-sensitive thiol proteins, particularly those involved in signal transduction pathway and protein translocation. Through immunoprecipitation/two-dimensional gel electrophoresis (IP-2DE), MALDI-TOF mass spectrometry, and a pull down assay, we found glutamine synthetase (GS; EC 6.3.1.2) as a candidate interacting protein with Gpx3. GS is a key enzyme in nitrogen metabolism and ammonium assimilation. It has been known that GS is non-enzymatically cleaved by ROS generated by MFO (thiol/ Fe(3+)/O(2) mixed-function oxidase) system. In this study, it is demonstrated that GS interacts with Gpx3 through its catalytic domain both in vivo and in vitro regardless of redox state. In addition, Gpx3 helps to protect GS from inactivation and degradation via oxidative stress in an activity-independent manner. Based on the results, it is suggested that Gpx3 protects GS from non-enzymatic proteolysis, thereby contributing to cell homeostasis when cell is exposed to oxidative stress.

  7. Effect of salinity tolerant PDH45 transgenic rice on physicochemical properties, enzymatic activities and microbial communities of rhizosphere soils.

    PubMed

    Sahoo, Ranjan Kumar; Tuteja, Narendra

    2013-08-01

    The effect of genetically modified (GM) plants on environment is now major concern worldwide. The plant roots of rhizosphere soil interact with variety of bacteria which could be influenced by the transgene in GM plants. The antibiotic resistance genes in GM plants may be transferred to soil microbes. In this study we have examined the effect of overexpression of salinity tolerant pea DNA helicase 45 (PDH45) gene on microbes and enzymatic activities in the rhizosphere soil of transgenic rice IR64 in presence and absence of salt stress in two different rhizospheric soils (New Delhi and Odisha, India). The diversity of the microbial community and soil enzymes viz., dehydrogenase, alkaline phosphatase, urease and nitrate reductase was assessed. The results revealed that there was no significant effect of transgene expression on rhizosphere soil of the rice plants. The isolated bacteria were phenotyped both in absence and presence of salt and no significant changes were found in their phenotypic characters as well as in their population. Overall, the overexpression of PDH45 in rice did not cause detectable changes in the microbial population, soil enzymatic activities and functional diversity of the rhizosphere soil microbial community.

  8. A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

    PubMed Central

    Matsunaga, Satoko; Masaoka, Takashi; Sawasaki, Tatsuya; Morishita, Ryo; Iwatani, Yasumasa; Tatsumi, Masashi; Endo, Yaeta; Yamamoto, Naoki; Sugiura, Wataru; Ryo, Akihide

    2015-01-01

    Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1. PMID:26583013

  9. Effect of salinity tolerant PDH45 transgenic rice on physicochemical properties, enzymatic activities and microbial communities of rhizosphere soils

    PubMed Central

    Sahoo, Ranjan Kumar; Tuteja, Narendra

    2013-01-01

    The effect of genetically modified (GM) plants on environment is now major concern worldwide. The plant roots of rhizosphere soil interact with variety of bacteria which could be influenced by the transgene in GM plants. The antibiotic resistance genes in GM plants may be transferred to soil microbes. In this study we have examined the effect of overexpression of salinity tolerant pea DNA helicase 45 (PDH45) gene on microbes and enzymatic activities in the rhizosphere soil of transgenic rice IR64 in presence and absence of salt stress in two different rhizospheric soils (New Delhi and Odisha, India). The diversity of the microbial community and soil enzymes viz., dehydrogenase, alkaline phosphatase, urease and nitrate reductase was assessed. The results revealed that there was no significant effect of transgene expression on rhizosphere soil of the rice plants. The isolated bacteria were phenotyped both in absence and presence of salt and no significant changes were found in their phenotypic characters as well as in their population. Overall, the overexpression of PDH45 in rice did not cause detectable changes in the microbial population, soil enzymatic activities and functional diversity of the rhizosphere soil microbial community. PMID:23733066

  10. The impact of element-element interactions on antioxidant enzymatic activity in the blood of white stork (Ciconia ciconia) chicks.

    PubMed

    Kamiński, Piotr; Kurhalyuk, Nataliya; Kasprzak, Mariusz; Jerzak, Leszek; Tkachenko, Halyna; Szady-Grad, Małgorzata; Klawe, Jacek J; Koim, Beata

    2009-02-01

    The aim of this work was to determine interrelationships among macroelements Na, K, Ca, Mg, and Fe, microelements Zn, Cu, Mn, and Co, and toxic heavy metals Pb and Cd in the blood of white stork Ciconia ciconia, during postnatal development, in different Polish environments, and their impact on the activity of antioxidant enzymes. We considered the content of thiobarbituric acid-reactive substances (TBARSs), i.e., malondialdehyde (MDA), and activity of superoxide dismutase (SOD), catalase (CAT), ceruloplasmine (CP), glutathione peroxidase (GPx), and glutathione reductase (GR). Blood samples were collected from storks developing at Odra meadows (Kłopot; southwestern Poland). They were compared with blood of chicks from several suburban sites located 20 km away from Zielona Góra (0.1 million inhabitants; southwestern Poland) and near Głogów, where a copper smelter is situated. We also conducted research in the Pomeranian region (Cecenowo; northern Poland). We collected blood samples via venipuncture of the brachial vein of chicks in 2005-2007. They were retrieved from the nest and placed in individual ventilated cotton sacks. The blood was collected using a 5-ml syringe washed with ethylenediaminetetraacetic acid (EDTA). We found significant interactions between macro- and microelements and enzymatic activity and TBARS products. We noticed the predominance of Cd and Pb participation in element-enzyme interactions. Simultaneously, we found interrelationships between cadmium and Na, K, Ca, Mg, and Fe and the activity of antioxidant enzymes SOD, CAT, CP, GR, and TBARS products in the blood of white stork chicks. In the case of lead these relationships were not numerous and they were significant for Ca, Mg, Cu, Mn, and Co. Correlations with enzymes were significant for Pb-CAT and Pb-TBARS. We noted that activities of most enzymes (SOD, CAT, CP, GR) and TBARS products are determined by their interactions with physiological elements Na, Ca, Mg, Fe, and Zn and toxic

  11. Effects of 24-epibrassinolide on enzymatic browning and antioxidant activity of fresh-cut lotus root slices.

    PubMed

    Gao, Hui; Chai, HongKang; Cheng, Ni; Cao, Wei

    2017-02-15

    Fresh-cut lotus root slices were treated with 80nM 24-epibrassinolide (EBR) and then stored at 4°C for 8days to investigate the effects on cut surface browning. The results showed that EBR treatment reduced cut surface browning in lotus root slices and alleviated membrane lipid peroxidation as reflected by low malondialdehyde content and lipoxygenase activity. EBR treatment inhibited the activity of phenylalanine ammonia lyase and polyphenol oxidase, and subsequently decreased phenolics accumulation and soluble quniones formation. The treatment also stimulated the activity of peroxidase, catalase and ascorbate peroxidase and delayed the loss of ascorbic acid, which would help prevent membrane lipid peroxidation, as a consequence, reducing decompartmentation of enzymes and substrates causing enzymatic browning. These results indicate that EBR treatment is a promising attempt to control browning at cut surface of fresh-cut lotus root slices.

  12. ALDH1 and podoplanin expression patterns predict the risk of malignant transformation in oral leukoplakia

    PubMed Central

    Habiba, Umma; Hida, Kyoko; Kitamura, Tetsuya; Matsuda, Aya Yanagawa; Higashino, Fumihiro; Ito, Yoichi M.; Ohiro, Yoichi; Totsuka, Yasunori; Shindoh, Masanobu

    2017-01-01

    Oral leukoplakia (OL) is a clinically diagnosed preneoplastic lesion of the oral cavity with an increased oral cancer risk. However, the risk of malignant transformation is still difficult to assess. The objective of the present study was to examine the expression patterns of aldehyde dehydrogenase 1 (ALDH1) and podoplanin in OL, and to determine their roles in predicting oral cancer development. In the present study, the expression patterns of ALDH1 and podoplanin were determined in samples from 79 patients with OL. The association between protein expression and clinicopathological parameters, including oral cancer-free survival, was analyzed during a mean follow-up period of 3.4 years. Expression of ALDH1 and podoplanin was observed in 61 and 67% patients, respectively. Kaplan-Meier analysis demonstrated that the expression of the proteins was correlated with the risk of progression to oral cancer. Multivariate analysis revealed that expression of ALDH1 and podoplanin was associated with 3.02- and 2.62-fold increased risk of malignant transformation, respectively. The malignant transformation risk of OL was considerably higher in cases with expression of both proteins. Point-prevalence analysis revealed that 66% of patients with co-expression of ALDH1 and podoplanin developed oral cancer. Taken together, our data indicate that ALDH1 and podoplanin expression patterns in OL are associated with oral cancer development, suggesting that ALDH1 and podoplanin may be useful biomarkers to identify OL patients with a substantially high oral cancer risk. PMID:28123562

  13. ALDH1 and podoplanin expression patterns predict the risk of malignant transformation in oral leukoplakia.

    PubMed

    Habiba, Umma; Hida, Kyoko; Kitamura, Tetsuya; Matsuda, Aya Yanagawa; Higashino, Fumihiro; Ito, Yoichi M; Ohiro, Yoichi; Totsuka, Yasunori; Shindoh, Masanobu

    2017-01-01

    Oral leukoplakia (OL) is a clinically diagnosed preneoplastic lesion of the oral cavity with an increased oral cancer risk. However, the risk of malignant transformation is still difficult to assess. The objective of the present study was to examine the expression patterns of aldehyde dehydrogenase 1 (ALDH1) and podoplanin in OL, and to determine their roles in predicting oral cancer development. In the present study, the expression patterns of ALDH1 and podoplanin were determined in samples from 79 patients with OL. The association between protein expression and clinicopathological parameters, including oral cancer-free survival, was analyzed during a mean follow-up period of 3.4 years. Expression of ALDH1 and podoplanin was observed in 61 and 67% patients, respectively. Kaplan-Meier analysis demonstrated that the expression of the proteins was correlated with the risk of progression to oral cancer. Multivariate analysis revealed that expression of ALDH1 and podoplanin was associated with 3.02- and 2.62-fold increased risk of malignant transformation, respectively. The malignant transformation risk of OL was considerably higher in cases with expression of both proteins. Point-prevalence analysis revealed that 66% of patients with co-expression of ALDH1 and podoplanin developed oral cancer. Taken together, our data indicate that ALDH1 and podoplanin expression patterns in OL are associated with oral cancer development, suggesting that ALDH1 and podoplanin may be useful biomarkers to identify OL patients with a substantially high oral cancer risk.

  14. ALDH2 polymorphism is associated with fasting blood glucose through alcohol consumption in Japanese men

    PubMed Central

    Yin, Guang; Naito, Mariko; Wakai, Kenji; Morita, Emi; Kawai, Sayo; Hamajima, Nobuyuki; Suzuki, Sadao; Kita, Yoshikuni; Takezaki, Toshiro; Tanaka, Keitaro; Morita, Makiko; Uemura, Hirokazu; Ozaki, Etsuko; Hosono, Satoyo; Mikami, Haruo; Kubo, Michiaki; Tanaka, Hideo

    2016-01-01

    ABSTRACT Associations between alcohol consumption and type 2 diabetes risk are inconsistent in epidemiologic studies. This study investigated the associations of ADH1B and ALDH2 polymorphisms with fasting blood glucose levels, and the impact of the associations of alcohol consumption with fasting blood glucose levels in Japanese individuals. This cross-sectional study included 907 men and 912 women, aged 35–69 years. The subjects were selected from among the Japan Multi-institutional Collaborative Cohort study across six areas of Japan. The ADH1B and ALDH2 polymorphisms were genotyped by Invader Assays. The ALDH2 Glu504Lys genotypes were associated with different levels of fasting blood glucose in men (P = 0.04). Mean fasting glucose level was positively associated with alcohol consumption in men with the ALDH2 504 Lys allele (Ptrend = 0.02), but not in men with the ALDH2 504Glu/Glu genotype (Ptrend = 0.45), resulting in no statistically significant interaction (P = 0.38). Alcohol consumption was associated with elevated fasting blood glucose levels compared with non-consumers in men (Ptrend = 0.002). The ADH1B Arg48His polymorphism was not associated with FBG levels overall or after stratification for alcohol consumption. These findings suggest that the ALDH2 polymorphism is associated with different levels of fasting blood glucose through alcohol consumption in Japanese men. The interaction of ALDH2 polymorphisms in the association between alcohol consumption and fasting blood glucose warrants further investigation. PMID:27303105

  15. SPINK1, ADH2, and ALDH2 gene variants and alcoholic chronic pancreatitis in Japan.

    PubMed

    Shimosegawa, Tooru; Kume, Kiyoshi; Masamune, Atsushi

    2008-03-01

    The serine protease inhibitor Kazal type 1 (SPINK1) is a potent antiprotease and an important inactivation factor of intrapancreatic trypsin activity. Loss of function by the SPINK1 mutations leads to decreased inhibitory capacity. The significance of SPINK1 mutations in alcoholic chronic pancreatitis (CP) in Japan and its functional role remain unclear. The aim of the present study was to clarify the incidence of SPINK1, alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) variants in CP patients in Japan. One hundred and 86 patients with CP, and 527 healthy volunteers were enrolled. Mutational analyses were performed by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. Serum pancreatic secretory trypsin inhibitor (PSTI) level was measured by radioimmunoassay. The frequencies of N34S and IVS3 + 2T > C in the SPINK1 gene were significantly higher in patients with non-alcoholic CP (12.9% and 8.6%, respectively) than in normal subjects (0.37% and 0%). In total, 18 of 93 (19.4%) patients with non-alcoholic CP had at least one SPINK1 mutation. Concerning alcoholic CP, we found IVS3 + 2T > C in a small number of patients (3.9%). Serum PSTI concentration was decreased in patients with the IVS3 + 2T > C mutation. The frequency of the ADH2*2 allele in the alcoholic CP group was significantly higher than that in alcoholics without pancreatitis. The frequency of the ALDH2*2 allele was significantly low in patients with alcoholic CP compared with healthy controls. In conclusion, SPINK1 mutations were associated with non-alcoholic CP. Furthermore, we revealed the amount of wild-type PSTI was decreased in patients with IVS3 + 2T > C mutation. Variants of alcohol-metabolizing enzymes appeared in the relation to alcoholic CP.

  16. Bacterial Production and Enzymatic Activities in Deep-Sea Sediments of the Pacific Ocean: Biogeochemical Implications of Different Temperature Constraints

    NASA Astrophysics Data System (ADS)

    Danovaro, R.; Corinaldesi, C.; dell'Anno, A.

    2002-12-01

    The deep-sea bed, acting as the ultimate sink for organic material derived from the upper oceans primary production, is now assumed to play a key role in biogeochemical cycling of organic matter on global scale. Early diagenesis of organic matter in marine sediments is dependent upon biological processes (largely mediated by bacterial activity) and by molecular diffusion. Organic matter reaching the sea floor by sedimentation is subjected to complex biogeochemical transformations that make organic matter largely unsuitable for direct utilization by benthic heterotrophs. Extracellular enzymatic activities in the sediment is generally recognized as the key step in the degradation and utilization of organic polymers by bacteria and a key role in biopolymeric carbon mobilization is played by aminopeptidase, alkaline phosphatase and glucosidase activities. In the present study we investigated bacterial density, bacterial C production and exo-enzymatic activities (aminopeptidase, glucosidase and phosphatase activity) in deep-sea sediments of the Pacific Ocean in relation with the biochemical composition of sediment organic matter (proteins, carbohydrates and lipids), in order to gather information on organic matter cycling and diagenesis. Benthic viral abundance was also measured to investigate the potential role of viruses on microbial loop functioning. Sediment samples were collected at eight stations (depth ranging from 2070-3100 m) along two transects located at the opposite side (north and south) of ocean seismic ridge Juan Fernandez (along latitudes 33° 20' - 33° 40'), constituted by the submerged vulcanoes, which connects the Chilean coasts to Rapa Nui Island. Since the northern and southern sides of this ridge apparently displayed small but significant differences in deep-sea temperature (related to the general ocean circulation), this sampling strategy allowed also investigating the role of different temperature constraints on bacterial activity and

  17. Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

    SciTech Connect

    Doherty, R.E.; Haywood-Small, S.L.; Sisley, K.; Cross, N.A.

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Isolated ALDH{sup Hi} PC3 cells preferentially form primitive holoclone-type colonies. Black-Right-Pointing-Pointer Primitive holoclone colonies are predominantly ALDH{sup Lo} but contain rare ALDH{sup Hi} cells. Black-Right-Pointing-Pointer Holoclone-forming cells are not restricted to the ALDH{sup Hi} population. Black-Right-Pointing-Pointer ALDH phenotypic plasticity occurs in PC3 cells (ALDH{sup Lo} to ALDH{sup Hi} and vice versa). Black-Right-Pointing-Pointer ALDH{sup Hi} cells are observed but very rare in PC3 spheroids grown in stem cell medium. -- Abstract: Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH{sup Lo} cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH{sup Hi} population, or whether all ALDH{sup Hi} cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH{sup Hi} cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH{sup Hi} cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH{sup Lo} population can develop ALDH{sup Hi} populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH{sup Hi} cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in

  18. [Isolation of wood-decaying fungi and evaluation of their enzymatic activity (Quindío, Colombia)].

    PubMed

    Chaparro, Deisy Fernanda; Rosas, Diana Carolina; Varela, Amanda

    2009-12-31

    White rot fungi (Ascomycota and Basidiomycota) were collected on fallen trunks with different decay stages, in a subandean forest (La Montaña del Ocaso nature reserve), and it was evaluated their ligninolitic activity. They were cultured on malt extract agar. Then it was performed semiquantitative tests for laccase and cellobiose dehydrogenase (CDH) activity using ABTS and DCPIP as enzymatic inducers. Based on the results of these tests, the fungi with higher activities from trunks with different decay stages were selected: Cookeina sulcipes (for stage 1), a fungus from the family Corticiaceae (for stage 2), Xylaria polymorpha (for stage 3) and Earliella sp. (for stage 4). A fermentation was performed at 28 degrees C, during 11 days, in a rotatory shaker at 150 rpm. Biomass, glucose, proteins and enzyme activities measurements were performed daily. The fungi that were in the trunks with decay states from 1 to 3, showed higher laccase activity as the state of decay increased. A higher DCH activity was also associated with a higher. Also, there was a positive relationship between both enzymes' activities. Erliella was the fungus which presented the highest biomass production (1140,19 g/l), laccase activity (157 UL(-1)) and CDH activity (43,50 UL(-1)). This work is the first report of laccase and CDH activity for Cookeina sulcipes and Earliella sp. Moreover, it gives basis for the use of these native fungi in biotechnological applications and the acknowledgment of their function in the wood decay process in native forest.

  19. Alternations in the liver enzymatic activity of Common carp, Cyprinus carpio in response to parasites, Dactylogyrus spp. and Gyrodactylus spp.

    PubMed

    Rastiannasab, Abulhasan; Afsharmanesh, Shiva; Rahimi, Ruhollah; Sharifian, Iman

    2016-12-01

    The present study was carried out to investigate the effects of parasites, monogenea, Dactylogyrus spp. and Gyrodactylus spp. on some enzymatic and biochemical components of liver in healthy and infected common carp, Cyprinus carpio. For this purpose, 10 healthy and 10 infected fish were collected from farm. The blood samples were taken and after separation of serum, the values of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) enzymes activities as well as Creatinine and Urea were measured. Based on obtained results, the values of AST, ALT enzymes activities as well as Creatinine and Urea were higher in the infected fish compared to non-infected fish. In conclusion; our results reveals that infection with external parasites, Dactylogyrus spp. and Gyrodactylus spp. can causes some dysfunctions in liver and kidney of common carp.

  20. Monoclonal antibodies raised against 167-180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity.

    PubMed

    Dekaminaviciute, Dovile; Kairys, Visvaldas; Zilnyte, Milda; Petrikaite, Vilma; Jogaite, Vaida; Matuliene, Jurgita; Gudleviciene, Zivile; Vullo, Daniela; Supuran, Claudiu T; Zvirbliene, Aurelija

    2014-12-01

    Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application.

  1. ALDH1A1 Deficiency in Gorlin Syndrome Suggests a Central Role for Retinoic Acid and ATM Deficits in Radiation Carcinogenesis.

    PubMed

    Weber, Thomas J; Magnaldo, Thierry; Xiong, Yijia

    2014-09-11

    We hypothesize that aldehyde dehydrogenase 1A1 (ALDH1A1) deficiency will result in impaired ataxia-telangiectasia mutated (ATM) activation in a retinoic acid-sensitive fashion. Data supporting this hypothesis include (1) reduced ATM activation in irradiated primary dermal fibroblasts from ALDH1A1-deficient Gorlin syndrome patients (GDFs), relative to ALDH1A1-positive normal human dermal fibroblasts (NHDFs) and (2) increased ATM activation by X-radiation in GDFs pretreated with retinoic acid, however, the impact of donor variability on ATM activation in fibroblasts was not assessed and is a prudent consideration in future studies. Clonogenic survival of irradiated cells showed differential responses to retinoic acid as a function of treatment time. Long-term (5 Day) retinoic acid treatment functioned as a radiosensitizer and was associated with downregulation of ATM protein levels. Short-term (7 h) retinoic acid treatment showed a trend toward increased survival of irradiated cells and did not downregulate ATM protein levels. Using a newly developed IncubATR technology, which defines changes in bulk chemical bond patterns in live cells, we can discriminate between the NHDF and GDF phenotypes, but treatment of GDFs with retinoic acid does not induce reversion of bulk chemical bond patterns associated with GDFs toward the NHDF phenotype. Collectively, our preliminary investigation of the Gorlin phenotype has identified deficient ALDH1A1 expression associated with deficient ATM activation as a possible susceptibility factor that is consistent with the high incidence of spontaneous and radiation-induced carcinogenesis in these patients. The IncubATR technology exhibits sufficient sensitivity to detect phenotypic differences in live cells that may be relevant to radiation health effects.

  2. ALDH1A1 Deficiency in Gorlin Syndrome Suggests a Central Role for Retinoic Acid and ATM Deficits in Radiation Carcinogenesis

    PubMed Central

    Weber, Thomas J.; Magnaldo, Thierry; Xiong, Yijia

    2014-01-01

    We hypothesize that aldehyde dehydrogenase 1A1 (ALDH1A1) deficiency will result in impaired ataxia-telangiectasia mutated (ATM) activation in a retinoic acid-sensitive fashion. Data supporting this hypothesis include (1) reduced ATM activation in irradiated primary dermal fibroblasts from ALDH1A1-deficient Gorlin syndrome patients (GDFs), relative to ALDH1A1-positive normal human dermal fibroblasts (NHDFs) and (2) increased ATM activation by X-radiation in GDFs pretreated with retinoic acid, however, the impact of donor variability on ATM activation in fibroblasts was not assessed and is a prudent consideration in future studies. Clonogenic survival of irradiated cells showed differential responses to retinoic acid as a function of treatment time. Long-term (5 Day) retinoic acid treatment functioned as a radiosensitizer and was associated with downregulation of ATM protein levels. Short-term (7 h) retinoic acid treatment showed a trend toward increased survival of irradiated cells and did not downregulate ATM protein levels. Using a newly developed IncubATR technology, which defines changes in bulk chemical bond patterns in live cells, we can discriminate between the NHDF and GDF phenotypes, but treatment of GDFs with retinoic acid does not induce reversion of bulk chemical bond patterns associated with GDFs toward the NHDF phenotype. Collectively, our preliminary investigation of the Gorlin phenotype has identified deficient ALDH1A1 expression associated with deficient ATM activation as a possible susceptibility factor that is consistent with the high incidence of spontaneous and radiation-induced carcinogenesis in these patients. The IncubATR technology exhibits sufficient sensitivity to detect phenotypic differences in live cells that may be relevant to radiation health effects. PMID:28250390

  3. Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling.

    PubMed

    Sullivan, James P; Spinola, Monica; Dodge, Michael; Raso, Maria G; Behrens, Carmen; Gao, Boning; Schuster, Katja; Shao, Chunli; Larsen, Jill E; Sullivan, Laura A; Honorio, Sofia; Xie, Yang; Scaglioni, Pier P; DiMaio, J Michael; Gazdar, Adi F; Shay, Jerry W; Wistuba, Ignacio I; Minna, John D

    2010-12-01

    Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for ALDH1A1, ALDH3A1, and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity, and that this activity is associated with ALDH1A1 expression. Isolated ALDH(+) lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared with their ALDH(-) counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH(+) cells. Suppression of the Notch pathway by treatment with either a γ-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH(+) lung cancer cells, commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together, these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential, that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis, and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH(+) component, implicating Notch signaling in lung cancer stem cell maintenance.

  4. Different enzymatic activities in carp (cyprinus carpio L.) as potential biomarkers of exposure to the pesticide methomyl.

    PubMed

    Hernández-Moreno, David; de la Casa-Resino, Irene; Maria Flores, José; González-Gómez, Manuel José; María Neila, Carlos; Soler, Francisco; Pérez-López, Marcos

    2014-09-29

    This study investigated the influence of the pesticide methomyl on different enzymatic activities in carp. The fish were exposed to a sub-lethal concentration (0.34 mg L-1) of methomyl for 15 days. On days 4 and 15, catalase (CAT) and glutathione-S-transferase (GST) activities were measured in the liver and gills. Acetylcholinesterase (AChE) activity in brain and muscle was also determined. Liver catalase activity slightly increased in exposed fish when compared to controls, but it was statistically significant only at the beginning of the experiment. No changes in CAT activity in the gills of exposed and control animals were observed (mean values were in the range 10.7-11.7 nmol min-1 per mg of protein). Liver GST activity was slightly inhibited in the exposed animals at the beginning of the study; however, it was significantly inhibited in the gills. Brain AChE activity was diminished throughout the experiment and significantly decreased after 96 h of exposure compared to controls (0.041 vs. 0.075 nmol min1 per mg of protein; p<0.001). Our findings suggest that CAT, GST, and AChE are reliable biomarkers of effect after exposure to methomyl.

  5. Amendment application in a multi-contaminated mine soil: effects on soil enzymatic activities and ecotoxicological characteristics.

    PubMed

    Manzano, Rebeca; Esteban, Elvira; Peñalosa, Jesús M; Alvarenga, Paula

    2014-03-01

    Several amendments were tested on soils obtained from an arsenopyrite mine, further planted with Arrhenatherum elatius and Festuca curvifolia, in order to assess their ability to improve soil's ecotoxicological characteristics. The properties used to assess the effects were: soil enzymatic activities (dehydrogenase, β-glucosidase, acid phosphatase, urease, protease and cellulase), terrestrial bioassays (Eisenia fetida mortality and avoidance behaviour), and aquatic bioassays using a soil leachate (Daphnia magna immobilisation and Vibrio fischeri bioluminescence inhibition). The treatment with FeSO4 1 % w/w was able to reduce extractable As in soil, but increased the extractable Cu, Mn and Zn concentrations, as a consequence of the decrease in soil pH, in relation to the unamended soil, from 5.0 to 3.4, respectively. As a consequence, this treatment had a detrimental effect in some of the soil enzymatic activities (e.g. dehydrogenase, acid phosphatase, urease and cellulase), did not allow plant growth, induced E. fetida mortality in the highest concentration tested (100 % w/w), and its soil leachate was very toxic towards D. magna and V. fischeri. The combined application of FeSO4 1 % w/w with other treatments (e.g. CaCO3 1 % w/w and paper mill 1 % w/w) allowed a decrease in extractable As and metals, and a soil pH value closer to neutrality. As a consequence, dehydrogenase activity, plant growth and some of the bioassays identified those as better soil treatments to this type of multi-contaminated soil.

  6. Cloning and molecular evolution of the aldehyde dehydrogenase 2 gene (Aldh2) in bats (Chiroptera).

    PubMed

    Chen, Yao; Shen, Bin; Zhang, Junpeng; Jones, Gareth; He, Guimei

    2013-02-01

    Old World fruit bats (Pteropodidae) and New World fruit bats (Phyllostomidae) ingest significant quantities of ethanol while foraging. Mitochondrial aldehyde dehydrogenase (ALDH2, encoded by the Aldh2 gene) plays an important role in ethanol metabolism. To test whether the Aldh2 gene has undergone adaptive evolution in frugivorous and nectarivorous bats in relation to ethanol elimination, we sequenced part of the coding region of the gene (1,143 bp, ~73 % coverage) in 14 bat species, including three Old World fruit bats and two New World fruit bats. Our results showed that the Aldh2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to Old World fruit bats and New World fruit bats. Further research is needed to determine whether other genes involved in ethanol metabolism have been the targets of positive selection in frugivorous and nectarivorous bats.

  7. Lactones 42. Stereoselective enzymatic/microbial synthesis of optically active isomers of whisky lactone.

    PubMed

    Boratyński, Filip; Smuga, Małgorzata; Wawrzeńczyk, Czesław

    2013-11-01

    Two different methods, enzyme-mediated reactions and biotrasformations with microorganisms, were applied to obtain optically pure cis- and trans-isomers of whisky lactone 4a and 4b. In the first method, eight alcohol dehydrogenases were investigated as biocatalysts to enantioselective oxidation of racemic erythro- and threo-3-methyloctane-1,4-diols (1a and 1b). Oxidation processes with three of them, alcohol dehydrogenases isolated from horse liver (HLADH) as well as recombinant from Escherichia coli and primary alcohol dehydrogenase (PADH I), were characterized by the highest degree of conversion with moderate enantioselectivity (ee=27-82%) of the reaction. In all enzymatic reactions enantiomerically enriched not naturally occurring isomers of trans-(-)-(4R,5S)-4b or cis-(+)-(4R,5R)-4a were formed preferentially. In the second strategy, based on microbial lactonization of γ-oxoacids, naturally occurring opposite isomers of whisky lactones were obtained. Trans-(+)-(4S,5R)-isomer (ee=99%) of whisky lactone 4b was stereoselectively formed as the only product of biotransformations of 3-methyl-4-oxooctanoic acid (5) catalyzed by Didimospheria igniaria KCH6651, Laetiporus sulphurens AM525, Chaetomium sp.1 KCH6670 and Saccharomyces cerevisiae AM464. Biotransformation of γ-oxoacid 5, in the culture of Beauveria bassiana AM278 and Pycnidiella resinae KCH50 afforded a mixtures of trans-(+)-(4S,5R)-4b with enantiomeric excess ee=99% and cis-(-)-(4S,5S)-4a with enantiomeric excesses ee=77% and ee=45% respectively.

  8. ALDH Enzyme Expression Is Independent of the Spermatogenic Cycle, and Their Inhibition Causes Misregulation of Murine Spermatogenic Processes1

    PubMed Central

    Kent, Travis; Arnold, Samuel L.; Fasnacht, Rachael; Rowsey, Ross; Mitchell, Debra; Hogarth, Cathryn A.; Isoherranen, Nina; Griswold, Michael D.

    2015-01-01

    Perturbations in the vitamin A metabolism pathway could be a significant cause of male infertility, as well as a target toward the development of a male contraceptive, necessitating the need for a better understanding of how testicular retinoic acid (RA) concentrations are regulated. Quantitative analyses have recently demonstrated that RA is present in a pulsatile manner along testis tubules. However, it is unclear if the aldehyde dehydrogenase (ALDH) enzymes, which are responsible for RA synthesis, contribute to the regulation of these RA concentration gradients. Previous studies have alluded to fluctuations in ALDH enzymes across the spermatogenic cycle, but these inferences have been based primarily on qualitative transcript localization experiments. Here, we show via various quantitative methods that the three well-known ALDH enzymes (ALDH1A1, ALDH1A2, and ALDH1A3), and an ALDH enzyme previously unreported in the murine testis (ALDH8A1), are not expressed in a stage-specific manner in the adult testis, but do fluctuate throughout juvenile development in perfect agreement with the first appearance of each advancing germ cell type. We also show, via treatments with a known ALDH inhibitor, that lowered testicular RA levels result in an increase in blood-testis barrier permeability, meiotic recombination, and meiotic defects. Taken together, these data further our understanding of the complex regulatory actions of RA on various spermatogenic events and, in contrast with previous studies, also suggest that the ALDH enzymes are not responsible for regulating the recently measured RA pulse. PMID:26632609

  9. Aldh2 knockout mice were more sensitive to DNA damage in leukocytes due to ethyl tertiary butyl ether exposure.

    PubMed

    Weng, Zuquan; Suda, Megumi; Ohtani, Katsumi; Mei, Nan; Kawamoto, Toshihiro; Nakajima, Tamie; Wang, Rui-Sheng

    2011-01-01

    To clarify the genotoxicity of ethyl tertiary butyl ether (ETBE), a gasoline additive, male and female C57BL/6 mice of Aldh2+/+ and Aldh2-/- genotypes, aged 8 wk, were exposed to 0, 500, 1,750, or 5,000 ppm ETBE for 6 h/day, 5 d per week for 13 wk. DNA damage in leukocytes was measured by the alkaline comet assay and expressed quantitatively as Tail Intensity (TI). For male mice, TI was significantly higher in all three groups exposed to ETBE than in those without exposure within Aldh2-/- mice, whereas within Aldh2+/+ mice, TI increased only in those exposed to 5,000 ppm of ETBE as compared with mice without exposure. For female mice, a significant increase in TI values was observed in the group exposed to 5,000 ppm of ETBE as compared with those without exposure within Aldh2-/- mice; TI in Aldh2-/- mice exposed to 1,750 and 5,000 ppm was significantly higher than in Aldh2+/+ mice without exposure. TI did not significantly increase in any of the groups exposed to ETBE within female Aldh2+/+ mice. Based on the results we suggest that Aldh2-/- mice are more sensitive to DNA damage caused by ETBE than Aldh2+/+ mice and that males seem more susceptible to this effect than females.

  10. Protein expression of CYP1A1, CYP1B1, ALDH1A1, and ALDH2 in young patients with oral squamous cell carcinoma.

    PubMed

    Kaminagakura, E; Caris, A; Coutinho-Camillo, C; Soares, F A; Takahama-Júnior, A; Kowalski, L P

    2016-06-01

    The purpose of this study was to evaluate the expression of the enzymes involved in the biotransformation of tobacco and alcohol. A study group of 41 young patients (≤40 years old) with oral squamous cell carcinoma (OSCC) was compared to 59 control subjects (≥50 years old) with tumours of similar clinical stages and topographies. The immunohistochemical expression of CYP1A1, CYP1B1, ALDH1A1, and ALDH2 was evaluated using the tissue microarray technique. There was a predominance of males, smokers, and alcohol drinkers in both groups. Most tumours were located in the tongue (43.9% vs. 50.8%), were well-differentiated (63.4% vs. 56.6%), and were in clinical stages III or IV (80.5% vs. 78.0%). No difference was observed in the expression of CYP1A1, ALDH1A1, or ALDH2 between the two groups. CYP1A1 and ALDH2 protein expression had no influence on the prognosis. The immunoexpression of CYP1B1 was significantly higher in the control group than in the young group (P<0.001). The 5-year relapse-free survival was better in patients with CYP1B1 overexpression vs. protein underexpression (64% vs. 25%; P<0.05), regardless of age. ALDH1A1 expression improved relapse-free survival in young patients. These results suggest a lower risk of recurrence with increased metabolism of carcinogens by CYP1B1. Further studies involving other genes and proteins are necessary to complement the results of this research.

  11. An enzymatic assay based on luciferase Ebola virus-like particles for evaluation of virolytic activity of antimicrobial peptides.

    PubMed

    Peskova, Marie; Heger, Zbynek; Janda, Petr; Adam, Vojtech; Pekarik, Vladimir

    2017-02-01

    Antimicrobial peptides are currently considered as promising antiviral compounds. Current assays to evaluate the effectivity of peptides against enveloped viruses based on liposomes or hemolysis are encumbered by the artificial nature of liposomes or distinctive membrane composition of used erythrocytes. We propose a novel assay system based on enzymatic Ebola virus-like particles containing sensitive luciferase reporter. The assay was validated with several cationic and anionic peptides and compared with lentivirus inactivation and hemolytic assays. The assay is sensitive and easy to perform in standard biosafety level laboratory with potential for high-throughput screens. The use of virus-like particles in the assay provides a system as closely related to the native viruses as possible eliminating some issues associated with other more artificial set ups. We have identified CAM-W (KWKLWKKIEKWGQGIGAVLKWLTTWL) as a peptide with the greatest antiviral activity against infectious lentiviral vectors and filoviral virus-like particles.

  12. Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis.

    PubMed

    Feder, Vanessa; Kmetzsch, Lívia; Staats, Charley Christian; Vidal-Figueiredo, Natalia; Ligabue-Braun, Rodrigo; Carlini, Célia Regina; Vainstein, Marilene Henning

    2015-04-01

    Ureases (EC 3.5.1.5) are Ni(2+) -dependent metalloenzymes produced by plants, fungi and bacteria that hydrolyze urea to produce ammonia and CO2 . The insertion of nickel atoms into the apo-urease is better characterized in bacteria, and requires at least three accessory proteins: UreD, UreF, and UreG. Our group has demonstrated that ureases possess ureolytic activity-independent biological properties that could contribute to the pathogenicity of urease-producing microorganisms. The presence of urease in pathogenic bacteria strongly correlates with pathogenesis in some human diseases. Some medically important fungi also produce urease, including Cryptococcus neoformans and Cryptococcus gattii. C. gattii is an etiological agent of cryptococcosis, most often affecting immunocompetent individuals. The cryptococcal urease might play an important role in pathogenesis. It has been proposed that ammonia produced via urease action might damage the host endothelium, which would enable yeast transmigration towards the central nervous system. To analyze the role of urease as a virulence factor in C. gattii, we constructed knockout mutants for the structural urease-coding gene URE1 and for genes that code the accessory proteins Ure4 and Ure6. All knockout mutants showed reduced multiplication within macrophages. In intranasally infected mice, the ure1Δ (lacking urease protein) and ure4Δ (enzymatically inactive apo-urease) mutants caused reduced blood burdens and a delayed time of death, whereas the ure6Δ (enzymatically inactive apo-urease) mutant showed time and dose dependency with regard to fungal burden. Our results suggest that C. gattii urease plays an important role in virulence, in part possibly through enzyme activity-independent mechanism(s).

  13. ADH and ALDH polymorphisms among Alaska Natives entering treatment for alcoholism.

    PubMed

    Segal, B

    1999-01-01

    The alcohol dehydrogenase (ADHs) and aldehyde dehydrogenases (ALDHs) involved in alcohol metabolism are polymorphic. Different alleles encode subunits of the enzymes that are related to differences in alcohol metabolism with different ethnic groups. This study examined the allele frequencies at the ADH1, ADH2, ADH3 and ALDH2 loci in Alaska Natives entering treatment for alcoholism to determine if allele frequencies at these loci differ among five distinct Alaska Native groups: Yupik and Inupiat Eskimos, Athabascan, Tlingit and Aleut. It was found that all persons were homozygous for the ADH1*1, ADH2*1 and ALDH2*1 alleles. Variations, however, were found for the allele distribution of the ADH3 genotype. Comparison with a general population sample found no differences in allele distributions for ADHs and ALDH2*1, but differences were found when comparisons were made with four Asian Groups. The study's findings suggest that the Alaska Natives are not protected from the risk of alcoholism in the same way that Asians who possess the ALDH2*2 genotype are considered to have a negative risk factor. Nor, does there appear to be any generalized differences between Alaska Native alcoholics and members of the general population with respect to the ALDH and ADH polymorphisms studied herein.

  14. Enhancing phytochemical levels, enzymatic and antioxidant activity of spinach leaves by chitosan treatment and an insight into the metabolic pathway using DART-MS technique.

    PubMed

    Singh, Shachi

    2016-05-15

    Phytochemicals are health promoting compounds, synthesized by the plants to protect them against biotic or abiotic stress. The metabolic pathways leading to the synthesis of these phytochemicals are highly inducible; therefore methods could be developed to enhance their production by the exogenous application of chemical inducers/elicitors. In the present experiment, chitosan was used as an elicitor molecule to improve the phytochemical content of spinach plant. When applied at a concentration of 0.01 mg/ml as a foliar spray, chitosan was able to cause an increase in the enzymatic (peroxidase, catalase and phenylalanine ammonium lyase (PAL)) and non enzymatic (total phenolics, flavonoids and proteins) defensive metabolites, as well as, in the total antioxidant activity of the spinach leaves. A 1.7-fold increase in the total phenolics, a 2-fold increase in total flavonoid and a 1.6-fold increase in total protein were achieved with the treatment. A higher level of enzymatic activity was observed with a 4-fold increase in peroxidase and approximately 3-fold increases in catalase and phenylalanine ammonium lyase activity. Antioxidant activity showed a positive correlation between phenolic compounds and the enzymatic activity. Direct analysis in real time mass spectrometry (DART-MS) was applied to generate the metabolite profile of control and treated leaves. DART analysis revealed the activation of phenylpropanoid pathway by chitosan molecule, targeting the synthesis of diverse classes of flavonoids and their glycosides. Important metabolites of stress response were also visible in the DART spectra, including proline and free sugars.

  15. Quantification of ricin, RCA and comparison of enzymatic activity in 18 Ricinus communis cultivars by isotope dilution mass spectrometry.

    PubMed

    Schieltz, David M; McWilliams, Lisa G; Kuklenyik, Zsuzsanna; Prezioso, Samantha M; Carter, Andrew J; Williamson, Yulanda M; McGrath, Sara C; Morse, Stephen A; Barr, John R

    2015-03-01

    The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) are two antibody-based methods commonly used to quantify ricin and RCA; however, antibodies currently used in these methods cannot distinguish between ricin and RCA due to the high sequence homology of the respective proteins. In this study, a technique combining antibody-based affinity capture with liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry (MS) was used to quantify the amounts of ricin and RCA independently in extracts prepared from the seeds of eighteen representative cultivars of R. communis which were propagated under identical conditions. Additionally, liquid chromatography and MRM-MS was used to determine rRNA N-glycosidase activity for each cultivar and the overall activity in these cultivars was compared to a purified ricin standard. Of the cultivars studied, the average ricin content was 9.3 mg/g seed, the average RCA content was 9.9 mg/g seed, and the enzymatic activity agreed with the activity of a purified ricin reference within 35% relative activity.

  16. Trapping RNase A on MCM41 pores: effects on structure stability, product inhibition and overall enzymatic activity.

    PubMed

    Matlahov, Irina; Geiger, Yasmin; Goobes, Gil

    2014-05-21

    Catalytic activity of enzymes can be drastically modified by immobilization on surfaces of different materials. It is particularly effective when the dimensions of the biomolecules and adsorption sites on the material surfaces are commensurate. This can be utilized to hinder the biological activity of degradation enzymes and switch off undesired biological processes. Ribonucleases are particularly attractive targets for complete sequestration being efficient at disintegrating viable RNA molecules. Here we show that efficient quenching of ribonuclease A activity can be achieved by immobilization on the surface of MCM41 porous silica. Electron microscopy, isothermal titration calorimetry, differential scanning calorimetry and adsorption isotherm measurements of ribonuclease A on the MCM41 surface are used to demonstrate that the enzyme adsorbs on the external surface of the porous silica through electrostatic interactions that overcome the unfavorable entropy change as the protein gets trapped on the surface, and that immobilization shifts up its denaturation temperature by 20-25 °C. Real-time kinetic measurements, using single injection titration calorimetry, demonstrate that enzymatic activity towards hydrolysis of cyclic nucleotides is lowered by nearly two orders of magnitude on MCM41 and that active inhibition by the formed product is much less effective on the surface than in solution.

  17. Quantification of ricin, RCA and comparison of enzymatic activity in 18 Ricinus communis cultivars by isotope dilution mass spectrometry

    PubMed Central

    Schieltz, David M.; McWilliams, Lisa G.; Kuklenyik, Zsuzsanna; Prezioso, Samantha M.; Carter, Andrew J.; Williamson, Yulanda M.; McGrath, Sara C.; Morse, Stephen A.; Barr, John R.

    2016-01-01

    The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) are two antibody-based methods commonly used to quantify ricin and RCA; however, antibodies currently used in these methods cannot distinguish between ricin and RCA due to the high sequence homology of the respective proteins. In this study, a technique combining antibody-based affinity capture with liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry (MS) was used to quantify the amounts of ricin and RCA independently in extracts prepared from the seeds of eighteen representative cultivars of R. communis which were propagated under identical conditions. Additionally, liquid chromatography and MRM-MS was used to determine rRNA N-glycosidase activity for each cultivar and the overall activity in these cultivars was compared to a purified ricin standard. Of the cultivars studied, the average ricin content was 9.3 mg/g seed, the average RCA content was 9.9 mg/g seed, and the enzymatic activity agreed with the activity of a purified ricin reference within 35% relative activity. PMID:25576235

  18. Assessment of cathepsin mRNA expression and enzymatic activity during early embryonic development in the yellowtail kingfish Seriola lalandi.

    PubMed

    Palomino, Jaime; Herrera, Giannina; Torres-Fuentes, Jorge; Dettleff, Phillip; Patel, Alok; Martínez, Víctor

    2017-02-21

    In pelagic species such as Seriola lalandi, survival of both the eggs and embryos depends on yolk processing during oocyte maturation and embryo development. The main enzymes involved in these processes are the cathepsins, which are essential for the hydration process, acquiring buoyancy and nutrition of the embryo before hatching. This study aimed to investigate the mRNA expression profiles of cathepsins B, D and L (catb, catd and catl) and the activity of these enzymes during early development in S. lalandi. We included previtellogenic oocytes (PO). All three enzymes were highly expressed in PO, but the expression was reduced throughout development. Between PO and recently spawned eggs (E1) the transcript to catb and catd decreased, unlike catl. Cathepsin B activity, showed stable levels between PO until blastula stage (E4). High activities levels of cathepsins D and L were observed in E1 in comparison with later developmental stages. Cathepsin L activity remained constant until E1, consistent with observations in other pelagic spawners, where its participation in a second protolithic cleavage of the yolk proteins, has been proposed for this enzyme. Their profiles of both mRNA expression and enzymatic activity indicate the importance of these enzymes during early development and suggest different roles in egg yolk processing for the hydration process and nutrition in early embryos in this species.

  19. Lactogenic Activity of an Enzymatic Hydrolysate from Octopus vulgaris and Carica papaya in SD Rats.

    PubMed

    Cai, Bingna; Chen, Hua; Sun, Han; Sun, Huili; Wan, Peng; Chen, Deke; Pan, Jianyu

    2015-11-01

    The traditional Chinese medicine theory believes that octopus papaya soup can stimulate milk production in lactating women. The objective of this study was to determine whether dietary supplementation with an enzymatic hydrolysate of Octopus vulgaris and Carica papaya (EHOC) could increase milk production and nutritional indexes in Sprague Dawley (SD) rats. Female SD rats (n = 24) were fed a control diet (n = 8), EHOC-supplemented diet, or a positive control diet (Shengruzhi) from day 10 of pregnancy to day 10 of lactation. Maternal serum, mammary gland (day 10 of lactation), milk, and pup weight (daily) were collected for analysis. Results showed that the EHOC diet obviously elevated daily milk yield and pup weight compared to the control group (P < .05). The EHOC diet was found to increase the concentration of prolactin (PRL), progesterone (P), estradiol (E2), and growth hormone (GH) significantly in the circulation and mammary gland. Mammary glands of EHOC-treated dams showed clear lobuloalveolar development and proliferation of myoepithelial cells, but no striking variations were observed among the groups. Furthermore, the nutrition content and immune globulin concentration in the milk of EHOC-supplemented dams were higher than those of the control group, especially the cholesterol, glucose, and IgG were higher by 44.98% (P < .001), 42.76% (P < .01), and 42.23% (P < .01), respectively. In conclusion, this article demonstrates that EHOC administration has beneficial effects on milk production in the dams and on performance of the dam and pup. These results indicate that EHOC could be explored as a potentially lactogenic nutriment for lactating women.

  20. Protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves: enzymatic characterization and procoagulant and fibrinogenolytic activities.

    PubMed

    de Menezes, Yamara A S; Félix-Silva, Juliana; da Silva-Júnior, Arnóbio A; Rebecchi, Ivanise M M; de Oliveira, Adeliana S; Uchoa, Adriana F; Fernandes-Pedrosa, Matheus de F

    2014-03-21

    Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogen)olytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.

  1. Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats.

    PubMed

    Nishiyama, Yoshihiro; Nakayama, Shouta M M; Watanabe, Kensuke P; Kawai, Yusuke K; Ohno, Marumi; Ikenaka, Yoshinori; Ishizuka, Mayumi

    2016-05-03

    Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.

  2. Allopregnanolone prevents memory impairment: effect on mRNA expression and enzymatic activity of hippocampal 3-α hydroxysteroid oxide-reductase.

    PubMed

    Escudero, Carla; Casas, Sebastián; Giuliani, Fernando; Bazzocchini, Vanesa; García, Sebastián; Yunes, Roberto; Cabrera, Ricardo

    2012-02-10

    In this work we investigated how the neurosteroid allopregnanolone can modulate learning and memory processes. For this purpose, we used ovariectomized (OVX) rats subcutaneously injected with oestradiol benzoate (E) alone or E and progesterone (P). Then, rats were injected in dorsal hippocampus with allopregnanolone or vehicle. Animals were tested in inhibitory avoidance task (IA task). After behavioural test hippocampal mRNA expression and enzymatic activity of 3α-HOR, the enzyme responsible of allopregnanolone synthesis, were analysed. In IA task OVX-EP rats spent less time on platform, compared to those OVX or OVX-E. Regression analyses revealed that there was a significant negative relationship between E-P infusion and performance in this task. Pre-training allopregnanolone administration to OVX-EP rats increased the time spent on the platform. Interestingly, when enzymatic activity of 3α-HOR was tested, OVX-EP rats showed a significant decrease in the enzymatic activity, compared with OVX and OVX-E rats. In addition, OVX-EP group showed a significant increase in the enzymatic activity after intrahippocampal infusion of allopregnanolone. On the other hand, when mRNA expression of 3α-HOR was analysed no differences were observed when the hippocampal allopregnanolone injection was done. These results suggest that E and P have amnesic effects on female rats, being reversed by allopregnanolone through its modulation on hippocampal 3α-HOR activity.

  3. Antifungal hydroxy fatty acids produced during sourdough fermentation: microbial and enzymatic pathways, and antifungal activity in bread.

    PubMed

    Black, Brenna A; Zannini, Emanuele; Curtis, Jonathan M; Gänzle, Michael G

    2013-03-01

    Lactobacilli convert linoleic acid to hydroxy fatty acids; however, this conversion has not been demonstrated in food fermentations and it remains unknown whether hydroxy fatty acids produced by lactobacilli have antifungal activity. This study aimed to determine whether lactobacilli convert linoleic acid to metabolites with antifungal activity and to assess whether this conversion can be employed to delay fungal growth on bread. Aqueous and organic extracts from seven strains of lactobacilli grown in modified De Man Rogosa Sharpe medium or sourdough were assayed for antifungal activity. Lactobacillus hammesii exhibited increased antifungal activity upon the addition of linoleic acid as a substrate. Bioassay-guided fractionation attributed the antifungal activity of L. hammesii to a monohydroxy C(18:1) fatty acid. Comparison of its antifungal activity to those of other hydroxy fatty acids revealed that the monohydroxy fraction from L. hammesii and coriolic (13-hydroxy-9,11-octadecadienoic) acid were the most active, with MICs of 0.1 to 0.7 g liter(-1). Ricinoleic (12-hydroxy-9-octadecenoic) acid was active at a MIC of 2.4 g liter(-1). L. hammesii accumulated the monohydroxy C(18:1) fatty acid in sourdough to a concentration of 0.73 ± 0.03 g liter(-1) (mean ± standard deviation). Generation of hydroxy fatty acids in sourdough also occurred through enzymatic oxidation of linoleic acid to coriolic acid. The use of 20% sourdough fermented with L. hammesii or the use of 0.15% coriolic acid in bread making increased the mold-free shelf life by 2 to 3 days or from 2 to more than 6 days, respectively. In conclusion, L. hammesii converts linoleic acid in sourdough and the resulting monohydroxy octadecenoic acid exerts antifungal activity in bread.

  4. Site-specific bioconjugation of an organometallic electron mediator to an enzyme with retained photocatalytic cofactor regenerating capacity and enzymatic activity.

    PubMed

    Lim, Sung In; Yoon, Sungho; Kim, Yong Hwan; Kwon, Inchan

    2015-04-07

    Photosynthesis consists of a series of reactions catalyzed by redox enzymes to synthesize carbohydrates using solar energy. In order to take the advantage of solar energy, many researchers have investigated artificial photosynthesis systems mimicking the natural photosynthetic enzymatic redox reactions. These redox reactions usually require cofactors, which due to their high cost become a key issue when constructing an artificial photosynthesis system. Combining a photosensitizer and an Rh-based electron mediator (RhM) has been shown to photocatalytically regenerate cofactors. However, maintaining the high concentration of cofactors available for efficient enzymatic reactions requires a high concentration of the expensive RhM; making this process cost prohibitive. We hypothesized that conjugation of an electron mediator to a redox enzyme will reduce the amount of electron mediators necessary for efficient enzymatic reactions. This is due to photocatalytically regenerated NAD(P)H being readily available to a redox enzyme, when the local NAD(P)H concentration near the enzyme becomes higher. However, conventional random conjugation of RhM to a redox enzyme will likely lead to a substantial loss of cofactor regenerating capacity and enzymatic activity. In order to avoid this issue, we investigated whether bioconjugation of RhM to a permissive site of a redox enzyme retains cofactor regenerating capacity and enzymatic activity. As a model system, a RhM was conjugated to a redox enzyme, formate dehydrogenase obtained from Thiobacillus sp. KNK65MA (TsFDH). A RhM-containing azide group was site-specifically conjugated to p-azidophenylalanine introduced to a permissive site of TsFDH via a bioorthogonal strain-promoted azide-alkyne cycloaddition and an appropriate linker. The TsFDH-RhM conjugate exhibited retained cofactor regenerating capacity and enzymatic activity.

  5. Developmental regulation of the adhesive and enzymatic activity of vascular adhesion protein-1 (VAP-1) in humans.

    PubMed

    Salmi, Marko; Jalkanen, Sirpa

    2006-09-01

    Vascular adhesion protein-1 (VAP-1) is a homodimeric glycoprotein that belongs to a unique subgroup of cell-surface-expressed oxidases. In adults, endothelial VAP-1 supports leukocyte rolling, firm adhesion, and transmigration in both enzyme activity-dependent and enzyme activity-independent manner. Here we studied the induction and function of VAP-1 during human ontogeny. We show that VAP-1 is already found in the smooth muscle at embryonic week 7. There are marked time-dependent switches in VAP-1 expression in the sinusoids of the liver, in the peritubular capillaries of the kidney, in the capillaries of the heart, and in the venules in the lamina propria of the gut. Fetal VAP-1 is dimerized, and it is enzymatically active. VAP-1 in fetal-type venules is able to bind cord blood lymphocytes. Also, adenovirally transfected VAP-1 on human umbilical vein endothelial cells is involved in rolling and firm adhesion of cord blood lymphocytes under conditions of physiologic shear stress. We conclude that VAP-1 is synthesized from early on in human vessels and it is functionally intact already before birth. Thus, VAP-1 may contribute critically to the oxidase activities in utero, and prove important for lymphocyte trafficking during human ontogeny.

  6. Effect of molecular surface packing on the enzymatic activity modulation of an anchored protein on phospholipid Langmuir monolayers.

    PubMed

    Caseli, Luciano; Oliveira, Rafael G; Masui, Douglas C; Furriel, Rosa P M; Leone, Francisco A; Maggio, Bruno; Zaniquelli, M Elisabete D

    2005-04-26

    The catalytic activity of a glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase has been studied in Langmuir phospholipid monolayers at different surface pressures. The enzyme substrate, p-nitrophenyl phosphate, was injected into the subphase of mixed enzyme/lipid Langmuir monolayers. Its hydrolysis product was followed by monitoring the absorbance at 410 nm in situ in the monolayer subphase of the Langmuir trough. Several surface pressures, corresponding to different molecular surface densities, were attained by lateral compression of the monolayers. The morphology of the monolayers, observed by fluorescence microscopy, showed three different types of domains owing to the heterogeneous partition of the enzyme within the mixed enzyme/lipid film. The catalytic activity was modulated by the enzyme surface density, and it increased until a pressure of 18 mN/m was reached, but it decreased significantly when the equilibrium in-plane elasticity (surface compressional modulus) increased more noticeably, resulting in alterations in the interface morphology. A model for the modulation of the enzyme orientation and catalytic activity by lipid/enzyme surface morphology and enzyme surface packing at the air/liquid interface is proposed. The results might have an important impact on the comprehension of the enzymatic activity regulation of GPI-anchored proteins in biomembranes.

  7. Dynamics of microbiological parameters, enzymatic activities and worm biomass production during vermicomposting of effluent treatment plant sludge of bakery industry.

    PubMed

    Yadav, Anoop; Suthar, S; Garg, V K

    2015-10-01

    This paper reports the changes in microbial parameters and enzymatic activities during vermicomposting of effluent treatment plant sludge (ETPS) of bakery industry spiked with cow dung (CD) by Eisenia fetida. Six vermibins containing different ratios of ETPS and CD were maintained under controlled laboratory conditions for 15 weeks. Total bacterial and total fungal count increased upto 7th week and declined afterward in all the bins. Maximum bacterial and fungal count was 31.6 CFU × 10(6) g(-1) and 31 CFU × 10(4) g(-1) in 7th week. Maximum dehydrogenase activity was 1921 μg TPF g(-1) h(-1) in 9th week in 100 % CD containing vermibin, whereas maximum urease activity was 1208 μg NH4 (-)N g(-1) h(-1) in 3rd week in 100 % CD containing vermibin. The enzyme activity and microbial counts were lesser in ETPS containing vermibins than control (100 % CD). The growth and fecundity of the worms in different vermibins were also investigated. The results showed that initially biomass and fecundity of the worms increased but decreased at the later stages due to non-availability of the palatable feed. This showed that quality and palatability of food directly affect biological parameters of the system.

  8. The Enzymatic Activity of Drosophila AWD/NDP Kinase Is Necessary but Not Sufficient for Its Biological Function

    PubMed

    Xu; Liu; Deng; Timmons; Hersperger; Steeg; Veron; Shearn

    1996-08-01

    The Drosophila abnormal wing discs (awd) gene encodes the subunit of a protein that has nucleoside diphosphate kinase (NDP kinase) activity. Null mutations of the awd gene cause lethality after puparium formation. Larvae homozygous for such mutations have small imaginal discs, lymph glands, and brain lobes. Neither the imaginal discs nor the ovaries from such null mutant larvae are capable of further growth or normal differentiation when transplanted into suitable host larvae. This null mutant phenotype can be entirely rescued by one copy of a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA. Tissue-specific expression of AWD protein from this rescue transgene is identical to tissue-specific expression of beta-galactosidase from a reporter transgene that has the same regulatory region fused to the bacterial lac Z gene. However, this rescue transgene or reporter transgene expression pattern is only a subset of the endogenous pattern of expression detected by either in situ hybridization or immunohistochemistry. This suggests that awd is normally expressed in some tissues where it is not required. The null mutant phenotype cannot be rescued at all by a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA with a mutation that eliminates NDP kinase activity by replacement of the active site histidine with alanine. This suggests that the enzymatic activity of the AWD protein is necessary for its biological function. The human genes nm23-H1 and nm23-H2 encode NDP kinase A and B subunits, respectively. The protein subunit encoded by either human nm23 gene is 78% identical to that encoded by the Drosophila awd gene. Transgenes that have the 750-bp awd upstream regulatory DNA fused to human nm23-H2 cDNA but not to nm23-H1 cDNA can rescue the imaginal disc phenotype and the zygotic lethality caused by homozygosis for an awd null mutation as efficiently as an awd transgene. However, rescue of female

  9. Effects of point mutation on enzymatic activity: correlation between protein electronic structure and motion in chorismate mutase reaction.

    PubMed

    Ishida, Toyokazu

    2010-05-26

    Assignment of particular roles to catalytic residues is an important requirement in clearly understanding enzyme functions. Therefore, predicting the catalytic activities of mutant variants is a fundamental challenge in computational biochemistry. Although site-directed mutagenesis is widely used for studying enzymatic activities and other important classes of protein function, interpreting mutation experiments is usually difficult mainly due to side effects induced by point mutations. Because steric and, in many cases, electrostatic effects may affect the local, fine geometries conserved in wild-type proteins that are usually believed to be thermodynamically stable, simply reducing a loss in catalytic activity into clear elements is difficult. To address these important but difficult issues, we performed a systematic ab initio QM/MM computational analysis combined with MD-FEP simulations and all-electron QM calculations for the entire protein matrix. We selected chorismate mutase, one of the simplest and well-known enzymes, to discuss the details of mutational effects on the enzymatic reaction process. On the basis of the reliable free energy profiles of the wild-type enzyme and several mutant variants, we analyzed the effects of point mutations relative to electronic structure and protein dynamics. In general, changes in geometrical parameters introduced by a mutation were usually limited to the local mutational site. However, this local structural modification could affect the global protein dynamics through correlated motions of particular amino acid residues even far from the mutation site. Even for mutant reactions with low catalytic activity, transition state stabilization was observed as a result of conformational modifications and reorganization around the active site. As for the electrostatic effect created by the polar protein environment, the wild-type enzyme was most effectively designed to stabilize the transition state of the reactive substrate, and

  10. ALDH1A2 (RALDH2) genetic variation in human congenital heart disease

    PubMed Central

    2009-01-01

    Background Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus. Methods One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay. Results We describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls. Conclusion In summary, our screen indicates that ALDH1A2 genetic

  11. Longitudinal changes in PON1 enzymatic activities in Mexican-American mothers and children with different genotypes and haplotypes

    SciTech Connect

    Huen, Karen; Harley, Kim; Bradman, Asa; Eskenazi, Brenda; Holland, Nina

    2010-04-15

    The paraoxonase 1 (PON1) enzyme prevents low-density lipoprotein oxidation and also detoxifies the oxon derivatives of certain neurotoxic organophosphate (OP) pesticides. PON1 activity in infants is low compared to adults, rendering them with lower metabolic and antioxidant capacities. We made a longitudinal comparison of the role of genetic variability on control of PON1 phenotypes in Mexican-American mothers and their children at the time of delivery (n = 388 and 338, respectively) and again 7 years later (n = 280 and 281, respectively) using generalized estimating equations models. At age 7, children's mean PON1 activities were still lower than those of mothers. This difference was larger in children with genotypes associated with low PON1 activities (PON1{sub -108TT}, PON1{sub 192QQ}, and PON1{sub -909CC}). In mothers, PON1 activities were elevated at delivery and during pregnancy compared to 7 years later when they were not pregnant (p < 0.001). In non-pregnant mothers, PON1 polymorphisms and haplotypes accounted for almost 2-fold more variation of arylesterase (AREase) and chlorpyrifos-oxonase (CPOase) activity than in mothers at delivery. In both mothers and children, the five PON1 polymorphisms (192, 55, -108, -909, -162) explained a noticeably larger proportion of variance of paraoxonase activity (62-78%) than AREase activity (12.3-26.6%). Genetic control of PON1 enzymatic activity varies in children compared to adults and is also affected by pregnancy status. In addition to known PON1 polymorphisms, unidentified environmental, genetic, or epigenetic factors may also influence variability of PON1 expression and therefore susceptibility to OPs and oxidative stress.

  12. Lung cancer tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi) tumor initiating cells.

    PubMed

    Liu, Jing; Xiao, Zhijie; Wong, Sunny Kit-Man; Tin, Vicky Pui-Chi; Ho, Ka-Yan; Wang, Junwen; Sham, Mai-Har; Wong, Maria Pik

    2013-10-01

    Limited improvement in long term survival of lung cancer patients has been achieved by conventional chemotherapy or targeted therapy. To explore the potentials of tumor initiating cells (TIC)-directed therapy, it is essential to identify the cell targets and understand their maintenance mechanisms. We have analyzed the performance of ALDH/CD44 co-expression as TIC markers and treatment targets of lung cancer using well-validated in vitro and in vivo analyses in multiple established and patient-derived lung cancer cells. The ALDH(hi)CD44(hi) subset showed the highest enhancement of stem cell phenotypic properties compared to ALDH(hi)CD44(lo), ALDH(lo)CD44(hi), ALDH(lo)CD44(lo) cells and unsorted controls. They showed higher invasion capacities, pluripotency genes and epithelial-mesenchymal transition transcription factors expression, lower intercellular adhesion protein expression and higher G2/M phase cell cycle fraction. In immunosuppressed mice, the ALDH(hi)CD44(hi)xenografts showed the highest tumor induction frequency, serial transplantability, shortest latency, largest volume and highest growth rates. Inhibition of sonic Hedgehog and Notch developmental pathways reduced ALDH+CD44+ compartment. Chemotherapy and targeted therapy resulted in higher AALDH(hi)CD44(hi) subset viability and ALDH(lo)CD44(lo) subset apoptosis fraction. ALDH inhibition and CD44 knockdown led to reduced stemness gene expression and sensitization to drug treatment. In accordance, clinical lung cancers containing a higher abundance of ALDH and CD44-coexpressing cells was associated with lower recurrence-free survival. Together, results suggested theALDH(hi)CD44(hi)compartment was the cellular mediator of tumorigenicity and drug resistance. Further investigation of the regulatory mechanisms underlying ALDH(hi)CD44(hi)TIC maintenance would be beneficial for the development of long term lung cancer control.

  13. Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance.

    PubMed

    de Oliveira, Leandro C; da Silva, Viviam M; Colussi, Francieli; Cabral, Aline D; de Oliveira Neto, Mario; Squina, Fabio M; Garcia, Wanius

    2015-01-01

    Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features.

  14. Conformational Changes in a Hyperthermostable Glycoside Hydrolase: Enzymatic Activity Is a Consequence of the Loop Dynamics and Protonation Balance

    PubMed Central

    de Oliveira, Leandro C.; da Silva, Viviam M.; Colussi, Francieli; Cabral, Aline D.; de Oliveira Neto, Mario; Squina, Fabio M.; Garcia, Wanius

    2015-01-01

    Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features. PMID:25723179

  15. Common HEXB polymorphisms reduce serum HexA and HexB enzymatic activities, potentially masking Tay-Sachs disease carrier identification.

    PubMed

    Vallance, Hilary; Morris, Tara J; Coulter-Mackie, Marion; Lim-Steele, Joyce; Kaback, Michael

    2006-02-01

    A DNA-proven Tay-Sachs disease (TSD) carrier and his brother were found to have serum percent Hexosaminidase A (%HexA) enzymatic activities in the non-carrier range, while the leukocyte %HexA profiles clearly identified them as TSD heterozygotes. Both their serum HexA and HexB enzymatic activities were below reference range, suggesting inheritance of mutations in both the HEXA (alpha-subunit) and HEXB (beta-subunit) genes. DNA sequencing revealed that both individuals, carried the common HEXA 1277_1278insTATC mutation, and two common HEXB polymorphisms: [619A>G (+) delTG]. To determine if these HEXB polymorphisms reduce HexA and HexB enzymatic activities, 69 DNA samples from subjects previously screened enzymatically in both serum and leukocytes for TSD carrier status were selected for either high, mid-range or low serum Total Hex (defined as the sum of HexA and HexB) activities and were tested for the HEXB mutations. Further, three additional TSD carriers ascertained by the atypical pattern of normal serum %HexA but carrier leukocyte %HexA, were found to have the [delTG (+) 619A>G] genotype. In addition, the frequency of the [delTG (+) 619A>G] genotype was significantly higher (P < 0.01) in subjects with low serum HexB enzymatic activities. Given the high frequency of the [delTG (+) 619A>G] haplotype in the Ashkenazi Jewish population (approximately 10%), up to 10% of TSD carriers may have normal serum %HexA values with low total Hex. Accordingly, serum %HexA should not be the sole criterion used for carrier status determination. Where total Hex activity is reduced, further testing with leukocyte Hex profiles is indicated.

  16. Inherited disorders of gamma-aminobutyric acid metabolism and advances in ALDH5A1 mutation identification.

    PubMed

    Pearl, Phillip L; Parviz, Mahsa; Vogel, Kara; Schreiber, John; Theodore, William H; Gibson, K Michael

    2014-12-29

    Inherited disorders of gamma-aminobutyric acid (GABA) metabolism include succinic semialdehyde dehydrogenase (SSADH) and gamma-aminobutyric acid transaminase (GABA-T) deficiencies. The clinical features, pathophysiology, diagnosis, and management of both, and an updated list of mutations in the ALDH5A1 gene, which cause SSADH deficiency, are discussed. A database of 112 individuals (71 children and adolescents, and 41 adults) indicates that developmental delay and hypotonia are the most common symptoms arising from SSADH deficiency. Furthermore, epilepsy is present in two-thirds of SSADH-deficient individuals by adulthood. Research with murine genetic models and human participants, using [(11) C] flumazenil positron emission tomography (FMZ-PET) and transcranial magnetic stimulation, have led to therapeutic trials, and the identification of additional disruptions to GABA metabolism. Suggestions for new therapies have arisen from findings of GABAergic effects on autophagy, with enhanced activation of the mammalian target of rapamycin (mTOR) pathway. Details of known pathogenic mutations in the ALDH5A1 gene, three of which have not previously been reported, are summarized here. Investigations into disorders of GABA metabolism provide fundamental insights into the mechanisms underlying epilepsy, and support the importance of developing biomarkers and clinical trials. Comprehensive definition of phenotypes arising as a result of deficiencies in both SSADH and GABA-T may increase our understanding of the neurophysiological consequences of a hyper-GABAergic state.

  17. Non-covalent forces tune the electron transfer complex between ferredoxin and sulfite reductase to optimize enzymatic activity.

    PubMed

    Kim, Ju Yaen; Kinoshita, Misaki; Kume, Satoshi; Gt, Hanke; Sugiki, Toshihiko; Ladbury, John E; Kojima, Chojiro; Ikegami, Takahisa; Kurisu, Genji; Goto, Yuji; Hase, Toshiharu; Lee, Young-Ho

    2016-11-01

    Although electrostatic interactions between negatively charged ferredoxin (Fd) and positively charged sulfite reductase (SiR) have been predominantly highlighted to characterize complex formation, the detailed nature of intermolecular forces remains to be fully elucidated. We investigated interprotein forces for the formation of an electron transfer complex between Fd and SiR and their relationship to SiR activity using various approaches over NaCl concentrations between 0 and 400 mM. Fd-dependent SiR activity assays revealed a bell-shaped activity curve with a maximum ∼40-70 mM NaCl and a reverse bell-shaped dependence of interprotein affinity. Meanwhile, intrinsic SiR activity, as measured in a methyl viologen-dependent assay, exhibited saturation above 100 mM NaCl. Thus, two assays suggested that interprotein interaction is crucial in controlling Fd-dependent SiR activity. Calorimetric analyses showed the monotonic decrease in interprotein affinity on increasing NaCl concentrations, distinguished from a reverse bell-shaped interprotein affinity observed from Fd-dependent SiR activity assay. Furthermore, Fd:SiR complex formation and interprotein affinity were thermodynamically adjusted by both enthalpy and entropy through electrostatic and non-electrostatic interactions. A residue-based NMR investigation on the addition of SiR to (15)N-labeled Fd at the various NaCl concentrations also demonstrated that a combination of electrostatic and non-electrostatic forces stabilized the complex with similar interfaces and modulated the binding affinity and mode. Our findings elucidate that non-electrostatic forces are also essential for the formation and modulation of the Fd:SiR complex. We suggest that a complex configuration optimized for maximum enzymatic activity near physiological salt conditions is achieved by structural rearrangement through controlled non-covalent interprotein interactions.

  18. An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity.

    PubMed Central

    Graves, M C; Lim, J J; Heimer, E P; Kramer, R A

    1988-01-01

    In order to define the protease domain of human immunodeficiency virus 1, various regions of the pol open reading frame were cloned and expressed in Escherichia coli. Antiserum directed against the conserved retroviral protease active site was used to identify pol precursor and processed species containing the presumed protease domain. The smallest product that accumulates is about 11 kDa as measured by NaDodSO4/PAGE. This size agrees with that predicted from the presence in this region of two Phe-Pro sequences, which is one of the cleavage sites recognized by HIV protease. DNA encoding only the predicted 11-kDa protein was cloned, bypassing the need for autoprocessing, and the protein was expressed to a high level in E. coli. This form is active as demonstrated by its ability to specifically cleave protease-deficient pol protein in vivo in E. coli. Extracts of E. coli containing the 11-kDa protease also process human immunodeficiency virus gag substrates in vitro. These results demonstrate that the 11-kDa protease is sufficient for enzymatic activity and are consistent with a major role for this form in virus maturation. Images PMID:3282230

  19. The effect of ultraviolet treatment on enzymatic activity and total phenolic content of minimally processed potato slices.

    PubMed

    Teoh, Li Shing; Lasekan, Ola; Adzahan, Noranizan Mohd; Hashim, Norhashila

    2016-07-01

    In this work, potato slices were exposed to different doses of UV-C irradiation (i.e. 2.28, 6.84, 11.41, and 13.68 kJ m(-2)) with or without pretreatment [i.e. ascorbic acid and calcium chloride (AACCl) dip] and stored at 4 ± 1 °C. Changes in enzymatic activities of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase (PAL), as well as total phenolic content (TPC) were investigated after 0, 3, 7 and 10 days of storage. Results showed that untreated and UV-C treated potato slices at 13.68 kJ m(-2) dosage level showed significantly higher PPO, POD and PAL activities. Conversely, untreated potato slices showed the lowest TPC during storage period. Potato slices subjected to AACCl dip plus UV-C at 6.84 kJ m(-2) produced lower PPO, POD and PAL activities, as well as maintained a high TPC during storage.

  20. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

    SciTech Connect

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

    2015-04-22

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

  1. Intramolecular long-distance nucleophilic reactions as a rapid fluorogenic switch applicable to the detection of enzymatic activity.

    PubMed

    Baba, Reisuke; Hori, Yuichiro; Kikuchi, Kazuya

    2015-03-16

    Long-distance intramolecular nucleophilic reactions are promising strategies for the design of fluorogenic probes to detect enzymatic activity involved in lysine modifications. However, such reactions have been challenging and hence have not been established. In this study, we have prepared fluorogenic peptides that induce intramolecular reactions between lysine nucleophiles and electrophiles in distal positions. These peptides contain a lysine and fluorescence-quenched fluorophore with a carbonate ester, which triggers nucleophilic transesterification resulting in fluorogenic response. Transesterification occurred under mild aqueous conditions despite the presence of a long nine-amino-acid spacer between the lysine and fluorophore. In addition, one of the peptides showed the fastest reaction kinetics with a half-life time of 3.7 min. Furthermore, the incorporation of this fluorogenic switch into the probes allowed rapid fluorogenic detection of histone deacetylase (HDAC) activity. These results indicate that the transesterification reaction has great potential for use as a general fluorogenic switch to monitor the activity of lysine-targeting enzymes.

  2. Overexpression of ALDH10A8 and ALDH10A9 Genes Provides Insight into Their Role in Glycine Betaine Synthesis and Affects Primary Metabolism in Arabidopsis thaliana.

    PubMed

    Missihoun, Tagnon D; Willée, Eva; Guegan, Jean-Paul; Berardocco, Solenne; Shafiq, Muhammad R; Bouchereau, Alain; Bartels, Dorothea

    2015-09-01

    Betaine aldehyde dehydrogenases oxidize betaine aldehyde to glycine betaine in species that accumulate glycine betaine as a compatible solute under stress conditions. In contrast, the physiological function of betaine aldehyde dehydrogenase genes is at present unclear in species that do not accumulate glycine betaine, such as Arabidopsis thaliana. To address this question, we overexpressed the Arabidopsis ALDH10A8 and ALDH10A9 genes, which were identified to code for betaine aldehyde dehydrogenases, in wild-type A. thaliana. We analysed changes in metabolite contents of transgenic plants in comparison with the wild type. Using exogenous or endogenous choline, our results indicated that ALDH10A8 and ALDH10A9 are involved in the synthesis of glycine betaine in Arabidopsis. Choline availability seems to be a factor limiting glycine betaine synthesis. Moreover, the contents of diverse metabolites including sugars (glucose and fructose) and amino acids were altered in fully developed transgenic plants compared with the wild type. The plant metabolic response to salt and the salt stress tolerance were impaired only in young transgenic plants, which exhibited a delayed growth of the seedlings early after germination. Our results suggest that a balanced expression of the betaine aldehyde dehydrogenase genes is important for early growth of A. thaliana seedlings and for salt stress mitigation in young seedlings.

  3. Anti-diabetic activity of beta-glucans and their enzymatically hydrolyzed oligosaccharides from Agaricus blazei.

    PubMed

    Kim, Yea-Woon; Kim, Ki-Hoon; Choi, Hyun-Ju; Lee, Dong-Seok

    2005-04-01

    Beta-glucans were prepared from Agaricus blazei Murill by repeated extraction with hot water. The average molecular weights of beta-glucans were 30-50 kDa by gel filtration chromatography. Oligosaccharides (AO), derived from hydrolyzing beta-glucans with an endo-beta-(1-->6)-glucanase from Bacillus megaterium, were mainly di- and tri-saccharides. Though beta-glucans and AO both showed anti-hyperglycemic, anti-hypertriglyceridemic, anti-hypercholesterolemic, and anti-arteriosclerotic activity indicating overall anti-diabetic activity in diabetic rats, AO had about twice the activity of beta-glucans with respect to anti-diabetic activity.

  4. ALDH2 polymorphism and alcohol-related cancers in Asians: a public health perspective.

    PubMed

    Chang, Jeffrey S; Hsiao, Jenn-Ren; Chen, Che-Hong

    2017-03-03

    The occurrence of more than 200 diseases, including cancer, can be attributed to alcohol drinking. The global cancer deaths attributed to alcohol-consumption rose from 243,000 in 1990 to 337,400 in 2010. In 2010, cancer deaths due to alcohol consumption accounted for 4.2% of all cancer deaths. Strong epidemiological evidence has established the causal role of alcohol in the development of various cancers, including esophageal cancer, head and neck cancer, liver cancer, breast cancer, and colorectal cancer. The evidence for the association between alcohol and other cancers is inconclusive. Because of the high prevalence of ALDH2*2 allele among East Asian populations, East Asians may be more susceptible to the carcinogenic effect of alcohol, with most evidence coming from studies of esophageal cancer and head and neck cancer, while data for other cancers are more limited. The high prevalence of ALDH2*2 allele in East Asian populations may have important public health implications and may be utilized to reduce the occurrence of alcohol-related cancers among East Asians, including: 1) Identification of individuals at high risk of developing alcohol-related cancers by screening for ALDH2 polymorphism; 2) Incorporation of ALDH2 polymorphism screening into behavioral intervention program for promoting alcohol abstinence or reducing alcohol consumption; 3) Using ALDH2 polymorphism as a prognostic indicator for alcohol-related cancers; 4) Targeting ALDH2 for chemoprevention; and 5) Setting guidelines for alcohol consumption among ALDH2 deficient individuals. Future studies should evaluate whether these strategies are effective for preventing the occurrence of alcohol-related cancers.

  5. Immunohistochemical Expression of CD56 and ALDH1 in Common Salivary Gland Tumors

    PubMed Central

    Seifi, Safoura; Seyedmajidi, Maryam; Salehinejad, Jahanshah; Gholinia, Hemmat; Aliakbarpour, Fatemeh

    2016-01-01

    Introduction: Natural killer (NK) cells, of which CD56 is a specific marker, play an important role in host defense against tumors. Cancer stem cells, of which aldehyde dehydrogenase isoform 1 (ALDH1) is an immunohistochemical marker, are a group of tumorigenic cells which are involved in migration and tumor recurrences. We aimed to evaluate the expression of ALDH1 and CD56 in common salivary gland tumors, as well as their relationship with each other and with a number of clinicopathologic factors. Materials and Methods: Forty-five paraffin blocks of salivary gland tumors (pleomorphic adenoma, mucoepidermoid carcinoma and adenoid cystic carcinoma, 15 samples each) were selected. Malignant tumors were classified into two groups: low-grade (including mucoepidermoid carcinoma grade I) and high-grade (including mucoepidermoid carcinoma grade III and adenoid cystic carcinoma). Immunohistochemical staining for ALDH1 and CD56 markers was performed. Data were analyzed using SPSS (20) and the Chi-square test. Results: CD56 expression was significantly higher in benign and high-grade malignant tumors (P=0.01). ALDH1 overexpressed in all three salivary tumors, but not to statistically significant degree (P=0.54). There was no statistically significant correlation between ALDH1 and CD56 expression with demographic factors (age, gender, or location of tumor; P>0.05). Conclusion: It appears that the number of NK cells and their function change in different types of salivary gland tumors (benign/malignant) and stroma. NK cells are important components of the anti-tumor system; therefore immune dysfunction is associated with tumor progression in tumors of the salivary gland. ALDH1 overexpression suggests its role in tumorogenesis, but ALDH1 is not involved in the morphogenesis of salivary gland tumors. PMID:28008389

  6. Seasonal Dynamics of Enzymatic Activities and Functional Diversity in Soils under Different Organic Management

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil microbial activity and diversity fluctuate seasonally under annual organic amendment for improving soil quality. We investigated the effects of municipal compost (MC), poultry litter (PL), and cover crops of spring oats and red clover (RC) on soil enzyme activities, and soil bacterial community...

  7. Enzymatic characterizations and activity regulations of N-acetyl-β-D-glucosaminidase from the spermary of Nile tilapia (Oreochromis niloticus).

    PubMed

    Zhang, Wei-Ni; Bai, Ding-Ping; Huang, Yi-Fan; Hu, Chong-Wei; Chen, Qing-Xi; Huang, Xiao-Hong

    2014-02-01

    N-Acetyl-β-D-glucosaminidase (NAGase) is proved to be correlated with reproduction of male animals. In this study, enzymatic characterizations of NAGase from spermary of Nile tilapia (Oreochromis niloticus) were investigated in order to further study its reproductive function in fish. Tilapia NAGase was purified to be PAGE homogeneous by the following techniques: (NH4)2SO4 fractionation (40-55%), DEAE-cellulose (DE-32) ion exchange chromatography, Sephadex G-200 gel filtration and DEAE-Sephadex (A-50). The specific activity of the purified enzyme was 4100 U/mg. The enzyme molecular weight was estimated as 118.0 kD. Kinetic studies showed that the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) by the enzyme followed Michaelis-Menten kinetics. The Michaelis-Menten constant (Km) and maximum velocity (Vm) were determined to be 0.67 mM and 23.26 μM/min, respectively. The optimum pH and optimum temperature of the enzyme for hydrolysis of pNP-NAG was to be at pH 5.7 and 55°C, respectively. The enzyme was stable in a pH range from 3.3 to 8.1 at 37°C, and inactive at temperature above 45°C. The enzyme activity was regulated by the following ions in decreasing order: Hg(2+) > Zn(2+) > Cu(2+) > Pb(2+) > Mn(2+). The IC50 of Cu(2+), Zn(2+) and Hg(2+) was 1.23, 0.28, and 0.0027 mM, respectively. However, the ions Li(+), Na(+), K(+), Mg(2+) and Ca(2+) had almost no influence on enzyme activity. In conclusion, the enzymatic characterizations of NAGase from tilapia were special to the other animals, which were correlated with its living habit; besides, CuSO4 and ZnSO4 should used very carefully as insecticides in tilapia cultivation since they both had strong regulations on the enzyme.

  8. Aldehyde PEGylation of laccase from Trametes versicolor in route to increase its stability: effect on enzymatic activity.

    PubMed

    Mayolo-Deloisa, Karla; González-González, Mirna; Simental-Martínez, Jesús; Rito-Palomares, Marco

    2015-03-01

    Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic compounds. Laccase can be used in bioremediation, beverage (wine, fruit juice, and beer) processing, ascorbic acid determination, sugar beet pectin gelation baking, and as a biosensor. Recently, the antiproliferative activity of laccase toward tumor cells has been reported. Because of the potential applications of this enzyme, the efforts for enhancing and stabilizing its activity have increased. Thus, the PEGylation of laccase can be an alternative. PEGylation is the covalent attachment of one or more molecules of methoxy poly(ethylene glycol) (mPEG) to a protein. Normally, during the PEGylation reaction, the activity is reduced but the stability increases; thus, it is important to minimize the loss of activity. In this work, the effects of molar ratio (1:4, 1:8, and 1:12), concentration of laccase (6 and 12 mg/ml), reaction time (4 and 17 h), molecular weight, and type of mPEG (20, 30, 40 kDa and 40 kDa-branched) were analyzed. The activity was measured using three substrates: ABTS, 2,6-dimethoxyphenol, and syringaldazine. The best conditions for laccase PEGylation were 12 mg/ml of laccase, molar ratio 1:4, and 4 h reaction time. Under these conditions, the enzyme was able to maintain nearly 100% of its enzymatic activity with ABTS. The PEGylation of laccase has not been extensively explored, so it is important to analyze the effects of this bioconjugation in route to produce a robust modified enzyme.

  9. Plasmon-Enhanced Enzymatic Reactions 2:Optimization of Enzyme Activity by Surface Modification of Silver Island Films with Biotin-Poly (Ethylene-glycol)-Amine.

    PubMed

    Abel, Biebele; Aslan, Kadir

    2012-01-01

    Surface modification of silver island films (SIFs) was carried out with Biotin-Poly (Ethylene-glycol)-Amine (BEA), which acts as a cross-linker between the silver surface and horse radish peroxidase (HRP) enzyme for optimum plasmon-enhanced enzymatic activity. SIFs-deposited blank glass slides and SIFs-deposited 3-Aminopropyltriethoxysilane(APTES)-coated glass slides were used as our plasmonic surfaces.In this regard, three different extent of loading of SIFs were also prepared (low, medium and high) on APTES-coated glass slides. Streptavidin-linked HRP enzyme was attached to SIFs-deposited blank glass slides and SIFs-deposited APTES-coated glass slides through the well-known biotin-streptavidin interactions. The characterization of these surfaces was done using optical absorption spectroscopy. The loading of SIFs on glass slides was observed to have significant effect on the efficiency of plasmon-enhanced enzymatic activity, where an enhancement of 200% in the enzymatic activity was observed when compared to our previously used strategies for enzyme immobilization in our preceding work[1]. In addition, SIFs-deposited on APTES-coated glass slides were found to be re-usable for plasmon-enhanced enzymatic reactions unlike SIFs deposited on to blank glass slides.

  10. Sam68 promotes self-renewal and glycolytic metabolism in mouse neural progenitor cells by modulating Aldh1a3 pre-mRNA 3'-end processing

    PubMed Central

    La Rosa, Piergiorgio; Bielli, Pamela; Compagnucci, Claudia; Cesari, Eleonora; Volpe, Elisabetta; Farioli Vecchioli, Stefano; Sette, Claudio

    2016-01-01

    The balance between self-renewal and differentiation of neural progenitor cells (NPCs) dictates neurogenesis and proper brain development. We found that the RNA- binding protein Sam68 (Khdrbs1) is strongly expressed in neurogenic areas of the neocortex and supports the self-renewing potential of mouse NPCs. Knockout of Khdrbs1 constricted the pool of proliferating NPCs by accelerating their cell cycle exit and differentiation into post-mitotic neurons. Sam68 function was linked to regulation of Aldh1a3 pre-mRNA 3'-end processing. Binding of Sam68 to an intronic polyadenylation site prevents its recognition and premature transcript termination, favoring expression of a functional enzyme. The lower ALDH1A3 expression and activity in Khdrbs1-/- NPCs results in reduced glycolysis and clonogenicity, thus depleting the embryonic NPC pool and limiting cortical expansion. Our study identifies Sam68 as a key regulator of NPC self-renewal and establishes a novel link between modulation of ALDH1A3 expression and maintenance of high glycolytic metabolism in the developing cortex. DOI: http://dx.doi.org/10.7554/eLife.20750.001 PMID:27845622

  11. Methods, microfluidic devices, and systems for detection of an active enzymatic agent

    DOEpatents

    Sommer, Gregory J; Hatch, Anson V; Singh, Anup K; Wang, Ying-Chih

    2014-10-28

    Embodiments of the present invention provide methods, microfluidic devices, and systems for the detection of an active target agent in a fluid sample. A substrate molecule is used that contains a sequence which may cleave in the presence of an active target agent. A SNAP25 sequence is described, for example, that may be cleaved in the presence of Botulinum Neurotoxin. The substrate molecule includes a reporter moiety. The substrate molecule is exposed to the sample, and resulting reaction products separated using electrophoretic separation. The elution time of the reporter moiety may be utilized to identify the presence or absence of the active target agent.

  12. Influence of the isolation procedure on coriander leaf volatiles with some correlation to the enzymatic activity.

    PubMed

    To Quynh, Cung Thi; Iijima, Yoko; Kubota, Kikue

    2010-01-27

    Coriander leaves (Coriandrum sativum L.) have become popular worldwide because of their pleasant and delicate aroma. By a hot water extraction method, in which coriander leaves were cut before suspending in boiling water for 2 min, the contents of the main volatile compounds such as alkanals and 2-alkenals from C10 to C14 decreased, while the levels of corresponding alcohols increased in comparison to those obtained by solvent extraction. To investigate the reasons for this variation, an enzyme activity was assayed. By using aliphatic aldehyde as a substrate and NADPH as a coenzyme, strong activity of an aliphatic aldehyde reductase was found for the first time in this herb in the relatively wide pH range of 5.0-9.0, with the maximum activity at pH 8.5. Additionally, the aliphatic aldehyde dehydrogenase, responsible for acid formation, was also found to have a relatively weak activity compared to that of reductase.

  13. A methodology for preparing nanostructured biomolecular interfaces with high enzymatic activity

    NASA Astrophysics Data System (ADS)

    Wong, Lu Shin; Karthikeyan, Chinnan V.; Eichelsdoerfer, Daniel J.; Micklefield, Jason; Mirkin, Chad A.

    2012-01-01

    The development of a novel method for functionalizing nanopatterned surfaces with catalytically active proteins is reported. This method involves using dip-pen nanolithography (DPN) and polymer pen lithography (PPL) to generate nanoscale patterns of coenzyme A, followed by a phosphopantetheinyl transferase-mediated coupling between coenzyme A and proteins fused to the ybbR-tag. By exploiting the ability to generate protein features over large areas afforded by DPN and PPL, it was now possible to measure protein activity directly on these surfaces. It was found that proteins immobilized on the nanoscale features not only display higher activity per area with decreasing feature size, but are also robust and can be used for repeated catalytic cycles. The immobilization method is applicable to a variety of proteins and gives rise to superior activity compared to proteins attached in random orientations on the surface.

  14. Gene expression and enzymatic activity of pectin methylesterase during fruit development and ripening in Coffea arabica L.

    PubMed

    Cação, S M B; Leite, T F; Budzinski, I G F; dos Santos, T B; Scholz, M B S; Carpentieri-Pipolo, V; Domingues, D S; Vieira, L G E; Pereira, L F P

    2012-09-03

    Coffee quality is directly related to the harvest and post harvest conditions. Non-uniform maturation of coffee fruits, combined with inadequate harvest, negatively affects the final quality of the product. Pectin methylesterase (PME) plays an important role in fruit softening due to the hydrolysis of methylester groups in cell wall pectins. In order to characterize the changes occurring during coffee fruit maturation, the enzymatic activity of PME was measured during different stages of fruit ripening. PME activity progressively increased from the beginning of the ripening process to the cherry fruit stage. In silico analysis of expressed sequence tags of the Brazilian Coffee Genome Project database identified 5 isoforms of PME. We isolated and cloned a cDNA homolog of PME for further characterization. CaPME4 transcription was analyzed in pericarp, perisperm, and endosperm tissues during fruit development and ripening as well as in other plant tissues. Northern blot analysis revealed increased transcription of CaPME4 in the pericarp 300 days after flowering. Low levels of CaPME4 mRNAs were observed in the endosperm 270 days after flowering. Expression of CaPME4 transcripts was strong in the branches and lower in root and flower tissues. We showed that CaPME4 acts specifically during the later stages of fruit ripening and possibly contributes to the softening of coffee fruit, thus playing a significant role in pectin degradation in the fruit pericarp.

  15. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

    PubMed Central

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  16. Cell-free extracellular enzymatic activity is linked to seasonal temperature changes: a case study in the Baltic Sea

    NASA Astrophysics Data System (ADS)

    Baltar, Federico; Legrand, Catherine; Pinhassi, Jarone

    2016-05-01

    Extracellular enzymatic activities (EEAs) are a crucial step in the degradation of organic matter. Dissolved (cell-free) extracellular enzymes in seawater can make up a significant contribution of the bulk EEA. However, the factors controlling the proportion of dissolved EEA in the marine environment remain unknown. Here we studied the seasonal changes in the proportion of dissolved relative to total EEA (of alkaline phosphatase (APase), β-glucosidase (BGase), and leucine aminopeptidase (LAPase)), in the Baltic Sea for 18 months. The proportion of dissolved EEA ranged between 37 and 100, 0 and 100, and 34 and 100 % for APase, BGase, and LAPase, respectively. A consistent seasonal pattern in the proportion of dissolved EEA was found among all the studied enzymes, with values up to 100 % during winter and < 40 % during summer. A significant negative relation was found between the proportion of dissolved EEA and temperature, indicating that temperature might be a critical factor controlling the proportion of dissolved relative to total EEA in marine environments. Our results suggest a strong decoupling of hydrolysis rates from microbial dynamics in cold waters. This implies that under cold conditions, cell-free enzymes can contribute to substrate availability at large distances from the producing cell, increasing the dissociation between the hydrolysis of organic compounds and the actual microbes producing the enzymes. This might also suggest a potential effect of global warming on the hydrolysis of organic matter via a reduction of the contribution of cell-free enzymes to the bulk hydrolytic activity.

  17. Nanonets Derived from Turnip Mosaic Virus as Scaffolds for Increased Enzymatic Activity of Immobilized Candida antarctica Lipase B.

    PubMed

    Cuenca, Sol; Mansilla, Carmen; Aguado, Marta; Yuste-Calvo, Carmen; Sánchez, Flora; Sánchez-Montero, Jose M; Ponz, Fernando

    2016-01-01

    Elongated flexuous plant viral nanoparticles (VNPs) represent an interesting platform for developing different applications in nanobiotechnology. In the case of potyviruses, the virion external surface is made up of helically arrayed domains of the viral structural coat protein (CP), repeated over 2000 times, in which the N- and C-terminal domains of each CP are projected toward the exterior of the external virion surface. These characteristics provide a chemical environment rich in functional groups susceptible to chemical conjugations. We have conjugated Candida antarctica lipase B (CALB) onto amino groups of the external surface of the potyvirus turnip mosaic virus (TuMV) using glutaraldehyde as a conjugating agent. Using this approach, TuMV virions were transformed into scaffolds for CALB nanoimmobilization. Analysis of the resulting structures revealed the formation of TuMV nanonets onto which large CALB aggregates were deposited. The functional enzymatic characterization of the CALB-bearing TuMV nanonets showed that CALB continued to be active in the nanoimmobilized form, even gaining an increased relative specific activity, as compared to the non-immobilized form. These novel virus-based nanostructures may provide a useful new approach to enzyme nanoimmobilization susceptible to be industrially exploited.

  18. Quantifying protein adsorption and function at nanostructured materials: enzymatic activity of glucose oxidase at GLAD structured electrodes.

    PubMed

    Jensen, Uffe B; Ferapontova, Elena E; Sutherland, Duncan S

    2012-07-31

    Nanostructured materials strongly modulate the behavior of adsorbed proteins; however, the characterization of such interactions is challenging. Here we present a novel method combining protein adsorption studies at nanostructured quartz crystal microbalance sensor surfaces (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical methods (cyclic voltammetry CV) allowing quantification of both bound protein amount and activity. The redox enzyme glucose oxidase is studied as a model system to explore alterations in protein functional behavior caused by adsorption onto flat and nanostructured surfaces. This enzyme and such materials interactions are relevant for biosensor applications. Novel nanostructured gold electrode surfaces with controlled curvature were fabricated using colloidal lithography and glancing angle deposition (GLAD). The adsorption of enzyme to nanostructured interfaces was found to be significantly larger compared to flat interfaces even after normalization for the increased surface area, and no substantial desorption was observed within 24 h. A decreased enzymatic activity was observed over the same period of time, which indicates a slow conformational change of the adsorbed enzyme induced by the materials interface. Additionally, we make use of inherent localized surface plasmon resonances in these nanostructured materials to directly quantify the protein binding. We hereby demonstrate a QCM-D-based methodology to quantify protein binding at complex nanostructured materials. Our approach allows label free quantification of protein binding at nanostructured interfaces.

  19. Label-free electrochemical detection of botulinum neurotoxin type E based on its enzymatic activity using interdigitated electrodes

    NASA Astrophysics Data System (ADS)

    Hyun, Sang Hwa; Park, Dae Keun; Kang, Aeyeon; Kim, Soohyun; Kim, Daehee; Shin, Yu Mi; Song, Ji-Joon; Yun, Wan Soo

    2016-02-01

    We report a simple label-free electrochemical method of detecting low concentrations of botulinum neurotoxin type E light chain (BoNT/E LC) based on its peptide cleavage activity. Dual-mode cyclic voltammetry was employed to observe changes in the redox signal of ferri-/ferro-cyanide on interdigitated microelectrodes, whose surfaces were covered by peptides designed from synaptosomal-associated protein 25 to be cleaved by BoNT/E LC. With the introduction of BoNT/E LC, the redox signal showed a time-dependent increase due to cleavage of the immobilized peptide molecules. In addition to the increased redox signal intensity, its time-dependence can be considered as a strong evidence of BoNT/E sensing, since the time-dependent increase can only result from the enzymatic activity of BoNT/E LC. Using this method, BoNT/E LC, at concentrations as low as 5 pg/ml, was readily measurable with only an hour of incubation.

  20. Nanonets Derived from Turnip Mosaic Virus as Scaffolds for Increased Enzymatic Activity of Immobilized Candida antarctica Lipase B

    PubMed Central

    Cuenca, Sol; Mansilla, Carmen; Aguado, Marta; Yuste-Calvo, Carmen; Sánchez, Flora; Sánchez-Montero, Jose M.; Ponz, Fernando

    2016-01-01

    Elongated flexuous plant viral nanoparticles (VNPs) represent an interesting platform for developing different applications in nanobiotechnology. In the case of potyviruses, the virion external surface is made up of helically arrayed domains of the viral structural coat protein (CP), repeated over 2000 times, in which the N- and C-terminal domains of each CP are projected toward the exterior of the external virion surface. These characteristics provide a chemical environment rich in functional groups susceptible to chemical conjugations. We have conjugated Candida antarctica lipase B (CALB) onto amino groups of the external surface of the potyvirus turnip mosaic virus (TuMV) using glutaraldehyde as a conjugating agent. Using this approach, TuMV virions were transformed into scaffolds for CALB nanoimmobilization. Analysis of the resulting structures revealed the formation of TuMV nanonets onto which large CALB aggregates were deposited. The functional enzymatic characterization of the CALB-bearing TuMV nanonets showed that CALB continued to be active in the nanoimmobilized form, even gaining an increased relative specific activity, as compared to the non-immobilized form. These novel virus-based nanostructures may provide a useful new approach to enzyme nanoimmobilization susceptible to be industrially exploited. PMID:27148295

  1. Enzymatic activity and thermal stability of metallo proteins in hydrated ionic liquids.

    PubMed

    Fujita, Kyoko; Ohno, Hiroyuki

    2010-12-01

    Hydrated choline dihydrogen phosphate (Hy[ch][dhp]) containing 30 wt% water was investigated as a novel protein solvent. The Hy[ch][dhp] dissolved some metallo proteins (cytochrome c, peroxidase, ascorbate oxidase, azurin, pseudoazurin and fructose dehydrogenase) without any modification. These proteins retained the surroundings of the active site after dissolution in Hy[ch][dhp]. Some metallo proteins were found to retain their activity in the Hy[ch][dhp].

  2. Absorption of enzymatically active sup 125 I-labeled bovine milk xanthine oxidase fed to rabbits

    SciTech Connect

    Rzucidlo, S.J. ); Zikakis, J.P. )

    1990-05-01

    Rabbits fed a regular laboratory diet supplemented with a high-fat milk containing xanthine oxidase (XO) were studied to determine the presence of active XO in the blood. A pilot feeding study, where rabbits consumed a high-fat diet containing xanthine oxidase, showed a correlation between dairy food consumption and XO activity in the blood. Antibody to dietary XO was also found. In a second study, rabbits were fed ad libitum the high-fat milk and blood serum samples were tested weekly for XO activity. No elevation in serum XO activity was found. A third study showed that serum XO activity was increased when rabbits were force fed the high-fat milk. The final study consisted of force feeding {sup 125}I-labeled XO to one rabbit to ascertain whether the observed increase in serum XO was due to dietary or endogenous XO. Isoelectric focusing of sera collected from the test rabbit strongly suggested that at least a portion of the serum XO contained the radioactive label. This is the first direct evidence showing the uptake of dietary active XO from the gut.

  3. Effects of various salts on the steady-state enzymatic activity of E. coli alkaline phosphatase.

    PubMed

    Poe, R W; Sangadala, V S; Brewer, J M

    1993-05-15

    Seventeen salts were tested at various concentrations for their effects on E. coli alkaline phosphatase steady-state activity. Three effects were distinguished: a general ionic strength effect, and weaker cation and anion effects. 1. All salts tested, including those with "noninteracting" cations and anions, stimulate alkaline phosphatase activity usually ca. 100% at moderate (0.05-0.3 M) concentrations. 2. Cations such as Na+ and Li+ produce further increases in activity at concentrations up to 1 M. The noninteracting cations tetramethylammonium and tetrapropylammonium produce lower activities at these concentrations. These do not provide the secondary stimulatory effect of cations such as Na+ or Li+. 3. Anions associated with greater "salting in" effectiveness such as thiocyanate also reduce activity at ca. 1 M concentrations. These latter effects are not dependent on protein concentration so they probably do not involve subunit dissociation. There is little effect on the fluorescence or fluorescence-polarization spectrum of the enzyme so there is no general effect of 1 M salts on the conformation of the protein. The Michaelis constant for the substrate, p-nitrophenylphosphate, and inhibition constant for inorganic phosphate are increased to some extent by salts, but the increase in activity is due to an increase in Vmax. Our working hypothesis is that increased ionic strength weakens electrostatic interactions, enabling noncovalently bound phosphate to dissociate more rapidly.

  4. Development of APE1 enzymatic DNA repair assays: low APE1 activity is associated with increase lung cancer risk.

    PubMed

    Sevilya, Ziv; Leitner-Dagan, Yael; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Lejbkowicz, Flavio; Rennert, Hedy S; Freedman, Laurence S; Rennert, Gad; Paz-Elizur, Tamar; Livneh, Zvi

    2015-09-01

    The key role of DNA repair in removing DNA damage and minimizing mutations makes it an attractive target for cancer risk assessment and prevention. Here we describe the development of a robust assay for apurinic/apyrimidinic (AP) endonuclease 1 (APE1; APEX1), an essential enzyme involved in the repair of oxidative DNA damage. APE1 DNA repair enzymatic activity was measured in peripheral blood mononuclear cell protein extracts using a radioactivity-based assay, and its association with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean APE1 enzyme activity in case patients was 691 [95% confidence interval (CI) = 655-727] units/ng protein, significantly lower than in control subjects (mean = 793, 95% CI = 751-834 units/ng protein, P = 0.0006). The adjusted odds ratio for lung cancer associated with 1 SD (211 units) decrease in APE1 activity was 2.0 (95% CI = 1.3-3.1; P = 0.002). Comparison of radioactivity- and fluorescence-based assays showed that the two are equivalent, indicating no interference by the fluorescent tag. The APE1Asp148Glu SNP was associated neither with APE1 enzyme activity nor with lung cancer risk. Taken together, our results indicate that low APE1 activity is associated with lung cancer risk, consistent with the hypothesis that 'bad DNA repair', rather than 'bad luck', is involved in cancer etiology. Such assays may be useful, along with additional DNA repair biomarkers, for risk assessment of lung cancer and perhaps other cancers, and for selecting individuals to undergo early detection techniques such as low-dose CT.

  5. Critical Role of the Secondary Binding Pocket in Modulating the Enzymatic Activity of DUSP5 toward Phosphorylated ERKs.

    PubMed

    Talipov, Marat R; Nayak, Jaladhi; Lepley, Michael; Bongard, Robert D; Sem, Daniel S; Ramchandran, Ramani; Rathore, Rajendra

    2016-11-08

    DUSP5 is an inducible nuclear dual-specificity phosphatase that specifically interacts with and deactivates extracellular signal-regulated kinases ERK1 and ERK2, which are responsible for cell proliferation, differentiation, and survival. The phosphatase domain (PD) of DUSP5 has unique structural features absent from other nuclear DUSPs, such as the presence of a secondary anion-binding site in the proximity of the reaction center and a glutamic acid E264 positioned next to the catalytic cysteine C263, as well as a remote intramolecular disulfide linkage. The overall 400 ns molecular dynamics simulations indicate that the secondary binding site of DUSP5 PD acts as an allosteric regulator of the phosphatase activity of DUSP5. Our studies have identified E264 as a critical constituent of the dual binding pocket, which regulates the catalytic activity of DUSP5 by forming a salt bridge with arginine R269. Molecular dynamics studies showed that initial occupation of the secondary binding pocket leads to the breakage of the salt bridge, which then allows the occupation of the active site. Indeed, biochemical analysis using the pERK assay on mutant E264Q demonstrated that mutation of glutamic acid E264 leads to an increase in the DUSP5 catalytic activity. The role of the secondary binding site in assembling the DUSP5-pERK pre-reactive complex was further demonstrated by molecular dynamics simulations that showed that the remote C197-C219 disulfide linkage controls the structure of the secondary binding pocket based on its redox state (i.e., disulfide/dithiol) and, in turn, the enzymatic activity of DUSP5.

  6. Aldehyde dehydrogenase 2 activation in aged heart improves the autophagy by reducing the carbonyl modification on SIRT1.

    PubMed

    Wu, Bing; Yu, Lu; Wang, Yishi; Wang, Hongtao; Li, Chen; Yin, Yue; Yang, Jingrun; Wang, Zhifa; Zheng, Qiangsun; Ma, Heng

    2016-01-19

    Cardiac aging is characterized by accumulation of damaged proteins and decline of autophagic efficiency. Here, by forestalling SIRT1 carbonylated inactivation in aged heart, we determined the benefits of activation of aldehyde dehydrogenase 2 (ALDH2) on the autophagy. In this study, the ALDH2 KO mice progressively developed age-related heart dysfunction and showed reduction in the life span, which strongly suggests that ALDH2 ablation leads to cardiac aging. What's more, aged hearts displayed a significant decrease ALDH2 activity, resulting in accumulation of 4-HNE-protein adducts and protein carbonyls, impairment in the autophagy flux, and, consequently, deteriorated cardiac function after starvation. Sustained Alda-1 (selective ALDH2 activator) treatment increased cardiac ALDH2 activity and abrogated these effects. Using SIRT1 deficient heterozygous (Sirt1+/-) mice, we found that SIRT1 was necessary for ALDH2 activation-induced autophagy. We further demonstrated that ALDH2 activation attenuated SIRT1 carbonylation and improved SIRT1 activity, thereby increasing the deacetylation of nuclear LC3 and FoxO1. Sequentially, ALDH2 enhanced SIRT1 regulates LC3-Atg7 interaction and FoxO1 increased Rab7 expression, which were both necessary and sufficient for restoring autophagy flux. These results highlight that both accumulation of proteotoxic carbonyl stress linkage with autophagy decline contribute to heart senescence. ALDH2 activation is adequate to improve the autophagy flux by reducing the carbonyl modification on SIRT1, which in turn plays an important role in maintaining cardiac health during aging.

  7. Snapshots of enzymatic Baeyer-Villiger catalysis: oxygen activation and intermediate stabilization.

    PubMed

    Orru, Roberto; Dudek, Hanna M; Martinoli, Christian; Torres Pazmiño, Daniel E; Royant, Antoine; Weik, Martin; Fraaije, Marco W; Mattevi, Andrea

    2011-08-19

    Baeyer-Villiger monooxygenases catalyze the oxidation of carbonylic substrates to ester or lactone products using NADPH as electron donor and molecular oxygen as oxidative reactant. Using protein engineering, kinetics, microspectrophotometry, crystallography, and intermediate analogs, we have captured several snapshots along the catalytic cycle which highlight key features in enzyme catalysis. After acting as electron donor, the enzyme-bound NADP(H) forms an H-bond with the flavin cofactor. This interaction is critical for stabilizing the oxygen-activating flavin-peroxide intermediate that results from the reaction of the reduced cofactor with oxygen. An essential active-site arginine acts as anchoring element for proper binding of the ketone substrate. Its positively charged guanidinium group can enhance the propensity of the substrate to undergo a nucleophilic attack by the flavin-peroxide intermediate. Furthermore, the arginine side chain, together with the NADP(+) ribose group, forms the niche that hosts the negatively charged Criegee intermediate that is generated upon reaction of the substrate with the flavin-peroxide. The fascinating ability of Baeyer-Villiger monooxygenases to catalyze a complex multistep catalytic reaction originates from concerted action of this Arg-NADP(H) pair and the flavin subsequently to promote flavin reduction, oxygen activation, tetrahedral intermediate formation, and product synthesis and release. The emerging picture is that these enzymes are mainly oxygen-activating and "Criegee-stabilizing" catalysts that act on any chemically suitable substrate that can diffuse into the active site, emphasizing their potential value as toolboxes for biocatalytic applications.

  8. Enzymatic activity of a mine soil varies according to vegetation cover and level of compost applied.

    PubMed

    de Varennes, Amerilis; Abreu, Maria Manuela; Qu, Guiwei; Cunha-Queda, Cristina

    2010-01-01

    We applied three doses of compost from mixed municipal solid waste (0, 15, and 30 g kg(-1) of soil) to a soil developed on pyrite mine wastes. Part of the soil was planted with young Erica australis L. collected at the mine; part was fertilized with N-P-K-Mg and sown with Dactylis glomerata L .Bare soil without mineral fertilization was included in the experiment, as well. Compost application to bare soil increased pH, provided plant nutrients, and enhanced the activity of the six soil enzymes tested. Growth of D. glomerata, and E. australis was stimulated in compost-amended soil compared with unamended controls. The presence of D. glomerata led to the greatest activities of soil acid phosphatase, beta-glucosidase, and cellulase compared with bare soil or with soil with E. australis. The presence of E. australis increased the activities of protease and cellulase in amended soil, compared with control, but it impaired dehydrogenase, fl-glucosidase, and acid phosphatase activities. These negative impacts probably derived from phenolic compounds known to be released from roots of this species. The survival strategy of this species seems to include a small need for P in the shoots, and the release of exudates that impair microbial activity and P cycling.

  9. Influence of Linker Length and Composition on Enzymatic Activity and Ribosomal Binding of Neomycin Dimers

    PubMed Central

    Watkins, Derrick; Kumar, Sunil; Green, Keith D.

    2015-01-01

    The human and bacterial A site rRNA binding as well as the aminoglycoside-modifying enzyme (AME) activity against a series of neomycin B (NEO) dimers is presented. The data indicate that by simple modifications of linker length and composition, substantial differences in rRNA selectivity and AME activity can be obtained. We tested five different AMEs with dimeric NEO dimers that were tethered via triazole, urea, and thiourea linkages. We show that triazole-linked dimers were the worst substrates for most AMEs, with those containing the longer linkers showing the largest decrease in activity. Thiourea-linked dimers that showed a decrease in activity by AMEs also showed increased bacterial A site binding, with one compound (compound 14) even showing substantially reduced human A site binding. The urea-linked dimers showed a substantial decrease in activity by AMEs when a conformationally restrictive phenyl linker was introduced. The information learned herein advances our understanding of the importance of the linker length and composition for the generation of dimeric aminoglycoside antibiotics capable of avoiding the action of AMEs and selective binding to the bacterial rRNA over binding to the human rRNA. PMID:25896697

  10. ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes.

    PubMed

    Boschin, Giovanna; Scigliuolo, Graziana Maria; Resta, Donatella; Arnoldi, Anna

    2014-02-15

    The objective of this investigation was to compare the angiotensin converting enzyme (ACE)-inhibitory activity of the hydrolysates obtained by pepsin digestion of proteins of some legumes, such as chickpea, common bean, lentil, lupin, pea, and soybean, by using the same experimental procedure. The ACE-inhibitory activity was measured by using the tripeptide hippuryl-histidyl-leucine (HHL), as model peptide, and HPLC-DAD, as analytical method. The peptide mixtures of all legumes were active, with soybean and lupin the most efficient, with IC50 values of 224 and 226 μg/ml, respectively. Considering the promising results obtained with lupin, and aiming to identify the protein(s) that release(s) the peptides responsible for the activity, the peptides obtained from the pepsin digestion of some industrial lupin protein isolates and purified protein fractions were tested. The most active mixture, showing an IC50 value of 138 μg/ml, was obtained hydrolysing a mixture of lupin α+β conglutin.

  11. Two-Photon Enzymatic Probes Visualizing Sub-cellular/Deep-brain Caspase Activities in Neurodegenerative Models

    PubMed Central

    Qian, Linghui; Zhang, Cheng-Wu; Mao, Yanli; Li, Lin; Gao, Nengyue; Lim, Kah-Leong; Xu, Qing-Hua; Yao, Shao Q.

    2016-01-01

    Caspases work as a double-edged sword in maintaining cell homeostasis. Highly regulated caspase activities are essential during animal development, but dysregulation might lead to different diseases, e.g. extreme caspase activation is known to promote neurodegeneration. At present, visualization of caspase activation has mostly remained at the cellular level, in part due to a lack of cell-permeable imaging probes capable of direct, real-time investigations of endogenous caspase activities in deep tissues. Herein, we report a suite of two-photon, small molecule/peptide probes which enable sensitive and dynamic imaging of individual caspase activities in neurodegenerative models under physiological conditions. With no apparent toxicity and the ability of imaging endogenous caspases both in different subcellular organelles of mammalian cells and in brain tissues, these probes serve as complementary tools to conventional histological analysis. They should facilitate future explorations of caspases at molecular, cellular and organism levels and inspire development of novel two-photon probes against other enzymes. PMID:27210613

  12. Enzymatic activities in brains of diabetic rats treated with vanadyl sulphate and sodium tungstate.

    PubMed

    Lemberg, A; Fernández, M A; Ouviña, G; Rodríguez, R R; Peredo, H A; Susemihl, C; Villarreal, I; Filinger, E J

    2007-12-01

    The hypothesis of the present study was that diabetes mellitus might affect brain metabolism. Streptozotocin (STZ)-induced diabetic rats, treated with vanadyl sulphate (V) and sodium tungstate (T) were employed to observe the aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine kinase (CK) activities in brain homogenates. Significant increases in AST, ALT and CK activities were found in diabetic brain homogenates against controls, suggesting increments of transamination in brain and/or increases in cell membrane permeability to these enzymes. The increase in brain CK possibly expresses alterations in energy production. The decrease in CK activity caused by V and T treatment in diabetic rats suggests that both agents tend to normalize energy consumption. It is also possible that V and T-induced hypoglycemic effects cause metabolic alterations in brain.

  13. Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

    PubMed Central

    Dolinska, Monika B.; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2014-01-01

    Background Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Methodology/Principal Findings The intra-melanosomal domain of human tyrosinase (residues 19–469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Conclusions/Significance The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1. PMID:24392141

  14. Long-term effects of nickel oxide nanoparticles on performance, microbial enzymatic activity, and microbial community of a sequencing batch reactor.

    PubMed

    Wang, Sen; Li, Zhiwei; Gao, Mengchun; She, Zonglian; Guo, Liang; Zheng, Dong; Zhao, Yangguo; Ma, Bingrui; Gao, Feng; Wang, Xuejiao

    2017-02-01

    The nitrogen and phosphorus removal, microbial enzymatic activity, and microbial community of a sequencing batch reactor (SBR) were evaluated under long-term exposure to nickel oxide nanoparticles (NiO NPs). High NiO NP concentration (over 5 mg L(-1)) affected the removal of chemical oxygen demand, nitrogen, and phosphorus. The presence of NiO NP inhibited the microbial enzymatic activities and reduced the nitrogen and phosphorus removal rates of activated sludge. The microbial enzymatic activities of the activated sludge showed a similar variation trend to the nitrogen and phosphorus removal rates with the increase in NiO NP concentration from 0 to 60 mg L(-1). The Ni content in the effluent and activated sludge showed an increasing trend with the increase in NiO NP concentration. Some NiO NPs were absorbed on the sludge surface or penetrate the cell membrane into the interior of microbial cells in the activated sludge. NiO NP facilitated the increase in reactive oxygen species by disturbing the balance between the oxidation and anti-oxidation processes, and the variation in lactate dehydrogenase demonstrated that NiO NP could destroy the cytomembrane and cause variations in the microbial morphology and physiological function. High-throughput sequencing demonstrated that the microbial community of SBR had some obvious changes at 0-60 mg L(-1) NiO NPs at the phyla, class and genus levels.

  15. Coupling Binding to Catalysis – Using Yeast Cell Surface Display to Select Enzymatic Activities

    PubMed Central

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    Summary We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence activated cell sorting. PMID:26060080

  16. FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE

    EPA Science Inventory

    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  17. Arid soil microbial enzymatic activity profile as affected by geographical location and soil degradation status

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evaluating soil health is critical for any successful remediation effort. Arid lands, with their minimal carbon and water contents, low nutritional status and restricted, seasonal microbial activity pose specific challenges to soil health restoration and by extension, restoration of ecosystem repr...

  18. Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

    PubMed

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting.

  19. Glycolytic enzymatic activities in developing seeds involved in the differences between standard and low oil content sunflowers (Helianthus annuus L.).

    PubMed

    Troncoso-Ponce, M Adrián; Garcés, Rafael; Martínez-Force, Enrique

    2010-12-01

    As opposed to other oilseeds, developing sunflower seeds do not accumulate starch initially. They rely on the sucrose that comes from the mother plant to synthesise lipid precursors. Glycolysis is the principal source of carbon skeletons and reducing power for lipid biosynthesis. In this work, glycolytic initial metabolites and enzyme activities from developing seed of two different sunflower lines, of high and low oil content, were compared during storage lipid synthesis. These two lines showed different kinetic lipid accumulation in the developing embryos. Fatty acids levels during the initial and final stage of lipid synthesis were higher in CAS-6 than in ZEN-8. The analysis of the photosynthate and sugars content suggests that, although the hexoses levels were quite similar in both lines, the amount of sucrose produced by the mother plant and available for lipid synthesis was higher in CAS-6. Although, a smaller amount of sucrose is available in the ZEN-8 line, its seeds maintain the levels of intermediate sugars in the initial steps of glycolysis due to an increase in the levels of the invertase, hexokinase and phosphoglucose isomerase activities in ZEN-8, with respect to CAS-6. Also, a readjustment in the final part of this metabolic route took place, with the activities of phosphoglycerate kinase and enolase in CAS-6 being higher, allowing increased synthesis of phosphoenolpiruvate, the intermediate carbon donor for fatty acid synthesis. In addition, recently, it has been shown that Arabidopsis mutants with a lower fat content in their seeds have a higher amount of sucrose. These data together point to these last two enzymatic activities, phosphoglycerate kinase and enolase, as being responsible for the lower fat content in the ZEN-8 line.

  20. The cystine/glutamate antiporter regulates indoleamine 2,3-dioxygenase protein levels and enzymatic activity in human dendritic cells.

    PubMed

    Mattox, Mildred L; D'Angelo, June A; Grimes, Zachary M; Fiebiger, Edda; Dickinson, Bonny L

    2012-11-30

    Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the tryptophan-catabolizing pathway and a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are predominantly mediated by dendritic cells (DCs) and IDO-competent DCs promote long-term immunologic tolerance, a detailed understanding of how IDO expression and activity is regulated in these cells is central to the rational design of therapies to induce robust immune tolerance. We previously reported that the cystine/glutamate antiporter modulates the functional expression of IDO in human monocyte-derived DCs. Specifically, we showed that blocking antiporter uptake of cystine significantly increased both IDO mRNA and IDO enzymatic activity and that this correlated with impaired DC presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway. The antiporter regulates intracellular and extracellular redox by transporting cystine into the cell in exchange for glutamate. Intracellular cystine is reduced to cysteine to support biosynthesis of the major cellular antioxidant glutathione and cysteine is exported from the cell where it functions as an extracellular antioxidant. Here we show that antiporter control of IDO expression in DCs is reversible, independent of interferon-γ, regulated by redox, and requires active protein synthesis. These findings highlight a role for antiporter regulation of cellular redox as a critical control point for modulating IDO expression and activity in DCs. Thus, systemic disease and aging, processes that perturb redox homeostasis, may adversely affect immunity by promoting the generation of IDO-competent DCs.

  1. Enzymatic activity and motility of recombinant Arabidopsis myosin XI, MYA1.

    PubMed

    Hachikubo, You; Ito, Kohji; Schiefelbein, John; Manstein, Dietmar J; Yamamoto, Keiichi

    2007-06-01

    We expressed recombinant Arabidopsis myosin XI (MYA1), in which the motor domain of MYA1 was connected to an artificial lever arm composed of triple helical repeats of Dictyostelium alpha-actinin, in order to understand its motor activity and intracellular function. The V(max) and K(actin) of the actin-activated Mg(2+) ATPase activity of the recombinant MYA1 were 50.7 Pi head(-1) s(-1) and 30.2 microM, respectively, at 25 degrees C. The recombinant MYA1 could translocate actin filament at the maximum velocity of 1.8 microm s(-1) at 25 degrees C in the in vitro motility assay. The value corresponded to a motility of 3.2 microm s(-1) for native MYA1 if we consider the difference in the lever arm length, and this value was very close to the velocity of cytoplasmic streaming in Arabidopsis hypocotyl epidermal cells. The extent of inhibition by ADP of the motility of MYA1 was similar to that of the well-known processive motor, myosin V, suggesting that MYA1 is a processive motor. The dissociation rate of the actin-MYA1-ADP complex induced by ATP (73.5 s(-1)) and the V(max) value of the actin-activated Mg(2+) ATPase activity revealed that MYA1 stays in the actin-bound state for about 70% of its mechanochemical cycle time. This high ratio of actin-bound states is also a characteristic of processive motors. Our results strongly suggest that MYA1 is a processive motor and involved in vesicle transport and/or cytoplasmic streaming.

  2. Intestinal morphology and enzymatic activity in newly weaned pigs fed contrasting fiber concentrations and fiber properties.

    PubMed

    Hedemann, M S; Eskildsen, M; Laerke, H N; Pedersen, C; Lindberg, J E; Laurinen, P; Knudsen, K E Bach

    2006-06-01

    The main objective of this study was to determine the effect of fiber source and concentration on morphological characteristics, mucin staining pattern, and mucosal enzyme activities in the gastrointestinal tract of pigs. The experiment included 50 pigs from 10 litters weaned at 4 wk of age (BW 8.6 +/- 1.4 kg) and divided into 5 treatment groups. Diets containing fiber of various physico-chemical properties and concentrations were formulated to contain 73, 104, or 145 g of dietary fiber/kg of DM. The diets were based on raw wheat and barley flours. Pectin and barley hulls, representing soluble and insoluble fiber sources, respectively, were used to increase the fiber concentration. The pigs were fed the experimental diets for 9 d, and then the pigs were euthanized and the entire gastrointestinal tract was removed. Tissue samples were taken from the mid and distal small intestine and from the mid colon. Inclusion of pectin in the diets significantly decreased (P < 0.001) ADFI and ADG compared with pigs fed no pectin. The villi and the crypts were shorter in pigs fed pectin-containing diets, but the villous height/crypt depth ratio was unaltered. Pectin significantly decreased the area of mucins in the crypts of the small intestine, indicating that the pigs fed the pectin-containing diet would probably be more susceptible to pathogenic bacteria, although this cannot be separated from the impact on ADFI. The lectin-binding pattern of the intestinal mucosa was unaffected by diet. The activity of lactase and maltase was increased in pigs fed diets with high fiber content, whereas sucrase activity was increased in pigs fed the pectin-containing diets. The activity of the peptidases, aminopeptidase N and dipeptidylpeptidase IV, was increased when feeding high fiber diets, whereas the activity of gamma-glutamyl transpeptidase remained unaffected by the experimental diets. In conclusion, the reduced feed intake observed with the pectin-containing diets could explain the

  3. Mitomycin C-DNA adducts generated by DT-diaphorase. Revised mechanism of the enzymatic reductive activation of mitomycin C.

    PubMed

    Suresh Kumar, G; Lipman, R; Cummings, J; Tomasz, M

    1997-11-18

    Mitomycin C (MC) was reductively activated by DT-diaphorase [DTD; NAD(P)H:quinone oxidoreductase] from rat liver carcinoma cells in the presence of Micrococcus lysodeicticus DNA at pH 5.8 and 7.4. The resulting alkylated MC-DNA complexes were digested to the nucleoside level and the covalent MC-nucleoside adducts were separated, identified, and quantitatively analyzed by HPLC. In analogous experiments, two other flavoreductases, NADH-cytochrome c reductase and NADPH-cytochrome c reductase, as well as two chemical reductive activating agents Na2S2O4 and H2/PtO2 were employed as activators for the alkylation of DNA by MC. DTD as well as all the other activators generated the four known major guanine-N2-MC adducts at both pHs. In addition, at the lower pH, the guanine-N7-linked adducts of 2,7-diaminomitosene were detectable in the adduct patterns. At a given pH all the enzymatic and chemical reducing agents generated very similar adduct patterns which, however, differed dramatically at the acidic as compared to the neutral pH. Overall yield of MC adducts was 3-4-fold greater at pH 7.4 than at 5. 8 except in the case of DTD when it was 4-fold lower. Without exception, however, cross-link adduct yields were greater at the acidic pH (2-10-fold within the series). The ratio of adducts of bifunctional activation to those of monofunctional activation was 6-20-fold higher at the acidic as compared to the neutral pH. A comprehensive mechanism of the alkylation of DNA by activated MC was derived from the DNA adduct analysis which complements earlier model studies of the activation of MC. The mechanism consists of three competing activation pathways yielding three different DNA-reactive electrophiles 11, 12, and 17 which generate three unique sets of DNA adducts as endproducts. The relative amounts of these adducts are diagnostic of the relative rates of the competing pathways in vitro, and most likely, in vivo. Factors that influence the relative rates of individual pathways

  4. Gamma irradiation effect on the enzymatic activities of horseradish and apple peroxidases

    NASA Astrophysics Data System (ADS)

    Constantinovici, M.; Oancea, D.; Zaharescu, T.

    2009-01-01

    The behavior at low-dose exposure (0.033-0.4 kGy) of horseradish peroxidase (HRP) and of two different purified fractions of apple (Jonathan cultivar) peroxidases (named APR 1S and APR 2S) was studied. The HRP solutions were added with either 0.32 M fructose or glucose in order to study their effect on enzymes activity response under γ ( 137Cs, dose rate 0.4 kGy/h) irradiation. The obtained results showed similar behavior between HRP-sugar-added solution and apple fraction with higher oligosaccharides content (APR 2S) undergoing low-dose treatment. The same pattern was observed between unglycosylated HRP and APR 1S with lower oligosaccharides content. These similarities gave us the possibility to conclude that the presence of oligosaccharides, in more or less quantities, influences in the same way the peroxidases activity, from different plant species, exposed to γ irradiation.

  5. Incorporating mobile nanospheres in the lumen of hybrid microcapsules for enhanced enzymatic activity.

    PubMed

    Shi, Jiafu; Zhang, Xiaoman; Zhang, Shaohua; Wang, Xiaoli; Jiang, Zhongyi

    2013-11-13

    Physical encapsulation of enzymes in microcapsules, as a mild, controllable method, has been widely utilized for enzyme immobilization. However, this method often suffers from the big mass transfer resistance from the capsule lumen. In this study, a novel biocatalysis system with enhanced catalytic activity is constructed through coencapsulating enzymes and nanospheres in the lumen of protamine/silica hybrid microcapsules, which are synthesized through the synergy of biomimetic silicification and layer-by-layer (LbL) assembly. When utilized as the host for catalase (CAT) encapsulation, the hybrid microcapsules maintain high mechanical stability, high enzyme loading, and low enzyme leaching. Particularly, because of the existence of mobile nanospheres, the mass transfer resistance in the microcapsules is significantly reduced because of the vigorous agitation, thus acquiring an enhanced catalytic activity. Our strategy may also find applications in drug delivery and biosensor fields.

  6. Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila.

    PubMed

    Aragon, V; Kurtz, S; Flieger, A; Neumeister, B; Cianciotto, N P

    2000-04-01

    Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by a pilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.

  7. Probing Mechanisms for Enzymatic Activity Enhancement of Organophosphorus Hydrolase in Functionalized Mesoporous Silica

    SciTech Connect

    Chen, Baowei; Lei, Chenghong; Shin, Yongsoon; Liu, Jun

    2009-12-25

    We have previously reported that organophosphorus hydrolase (OPH) can be spontaneously entrapped in functionalized mesoporous silica (FMS) with HOOC - as the functional groups and the entrapped OPH in HOOC-FMS showed enhanced enzyme specific activity. This work is to study the mechanisms that why OPH entrapped in FMS displayed the enhanced activity in views of OPH-FMS interactions using spectroscopic methods. The circular dichroism (CD) spectra show that, comparing to the secondary structure of OPH free in solution, OPH in HOOC-FMS displayed increased a-helix/b-strand transition of OPH with increased OPH loading density. The fluorescence emission spectra of Trp residues were used to assess the tertiary structural changes of the enzyme. There was a 42% increase in fluorescence. This is in agreement with the fact that the fluorescence intensity of OPH was increased accompanying with the increased OPH activity when decreasing urea concentrations in solution. The steady-state anisotropy was increased after OPH entrapping in HOOC-FMS comparing to the free OPH in solution, indicating that protein mobility was reduced upon entrapment. The solvent accessibility of Trp residues of OPH was probed by using acrylamide as a collisional quencher. Trp residues of OPH-FMS had less solvent exposure comparing with free OPH in solution due to its electrostatical binding to HOOC-FMS thereby displaying the increased fluorescence intensity. These results suggest the interactions of OPH with HOOC-FMS resulted in the protein immobilization and a favorable conformational change for OPH in the crowded confinement space and accordingly the enhanced activity.

  8. Sulfonate derived phosphoramidates as active intermediates in the enzymatic primer-extension of DNA.

    PubMed

    De, S; Groaz, E; Margamuljana, L; Abramov, M; Marlière, P; Herdewijn, P

    2015-04-07

    Novel unnatural 5'-phosphoramidate nucleosides, capable of being processed as substrates by DNA polymerases for multiple nucleotide incorporations, have been designed. The mimics feature metabolites such as taurine and a broad range of aliphatic sulfonates coupled through a P-N bond to the 5'-phosphate position of deoxynucleotides, to allow binding interactions in the enzyme active site. The utility of all of the analogues as pyrophosphate mimics was demonstrated for the chain elongation of DNA, using both thermophilic and mesophilic microbial polymerases.

  9. Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases

    PubMed Central

    Goettig, Peter

    2016-01-01

    Posttranslational modifications are an important feature of most proteases in higher organisms, such as the conversion of inactive zymogens into active proteases. To date, little information is available on the role of glycosylation and functional implications for secreted proteases. Besides a stabilizing effect and protection against proteolysis, several proteases show a significant influence of glycosylation on the catalytic activity. Glycans can alter the substrate recognition, the specificity and binding affinity, as well as the turnover rates. However, there is currently no known general pattern, since glycosylation can have both stimulating and inhibiting effects on activity. Thus, a comparative analysis of individual cases with sufficient enzyme kinetic and structural data is a first approach to describe mechanistic principles that govern the effects of glycosylation on the function of proteases. The understanding of glycan functions becomes highly significant in proteomic and glycomic studies, which demonstrated that cancer-associated proteases, such as kallikrein-related peptidase 3, exhibit strongly altered glycosylation patterns in pathological cases. Such findings can contribute to a variety of future biomedical applications. PMID:27898009

  10. Effects of cerium oxide nanoparticles on soil enzymatic activities and wheat grass nutrients uptake

    NASA Astrophysics Data System (ADS)

    Li, Biting; Chen, Yirui; Bai, Lingyun; Jacobson, Astrid; Darnault, Christophe

    2015-04-01

    The US National Science Foundation estimated that the use of nanomaterials and nanotechnology would reach a global market value of 1 million this year. Concomitant with the wide applications of nanoparticles is an increasing risk of adverse effects to the environment and human health. As a common nanomaterial used as a fuel catalyst and polish material, cerium (IV) oxide nanoparticles (CeO2 NP) were tested for their potential impact on soil health and plant growth. Through exposure by air, water, and solid deposition, nanoparticles may accumulate in soils and impact agricultural systems. The objectives of this research were to determine whether CeO2 NPs affect the growth of wheat grass and selected soil enzyme activities chose as indicators of soil health. Wheat grass was grown in plant boxes containing CeO2 NPs mixed with agricultural soil at different concentrations. Two control groups were included: one consisting of soil with plants but no CeO2 NPs, and one containing only soil, i.e., no NP or wheat plants added. The plants were grown for 10 weeks and harvested every two weeks in a laboratory under sodium growth lights. At the end of the each growing period, two weeks, soils were assayed for phosphatase, β-glucosidase, and urease activities, and NPK values. Spectrophotometer analyses were used to assess enzyme activities, and NPK values were tested by Clemson Agricultural Center. Wheat yields were estimated by shoot and root lengths and weights.

  11. Effect of high pressures on the enzymatic activity of commercial milk protein coagulants

    NASA Astrophysics Data System (ADS)

    Wiśniewska, Krystyna; Reps, Arnold; Jankowska, Agnieszka

    2014-04-01

    This study was aimed at determining the effect of high pressures in the range of 100-1000 MPa/15 min, applied in 100 MPa increments, on the coagulating and proteolytic activity of commercial coagulants produced with genetic engineering methods: Maxiren, Chymogen, Chymax and of a natural rennin preparation, Hala. The coagulating activity of Hala preparation differed compared with the other preparations, due to greater resistance to high pressures, especially in the range of 500-600 MPa. The preparations produced with genetic engineering methods lost their capability for milk protein coagulation by 500 MPa. Pressurization at 200 MPa contributed to their reduced capability for casein macroproteolysis. In contrast, an increase in Chymax, Chymogen, Maxiren and Hala preparations' hydrolytic capability for the macroproteolysis of isoelectric casein was observed upon pressure treatment at 100 and 400 MPa and for microproteolysis after pressure treatment at 200 MPa. Storage (48 h/5°C) of the pressurized preparations had an insignificant effect on their coagulating and proteolytic activities.

  12. Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II.

    PubMed

    Verma, Shikha; Mehta, Ranjit Kumar; Maiti, Prasanta; Röhm, Klaus-Heinrich; Sonawane, Avinash

    2014-07-01

    Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer-dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia.

  13. Antimicrobial and antioxidant activities of clove essential oil and eugenyl acetate produced by enzymatic esterification.

    PubMed

    Vanin, Adriana B; Orlando, Tainara; Piazza, Suelen P; Puton, Bruna M S; Cansian, Rogério L; Oliveira, Debora; Paroul, Natalia

    2014-10-01

    This work reports the maximization of eugenyl acetate production by esterification of essential oil of clove in a solvent-free system using Novozym 435 as catalyst. The antimicrobial and antioxidant activities of clove essential oil and eugenyl acetate produced were determined. The conditions that maximized eugenyl acetate production were 60 °C, essential oil of clove to acetic anhydride ratio of 1:5, 150 rpm, and 10 wt% of enzyme, with a conversion of 99.87 %. A kinetic study was performed to assess the influence of substrates' molar ratio, enzyme concentration, and temperature on product yield. Results show that an excess of anhydride, enzyme concentration of 5.5 wt%, 50 °C, and essential oil of clove to acetic anhydride ratio of 1:5 afforded nearly a complete conversion after 2 h of reaction. Comparing the antibacterial activity of the essential oil of clove before and after esterification, we observed a decrease in the antimicrobial activity of eugenyl acetate, particularly with regard to minimum inhibitory concentration (MIC). Both eugenyl acetate and clove essential oil were most effective to the gram-negative than gram-positive bacteria group. The results showed a high antioxidant potential for essential oil before and particularly after the esterification reaction thus becoming an option for the formulation of new antioxidant products.

  14. EphB4 cellular kinase activity assayed using an enzymatic protein interaction system.

    PubMed

    Wehrman, Tom; Nguyen, Mimi; Feng, Wei; Bader, Benjamin

    2013-05-01

    Receptor tyrosine kinases (RTKs) are important players in various cellular processes, including proliferation, migration, metabolism, and neuronal development. EphB4 RTK is essential for the development of a functional arterial-venous network in embryonic and adult neoangiogenesis. To develop novel inhibitors of EphB4 that might have applications in severe diseases like cancer and retinopathies, assays need to be in place that resemble, in a most physiological fashion, the activation and downstream function of the kinase. In addition, such assays need to be amenable to high-throughput screening to serve efficiently the modern drug discovery processes in the pharmaceutical industry. The authors have developed an enzyme fragment complementation assay that measures the interaction of a downstream docking protein to the activated and phosphorylated full-length EphB4 kinase in cells. The assay is specific, robust, and amenable to miniaturization and high-throughput screening. It covers most steps in the activation process of EphB4, including ligand binding, autophosphorylation, and docking of a downstream interactor. This assay format can be transferred to other RTKs and adds an important cell-based kinase assay option to researchers in the field.

  15. Investigation of trypsin-CdSe quantum dot interactions via spectroscopic methods and effects on enzymatic activity.

    PubMed

    Kaur, Gurvir; Tripathi, S K

    2015-01-05

    The paper presents the interactions between trypsin and water soluble cadmium selenide (CdSe) quantum dots investigated by spectrophotometric methods. CdSe quantum dots have strong ability to quench the intrinsic fluorescence of trypsin by a static quenching mechanism. The quenching has been studied at three different temperatures where the results revealed that electrostatic interactions exist between CdSe quantum dots and trypsin and are responsible to stabilize the complex. The Scatchard plot from quenching revealed 1 binding site for quantum dots by trypsin, the same has been confirmed by making isothermal titrations of quantum dots against trypsin. The distance between donor and acceptor for trypsin-CdSe quantum dot complexes is calculated to be 2.8 nm by energy transfer mechanisms. The intrinsic fluorescence of CdSe quantum dots has also been enhanced by the trypsin, and is linear for concentration of trypsin ranging 1-80 μl. All the observations evidence the formation of trypsin-CdSe quantum dot conjugates, where trypsin retains the enzymatic activity which in turn is temperature and pH dependent.

  16. Differential coral bleaching-Contrasting the activity and response of enzymatic antioxidants in symbiotic partners under thermal stress.

    PubMed

    Krueger, Thomas; Hawkins, Thomas D; Becker, Susanne; Pontasch, Stefanie; Dove, Sophie; Hoegh-Guldberg, Ove; Leggat, William; Fisher, Paul L; Davy, Simon K

    2015-12-01

    Mass coral bleaching due to thermal stress represents a major threat to the integrity and functioning of coral reefs. Thermal thresholds vary, however, between corals, partly as a result of the specific type of endosymbiotic dinoflagellate (Symbiodinium sp.) they harbour. The production of reactive oxygen species (ROS) in corals under thermal and light stress has been recognised as one mechanism that can lead to cellular damage and the loss of their symbiont population (Oxidative Theory of Coral Bleaching). Here, we compared the response of symbiont and host enzymatic antioxidants in the coral species Acropora millepora and Montipora digitata at 28°C and 33°C. A. millepora at 33°C showed a decrease in photochemical efficiency of photosystem II (PSII) and increase in maximum midday excitation pressure on PSII, with subsequent bleaching (declining photosynthetic pigment and symbiont density). M. digitata exhibited no bleaching response and photochemical changes in its symbionts were minor. The symbiont antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and catalase peroxidase showed no significant upregulation to elevated temperatures in either coral, while only catalase was significantly elevated in both coral hosts at 33°C. Increased host catalase activity in the susceptible coral after 5days at 33°C was independent of antioxidant responses in the symbiont and preceded significant declines in PSII photochemical efficiencies. This finding suggests a potential decoupling of host redox mechanisms from symbiont photophysiology and raises questions about the importance of symbiont-derived ROS in initiating coral bleaching.

  17. A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation.

    PubMed

    Pucher, Marion E; Steiger, Matthias G; Mach, Robert L; Mach-Aigner, Astrid R

    2011-06-10

    Hypocrea jecorina (anamorph Trichoderma reesei) is a saprophytic fungus that produces hydrolases, which are applied in different types of industries and used for the production of biofuel. A recombinant Hypocrea strain, which constantly expresses the main transcription activator of hydrolases (Xylanase regulator 1), was found to grow faster on xylan and its monomeric backbone molecule d-xylose. This strain also showed improved ability of clearing xylan medium on plates. Furthermore, this strain has a changed transcription profile concerning genes encoding for hydrolases and enzymes associated with degradation of (hemi)celluloses. We demonstrated that enzymes of this strain from a xylan cultivation favoured break down of hemicelluloses to the monomer d-xylose compared to the parental strain, while the enzymes of the latter one formed more xylobiose. Applying supernatants from cultivation on carboxymethylcellulose in enzymatic conversion of hemicelluloses, the enzymes of the recombinant strain were clearly producing more of both, d-xylose and xylobiose, compared to the parental strain. Altogether, these results point to a changed hydrolase expression profile, an enhanced capability to form the xylan-monomer d-xylose and the assumption that there is a disordered induction pattern if the Xylanase regulator 1 is de-regulated in Hypocrea.

  18. Investigation of trypsin-CdSe quantum dot interactions via spectroscopic methods and effects on enzymatic activity

    NASA Astrophysics Data System (ADS)

    Kaur, Gurvir; Tripathi, S. K.

    2015-01-01

    The paper presents the interactions between trypsin and water soluble cadmium selenide (CdSe) quantum dots investigated by spectrophotometric methods. CdSe quantum dots have strong ability to quench the intrinsic fluorescence of trypsin by a static quenching mechanism. The quenching has been studied at three different temperatures where the results revealed that electrostatic interactions exist between CdSe quantum dots and trypsin and are responsible to stabilize the complex. The Scatchard plot from quenching revealed 1 binding site for quantum dots by trypsin, the same has been confirmed by making isothermal titrations of quantum dots against trypsin. The distance between donor and acceptor for trypsin-CdSe quantum dot complexes is calculated to be 2.8 nm by energy transfer mechanisms. The intrinsic fluorescence of CdSe quantum dots has also been enhanced by the trypsin, and is linear for concentration of trypsin ranging 1-80 μl. All the observations evidence the formation of trypsin-CdSe quantum dot conjugates, where trypsin retains the enzymatic activity which in turn is temperature and pH dependent.

  19. Long-term effects of cupric oxide nanoparticles (CuO NPs) on the performance, microbial community and enzymatic activity of activated sludge in a sequencing batch reactor.

    PubMed

    Wang, Sen; Li, Zhiwei; Gao, Mengchun; She, Zonglian; Ma, Bingrui; Guo, Liang; Zheng, Dong; Zhao, Yangguo; Jin, Chunji; Wang, Xuejiao; Gao, Feng

    2017-02-01

    The long-term effects of cupric oxide nanoparticles (CuO NPs) on the performance, microbial activity and microbial community of activated sludge were investigated in a sequencing batch reactor (SBR). The SBR performance had no evident change at 0-10 mg/L CuO NPs, whereas the CuO NPs concentration at 30-60 mg/L affected the COD, NH4(+)-N and soluble orthophosphate (SOP) removal, nitrogen and phosphorus removal rate and microbial enzymatic activity of activated sludge. Some CuO NPs might be absorbed on the surface of activated sludge or penetrate the microbial cytomembrane into the microbial cell interior of activated sludge. Compared to 0 mg/L CuO NPs, the reactive oxygen species (ROS) production and lactate dehydrogenase (LDH) release increased by 43.6% and 56.4% at 60 mg/L CuO NPs, respectively. The variations of ROS production and LDH release demonstrated that CuO NPs could induce the toxicity towards the microorganisms and destroy the integrity of microbial cytomembrane in the activated sludge. High throughput sequencing of 16S rDNA indicated that CuO NPs could evidently impact on the microbial richness, diversity and composition of activated sludge in the SBR.

  20. Relationships between microbial extracellular enzymatic activity and suspended and sinking particulate organic matter: seasonal transformations in the North Water

    NASA Astrophysics Data System (ADS)

    Huston, A. L.; Deming, J. W.

    Despite the importance of hydrolytic activities by bacterial extracellular enzymes (EE) in the temperate ocean, little is known about the role of extracellular enzymatic activity (EEA) in determining the fate of particulate organic matter (POM) in polar seas. To explore the issue further, we measured various chemical and bacterial parameters in the near-0°C waters of the North Water during the months of May and July of 1998. Seawater (SW) samples were collected by Niskin bottle at the depth of the chlorophyll fluorescence maximum (8-90 m), while samples of sinking particles and aggregates were collected in short-term (0.5-1.2 d), unpoisoned, floating sediment traps deployed at depths typically below the mixed layer (50-136 m). Samples were analyzed for POC, PON, and abundance of total and actively respiring bacteria. They were also incubated with fluorescently tagged substrate analogs to measure potential maximal rates of three classes of EE (leucine-aminopeptidase, chitobiase, and β-glucosidase) at -1°C. The percentage of actively respiring bacteria was always higher in sediment trap samples than in SW (medians of 38% and 24% versus 10% and 12% in May and July, respectively). Cell-specific rates of EEA were also higher in the trap samples and, for both sample types, similar to published rates from temperate waters. Rates of EEA when scaled to the abundance of actively respiring bacteria, however, did not differ between sample types, suggesting that the elevated EEA associated with sinking material is due to the greater abundance of metabolically active cells supported by such material and not due to enhanced enzyme expression in general, as suggested by previous studies. In this study, leucine-aminopeptidase activity was always much higher than the other classes of EEA, becoming even more dominant later in the season; it always correlated positively with the abundance of both total and actively respiring bacteria. Enzyme ratios indicating protease dominance

  1. The inositol 5-phosphatase SHIP1 is a nucleo-cytoplasmic shuttling protein and enzymatically active in cell nuclei.

    PubMed

    Nalaskowski, Marcus M; Metzner, Anja; Brehm, Maria A; Labiadh, Sena; Brauer, Helena; Grabinski, Nicole; Mayr, Georg W; Jücker, Manfred

    2012-03-01

    The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in hematopoietic cells. SHIP1 mediates its regulatory function after relocalization from the cytoplasm to the plasma membrane where it converts its substrate PI(3,4,5)P(3) to PI(3,4)P(2) thereby terminating PI3-kinase mediated signaling. In addition, SHIP1 converts Ins(1,3,4,5)P(4) to Ins(1,3,4)P(3) thereby regulating inositol phosphate metabolism. Here we report, that SHIP1 can be detected in nuclear puncta of Jurkat cells by confocal microscopy after expression of SHIP1 from a tetracycline inducible vector. SHIP1-containing nuclear puncta partially co-localize with FLASH, a multifunctional nuclear protein that has been linked to apoptotic signaling and transcriptional control. Nuclear localization was confirmed for endogenously expressed SHIP1 in the myeloid leukemia cell line TF1. In addition, enzymatically active SHIP1 was found in nuclear fractions of Jurkat cells with a similar specific activity as cytoplasmic SHIP1. Further analysis revealed that SHIP1 is a nucleocytoplasmic shuttling protein which is actively imported into and exported out of the nucleus. Nuclear import is mediated by two canonical nuclear localization signals (NLS) i.e. K(327)KSK and K(547)KLR. Mutational inactivation of each NLS motif inhibited nuclear import and reduced the proliferation of cells indicating a functional role of nuclear SHIP1 for cell growth. Our data indicate that SHIP1 is partly localized in the nucleus and suggest that SHIP1 plays a role for nuclear phosphoinositide and/or nuclear inositol phosphate signaling.

  2. Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components.

    PubMed

    Lu, Qiaosheng; Wierzbicki, Sara; Krasilnikov, Andrey S; Schmitt, Mark E

    2010-03-01

    RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.

  3. Induction of ferroxidase enzymatic activity by copper reduces MPP+-evoked neurotoxicity in rats.

    PubMed

    Rubio-Osornio, Moisés; Montes, Sergio; Heras-Romero, Yessica; Guevara, Jorge; Rubio, Carmen; Aguilera, Penélope; Rivera-Mancia, Susana; Floriano-Sánchez, Esaú; Monroy-Noyola, Antonio; Ríos, Camilo

    2013-03-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by decreased dopamine, intracellular inclusions (Lewy bodies) and brain iron deposits. PD has also been related with reduced ferroxidase activity, diminished antioxidant defenses and lipid peroxidation. Striatal injection of 1-methyl-4-phenylpyridinium (MPP(+)) into rodents reproduces the major biochemical characteristics of PD, including oxidative stress. Copper (Cu) plays an important role as prosthetic group of several proteins involved in iron metabolism and antioxidant responses, such as ceruloplasmin (Cp). In the present study, intraperitoneal CuSO4 injection (10μmol/kg) produced an insignificant increase of Cu content in striatum and midbrain (17.5% and 7%, respectively). After 10 and 11h, Cu induced 6- and 4-fold increase Cp mRNA in midbrain and striatum, respectively. Cu-supplement also produced a time-dependent increase ferroxidase activity in striatal tissue, reaching a maximum 16h after Cu treatment in midbrain; while, ferrous iron content diminished 18% in striatum and 8% in midbrain. In regard the PD model, we found that MPP(+) (10μg/8μL, intrastriatal), induced a significant (P<0.05) reduction of striatal ferroxidase activity; this effect was reverted by Cu pre-treatment 16h before MPP(+). Likewise, Cu-supplement prevented lipid fluorescent products formation in striatum, evaluated (P<0.01) 6h after MPP(+). In the long term, apomorphine-evoked circling behavior was evaluated 6 days after MPP(+) injury; Cu pre-treatment significantly reduced (P<0.05) the apomorphine-induced ipsilateral turns in MPP(+)-lesioned rats. These results suggest that Cu-induced expression of Cp could be an interesting scope against the deleterious effects of iron deposits in PD.

  4. Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst)

    PubMed Central

    Martins, Julia M.; DeMarco, Ricardo; Jameson, David M.; Castro, Aline M.; Bossolan, Nelma R. S.; Wallace, B. A.; Araujo, Ana P. U.

    2016-01-01

    Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required. PMID:27351338

  5. Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst).

    PubMed

    Lopes, Jose L S; Yoneda, Juliana S; Martins, Julia M; DeMarco, Ricardo; Jameson, David M; Castro, Aline M; Bossolan, Nelma R S; Wallace, B A; Araujo, Ana P U

    2016-01-01

    Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.

  6. Protoporphyrins Enhance Oligomerization and Enzymatic Activity of HtrA1 Serine Protease

    PubMed Central

    Jo, Hakryul; Patterson, Victoria; Stoessel, Sean; Kuan, Chia-Yi; Hoh, Josephine

    2014-01-01

    High temperature requirement protein A1 (HtrA1), a secreted serine protease of the HtrA family, is associated with a multitude of human diseases. However, the exact functions of HtrA1 in these diseases remain poorly understood. We seek to unravel the mechanisms of HtrA1 by elucidating its interactions with chemical or biological modulators. To this end, we screened a small molecule library of 500 bioactive compounds to identify those that alter the formation of extracellular HtrA1 complexes in the cell culture medium. An initial characterization of two novel hits from this screen showed that protoporphyrin IX (PPP-IX), a precursor in the heme biosynthetic pathway, and its metalloporphyrin (MPP) derivatives fostered the oligomerization of HtrA1 by binding to the protease domain. As a result of the interaction with MPPs, the proteolytic activity of HtrA1 against Fibulin-5, a specific HtrA1 substrate in age-related macular degeneration (AMD), was increased. This physical interaction could be abolished by the missense mutations of HtrA1 found in patients with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Furthermore, knockdown of HtrA1 attenuated apoptosis induced by PPP-IX. These results suggest that PPP-IX, or its derivatives, and HtrA1 may function as co-factors whereby porphyrins enhance oligomerization and the protease activity of HtrA1, while active HtrA1 elevates the pro-apoptotic actions of porphyrin derivatives. Further analysis of this interplay may shed insights into the pathogenesis of diseases such as AMD, CARASIL and protoporphyria, as well as effective therapeutic development. PMID:25506911

  7. Screening for genes coding for putative antitumor compounds, antimicrobial and enzymatic activities from haloalkalitolerant and haloalkaliphilic bacteria strains of Algerian Sahara Soils.

    PubMed

    Selama, Okba; Amos, Gregory C A; Djenane, Zahia; Borsetto, Chiara; Laidi, Rabah Forar; Porter, David; Nateche, Farida; Wellington, Elizabeth M H; Hacène, Hocine

    2014-01-01

    Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications.

  8. Lignin binding to pancreatic lipase and its influence on enzymatic activity.

    PubMed

    Zhang, Juan; Xiao, Lin; Yang, Yucai; Wang, Zhaoxia; Li, Genxi

    2014-04-15

    In this paper, we find that the effect of lignin on pancreatic lipase (PL) is dependent on reaction medium and substrate used. Experimental results reveal that lignin can gradually bind to PL to form a PL-lignin complex, resulting in an increased activity of the enzyme. The binding process is spontaneous and the PL-lignin complex formation is an endothermic reaction induced by hydrophobic and electrostatic interaction. There is a non-radiation energy transfer from PL to lignin during the binding process, and the binding of lignin to PL conforms to a secondary exponential decay function. Moreover, the α-helix content of the enzyme will be changed and the rigidity of its side chain will be enhanced due to the formation of lignin-PL complex. This study has not only provided the activation effect of lignin on PL, but also given an insight into the interaction between lignin and the enzyme, which would benefit the application of lignin in the pharmacy and food industry, as well as other fields.

  9. Cathepsin S of Sciaenops ocellatus: Identification, transcriptional expression and enzymatic activity.

    PubMed

    Sun, Bo-Guang; Chi, Heng

    2016-01-01

    Cathepsin S is a member of cysteine cathepsins and belongs to the cathepsin L-like family. In mammals, it is known to participate in various physiological processes and host immune defense. In teleost fish, the function of cathepsin S is less investigated. In the present work, we characterized a cathepsin S homologue (SoCatS) from red drum (Sciaenops ocellatus), a commercially valuable fish in Chinese mariculture. Like all cathepsin S, SoCatS possesses a peptidase domain with four catalytically essential residues (Gln140, Cys146, His285, and Asn305) conserved in the cathepsin S of different organisms. SoCatS shares 60-90% overall sequence identities with known teleost cathepsin S. Phylogenetic profiling indicated that SoCatS is evolutionally close to the cathepsin S of other teleost fish, especially Miichthys miiuy, a member of Sciaenidae family like red drum. SoCatS expression was detected in various tissues and was enhanced by bacterial infection. Purified recombinant SoCatS exhibited apparent peptidase activity with maximum at 50°C and pH 7.5. This activity depended on the catalytic residue Cys146 and was severely reduced by the cathepsin inhibitor E-64. Our results suggest that SoCatS functions as a cysteine protease which is probably involved in the antibacterial immunity of red drum.

  10. Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

    PubMed Central

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P.; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V.

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  11. A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions

    PubMed Central

    Schuster, Sabine; Roessler, Claudia; Meleshin, Marat; Zimmermann, Philipp; Simic, Zeljko; Kambach, Christian; Schiene-Fischer, Cordelia; Steegborn, Clemens; Hottiger, Michael O.; Schutkowski, Mike

    2016-01-01

    Sirtuins are NAD+ dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1–6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M−1s−1. These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM). PMID:26940860

  12. Changes in GDPase/UDPase enzymatic activity in response to oxidative stress in four Candida species.

    PubMed

    Delgado-Carmona, Jenny Daniela; Ramírez-Quijas, Mayra Denisse; Vega-González, Arturo; López-Romero, Everardo; Cuéllar-Cruz, Mayra

    2015-07-01

    The terminal processing of proteins and lipids occurs in the Golgi apparatus and involves the transport of sugar nucleotides into the Golgi lumen by specific carriers and the accumulation of nucleoside diphosphates (NDPs) as a result of oligosaccharide-protein glycosyltransferase activity. NDPs are converted into the corresponding nucleoside monophosphates (NMPs) by nucleoside diphosphatases (NDPases), thus relieving inhibition of sugar transferases. In addition, NMPs are then exchanged for equimolecular amounts of cytosolic sugar nucleotides by antiport transport systems. NDPases, commonly GDPase and UDPase, thus play a critical role in glycoprotein maturation and may influence fungal pathogenesis, morphogenesis, and cell wall properties. Interest of this laboratory has recently focused on the effect of reactive oxygen species (ROS) on enzymes involved in detoxification of these oxidants and on the metabolism of biomolecules such as lipids, nucleic acids, and proteins in human pathogenic Candida species. We therefore consider it important to extend these studies to determine how GDPase and UDPase are affected after exposure of cells to oxidants such as menadione, a superoxide (O2 (•-))-generator, and H2O2. Results indicate that activity of both enzymes decrease in response to these agents suggesting that ROS may also affect other critical cell functions such as protein glycosylation.

  13. The chromodomains of CHD1 are critical for enzymatic activity but less important for chromatin localization.

    PubMed

    Morettini, Stefano; Tribus, Martin; Zeilner, Anette; Sebald, Johanna; Campo-Fernandez, Beatriz; Scheran, Gabriele; Wörle, Hildegard; Podhraski, Valerie; Fyodorov, Dmitry V; Lusser, Alexandra

    2011-04-01

    The molecular motor protein CHD1 has been implicated in the regulation of transcription and in the transcription-independent genome-wide incorporation of H3.3 into paternal chromatin in Drosophila melanogaster. A key feature of CHD1 is the presence of two chromodomains, which can bind to histone H3 methylated at lysine 4 and thus might serve to recruit and/or maintain CHD1 at the chromatin. Here, we describe genetic and biochemical approaches to the study of the Drosophila CHD1 chromodomains. We found that overall localization of CHD1 on polytene chromosomes does not appreciably change in chromodomain-mutant flies. In contrast, the chromodomains are important for transcription-independent activities of CHD1 during early embryonic development as well as for transcriptional regulation of several heat shock genes. However, neither CHD1 nor its chromodomains are needed for RNA polymerase II localization and H3K4 methylation but loss of CHD1 decreases transcription-induced histone eviction at the Hsp70 gene in vivo. Chromodomain mutations negatively affect the chromatin assembly activities of CHD1 in vitro, and they appear to be involved in linking the ATP-dependent motor to the chromatin assembly function of CHD1.

  14. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Rhamnonate Dehydratase

    SciTech Connect

    Rakus,J.; Fedorov, A.; Fedorov, E.; Glaner, M.; Hubbard, B.; Delli, J.; Babbitt, P.; Almo, S.; Gerlt, J.

    2008-01-01

    The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy-l-mannonate) has the 'best' kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg2+; the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy-l-rhamnonate (obtained by reduction of the product with NaBH4). Like other members of the enolase superfamily, RhamD contains an N-terminal a + {beta} capping domain and a C-terminal ({beta}/a)7{beta}-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining '20s loop' in the capping domain is extended in length and the '50s loop' is truncated. The ligands for the Mg2+ are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth {beta}-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth {beta}-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg2+-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and delivers a proton to carbon-3

  15. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Fuconate Dehydratase from Xanthomonas campestris

    SciTech Connect

    Yew,W.; Fedorov, A.; Fedorov, E.; Rakus, J.; Pierce, R.; Almo, S.; Gerlt, J.

    2006-01-01

    Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report the authors use both genome (operon) context and screening of a library of acid sugars to assign the L-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI: 21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups: three carboxylate ligands for the essential Mg2+ located at the ends of th third, fourth, and fifth-strands in the (/)7-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and sixth-strands (His 351 and Asp 324, respectively), and a Glue at the end of the eighth-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting 2-keto-3-deoxy-L-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity: In addition to L-fuconate, FucD also catalyzes the dehydration of L-galactonate, D-arabinonate, D-altronate, L-talonate, and D-ribonate. The dehydrations of L-fuconate, L-galactonate, and D-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of L-talonate and D-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields L

  16. Phospholipase C produced by Clostridium botulinum types C and D: comparison of gene, enzymatic, and biological activities with those of Clostridium perfringens alpha-toxin.

    PubMed

    Fatmawati, Ni Nengah Dwi; Sakaguchi, Yoshihiko; Suzuki, Tomonori; Oda, Masataka; Shimizu, Kenta; Yamamoto, Yumiko; Sakurai, Jun; Matsushita, Osamu; Oguma, Keiji

    2013-01-01

    Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

  17. A comparison of glucose oxidase and aldose dehydrogenase as mediated anodes in printed glucose/oxygen enzymatic fuel cells usi