Sample records for aldh enzymatic activity

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

    PubMed

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

    2017-10-01

    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. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. 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. © 2014 UICC.

  3. 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.

  4. Structural and Functional Modifications of Corneal Crystallin ALDH3A1 by UVB Light

    PubMed Central

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

    2010-01-01

    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. PMID:21203538

  5. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. High ALDH1 expression correlates with better prognosis in tumorigenic malignant melanoma.

    PubMed

    Taylor, Laura A; Abraham, Ronnie M; Tahirovic, Emin; van Belle, Patricia; Li, Bin; Huang, Linfang; Elder, David E; Gimotty, Phyllis; Xu, Xiaowei

    2017-05-01

    Aldehyde dehydrogenase 1 (ALDH1) has been proposed as biomarker of stem cells for certain human cancers. ALDH1 expression has been correlated with poor patient outcomes in a variety of malignancies but better patient outcomes in others, and its prognostic significance in malignant melanoma is unclear. Thus, 68 melanoma patients with comprehensive clinical and pathologic follow-up data were used to construct a tissue microarray. A modified histological score (H-score) with a maximum score of 300 was used to quantify immunohistochemical staining for ALDH1. Survival time was defined as the time between diagnosis and melanoma-specific death. Using univariate logistic regression, a low (<80 H-score) ALDH1 score showed 3.7-fold increase in risk for melanoma-specific death within 10 years when compared with high (>80) ALDH1 levels (P=0.017). Odds of MSD were lower by a factor of ~0.9 for each 10-point increase in H-Score. Median survival time was 44.1 months and 180.9 months for patients with low and high ALDH1 expression, respectively. Using multivariate analysis, ALDH1 H-score was found to be an independent prognostic factor. These findings suggest that ALDH1 expression in malignant melanoma has a favorable effect on patient survival. Further study is needed elucidate the function of this enzymatic protein in melanoma progression.

  7. 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

  8. 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.

  9. ALDH2 Activator Inhibits Increased Myocardial Infarction Injury by Nitroglycerin Tolerance

    PubMed Central

    Sun, Lihan; Ferreira, Julio Cesar Batista; Mochly-Rosen, Daria

    2012-01-01

    Nitroglycerin, which helps impaired cardiac function as it is converted to nitric oxide, is used worldwide to treat patients with various ischemic and congestive cardiac diseases, including angina pectoris. Nevertheless, after continuous treatment, the benefits of nitroglycerin are limited by the development of tolerance to the drug. Nitroglycerin tolerance is a result of inactivation of aldehyde dehydrogenase 2 (ALDH2), an enzyme essential for cardioprotection in animals subjected to myocardial infarction (MI). Here we tested the hypothesis that the tolerance that develops as a result of sustained nitroglycerin treatment increases cardiac injury by subsequent MI. In a rat model of MI, 16 hours of prior, sustained nitroglycerin treatment (7.2 mg/kg/day) resulted in infarcts that were twice as large as those in untreated control animals and in diminished cardiac function at 3 days and 2 weeks after the MI. We also sought to identify a potential treatment to protect against this increased cardiac damage. Nitroglycerin inhibited ALDH2 activity in vitro, an effect that was blocked by Alda-1, an activator of ALDH2. Co-administration of Alda-1 (16 mg/kg/day) with the nitroglycerin prevented the nitroglycerin-induced increase in cardiac dysfunction after MI in rats, at least in part by enhancing metabolism of reactive aldehyde adducts that impair normal protein functions. If our animal studies showing that nitroglycerin tolerance increases cardiac injury upon ischemic insult are corroborated in humans, activators of ALDH2 such as Alda-1 may help to protect MI patients from this nitroglycerin-induced increase in cardiac injury, while maintaining the cardiac benefits of the increased nitric oxide concentrations produced by nitroglycerin. PMID:22049071

  10. 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

  11. Verification of ALDH Activity as a Biomarker in Colon Cancer Stem Cells-Derived HT-29 Cell Line.

    PubMed

    Khorrami, Samaneh; Zavaran Hosseini, Ahmad; Mowla, Seyed Javad; Malekzadeh, Reza

    2015-10-01

    Recent evidence has suggested that epithelial cancers including colorectal cancer (CRC) have driven by a small population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which could be responsible for recurrence of cancer. Aldehyde dehydrogenase 1 (ALDH1) activity has used as a functional stem cell biomarker to isolate CSCs in different cancers such as colorectal cancer. The main aim of this research was to determine the utility of ALDH1 activity along with CD44 and EPCAM in identifying stem cell-like cells in human HT-29 colonic adenocarcinoma cell line. In this experimental study, colon CSCs biomarkers including CD44, EPCAM and ALDH1 in colonospheres and parent cells have analyzed by flow cytometry. The expression levels of stemness genes in spheroid and parental cells have investigated using SYBR Green real-time PCR. In addition, in vivo xenografts assay has performed to determine tumorigenic potential of tumor spheroid cells in nude mice. According to results, over 92% of spheroids were CD44+/EpCAM+, while parent cells only have expressed 38% of CD44/EpCAM biomarkers (P < 0.001). Controversially, ALDH activity was about 2-fold higher in the parent cells than spheroid cells (P < 0.05). In comparison with the parental cells, expression levels of ''stemness'' genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 have significantly increased in colonosphere cells (P < 0.05). Further, administration of 2500 spheroids could be sufficient to initiate tumor growth in nude mice, while 1x106 of parental cells has needed to form tumor. For the first time, we have shown that colonospheres with low ALDH1 activity has indicated increased tumorigenic potential and stemness properties. So, it hasn't seemed that ALDH1 could become a useful biomarker to identify CSCs population in HT-29 cell line.

  12. ALDH2 restores exhaustive exercise-induced mitochondrial dysfunction in skeletal muscle

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Qiuping; Zheng, Jianheng; Qiu, Jun

    Background: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is highly expressed in heart and skeletal muscles, and is the major enzyme that metabolizes acetaldehyde and toxic aldehydes. The cardioprotective effects of ALDH2 during cardiac ischemia/reperfusion injury have been recognized. However, less is known about the function of ALDH2 in skeletal muscle. This study was designed to evaluate the effect of ALDH2 on exhaustive exercise-induced skeletal muscle injury. Methods: We created transgenic mice expressing ALDH2 in skeletal muscles. Male wild-type C57/BL6 (WT) and ALDH2 transgenic mice (ALDH2-Tg), 8-weeks old, were challenged with exhaustive exercise for 1 week to induce skeletal muscle injury. Animalsmore » were sacrificed 24 h post-exercise and muscle tissue was excised. Results: ALDH2-Tg mice displayed significantly increased treadmill exercise capacity compared to WT mice. Exhaustive exercise caused an increase in mRNA levels of the muscle atrophy markers, Atrogin-1 and MuRF1, and reduced mitochondrial biogenesis and fusion in WT skeletal muscles; these effects were attenuated in ALDH2-Tg mice. Exhaustive exercise also enhanced mitochondrial autophagy pathway activity, including increased conversion of LC3-I to LC3-II and greater expression of Beclin1 and Bnip3; the effects of which were mitigated by ALDH2 overexpression. In addition, ALDH2-Tg reversed the increase of an oxidative stress biomarker (4-hydroxynonenal) and decreased levels of mitochondrial antioxidant proteins, including manganese superoxide dismutase and NAD(P)H:quinone oxidoreductase 1, in skeletal muscle induced by exhaustive exercise. Conclusion: ALDH2 may reverse skeletal muscle mitochondrial dysfunction due to exhaustive exercise by regulating mitochondria dynamic remodeling and enhancing the quality of mitochondria. - Highlights: • Skeletal muscle ALDH2 expression and activity declines during exhaustive exercise. • ALDH2 overexpression enhances physical performance and restores

  13. Alda-1, an ALDH2 activator, protects against hepatic ischemia/reperfusion injury in rats via inhibition of oxidative stress.

    PubMed

    Zhang, Tao; Zhao, Qiang; Ye, Fang; Huang, Chan-Yan; Chen, Wan-Mei; Huang, Wen-Qi

    2018-04-13

    Previous studies have proved that activation of aldehyde dehydrogenase two (ALDH2) can attenuate oxidative stress through clearance of cytotoxic aldehydes, and can protect against cardiac, cerebral, and lung ischemia/reperfusion (I/R) injuries. In this study, we investigated the effects of the ALDH2 activator Alda-1 on hepatic I/R injury. Partial warm ischemia was performed in the left and middle hepatic lobes of Sprague-Dawley rats for 1 h, followed by 6 h of reperfusion. Rats received either Alda-1 or vehicle by intravenous injection 30 min before ischemia. Blood and tissue samples of the rats were collected after 6-h reperfusion. Histological injury, proinflammatory cytokines, reactive oxygen species (ROS), cellular apoptosis, ALDH2 expression and activity, 4-hydroxy-trans-2-nonenal (4-HNE) and malondialdehyde (MDA) were measured. BRL-3A hepatocytes were subjected to hypoxia/reoxygenation (H/R). Cell viability, ROS, and mitochondrial membrane potential were determined. Pretreatment with Alda-1 significantly alleviated I/R-induced elevations of alanine aminotransferase and aspartate amino transferase, and significantly blunted the pathological injury of the liver. Moreover, Alda-1 significantly inhibited ROS and proinflammatory cytokines production, 4-HNE and MDA accumulation, and apoptosis. Increased ALDH2 activity was found after Alda-1 administration. No significant changes in ALDH2 expression were observed after I/R. ROS was also higher in H/R cells than in control cells, which was aggravated upon treatment with 4-HNE, and reduced by Alda-1 treatment. Cell viability and mitochondrial membrane potential were inhibited in H/R cells, which was attenuated upon Alda-1 treatment. Activation of ALDH2 by Alda-1 attenuates hepatic I/R injury via clearance of cytotoxic aldehydes.

  14. Citral reduces breast tumor growth by inhibiting the cancer stem cell marker ALDH1A3.

    PubMed

    Thomas, Margaret Lois; de Antueno, Roberto; Coyle, Krysta Mila; Sultan, Mohammad; Cruickshank, Brianne Marie; Giacomantonio, Michael Anthony; Giacomantonio, Carman Anthony; Duncan, Roy; Marcato, Paola

    2016-11-01

    Breast cancer stem cells (CSCs) can be identified by increased Aldefluor fluorescence caused by increased expression of aldehyde dehydrogenase 1A3 (ALDH1A3), as well as ALDH1A1 and ALDH2. In addition to being a CSC marker, ALDH1A3 regulates gene expression via retinoic acid (RA) signaling and plays a key role in the progression and chemotherapy resistance of cancer. Therefore, ALDH1A3 represents a druggable anti-cancer target of interest. Since to date, there are no characterized ALDH1A3 isoform inhibitors, drugs that were previously described as inhibiting the activity of other ALDH isoforms were tested for anti-ALDH1A3 activity. Twelve drugs (3-hydroxy-dl-kynurenine, benomyl, citral, chloral hydrate, cyanamide, daidzin, DEAB, disulfiram, gossypol, kynurenic acid, molinate, and pargyline) were compared for their efficacy in inducing apoptosis and reducing ALDH1A3, ALDH1A1 and ALDH2-associated Aldefluor fluorescence in breast cancer cells. Citral was identified as the best inhibitor of ALDH1A3, reducing the Aldefluor fluorescence in breast cancer cell lines and in a patient-derived tumor xenograft. Nanoparticle encapsulated citral specifically reduced the enhanced tumor growth of MDA-MB-231 cells overexpressing ALDH1A3. To determine the potential mechanisms of citral-mediated tumor growth inhibition, we performed cell proliferation, clonogenic, and gene expression assays. Citral reduced ALDH1A3-mediated colony formation and expression of ALDH1A3-inducible genes. In conclusion, citral is an effective ALDH1A3 inhibitor and is able to block ALDH1A3-mediated breast tumor growth, potentially via blocking its colony forming and gene expression regulation activity. The promise of ALDH1A3 inhibitors as adjuvant therapies for patients with tumors that have a large population of high-ALDH1A3 CSCs is discussed. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  15. Establishment of a Quick and Highly Accurate Breath Test for ALDH2 Genotyping

    PubMed Central

    Aoyama, Ikuo; Ohashi, Shinya; Amanuma, Yusuke; Hirohashi, Kenshiro; Mizumoto, Ayaka; Funakoshi, Makiko; Tsurumaki, Mihoko; Nakai, Yukie; Tanaka, Katsuyuki; Hanada, Mariko; Uesaka, Aki; Chiba, Tsutomu; Muto, Manabu

    2017-01-01

    Objectives: Acetaldehyde, the first metabolite of ethanol, is a definite carcinogen for the esophagus, head, and neck; and aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that catalyzes the metabolism of acetaldehyde. The ALDH2 genotype exists as ALDH2*1/*1 (active ALDH2), ALDH2*1/*2 (heterozygous inactive ALDH2), and ALDH2*2/*2 (homozygous inactive ALDH2). Many epidemiological studies have reported that ALDH2*2 carriers are at high risk for esophageal or head and neck squamous cell carcinomas by habitual drinking. Therefore, identification of ALDH2*2 carriers would be helpful for the prevention of those cancers, but there have been no methods suitable for mass screening to identify these individuals. Methods: One hundred and eleven healthy volunteers (ALDH2*1/*1 carriers: 53; ALDH2*1/*2 carriers: 48; and ALDH2*2/*2 carriers: 10) were recruited. Breath samples were collected after drinking 100 ml of 0.5% ethanol using specially designed gas bags, and breath ethanol and acetaldehyde levels were measured by semiconductor gas chromatography. Results: The median (range) breath acetaldehyde levels at 1 min after alcohol ingestion were 96.1 (18.1–399.0) parts per billion (p.p.b.) for the ALDH2*1/*1 genotype, 333.5 (78.4–1218.4) p.p.b. for the ALDH2*1/*2 genotype, and 537.1 (213.2–1353.8) p.p.b. for the ALDH2*2/*2 genotype. The breath acetaldehyde levels in ALDH2*2 carriers were significantly higher than for the ALDH2*1/*1 genotype. Notably, the ratio of breath acetaldehyde level-to-breath ethanol level could identify carriers of the ALDH2*2 allele very accurately (whole accuracy; 96.4%). Conclusions: Our novel breath test is a useful tool for identifying ALDH2*2 carriers, who are at high risk for esophageal and head and neck cancers. PMID:28594397

  16. Spectroscopic characterisation of interaction of ferulic acid with aldehyde dehydrogenase (ALDH).

    PubMed

    Kolawole, Ayodele O; Agaba, Ruth J; Oluwole, Matthew O

    2017-05-01

    Interaction of a pharmacological important phenolic, ferulic acid, with Aldehyde dehydrogenase (ALDH) at the simulative pH condition, was studied using spectroscopic approach. Ferulic acid caused a decrease in the fluorescence intensity formed from ALDH-ferulic acid complex resulting in mixed inhibition of ALDH activity (IC 50 =30.65μM). The intrinsic quenching was dynamic and induced altered conformation of ALDH and made the protein less compact but might not unfold it. ALDH has two binding sites for ferulic acid at saturating concentrations having association constant of 1.35×10 3 Lmol -1 and a dissociation constant of 9.7×10 7 Lmol -1 at 25°C indicating ALDH-ferulic acid complex formation is more favourable than its dissociation. The interaction was not spontaneous and endothermic and suggests the involvement of hydrophobic interactions with a FRET binding distance of 4.49nm. Change in pH near and far from isoelectric points of ferulic acid did not affect the bonding interaction. Using trehalose as viscosogen, the result from Stoke-Einstein hypothesis showed that ferulic acid-ALDH binding and dissociation equilibrium was diffusion controlled. These results clearly suggest the unique binding properties and lipophilicity influence of ferulic acid. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. 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

  18. ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells.

    PubMed

    Moreb, Jan S; Baker, Henry V; Chang, Lung-Ji; Amaya, Maria; Lopez, M Cecilia; Ostmark, Blanca; Chou, Wayne

    2008-11-24

    Aldehyde dehydrogenase isozymes ALDH1A1 and ALDH3A1 are highly expressed in non small cell lung cancer. Neither the mechanisms nor the biologic significance for such over expression have been studied. We have employed oligonucleotide microarrays to analyze changes in gene profiles in A549 lung cancer cell line in which ALDH activity was reduced by up to 95% using lentiviral mediated expression of siRNA against both isozymes (Lenti 1+3). Stringent analysis methods were used to identify gene expression patterns that are specific to the knock down of ALDH activity and significantly different in comparison to wild type A549 cells (WT) or cells similarly transduced with green fluorescent protein (GFP) siRNA. We confirmed significant and specific down regulation of ALDH1A1 and ALDH3A1 in Lenti 1+3 cells and in comparison to 12 other ALDH genes detected. The results of the microarray analysis were validated by real time RT-PCR on RNA obtained from Lenti 1+3 or WT cells treated with ALDH activity inhibitors. Detailed functional analysis was performed on 101 genes that were significantly different (P < 0.001) and their expression changed by > or = 2 folds in the Lenti 1+3 group versus the control groups. There were 75 down regulated and 26 up regulated genes. Protein binding, organ development, signal transduction, transcription, lipid metabolism, and cell migration and adhesion were among the most affected pathways. These molecular effects of the ALDH knock-down are associated with in vitro functional changes in the proliferation and motility of these cells and demonstrate the significance of ALDH enzymes in cell homeostasis with a potentially significant impact on the treatment of lung cancer.

  19. Gender differences in the effects of ADH1B and ALDH2 polymorphisms on alcoholism.

    PubMed

    Kimura, Mitsuru; Miyakawa, Tomohiro; Matsushita, Sachio; So, Mirai; Higuchi, Susumu

    2011-11-01

    Gender differences are known to exist in the prevalence, characteristics, and course of alcohol dependence. Elucidating gender differences in the characteristics of alcohol dependence is important in gender-based medicine and may improve treatment outcomes. Many studies have shown that genetic factors are associated with the risk of alcohol dependence in both genders. Polymorphisms of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) are strong genetic determinants of alcohol dependence. This study aimed to clarify gender differences in the effects of ADH1B and ALDH2 polymorphism on the development of alcohol dependence. Subjects were 200 female alcoholics and 415 male alcoholics hospitalized in Kurihama Alcoholism Center. Clinical information and background data were obtained by chart review. ALDH2 and ADH1B genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism method. The onset age of female alcoholics with inactive ALDH2 genotype was significantly lower than those with active ALDH2 genotype, but the onset age did not differ between the inactive and active ALDH2 group in male alcoholics. The difference in onset age between the ADH1B genotype groups did not reach significant levels. The prevalence of comorbid psychiatric disorders, including major depression, eating disorder, panic disorder, and borderline personality disorder, was significantly higher in female alcoholics with inactive ALDH2 or superactive ADH1B than in those with active ALDH2 or normal ADH1B. ALDH2 polymorphism appears to have contrasting effects on the development of alcoholism in women and men. One possible reason for this gender difference may be the high prevalence of psychiatric comorbidities in female alcoholics with inactive ALDH2. Copyright © 2011 by the Research Society on Alcoholism.

  20. ALDH1B1 links alcohol consumption and diabetes.

    PubMed

    Singh, Surendra; Chen, Ying; Matsumoto, Akiko; Orlicky, David J; Dong, Hongbin; Thompson, David C; Vasiliou, Vasilis

    2015-08-07

    Aldehyde dehydrogenase 1B1 (ALDH1B1) is a mitochondrial enzyme sharing 65% and 72% sequence identity with ALDH1A1 and ALDH2 proteins, respectively. Compared to the latter two ALDH isozymes, little is known about the physiological functions of ALDH1B1. Studies in humans indicate that ALDH1B1 may be associated with alcohol sensitivity and stem cells. Our recent in vitro studies using human ALDH1B1 showed that it metabolizes acetaldehyde and retinaldehyde. To investigate the in vivo role of ALDH1B1, we generated and characterized a global Aldh1b1 knockout mouse line. These knockout (KO) mice are fertile and show overtly good health. However, ethanol pharmacokinetic analysis revealed ∼40% increase in blood acetaldehyde levels in KO mice. Interestingly, the KO mice exhibited higher fasting blood glucose levels. Collectively, we show for the first time the functional in vivo role of ALDH1B1 in acetaldehyde metabolism and in maintaining glucose homeostasis. This mouse model is a valuable tool to investigate the mechanism by which alcohol may promote the development of diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. A missense mutation in ALDH1A3 causes isolated microphthalmia/anophthalmia in nine individuals from an inbred Muslim kindred.

    PubMed

    Mory, Adi; Ruiz, Francesc X; Dagan, Efrat; Yakovtseva, Evgenia A; Kurolap, Alina; Parés, Xavier; Farrés, Jaume; Gershoni-Baruch, Ruth

    2014-03-01

    Nine affected individuals with isolated anophthalmia/microphthalmia from a large Muslim-inbred kindred were investigated. Assuming autosomal-recessive mode of inheritance, whole-genome linkage analysis, on DNA samples from four affected individuals, was undertaken. Homozygosity mapping techniques were employed and a 1.5-Mbp region, homozygous in all affected individuals, was delineated. The region contained nine genes, one of which, aldehyde dehydrogenase 1 (ALDH1A3), was a clear candidate. This gene seems to encode a key enzyme in the formation of a retinoic-acid gradient along the dorsoventral axis during an early eye development and the development of the olfactory system. Sanger sequence analysis revealed a missense mutation, causing a substitution of valine (Val) to methionine (Met) at position 71. Analyzing the p.Val71Met missense mutation using standard open access software (MutationTaster online, PolyPhen, SIFT/PROVEAN) predicts this variant to be damaging. Enzymatic activity, studied in vitro, showed no changes between the mutated and the wild-type ALDH1A3 protein.

  2. A missense mutation in ALDH1A3 causes isolated microphthalmia/anophthalmia in nine individuals from an inbred Muslim kindred

    PubMed Central

    Mory, Adi; Ruiz, Francesc X; Dagan, Efrat; Yakovtseva, Evgenia A; Kurolap, Alina; Parés, Xavier; Farrés, Jaume; Gershoni-Baruch, Ruth

    2014-01-01

    Nine affected individuals with isolated anophthalmia/microphthalmia from a large Muslim-inbred kindred were investigated. Assuming autosomal-recessive mode of inheritance, whole-genome linkage analysis, on DNA samples from four affected individuals, was undertaken. Homozygosity mapping techniques were employed and a 1.5-Mbp region, homozygous in all affected individuals, was delineated. The region contained nine genes, one of which, aldehyde dehydrogenase 1 (ALDH1A3), was a clear candidate. This gene seems to encode a key enzyme in the formation of a retinoic-acid gradient along the dorsoventral axis during an early eye development and the development of the olfactory system. Sanger sequence analysis revealed a missense mutation, causing a substitution of valine (Val) to methionine (Met) at position 71. Analyzing the p.Val71Met missense mutation using standard open access software (MutationTaster online, PolyPhen, SIFT/PROVEAN) predicts this variant to be damaging. Enzymatic activity, studied in vitro, showed no changes between the mutated and the wild-type ALDH1A3 protein. PMID:23881059

  3. ALDH2*2 and peer drinking in East Asian college students.

    PubMed

    O'Shea, Taryn; Thomas, Nathaniel; Webb, Bradley Todd; Dick, Danielle M; Kendler, Kenneth S; Chartier, Karen G

    2017-11-01

    The ALDH2*2 allele (A-allele) at rs671 is more commonly carried by Asians and is associated with alcohol-related flushing, a strong adverse reaction to alcohol that is protective against drinking. Social factors, such as having friends who binge drink, also contribute to drinking in Asian youth. This study examined the interplay between ALDH2*2, peer drinking, and alcohol consumption in college students. We hypothesized that the relationship between ALDH2*2 and standard grams of ethanol per month would vary based on the level of peer drinking. Subjects (N = 318, 63.25% female) were East Asian college students in the United States who reported drinking alcohol. Data were from the freshman year of a university survey that included a saliva DNA sample. ALDH2*2 status was coded ALDH2*2(+) (A/G and A/A genotypes) and ALDH2*2(-) (G/G genotype). Peer drinking was students' perception of how many of their friends "got drunk". Main effects of ALDH2*2(-) and having more friends who got drunk were associated with greater alcohol consumption. The ALDH2*2 × peer drunkenness interaction showed a stronger positive association with alcohol consumption for ALDH2*2(-) versus ALDH2*2(+) at increasing levels of peer drunkenness. Follow-up comparisons within each peer drunkenness level identified significantly higher alcohol consumption for ALDH2*2(-) compared to ALDH2*2(+) at the all friends got drunk level. There was evidence of a stronger effect for ALDH2*2(-) compared to ALDH2*2(+) with greater alcohol use when students were more exposed to peer drinking. Findings contribute to a growing literature on the interrelationships between genetic influences and more permissive environments for alcohol consumption.

  4. Comparative and evolutionary studies of vertebrate ALDH1A-like genes and proteins.

    PubMed

    Holmes, Roger S

    2015-06-05

    Vertebrate ALDH1A-like genes encode cytosolic enzymes capable of metabolizing all-trans-retinaldehyde to retinoic acid which is a molecular 'signal' guiding vertebrate development and adipogenesis. Bioinformatic analyses of vertebrate and invertebrate genomes were undertaken using known ALDH1A1, ALDH1A2 and ALDH1A3 amino acid sequences. Comparative analyses of the corresponding human genes provided evidence for distinct modes of gene regulation and expression with putative transcription factor binding sites (TFBS), CpG islands and micro-RNA binding sites identified for the human genes. ALDH1A-like sequences were identified for all mammalian, bird, lizard and frog genomes examined, whereas fish genomes displayed a more restricted distribution pattern for ALDH1A1 and ALDH1A3 genes. The ALDH1A1 gene was absent in many bony fish genomes examined, with the ALDH1A3 gene also absent in the medaka and tilapia genomes. Multiple ALDH1A1-like genes were identified in mouse, rat and marsupial genomes. Vertebrate ALDH1A1, ALDH1A2 and ALDH1A3 subunit sequences were highly conserved throughout vertebrate evolution. Comparative amino acid substitution rates showed that mammalian ALDH1A2 sequences were more highly conserved than for the ALDH1A1 and ALDH1A3 sequences. Phylogenetic studies supported an hypothesis for ALDH1A2 as a likely primordial gene originating in invertebrate genomes and undergoing sequential gene duplication to generate two additional genes, ALDH1A1 and ALDH1A3, in most vertebrate genomes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

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

    2016-12-01

    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.

  6. A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines.

    PubMed

    Wang, Fei; Guo, Tao; Jiang, Hongmei; Li, Ruobi; Wang, Ting; Zeng, Ni; Dong, Guanghui; Zeng, Xiaowen; Li, Daochuan; Xiao, Yongmei; Hu, Qiansheng; Chen, Wen; Xing, Xiumei; Wang, Qing

    2018-06-01

    Gene knockdown and knockout using RNAi and CRISPR/Cas9 allow for efficient evaluation of gene function, but it is unclear how the choice of technology can influence the results. To compare the phenotypes obtained using siRNA and CRISPR/Cas9 technologies, aldehyde dehydrogenase 2 (ALDH2) was selected as an example. In this study, we constructed one HepG2 cell line with a homozygous mutation in the fifth exon of ALDH2 (ALDH2-KO1 cell) using the eukaryotic CRISPR/Cas9 expression system followed by the limited dilution method and one HepG2 cell line with different mutations in the ALDH2 gene (ALDH2-KO2 cell) using the lentivirus CRISPR/Cas9 system. Additionally, one ALDH2-knockdown (KD) HepG2 cell line was created using siRNA. The reproducibility of these methods was further verified in the HEK293FT cell line. We found that the mRNA expression level of ALDH2 was significantly decreased and the protein expression level of ALDH2 was completely abolished in the ALDH2-KO cell lines, but not in ALDH2-KD cells. Furthermore, the functional activity of ALDH2 was also markedly disrupted in the two ALDH2-KO cell lines compared with ALDH2-KD and wild-type cells. The lack of ALDH2 expression mediated by CRIPSR/Cas9 resulted in a more dramatic increase in the cellular susceptibility to chemical-induced reactive oxygen species generation, cytotoxicity, apoptosis, and inflammation, especially at low concentrations compared with ALDH2-KD and WT cells. Therefore, we consider the gene knockout cell line created by CRISPR/Cas9 to be a more useful tool for identifying the function of a gene.

  7. Importance of inverse correlation between ALDH3A1 and PPARγ in tumor cells and tissue regeneration.

    PubMed

    Oraldi, M; Saracino, S; Maggiora, M; Chiaravalloti, A; Buemi, C; Martinasso, G; Paiuzzi, E; Thompson, D; Vasiliou, V; Canuto, R A

    2011-05-30

    Aldehyde dehydrogenase (ALDH) enzymes are involved in maintaining cellular homeostasis by metabolizing both endogenous and exogenous reactive aldehydes. They modulate several cell functions including proliferation, differentiation, survival as well as cellular response to oxidative stress. We previously reported that ALDH3A1 expression is inversely correlated with the activation of PPARs (Peroxisome Proliferators-Activated Receptors), a category of orphan nuclear hormone receptors, in both rat and human cells. PPARγ is involved in cell proliferation. In this study, we have used PPARγ transfection and inhibition to examine the relationship between ALDH3A1 and PPARγ and their role as regulators of cell proliferation. Induction of PPARγ in A549 and NCTC 2544 cells by transfection caused a decrease in ALDH3A1 and inhibition of cell proliferation, a result we obtained previously using ligands that induce PPARγ. A reduction of PPARγ expression using siRNA increased ALDH3A1 expression and cell proliferation. In cells induced to proliferate in a model of tissue regeneration, ALDH3A1 expression increased during the period of proliferation, whereas PPARγ expression decreased. In conclusion, through modulation of PPARγ or ALDH3A1, it may be possible to reduce cell proliferation in tumor cells or stimulate cell proliferation in normal cells during tissue regeneration. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  8. Aldehyde dehydrogenase 3A1 protects airway epithelial cells from cigarette smoke-induced DNA damage and cytotoxicity.

    PubMed

    Jang, Jun-Ho; Bruse, Shannon; Liu, Yushi; Duffy, Veronica; Zhang, Chunyu; Oyamada, Nathaniel; Randell, Scott; Matsumoto, Akiko; Thompson, David C; Lin, Yong; Vasiliou, Vasilis; Tesfaigzi, Yohannes; Nyunoya, Toru

    2014-03-01

    Aldehyde dehydrogenase 3A1 (ALDH3A1), an ALDH superfamily member, catalyzes the oxidation of reactive aldehydes, highly toxic components of cigarette smoke (CS). Even so, the role of ALDH3A1 in CS-induced cytotoxicity and DNA damage has not been examined. Among all of the ALDH superfamily members, ALDH3A1 mRNA levels showed the greatest induction in response to CS extract (CSE) exposure of primary human bronchial epithelial cells (HBECs). ALDH3A1 protein accumulation was accompanied by increased ALDH enzymatic activity in CSE-exposed immortalized HBECs. The effects of overexpression or suppression of ALDH3A1 on CSE-induced cytotoxicity and DNA damage (γH2AX) were evaluated in cultured immortalized HBECs. Enforced expression of ALDH3A1 attenuated cytotoxicity and downregulated γH2AX. SiRNA-mediated suppression of ALDH3A1 blocked ALDH enzymatic activity and augmented cytotoxicity in CSE-exposed cells. Our results suggest that the availability of ALDH3A1 is important for cell survival against CSE in HBECs. Published by Elsevier Inc.

  9. Origin and dispersal of atypical aldehyde dehydrogenase ALDH2487Lys.

    PubMed

    Luo, Huai-Rong; Wu, Gui-Sheng; Pakstis, Andrew J; Tong, Li; Oota, Hiroki; Kidd, Kenneth K; Zhang, Ya-Ping

    2009-04-15

    The East Asian respond with a marked facial flushing and mild to moderate symptoms of intoxication after drinking the amounts of alcohol that has no detectable effect on European. The alcohol sensitivity in Orientals is due to a delayed oxidation of acetaldehyde by an atypical aldehyde dehydrogenase ALDH2487Lys, which is resulted from a structural mutation in gene ALDH2. The atypical ALDH2487Lys allele has been associated with various phenotypic statuses, such as protective against alcohol dependence and the risk of alcohol-related digestive tract cancers. Here, we have examined this SNP, adjacent four non-coding SNPs, and one downstream STRP on ALDH2 gene, in total of 1072 unrelated healthy individuals from 14 Chinese populations and 130 Indian individuals. Five major haplotypes based on five SNPs across the ALDH2 gene 40 kb were found in all East Asian populations. The frequencies of the ancestral haplotype GCCTG and the East Asian special haplotype GCCTA containing the atypical ALDH2487Lys allele were 44.8% and 14.9%, respectively. The frequency of the atypical ALDH2487Lys allele or the East Asian specific haplotype GCCTA is high in Yunnan, South coastal, east coastal of China, and decreased gradually toward inland China, West, Northwest and North China. Combined with demographic history in East Asian, our results showed that the presence of ALDH2487Lys allele in peripheral regions of China might be the results of historical migration events from China to these regions. The origin of ALDH2487Lys could be possibly traced back to ancient Pai-Yuei tribe in South China.

  10. Expression and Interaction Analysis among Saffron ALDHs and Crocetin Dialdehyde.

    PubMed

    Gómez-Gómez, Lourdes; Pacios, Luis F; Diaz-Perales, Araceli; Garrido-Arandia, María; Argandoña, Javier; Rubio-Moraga, Ángela; Ahrazem, Oussama

    2018-05-09

    In saffron, the cleavage of zeaxanthin by means of CCD2 generates crocetin dialdehyde, which is then converted by an unknown aldehyde dehydrogenase to crocetin. A proteome from saffron stigma was released recently and, based on the expression pattern and correlation analyses, five aldehyde dehydrogenases (ALDHs) were suggested as possible candidates to generate crocetin from crocetin dialdehydes. We selected four of the suggested ALDHs and analyzed their expression in different tissues, determined their activity over crocetin dialdehyde, and performed structure modeling and docking calculation to find their specificity. All the ALDHs were able to convert crocetin dialdehyde to crocetin, but two of them were stigma tissue-specific. Structure modeling and docking analyses revealed that, in all cases, there was a high coverage of residues in the models. All of them showed a very close conformation, indicated by the low root-mean-square deviation (RMSD) values of backbone atoms, which indicate a high similarity among them. However, low affinity between the enzymes and the crocetin dialdehyde were observed. Phylogenetic analysis and binding affinities calculations, including some ALDHs from Gardenia jasmonoides , Crocus sieberi , and Buddleja species that accumulate crocetin and Bixa orellana synthetizing the apocarotenoid bixin selected on their expression pattern matching with the accumulation of either crocins or bixin, pointed out that family 2 C4 members might be involved in the conversion of crocetin dialdehyde to crocetin with high specificity.

  11. ALDH1A3, the Major Aldehyde Dehydrogenase Isoform in Human Cholangiocarcinoma Cells, Affects Prognosis and Gemcitabine Resistance in Cholangiocarcinoma Patients.

    PubMed

    Chen, Ming-Huang; Weng, Jing-Jie; Cheng, Chi-Tung; Wu, Ren-Chin; Huang, Shih-Chiang; Wu, Chiao-En; Chung, Yi-Hsiu; Liu, Chun-Yu; Chang, Mu-Hsin; Chen, Ming-Han; Chiang, Kun-Chun; Yeh, Ta-Sen; Su, Yeu; Yeh, Chun-Nan

    2016-08-15

    Intrahepatic cholangiocarcinoma is a fatal primary liver cancer resulting from diagnosis at an advanced stage. Understanding the mechanisms of drug resistance and metastasis of cholangiocarcinoma may improve the disease prognosis. Enhanced aldehyde dehydrogenase (ALDH) activity is suggested to be associated with increased drug resistance and the metastasis. This study aims to investigate the roles of the ALDH isoforms in cholangiocarcinoma. Aldefluor assays, RT-PCR, and Western blot analysis were used to identify the major ALDH isoforms contributing to Aldefluor activity in human cholangiocarcinoma cell lines. We manipulated isoform expression in HuCCT1 cells to elucidate the role of ALDH1A3 in the malignant progression of these cells. Finally, we used immunohistochemical staining to evaluate the clinical significance of ALDH1A3 in 77 hepatectomized cholangiocarcinoma patients and an additional 31 patients with advanced cholangiocarcinoma who received gemcitabine-based therapy. ALDH(high) cholangiocarcinoma cells not only migrated faster but were more resistant to gemcitabine. Among the 19 ALDH isoforms studied, ALDH1A3 was found to be the main contributor to Aldefluor activity. In addition, we also found that knockdown of ALDH1A3 expression in HuCCT1 cells markedly reduced not only their sensitivity to gemcitabine, which might be attributed to a decreased expression of ribonucleotide reductase M1, but also their migration. Most importantly, this enzyme was also identified as an independent poor prognostic factor for patients with intrahepatic cholangiocarcinoma, as well as a prognostic biomarker of gemcitabine-treated patients. ALDH1A3 plays an important role in enhancing malignant behavior of cholangiocarcinoma and serves as a new therapeutic target. Clin Cancer Res; 22(16); 4225-35. ©2016 AACR. ©2016 American Association for Cancer Research.

  12. Garcinol from Garcinia indica Downregulates Cancer Stem-like Cell Biomarker ALDH1A1 in Nonsmall Cell Lung Cancer A549 Cells through DDIT3 Activation.

    PubMed

    Wang, Jinhan; Wang, Liwen; Ho, Chi-Tang; Zhang, Kunsheng; Liu, Qiang; Zhao, Hui

    2017-05-10

    Nonsmall cell lung cancer (NSCLC) is the predominant type of lung cancer. Patients with NSCLC show high mortality rates because of failure to clean up cancer stem cells (CSCs). The anticancer activity of phytochemical garcinol has been identified in various cancer cell models. However, the effect of garcinol on NSCLC cell lines is still lacking. Of the NSCLC cell lines we tested, A549 cells were the most sensitive to garcinol. Interestingly, Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) was preferentially expressed in A549 cells and downregulated by the addition of garcinol. We also found that garcinol enriched DNA damage-inducible transcript 3 (DDIT3) and then altered DDIT3-CCAAT-enhancer-binding proteins beta (C/EBPβ) interaction resulting in a decreased binding of C/EBPβ to the endogenous ALDH1A1 promoter. Furthermore, garcinol's inhibition of ALDH1A1 was identified in a xenograft mice model. Garcinol repressed ALDH1A1 transcription in A549 cells through alterations in the interaction between DDIT3 and C/EBPβ. Garcinol could be a potential dietary phytochemical candidate for NSCLCs patients whose tumors harbored high ALDH1A1 expression.

  13. ALDH16A1 is a novel non-catalytic enzyme that may be involved in the etiology of gout via protein–protein interactions with HPRT1

    PubMed Central

    Vasiliou, Vasilis; Sandoval, Monica; Backos, Donald S.; Jackson, Brian C.; Chen, Ying; Reigan, Philip; Lanaspa, Miguel A.; Johnson, Richard J.; Koppaka, Vindhya; Thompson, David C.

    2013-01-01

    Gout, a common form of inflammatory arthritis, is strongly associated with elevated uric acid concentrations in the blood (hyperuricemia). A recent study in Icelanders identified a rare missense single nucleotide polymorphism (SNP) in the ALDH16A1 gene, ALDH16A1*2, to be associated with gout and serum uric acid levels. ALDH16A1 is a novel and rather unique member of the ALDH superfamily in relation to its gene and protein structures. ALDH16 genes are present in fish, amphibians, protista, bacteria but absent from archaea, fungi and plants. In most mammalian species, two ALDH16A1 spliced variants (ALDH16A1, long form and ALDH16A1_v2, short form) have been identified and both are expressed in HepG-2, HK-2 and HK-293 human cell lines. The ALDH16 proteins contain two ALDH domains (as opposed to one in the other members of the superfamily), four transmembrane and one coiled-coil domains. The active site of ALDH16 proteins from bacterial, frog and lower animals contain the catalytically important cysteine residue (Cys-302); this residue is absent from the mammalian and fish orthologs. Molecular modeling predicts that both the short and long forms of human ALDH16A1 protein would lack catalytic activity but may interact with the hypoxanthine-guanine phosphoribosyltransferase (HPRT1) protein, a key enzyme involved in uric acid metabolism and gout. Interestingly, such protein-protein interactions with HPRT1 are predicted to be impaired for the long or short forms of ALDH16A1*2. These results lead to the intriguing possibility that association between ALDH16A1 and HPRT1 may be required for optimal HPRT activity with disruption of this interaction possibly contributing to the hyperuricemia seen in ALDH16A1*2 carriers. PMID:23348497

  14. ALDH16A1 is a novel non-catalytic enzyme that may be involved in the etiology of gout via protein-protein interactions with HPRT1.

    PubMed

    Vasiliou, Vasilis; Sandoval, Monica; Backos, Donald S; Jackson, Brian C; Chen, Ying; Reigan, Philip; Lanaspa, Miguel A; Johnson, Richard J; Koppaka, Vindhya; Thompson, David C

    2013-02-25

    Gout, a common form of inflammatory arthritis, is strongly associated with elevated uric acid concentrations in the blood (hyperuricemia). A recent study in Icelanders identified a rare missense single nucleotide polymorphism (SNP) in the ALDH16A1 gene, ALDH16A1*2, to be associated with gout and serum uric acid levels. ALDH16A1 is a novel and rather unique member of the ALDH superfamily in relation to its gene and protein structures. ALDH16 genes are present in fish, amphibians, protista, bacteria but absent from archaea, fungi and plants. In most mammalian species, two ALDH16A1 spliced variants (ALDH16A1, long form and ALDH16A1_v2, short form) have been identified and both are expressed in HepG-2, HK-2 and HK-293 human cell lines. The ALDH16 proteins contain two ALDH domains (as opposed to one in the other members of the superfamily), four transmembrane and one coiled-coil domains. The active site of ALDH16 proteins from bacterial, frog and lower animals contain the catalytically important cysteine residue (Cys-302); this residue is absent from the mammalian and fish orthologs. Molecular modeling predicts that both the short and long forms of human ALDH16A1 protein would lack catalytic activity but may interact with the hypoxanthine-guanine phosphoribosyltransferase (HPRT1) protein, a key enzyme involved in uric acid metabolism and gout. Interestingly, such protein-protein interactions with HPRT1 are predicted to be impaired for the long or short forms of ALDH16A1*2. These results lead to the intriguing possibility that association between ALDH16A1 and HPRT1 may be required for optimal HPRT activity with disruption of this interaction possibly contributing to the hyperuricemia seen in ALDH16A1*2 carriers. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. MAOA interacts with the ALDH2 gene in anxiety-depression alcohol dependence.

    PubMed

    Lee, Sheng-Yu; Hahn, Cheng-Yi; Lee, Jia-Fu; Huang, San-Yuan; Chen, Shiou-Lan; Kuo, Po-Hsiu; Lee, I Hui; Yeh, Tzung Lieh; Yang, Yen Kuang; Chen, Shih-Heng; Ko, Huei-Chen; Lu, Ru-Band

    2010-07-01

    Alcohol dependence is usually comorbid with anxiety disorder, depressive disorder, or both; this comorbidity may increase drinking behavior. We previously hypothesized that anxiety-depressive alcohol dependence (ANX/DEP ALC) was a genetically specific subtype of alcohol dependence. ANX/DEP ALC may be related to dopamine and serotonin, which are catalyzed by monoamine oxidase A (MAOA) and acetaldehyde dehydrogenase 2 (ALDH2). The aim of this study was to determine whether the interaction between the MAOA and the ALDH2 genes is associated with ANX/DEP ALC. We recruited 383 Han Chinese men in Taiwan: 143 ANX/DEP ALC and 240 healthy controls. The diagnosis of ANX/DEP ALC (alcohol dependence with a past or current history of anxiety, depressive disorder, or both) was made using DSM-IV criteria. Genotypes of ALDH2 and MAOA-uVNTR (variable number of tandem repeat located upstream) were determined using PCR-RFLP. The ALDH2, but not the MAOA-uVNTR, polymorphism was associated with ANX/DEP ALC. After stratifying the MAOA-uVNTR polymorphism, we found a stronger association between the ALDH2*1/*2 and *2/*2 genotypes and the controls in the MAOA-uVNTR 4-repeat subgroup. Logistic regression significantly associated the interaction between ALDH2 and MAOA variants with ANX/DEP ALC. We conclude that the MAOA and ALDH2 genes interact in ANX/DEP ALC. Although the MAOA gene alone is not associated with ANX/DEP ALC, we hypothesize that different variants of MAOA-uVNTR polymorphisms modify the protective effects of the ALDH2*2 allele on ANX/DEP ALC in Han Chinese in Taiwan.

  16. Molecular profiling of ALDH1+ colorectal cancer stem cells reveals preferential activation of MAPK, FAK, and oxidative stress pro-survival signalling pathways.

    PubMed

    Vishnubalaji, Radhakrishnan; Manikandan, Muthurangan; Fahad, Mohamed; Hamam, Rimi; Alfayez, Musaad; Kassem, Moustapha; Aldahmash, Abdullah; Alajez, Nehad M

    2018-03-02

    Tumour heterogeneity leads to variable clinical response and inaccurate diagnostic and prognostic assessment. Cancer stem cells (CSCs) represent a subpopulation responsible for invasion, metastasis, therapeutic resistance, and recurrence in many human cancer types. However, the true identity of colorectal cancer (CRC) SCs remains elusive. Here, we aimed to characterize and define the gene expression portrait of CSCs in CRC-model SW403 cells. We found that ALDH + positive cells are clonogenic and highly proliferative; their global gene expression profiling-based molecular signature revealed gene enrichment related to DNA damage, MAPK, FAK, oxidative stress response, and Wnt signalling. ALDH + cells showed enhanced ROS stress resistance, whereas MAPK/FAK pathway pharmacologic inhibition limited their survival. Conversely, 5-fluorouracil increased the ALDH + cell fraction among the SW403, HCT116 and SW620 CRC models. Notably, analysis of ALDH1A1 and POU5F1 expression levels in cohorts of 462 or 420 patients for overall (OS) or disease-free (DFS) survival, respectively, obtained from the Cancer Genome Atlas CRC dataset, revealed strong association between elevated expression and poor OS ( p = 0.006) and poor DFS ( p = 0.05), thus implicating ALDH1A1 and POU5F1 in CRC prognosis. Our data reveal distinct molecular signature of ALDH + CSCs in CRC and suggest pathways relevant for successful targeted therapies and management of CRC.

  17. Molecular profiling of ALDH1+ colorectal cancer stem cells reveals preferential activation of MAPK, FAK, and oxidative stress pro-survival signalling pathways

    PubMed Central

    Vishnubalaji, Radhakrishnan; Manikandan, Muthurangan; Fahad, Mohamed; Hamam, Rimi; Alfayez, Musaad; Kassem, Moustapha; Aldahmash, Abdullah; Alajez, Nehad M.

    2018-01-01

    Tumour heterogeneity leads to variable clinical response and inaccurate diagnostic and prognostic assessment. Cancer stem cells (CSCs) represent a subpopulation responsible for invasion, metastasis, therapeutic resistance, and recurrence in many human cancer types. However, the true identity of colorectal cancer (CRC) SCs remains elusive. Here, we aimed to characterize and define the gene expression portrait of CSCs in CRC-model SW403 cells. We found that ALDH+ positive cells are clonogenic and highly proliferative; their global gene expression profiling-based molecular signature revealed gene enrichment related to DNA damage, MAPK, FAK, oxidative stress response, and Wnt signalling. ALDH+ cells showed enhanced ROS stress resistance, whereas MAPK/FAK pathway pharmacologic inhibition limited their survival. Conversely, 5-fluorouracil increased the ALDH+ cell fraction among the SW403, HCT116 and SW620 CRC models. Notably, analysis of ALDH1A1 and POU5F1 expression levels in cohorts of 462 or 420 patients for overall (OS) or disease-free (DFS) survival, respectively, obtained from the Cancer Genome Atlas CRC dataset, revealed strong association between elevated expression and poor OS (p = 0.006) and poor DFS (p = 0.05), thus implicating ALDH1A1 and POU5F1 in CRC prognosis. Our data reveal distinct molecular signature of ALDH+ CSCs in CRC and suggest pathways relevant for successful targeted therapies and management of CRC. PMID:29568377

  18. Correlation of ALDH1 and Notch3 Expression: Clinical implication in Ovarian Carcinomas.

    PubMed

    Kim, Mi Joung; Kim, A-Ram; Jeong, Ju-Yeon; Kim, Kwang-Il; Kim, Tae-Heon; Lee, Chan; Chung, Kwanghoe; Ko, Young-Hyeh; An, Hee-Jung

    2017-01-01

    Purpose : ALDH1 is a putative cancer stem cell marker, while the Notch signaling pathway is involved in regulation of cancer stem cell (CSC)s. This study aims to determine the expression of Notch signaling genes in ovarian CSCs, and to assess the clinical impact of expression of ALDH1 and Notch signaling genes in ovarian cancers. Methods : We examined expression of Notch signaling genes in FACS-sorted ALDH1(+) putative ovarian CSCs and expression of ALDH1 and Notch signaling genes in 86 ovarian epithelial tumors and various ovarian cancer cell lines by real-time RT-PCR, including Notch receptors ( Notch1-4 ), Notch ligands ( Jagged1 and Jagged2 ), and the downstream molecule, Hes1 . Furthermore, we correlated their expression with clinicopathological parameters and patient's survival in ovarian serous carcinoma (OSC)s, the most prevalent type of ovarian cancer. Results : The higher expression levels of ALDH1 and Notch related genes, especially Notch3 were associated with CSCs and with chemoresistant OSCs and paclitaxel-resistant SKpac ovarian cancer cells. Among the Notch signaling genes, high Notch3 expression was significantly associated with all the parameters of poor prognosis, i.e., advanced stage, lymph node and distant metastases, and chemoresistance, whereas other genes were less correlated with these parameters. A combined upregulation of ALDH1 and Notch3 was an independent poor prognostic factor in OSCs. Conclusions : ALDH1 correlates with Notch3 expression in ovarian carcinomas. ALDH1 and Notch3 overexpression is an independent poor prognostic indicator for worse patient's survival in this subset of OSCs.

  19. Characteristics and expression patterns of the aldehyde dehydrogenase (ALDH) gene superfamily of foxtail millet (Setaria italica L.).

    PubMed

    Chen, Zhu; Chen, Ming; Xu, Zhao-shi; Li, Lian-cheng; Chen, Xue-ping; Ma, You-zhi

    2014-01-01

    Recent genomic sequencing of the foxtail millet, an abiotic, stress-tolerant crop, has provided a great opportunity for novel gene discovery and functional analysis of this popularly-grown grass. However, few stress-mediated gene families have been studied. Aldehyde dehydrogenases (ALDHs) comprise a gene superfamily encoding NAD (P) +-dependent enzymes that play the role of "aldehyde scavengers", which indirectly detoxify cellular ROS and reduce the effect of lipid peroxidation meditated cellular toxicity under various environmental stresses. In the current paper, we identified a total of 20 ALDH genes in the foxtail millet genome using a homology search and a phylogenetic analysis and grouped them into ten distinct families based on their amino acid sequence identity. Furthermore, evolutionary analysis of foxtail millet reveals that both tandem and segmental duplication contributed significantly to the expansion of its ALDH genes. The exon-intron structures of members of the same family in foxtail millet or the orthologous genes in rice display highly diverse distributions of their exonic and intronic regions. Also, synteny analysis shows that the majority of foxtail millet and rice ALDH gene homologs exist in the syntenic blocks between the two, implying that these ALDH genes arose before the divergence of cereals. Semi-quantitative and real-time quantitative PCR data reveals that a few SiALDH genes are expressed in an organ-specific manner and that the expression of a number of foxtail millet ALDH genes, such as, SiALDH7B1, SiALDH12A1 and SiALDH18B2 are up-regulated by osmotic stress, cold, H2O2, and phytohormone abscisic acid (ABA). Furthermore, the transformation of SiALDH2B2, SiALDH10A2, SiALDH5F1, SiALDH22A1, and SiALDH3E2 into Escherichia coli (E.coli) was able to improve their salt tolerance. Taken together, our results show that genome-wide identification characteristics and expression analyses provide unique opportunities for assessing the functional

  20. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.

  1. 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

  2. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics.

    PubMed

    Brocker, Chad; Vasiliou, Melpomene; Carpenter, Sarah; Carpenter, Christopher; Zhang, Yucheng; Wang, Xiping; Kotchoni, Simeon O; Wood, Andrew J; Kirch, Hans-Hubert; Kopečný, David; Nebert, Daniel W; Vasiliou, Vasilis

    2013-01-01

    In recent years, there has been a significant increase in the number of completely sequenced plant genomes. The comparison of fully sequenced genomes allows for identification of new gene family members, as well as comprehensive analysis of gene family evolution. The aldehyde dehydrogenase (ALDH) gene superfamily comprises a group of enzymes involved in the NAD(+)- or NADP(+)-dependent conversion of various aldehydes to their corresponding carboxylic acids. ALDH enzymes are involved in processing many aldehydes that serve as biogenic intermediates in a wide range of metabolic pathways. In addition, many of these enzymes function as 'aldehyde scavengers' by removing reactive aldehydes generated during the oxidative degradation of lipid membranes, also known as lipid peroxidation. Plants and animals share many ALDH families, and many genes are highly conserved between these two evolutionarily distinct groups. Conversely, both plants and animals also contain unique ALDH genes and families. Herein we carried out genome-wide identification of ALDH genes in a number of plant species-including Arabidopsis thaliana (thale crest), Chlamydomonas reinhardtii (unicellular algae), Oryza sativa (rice), Physcomitrella patens (moss), Vitis vinifera (grapevine) and Zea mays (maize). These data were then combined with previous analysis of Populus trichocarpa (poplar tree), Selaginella moellindorffii (gemmiferous spikemoss), Sorghum bicolor (sorghum) and Volvox carteri (colonial algae) for a comprehensive evolutionary comparison of the plant ALDH superfamily. As a result, newly identified genes can be more easily analyzed and gene names can be assigned according to current nomenclature guidelines; our goal is to clarify previously confusing and conflicting names and classifications that might confound results and prevent accurate comparisons between studies.

  3. Depleted aldehyde dehydrogenase 1A1 (ALDH1A1) reverses cisplatin resistance of human lung adenocarcinoma cell A549/DDP.

    PubMed

    Wei, Yunyan; Wu, Shuangshuang; Xu, Wei; Liang, Yan; Li, Yue; Zhao, Weihong; Wu, Jianqing

    2017-01-01

    Cisplatin is the standard first-line chemotherapeutic agent for the treatment of non-small cell lung cancer (NSCLC). However, resistance to chemotherapy has been a major obstacle in the management of NSCLC. Aldehyde dehydrogenase 1A1 (ALDH1A1) overexpression has been observed in a variety of cancers, including lung cancer. The purpose of this study was to investigate the effect of ALDH1A1 expression on cisplatin resistance and explore the mechanism responsible. Reverse transcriptase-PCR was applied to measure the messenger RNA expression of ALDH1A1, while Western blot assay was employed to evaluate the protein expression of ALDH1A1, B-cell lymphoma 2, Bcl-2-like protein 4, phospho-protein kinase B (p-AKT) and AKT. A short hairpin RNA was used to knockdown ALDH1A1 expression. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the effect of ALDH1A1 decrease on cell viability. The cell apoptotic rate was tested using flow cytometry assay. ALDH1A1 is overexpressed in cisplatin resistant cell line A549/DDP, compared with A549. ALDH1A1 depletion significantly decreased A549/DDP proliferation, increased apoptosis, and reduced cisplatin resistance. In addition, the phosphoinositide 3-kinase (PI3K) / AKT pathway is activated in A549/DDP, and ALDH1A1 knockdown reduced the phosphorylation level of AKT. Moreover, the combination of ALDH1A1-short hairpin RNA and PI3K/AKT pathway inhibitor LY294002 markedly inhibited cell viability, enhanced apoptotic cell death, and increased cisplatin sensitivity. These results suggest that ALDH1A1 depletion could reverse cisplatin resistance in human lung cancer cell line A549/DDP, and may act as a potential target for the treatment of lung cancers resistant to cisplatin. © 2016 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

  4. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics

    PubMed Central

    Brocker, Chad; Vasiliou, Melpomene; Carpenter, Sarah; Carpenter, Christopher; Zhang, Yucheng; Wang, Xiping; Kotchoni, Simeon O.; Wood, Andrew J.; Kirch, Hans-Hubert; Kopečný, David; Nebert, Daniel W.

    2012-01-01

    In recent years, there has been a significant increase in the number of completely sequenced plant genomes. The comparison of fully sequenced genomes allows for identification of new gene family members, as well as comprehensive analysis of gene family evolution. The aldehyde dehydrogenase (ALDH) gene superfamily comprises a group of enzymes involved in the NAD+- or NADP+-dependent conversion of various aldehydes to their corresponding carboxylic acids. ALDH enzymes are involved in processing many aldehydes that serve as biogenic intermediates in a wide range of metabolic pathways. In addition, many of these enzymes function as ‘aldehyde scavengers’ by removing reactive aldehydes generated during the oxidative degradation of lipid membranes, also known as lipid peroxidation. Plants and animals share many ALDH families, and many genes are highly conserved between these two evolutionarily distinct groups. Conversely, both plants and animals also contain unique ALDH genes and families. Herein we carried outgenome-wide identification of ALDH genes in a number of plant species—including Arabidopsis thaliana (thale crest), Chlamydomonas reinhardtii (unicellular algae), Oryza sativa (rice), Physcomitrella patens (moss), Vitis vinifera (grapevine) and Zea mays (maize). These data were then combined with previous analysis of Populus trichocarpa (poplar tree), Selaginella moellindorffii (gemmiferous spikemoss), Sorghum bicolor (sorghum) and Volvox carteri (colonial algae) for a comprehensive evolutionary comparison of the plant ALDH superfamily. As a result, newly identified genes can be more easily analyzed and gene names can be assigned according to current nomenclature guidelines; our goal is to clarify previously confusing and conflicting names and classifications that might confound results and prevent accurate comparisons between studies. PMID:23007552

  5. 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.

  6. Number of nitrate groups determines reactivity and potency of organic nitrates: a proof of concept study in ALDH-2−/− mice

    PubMed Central

    Wenzel, P; Hink, U; Oelze, M; Seeling, A; Isse, T; Bruns, K; Steinhoff, L; Brandt, M; Kleschyov, A L; Schulz, E; Lange, K; Weiner, H; Lehmann, J; Lackner, K J; Kawamoto, T; Münzel, T; Daiber, A

    2007-01-01

    Background and purpose: Mitochondrial aldehyde dehydrogenase (ALDH-2) has been shown to provide a pathway for bioactivation of organic nitrates and to be prone to desensitization in response to highly potent, but not to less potent, nitrates. We therefore sought to support the hypothesis that bioactivation by ALDH-2 critically depends on the number of nitrate groups within the nitrovasodilator. Experimental approach: Nitrates with one (PEMN), two (PEDN; GDN), three (PETriN; glyceryl trinitrate, GTN) and four (pentaerithrityl tetranitrate, PETN) nitrate groups were investigated. Vasodilatory potency was measured in isometric tension studies using isolated aortic segments of wild type (WT) and ALDH-2−/− mice. Activity of the cGMP-dependent kinase-I (reflected by levels of phosphorylated VAsodilator Stimulated Phosphoprotein, P-VASP) was quantified by Western blot analysis, mitochondrial dehydrogenase activity by HPLC. Following incubation of isolated mitochondria with PETN, PETriN-chromophore and PEDN, metabolites were quantified using chemiluminescence nitrogen detection and mass spectrometry. Key results: Compared to WT, vasorelaxation in response to PETN, PETriN and GTN was attenuated about 10fold in ALDH-2−/− mice, identical to WT vessels preincubated with inhibitors of ALDH-2. Reduced vasodilator potency correlated with reduced P-VASP formation and diminished biotransformation of the tetranitrate- and trinitrate-compounds. None of these findings were observed for PEDN, GDN and PEMN. Conclusions and implications: Our results support the crucial role of ALDH-2 in bioactivating highly reactive nitrates like GTN, PETN and PETriN. ALDH-2-mediated relaxation by organic nitrates therefore depends mainly on the number of nitrate groups. Less potent nitrates like PEDN, GDN and PEMN are apparently biotransformed by other pathways. PMID:17220910

  7. 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.

  8. Effects of ALDH2*2 on Alcohol Problem Trajectories of Asian American College Students

    PubMed Central

    Luczak, Susan E.; Yarnell, Lisa M.; Prescott, Carol A.; Myers, Mark G.; Liang, Tiebing; Wall, Tamara L.

    2014-01-01

    The variant aldehyde dehydrogenase allele, ALDH2*2, consistently has been associated with protection against alcohol dependence, but the mechanism underlying this process is not known. This study examined growth trajectories of alcohol consumption (frequency, average quantity, binge drinking, maximum drinks) and problems over the college years and then tested whether the ALDH2 genotype mediated or moderated the relationship between alcohol consumption and problems. Asian American college students (N = 433) reported on their drinking behavior in their first year of college and then annually for 3 consecutive years. Alcohol consumption and problems increased over the college years for both those with and without ALDH2*2, but having an ALDH2*2 allele was associated with less of an increase in problems over time. A mediation model was supported, with ALDH2*2 group differences in problems fully accounted for by differences in frequency of binge drinking. Findings also supported a moderation hypothesis: All four alcohol consumption variables were significant predictors of subsequent alcohol problems, but these relationships were not as strong in those with ALDH2*2 as in those without ALDH2*2. Our findings suggest that the interplay between ALDH2*2 and drinking-related problems is complex, involving both mediation and moderation processes that reduce the likelihood of developing problems via reduction of heavy drinking as well as by altering the relationship between alcohol consumption and problems. Results of this longitudinal study provide evidence that what seems like a relatively straightforward effect of a diminished ability to metabolize alcohol on drinking behavior is actually dependent on behavior and developmental stage. PMID:24661165

  9. 9-cis Retinoic Acid is the ALDH1A1 Product that Stimulates Melanogenesis

    PubMed Central

    Paterson, Elyse K.; Ho, Hsiang; Kapadia, Rubina; Ganesan, Anand K.

    2013-01-01

    Aldehyde dehydrogenase 1A1 (ALDH1A1), an enzyme that catalyzes the conversion of lipid aldehydes to lipid carboxylic acids, plays pleiotropic roles in UV-radiation resistance, melanogenesis, and stem cell maintenance. In this study, a combination of RNAi and pharmacologic approaches were used to determine which ALDH1A1 substrates and products regulate melanogenesis. Initial studies revealed that neither the UV-induced lipid aldehyde 4-hydroxy-2-nonenal nor the ALDH1A1 product all-trans retinoic acid appreciably induced melanogenesis. In contrast, both the ALDH1A1 substrate 9-cis retinal and its corresponding product 9-cis retinoic acid potently induced the accumulation of MITF mRNA, Tyrosinase mRNA, and melanin. ALDH1A1 depletion inhibited the ability of 9-cis retinal but not 9-cis retinoic acid to stimulate melanogenesis, indicating that ALDH1A1 regulates melanogenesis by catalyzing the conversion of 9-cis retinal to 9-cis retinoic acid. The addition of potent ALDH1A inhibitors (cyanamide or Angeli’s salt) suppressed Tyrosinase and MITF mRNA accumulation in vitro and also melanin accumulation in skin equivalents, suggesting that 9-cis retinoids regulate melanogenesis in the intact epidermis. Taken together, these studies not only identify cyanamide as a potential novel treatment for hyperpigmentary disorders, but also identify 9-cis retinoic acid as a pigment stimulatory agent that may have clinical utility in the treatment of hypopigmentary disorders, such as vitiligo. PMID:23489423

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  12. Cancer stem-like cells of ovarian clear cell carcinoma are enriched in the ALDH-high population associated with an accelerated scavenging system in reactive oxygen species.

    PubMed

    Mizuno, T; Suzuki, N; Makino, H; Furui, T; Morii, E; Aoki, H; Kunisada, T; Yano, M; Kuji, S; Hirashima, Y; Arakawa, A; Nishio, S; Ushijima, K; Ito, K; Itani, Y; Morishige, K

    2015-05-01

    In ovarian cancer cases, recurrence after chemotherapy is frequently observed, suggesting the involvement of ovarian cancer stem-like cells (CSCs). The chemoresistance of ovarian clear cell carcinomas is particularly strong in comparison to other epithelial ovarian cancer subtypes. We investigated the relationship between a CSC marker, aldehyde dehydrogenase 1 (ALDH1), and clinical prognosis using ovarian clear cell carcinoma tissue samples. Furthermore, we investigated the antioxidant mechanism by which CSCs maintain a lower reactive oxygen species (ROS) level, which provides protection from chemotherapeutic agents. Immunohistochemical staining was performed to examine the CSC markers (CD133, CD44, ALDH1) using ovarian clear cell carcinoma tissue samples (n=81). Clear cell carcinoma cell lines (KOC-7C, OVTOKO) are separated into the ALDH-high and ALDH-low populations by ALDEFLUOR assay and fluorescence-activated cell sorting (FACS). We compared the intracellular ROS level, mRNA level of the antioxidant enzymes and Nrf2 expression of the two populations. High ALDH1 expression levels are related to advanced stage in clear cell carcinoma cases. ALDH1 expression significantly reduced progression free survival. Other markers are not related to clinical stage and prognosis. ALDH-high cells contained a lower ROS level than ALDH-low cells. Antioxidant enzymes were upregulated in ALDH-high cells. ALDH-high cells showed increased expression of Nrf2, a key transcriptional factor of the antioxidant system. ALDH-positive CSCs might have increased Nrf2-induced antioxidant scavengers, which lower ROS level relevant to chemoresistance in ovarian clear cell carcinoma. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Aldehyde dehydrogenase 1 (ALDH1) expression is an independent prognostic factor in triple negative breast cancer (TNBC).

    PubMed

    Ma, Fei; Li, Huihui; Li, Yiqun; Ding, Xiaoyan; Wang, Haijuan; Fan, Ying; Lin, Chen; Qian, Haili; Xu, Binghe

    2017-04-01

    Triple negative breast cancer (TNBC) is a subset of breast cancer that is highly aggressive and has a poor prognosis. Meanwhile, cancer stem cells (CSCs) are also characterized by a strong tumorigenic potential, which might be partly responsible for the aggressive behavior of TNBC. We previously showed that CSCs are enriched in TNBC cell lines and tissues. Further experiments in animal models revealed higher tumorigenicity of CSCs sorted from TNBC cell lines. In this study, we aimed to determine the clinical relationship between CSCs and TNBC by exploring the expression of aldehyde dehydrogenase 1 (ALDH1), which is a putative marker of breast CSCs, in TNBC tissues.ALDH1 levels in paraffin-embedded tumor tissues from 158 TNBC patients were evaluated by immunohistochemistry staining using an ALDH1A1 primary antibody. Staining evaluation was performed independently by two pathologists, and the expression level of ALDH1 was evaluated in terms of the percentage and intensity of positive cells. The association of immunohistochemistry staining of ALDH1 expression with clinical parameters was also analyzed.ALDH1 expression in tumor cells was observed in 88 out of 158 cases (55.7%). Analysis of clinicopathological parameters showed that the immunohistochemistry staining of ALDH1 was significantly correlated with tumor size (P = 0.02) and stage (P = 0.04). Survival analysis in patients with ALDH1 expression demonstrated shorter relapse-free survival (RFS) and overall survival (OS) times (P = 0.01; P = 0.001). Moreover, Cox multivariate analysis revealed that ALDH1 expression was an independent prognostic indicator of RFS and OS (P = 0.04; P = 0.04).Immunohistochemistry staining of ALDH1 in tumor cells is an independent prognostic indicator of RFS and OS in TNBC patients.

  14. 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.

  15. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. [Effect of ultraviolet radiation on ALDH1 expression in human lens epithelial cells].

    PubMed

    Shi, Jingming; Jia, Songbai; Chen, Xuan; Tang, Luosheng

    2012-06-01

    To determine the apoptosis-inducing effect of ultraviolet light (UV) on human lens epithelial cell (HLEC) and to explore the involvement of changes in ALDH1 folowing UV radiation. HLEC was exposed to the same UV light source and was subsequently divided into 6 groups according to UV radiation time of 0 (control group), 5, 10, 15, and 30 min. Apoptosis was detected by AO/EB staining. Changes of ALDH1 in HLEC were detected by immunohistochemical staining and Western blot. The intensity of immunohistochemical staining and the rate of positive cells decreased with increase of UV time (P<0.05). The rate of positive ALDH1 cells was negatively correlated with the rate of apoptosis (r= -0.92, P<0.05). Western blot showed the integrated absorbance values significantly decreased with the increase of UV time (P<0.05). ALDH1 in HLEC decreases with an increase of UV exposure, which may be related to UV induced apoptosis of HLEC.

  17. Evaluation of alcohol dehydrogenase and aldehyde dehydrogenase enzymes as bi-enzymatic anodes in a membraneless ethanol microfluidic fuel cell

    NASA Astrophysics Data System (ADS)

    Galindo-de-la-Rosa, J.; Arjona, N.; Arriaga, L. G.; Ledesma-García, J.; Guerra-Balcázar, M.

    2015-12-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldH) enzymes were immobilized by covalent binding and used as the anode in a bi-enzymatic membraneless ethanol hybrid microfluidic fuel cell. The purpose of using both enzymes was to optimize the ethanol electro-oxidation reaction (EOR) by using ADH toward its direct oxidation and AldH for the oxidation of aldehydes as by-products of the EOR. For this reason, three enzymatic bioanode configurations were evaluated according with the location of enzymes: combined, vertical and horizontally separated. In the combined configuration, a current density of 16.3 mA cm-2, a voltage of 1.14 V and a power density of 7.02 mW cm-2 were obtained. When enzymes were separately placed in a horizontal and vertical position the ocp drops to 0.94 V and to 0.68 V, respectively. The current density also falls to values of 13.63 and 5.05 mA cm-2. The decrease of cell performance of bioanodes with separated enzymes compared with the combined bioanode was of 31.7% and 86.87% for the horizontal and the vertical array.

  18. Tracing Jomon and Yayoi ancestries in Japan using ALDH2 and JC virus genotype distributions.

    PubMed

    Miyamori, Daisuke; Ishikawa, Noboru; Idota, Nozomi; Kakiuchi, Yasuhiro; McLean, Stuart; Kitamura, Tadaichi; Ikegaya, Hiroshi

    2015-01-01

    According to the dual structure model, the modern Japanese ethnic population consists of a mixture of the Jomon people, who have existed in Japan since at least the New Stone Age, and the Yayoi people, who migrated to western Japan from China around the year 300 bc Some reports show that the Yayoi are linked to a mutation of the aldehyde dehydrogenase 2 gene (ALDH2). Recent viral studies indicate two major groups found in the Japanese population: a group with the CY genotype JC virus (JCV) and a group with the MY genotype JCV. It is unclear whether either genotype of the JC virus is related to the Jomon or Yayoi. In this study, we attempted to detect JCV genotypes and ALDH2 mutations from the DNA of 247 Japanese urine samples to clarify the relationship between the dual structure model and the JCV genotype through ALDH2 mutation analysis and JCV genotyping. The ALDH2 polymorphism among 66 JC virus-positive samples was analyzed, and it was found that the ALDH2 variant is significantly higher in the population with CY genotype JCV (51.5 %) than in the population with the MY genotype (24.2 %) (p < 0.05). From these findings, it may be inferred that the ALDH2 mutation, which is related to the Yayoi, is related to CY genotype JCV. When the Yayoi migrated to the Japanese archipelago, they brought the ALDH2 mutation as well as the CY genotype JCV.

  19. Association between ALDH2 rs671 G>A polymorphism and gastric cancer susceptibility in Eastern Asia

    PubMed Central

    Jiang, You; Zhang, Jun; Wu, Yuee; Wang, Jian; Li, Liang

    2017-01-01

    To date, the relationship between the aldehyde dehydrogenases-2 (ALDH2) rs671 G>A (Glu504Lys) polymorphism and gastric cancer (GC) risk has not been thoroughly elucidated. To derive a more precise estimation of the effect of the ALDH2 rs671 G>A polymorphism on GC, we conducted this meta-analysis. We searched for qualified studies in the Embase, PubMed, Wang Fan and China National Knowledge Infrastructure databases. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the association. A total of 6,421 GC patients and 8,832 control subjects were included in the present study. The pooled results indicated no significant relationship between the ALDH2 rs671 G>A polymorphism and GC susceptibility in all genetic models. A stratified analysis by country showed that the ALDH2 rs671 G>A polymorphism might be a risk factor for GC in Japan (Allele model: P unadjusted = 0.034; Dominant model: P unadjusted = 0.040); however, the result was nonsignificant when the Bonferroni correction and false discovery rate (FDR) were applied. In subgroup analyses by drinking status in the dominant model, our study revealed that the ALDH2 rs671 G>A polymorphism significantly increased the risk of GC for drinkers (dominant model: P < 0.001). No relationship between the ALDH2 rs671 G>A polymorphism and GC risk was observed in any other subgroup. Our present study indicated no association between the ALDH2 rs671 G>A polymorphism and GC risk in Eastern Asian populations. However, the ALDH2 rs671 G>A polymorphism can significantly increase GC risk for drinkers. PMID:29254255

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, In-Gyu, E-mail: igkim@kaeri.re.kr; Department of Radiation Biotechnology and Applied Radioisotope, Korea University of Science and Technology; Lee, Jae-Ha

    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 cancermore » 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.« less

  1. Association between ALDH2 and ADH1B polymorphisms, alcohol drinking and gastric cancer: a replication and mediation analysis.

    PubMed

    Ishioka, Kuka; Masaoka, Hiroyuki; Ito, Hidemi; Oze, Isao; Ito, Seiji; Tajika, Masahiro; Shimizu, Yasuhiro; Niwa, Yasumasa; Nakamura, Shigeo; Matsuo, Keitaro

    2018-04-03

    Aldehyde dehydrogenase 2 (ALDH2; rs671, Glu504Lys) and alcohol dehydrogenase 1B (ADH1B; rs1229984, His47Arg) polymorphisms have a strong impact on carcinogenic acetaldehyde accumulation after alcohol drinking. To date, however, evidence for a significant ALDH2-alcohol drinking interaction and a mediation effect of ALDH2/ADH1B through alcohol drinking on gastric cancer have remained unclear. We conducted two case-control studies to validate the interaction and to estimate the mediation effect on gastric cancer. We calculated odds ratios (OR) and 95% confidence intervals (CI) for ALDH2/ADH1B genotypes and alcohol drinking using conditional logistic regression models after adjustment for potential confounding in the HERPACC-2 (697 cases and 1372 controls) and HERPACC-3 studies (678 cases and 678 controls). We also conducted a mediation analysis of the combination of the two studies to assess whether the effects of these polymorphisms operated through alcohol drinking or through other pathways. ALDH2 Lys alleles had a higher risk with increased alcohol consumption compared with ALDH2 Glu/Glu (OR for heavy drinking, 3.57; 95% CI 2.04-6.27; P for trend = 0.007), indicating a significant ALDH2-alcohol drinking interaction (P interaction  = 0.024). The mediation analysis indicated a significant positive direct effect (OR 1.67; 95% CI 1.38-2.03) and a protective indirect effect (OR 0.84; 95% CI 0.76-0.92) of the ALDH2 Lys alleles with the ALDH2-alcohol drinking interaction. No significant association of ADH1B with gastric cancer was observed. The observed ALDH2-alcohol drinking interaction and the direct effect of ALDH2 Lys alleles may suggest the involvement of acetaldehyde in the development of gastric cancer.

  2. ALDH2 genotype has no effect on salivary acetaldehyde without the presence of ethanol in the systemic circulation.

    PubMed

    Helminen, Andreas; Väkeväinen, Satu; Salaspuro, Mikko

    2013-01-01

    Acetaldehyde associated with alcoholic beverages was recently classified as carcinogenic (Group 1) to humans based on uniform epidemiological and biochemical evidence. ALDH2 (aldehyde dehydrogenase 2) deficient alcohol consumers are exposed to high concentrations of salivary acetaldehyde and have an increased risk of upper digestive tract cancer. However, this interaction is not seen among ALDH2 deficient non-drinkers or rare drinkers, regardless of their smoking status or consumption of edibles containing ethanol or acetaldehyde. Therefore, the aim of this study was to examine the effect of the ALDH2 genotype on the exposure to locally formed acetaldehyde via the saliva without ethanol ingestion. The ALDH2 genotypes of 17 subjects were determined by PCR-RFLP. The subjects rinsed out their mouths with 5 ml of 40 vol% alcohol for 5 seconds. Salivary ethanol and acetaldehyde levels were measured by gas chromatography. Acetaldehyde reached mutagenic levels rapidly and the exposure continued for up to 20 minutes. The mean salivary acetaldehyde concentrations did not differ between ALDH2 genotypes. For ALDH2 deficient subjects, an elevated exposure to endogenously formed acetaldehyde requires the presence of ethanol in the systemic circulation. Our findings provide a logical explanation for how there is an increased incidence of upper digestive tract cancers among ALDH2 deficient alcohol drinkers, but not among those ALDH2 deficient subjects who are locally exposed to acetaldehyde without bloodborne ethanol being delivered to the saliva. Thus, ALDH2 deficient alcohol drinkers provide a human model for increased local exposure to acetaldehyde derived from the salivary glands.

  3. Alcohol intake and folate antagonism via CYP2E1 and ALDH1: Effects on oral carcinogenesis

    PubMed Central

    Hwang, Phillip H.; Lian, Lisa; Zavras, Athanasios I.

    2011-01-01

    The interaction of folate and alcohol consumption has been shown to have an antagonistic effect on the risk of oral cancer. Studies have demonstrated that increased intake of folate decreases the risk of oral cancer, while greater alcohol consumption has an opposite effect. However, what is poorly understood is the biological interaction of these two dietary factors in relation to carcinogenesis. We hypothesize that cytochrome P450 2E1 (CYP2E1) and the family of aldehyde dehydrogenase 1 (ALDH1) enzymes may play a causal role in the occurrence of oral cancer. Chronic and high alcohol use has been implicated in the induction of CYP2E1, which oxidizes ethanol to acetaldehyde. Acetaldehyde is a known carcinogen. As the first metabolite of ethanol, it has been shown to interfere with DNA methylation, synthesis and repair, as well as bind to protein and DNA to form stable adducts, which lead to the eventual formation of damaged DNA and cell proliferation. Studies using liver cells have demonstrated that S-adenosyl methionine (SAM), which is a product of folate metabolism, regulates the expression and catalytic activity of CYP2E1. Our first hypothesis is that as increased levels of folate lead to higher concentrations of SAM, SAM antagonizes the expression of CYP2E1, which results in decreased conversion of ethanol into acetaldehyde. Thus, the lower levels of acetaldehyde may lower risk of oral cancer. There are also two enzymes within the ALDH1 family that play an important role both in ethanol metabolism and the folate one-carbon pathway. The first, ALDH1A1, converts acetaldehyde into its non-carcinogenic byproduct, acetate, as part of the second step in the ethanol metabolism pathway. The second, ALDH1L1, also known as FDH, is required for DNA nucleotide biosynthesis, and is upregulated at high concentrations of folate. ALDH1L1 appears to be a chief regulator of cellular metabolism as it is strongly downregulated at certain physiological and pathological conditions

  4. Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology

    PubMed Central

    Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J.; Salomons, Gajja S.

    2017-01-01

    Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease. PMID:29053735

  5. Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.

    PubMed

    Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J; Salomons, Gajja S; Mercimek-Andrews, Saadet

    2017-01-01

    Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease.

  6. Aldehyde dehydrogenase 7A1 (ALDH7A1) is a novel enzyme involved in cellular defense against hyperosmotic stress.

    PubMed

    Brocker, Chad; Lassen, Natalie; Estey, Tia; Pappa, Aglaia; Cantore, Miriam; Orlova, Valeria V; Chavakis, Triantafyllos; Kavanagh, Kathryn L; Oppermann, Udo; Vasiliou, Vasilis

    2010-06-11

    Mammalian ALDH7A1 is homologous to plant ALDH7B1, an enzyme that protects against various forms of stress, such as salinity, dehydration, and osmotic stress. It is known that mutations in the human ALDH7A1 gene cause pyridoxine-dependent and folic acid-responsive seizures. Herein, we show for the first time that human ALDH7A1 protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes. Human ALDH7A1 expression in Chinese hamster ovary cells attenuated osmotic stress-induced apoptosis caused by increased extracellular concentrations of sucrose or sodium chloride. Purified recombinant ALDH7A1 efficiently metabolized a number of aldehyde substrates, including the osmolyte precursor, betaine aldehyde, lipid peroxidation-derived aldehydes, and the intermediate lysine degradation product, alpha-aminoadipic semialdehyde. The crystal structure for ALDH7A1 supports the enzyme's substrate specificities. Tissue distribution studies in mice showed the highest expression of ALDH7A1 protein in liver, kidney, and brain, followed by pancreas and testes. ALDH7A1 protein was found in the cytosol, nucleus, and mitochondria, making it unique among the aldehyde dehydrogenase enzymes. Analysis of human and mouse cDNA sequences revealed mitochondrial and cytosolic transcripts that are differentially expressed in a tissue-specific manner in mice. In conclusion, ALDH7A1 is a novel aldehyde dehydrogenase expressed in multiple subcellular compartments that protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes.

  7. Aldehyde Dehydrogenase 7A1 (ALDH7A1) Is a Novel Enzyme Involved in Cellular Defense against Hyperosmotic Stress*

    PubMed Central

    Brocker, Chad; Lassen, Natalie; Estey, Tia; Pappa, Aglaia; Cantore, Miriam; Orlova, Valeria V.; Chavakis, Triantafyllos; Kavanagh, Kathryn L.; Oppermann, Udo; Vasiliou, Vasilis

    2010-01-01

    Mammalian ALDH7A1 is homologous to plant ALDH7B1, an enzyme that protects against various forms of stress, such as salinity, dehydration, and osmotic stress. It is known that mutations in the human ALDH7A1 gene cause pyridoxine-dependent and folic acid-responsive seizures. Herein, we show for the first time that human ALDH7A1 protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes. Human ALDH7A1 expression in Chinese hamster ovary cells attenuated osmotic stress-induced apoptosis caused by increased extracellular concentrations of sucrose or sodium chloride. Purified recombinant ALDH7A1 efficiently metabolized a number of aldehyde substrates, including the osmolyte precursor, betaine aldehyde, lipid peroxidation-derived aldehydes, and the intermediate lysine degradation product, α-aminoadipic semialdehyde. The crystal structure for ALDH7A1 supports the enzyme's substrate specificities. Tissue distribution studies in mice showed the highest expression of ALDH7A1 protein in liver, kidney, and brain, followed by pancreas and testes. ALDH7A1 protein was found in the cytosol, nucleus, and mitochondria, making it unique among the aldehyde dehydrogenase enzymes. Analysis of human and mouse cDNA sequences revealed mitochondrial and cytosolic transcripts that are differentially expressed in a tissue-specific manner in mice. In conclusion, ALDH7A1 is a novel aldehyde dehydrogenase expressed in multiple subcellular compartments that protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes. PMID:20207735

  8. Genome-wide identification and analysis of the aldehyde dehydrogenase (ALDH) gene superfamily in apple (Malus × domestica Borkh.).

    PubMed

    Li, Xiaoqin; Guo, Rongrong; Li, Jun; Singer, Stacy D; Zhang, Yucheng; Yin, Xiangjing; Zheng, Yi; Fan, Chonghui; Wang, Xiping

    2013-10-01

    Aldehyde dehydrogenases (ALDHs) represent a protein superfamily encoding NAD(P)(+)-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes. In plants, they are involved in many biological processes and play a role in the response to environmental stress. In this study, a total of 39 ALDH genes from ten families were identified in the apple (Malus × domestica Borkh.) genome. Synteny analysis of the apple ALDH (MdALDH) genes indicated that segmental and tandem duplications, as well as whole genome duplications, have likely contributed to the expansion and evolution of these gene families in apple. Moreover, synteny analysis between apple and Arabidopsis demonstrated that several MdALDH genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes appeared before the divergence of lineages that led to apple and Arabidopsis. In addition, phylogenetic analysis, as well as comparisons of exon-intron and protein structures, provided further insight into both their evolutionary relationships and their putative functions. Tissue-specific expression analysis of the MdALDH genes demonstrated diverse spatiotemporal expression patterns, while their expression profiles under abiotic stress and various hormone treatments indicated that many MdALDH genes were responsive to high salinity and drought, as well as different plant hormones. This genome-wide identification, as well as characterization of evolutionary relationships and expression profiles, of the apple MdALDH genes will not only be useful for the further analysis of ALDH genes and their roles in stress response, but may also aid in the future improvement of apple stress tolerance. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  9. The oxidation status of ALDH3A1 in human saliva and its correlation with antioxidant capacity measured by ORAC method.

    PubMed

    Bogucka, Małgorzata; Giebułtowicz, Joanna; Zawada, Katarzyna; Wroczyński, Piotr; Wierzchowski, Jacek; Pietrzak, Monika; Piekarczyk, Piotr; Romanowska, Katarzyna

    2009-01-01

    Oxidation status of the salivary aldehyde dehydrogenase (ALDH) was measured in healthy human population using two-assay fluorimetric method and compared with antioxidant capacity (ORAC) in non-smoking and heavy smokers group. Influence of high or low antioxidant diet was also examined. Except for the group of smokers, the salivary ALDH oxidation degree in human saliva was not correlated with antioxidant capacity. Simultaneously direct administration of the antioxidant-containing drug, Fluimucil, resulted in short-term, but statistically significant increase of the reduced (active) form of the enzyme, presumably due to a radical-scavenging activity of the drug.

  10. 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.

  11. Hangover symptoms in Asian Americans with variations in the aldehyde dehydrogenase (ALDH2) gene.

    PubMed

    Wall, T L; Horn, S M; Johnson, M L; Smith, T L; Carr, L G

    2000-01-01

    Hangovers are not experienced by all people and whether they contribute to the development of alcoholism is unclear. One population that might provide some insight into the role of hangover in the etiology of alcohol use disorders is that of individuals of Asian heritage. Certain Asians have lower rates of alcohol use and alcoholism, findings associated with a mutation in the aldehyde dehydrogenase (ALDH2) gene. Asians with ALDH2*2 alleles drink less and are less likely to be alcoholic than Asians without this mutation. Following alcohol ingestion, they exhibit more intense reactions to alcohol and generate higher levels of the metabolite acetaldehyde. This study evaluated hangover symptoms in Asian Americans with variations in the ALDH2 gene. Men and women of Chinese, Japanese and Korean heritage (N = 140) were asked about their drinking history and a blood sample was collected for genotyping at the ALDH2 locus. Subjects used a Likert-type scale to estimate their severity of hangover and completed a 13-item hangover scale assessing the frequency of hangover symptoms during the previous 6 months. With abstainers (n = 17) excluded and with the effects of gender and recent drinking history controlled, ALDH2 genotype accounted for a significant amount of additional variability in the estimated severity of hangover score with a similar, but nonsignificant, trend for a five-item subscale score derived from the hangover scale. These results suggest that Asian Americans with ALDH2*2 alleles may experience more severe hangovers that may contribute, in part, to protection against the development of excessive or problematic drinking in this population.

  12. 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

  13. The phenotype and clinical course of Japanese Fanconi Anaemia infants is influenced by patient, but not maternal ALDH2 genotype.

    PubMed

    Yabe, Miharu; Yabe, Hiromasa; Morimoto, Tsuyoshi; Fukumura, Akiko; Ohtsubo, Keisuke; Koike, Takashi; Yoshida, Kenichi; Ogawa, Seishi; Ito, Etsuro; Okuno, Yusuke; Muramatsu, Hideki; Kojima, Seiji; Matsuo, Keitaro; Hira, Asuka; Takata, Minoru

    2016-11-01

    Studies using Fanconi anaemia (FA) mutant mouse models suggested that the combination of a defective FA pathway and aldehyde dehydrogenase-2 (ALDH2) dysfunction could provoke bone marrow failure, leukaemia and developmental defects, and that both maternal and fetal aldehyde detoxification are crucial to protect the developing embryo from DNA damage. We studied the ALDH2 genotypes of 35 Japanese FA patients and their mothers. We found that a normal maternal ALDH2 allele was not essential for fetal development of ALDH2-deficient patients, and none of the post-natal clinical parameters were clearly affected by the maternal ALDH2 genotype in these patients. © 2016 John Wiley & Sons Ltd.

  14. 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.

  15. Inducible targeting of CNS astrocytes in Aldh1l1-CreERT2 BAC transgenic mice

    PubMed Central

    Winchenbach, Jan; Düking, Tim; Berghoff, Stefan A.; Stumpf, Sina K.; Hülsmann, Swen; Nave, Klaus-Armin; Saher, Gesine

    2016-01-01

    Background: Studying astrocytes in higher brain functions has been hampered by the lack of genetic tools for the efficient expression of inducible Cre recombinase throughout the CNS, including the neocortex. Methods: Therefore, we generated BAC transgenic mice, in which CreERT2 is expressed under control of the Aldh1l1 regulatory region. Results: When crossbred to Cre reporter mice, adult Aldh1l1-CreERT2 mice show efficient gene targeting in astrocytes. No such Cre-mediated recombination was detectable in CNS neurons, oligodendrocytes, and microglia. As expected, Aldh1l1-CreERT2 expression was evident in several peripheral organs, including liver and kidney. Conclusions: Taken together, Aldh1l1-CreERT2 mice are a useful tool for studying astrocytes in neurovascular coupling, brain metabolism, synaptic plasticity and other aspects of neuron-glia interactions. PMID:28149504

  16. Inducible targeting of CNS astrocytes in Aldh1l1-CreERT2 BAC transgenic mice.

    PubMed

    Winchenbach, Jan; Düking, Tim; Berghoff, Stefan A; Stumpf, Sina K; Hülsmann, Swen; Nave, Klaus-Armin; Saher, Gesine

    2016-01-01

    Background: Studying astrocytes in higher brain functions has been hampered by the lack of genetic tools for the efficient expression of inducible Cre recombinase throughout the CNS, including the neocortex. Methods: Therefore, we generated BAC transgenic mice, in which CreERT2 is expressed under control of the Aldh1l1 regulatory region. Results: When crossbred to Cre reporter mice, adult Aldh1l1-CreERT2 mice show efficient gene targeting in astrocytes. No such Cre-mediated recombination was detectable in CNS neurons, oligodendrocytes, and microglia. As expected, Aldh1l1-CreERT2 expression was evident in several peripheral organs, including liver and kidney. Conclusions: Taken together, Aldh1l1-CreERT2 mice are a useful tool for studying astrocytes in neurovascular coupling, brain metabolism, synaptic plasticity and other aspects of neuron-glia interactions.

  17. 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. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. ALDH1A3 mutations cause recessive anophthalmia and microphthalmia.

    PubMed

    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

    2013-02-07

    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. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  19. The ALDH21 gene found in lower plants and some vascular plants codes for a NADP+ -dependent succinic semialdehyde dehydrogenase.

    PubMed

    Kopečná, Martina; Vigouroux, Armelle; Vilím, Jan; Končitíková, Radka; Briozzo, Pierre; Hájková, Eva; Jašková, Lenka; von Schwartzenberg, Klaus; Šebela, Marek; Moréra, Solange; Kopečný, David

    2017-10-01

    Lower plant species including some green algae, non-vascular plants (bryophytes) as well as the oldest vascular plants (lycopods) and ferns (monilophytes) possess a unique aldehyde dehydrogenase (ALDH) gene named ALDH21, which is upregulated during dehydration. However, the gene is absent in flowering plants. Here, we show that ALDH21 from the moss Physcomitrella patens codes for a tetrameric NADP + -dependent succinic semialdehyde dehydrogenase (SSALDH), which converts succinic semialdehyde, an intermediate of the γ-aminobutyric acid (GABA) shunt pathway, into succinate in the cytosol. NAD + is a very poor coenzyme for ALDH21 unlike for mitochondrial SSALDHs (ALDH5), which are the closest related ALDH members. Structural comparison between the apoform and the coenzyme complex reveal that NADP + binding induces a conformational change of the loop carrying Arg-228, which seals the NADP + in the coenzyme cavity via its 2'-phosphate and α-phosphate groups. The crystal structure with the bound product succinate shows that its carboxylate group establishes salt bridges with both Arg-121 and Arg-457, and a hydrogen bond with Tyr-296. While both arginine residues are pre-formed for substrate/product binding, Tyr-296 moves by more than 1 Å. Both R121A and R457A variants are almost inactive, demonstrating a key role of each arginine in catalysis. Our study implies that bryophytes but presumably also some green algae, lycopods and ferns, which carry both ALDH21 and ALDH5 genes, can oxidize SSAL to succinate in both cytosol and mitochondria, indicating a more diverse GABA shunt pathway compared with higher plants carrying only the mitochondrial ALDH5. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  20. Neural retina-specific Aldh1a1 controls dorsal choroidal vascular development via Sox9 expression in retinal pigment epithelial cells.

    PubMed

    Goto, So; Onishi, Akishi; Misaki, Kazuyo; Yonemura, Shigenobu; Sugita, Sunao; Ito, Hiromi; Ohigashi, Yoko; Ema, Masatsugu; Sakaguchi, Hirokazu; Nishida, Kohji; Takahashi, Masayo

    2018-04-03

    VEGF secreted from retinal pigment epithelial (RPE) cells is responsible for the choroidal vascular development; however, the molecular regulatory mechanism is unclear. We found that Aldh1a1 -/- mice showed choroidal hypoplasia with insufficient vascularization in the dorsal region, although Aldh1a1, an enzyme that synthesizes retinoic acids (RAs), is expressed in the dorsal neural retina, not in the RPE/choroid complex. The level of VEGF in the RPE/choroid was significantly decreased in Aldh1a1 -/- mice, and RA-dependent enhancement of VEGF was observed in primary RPE cells. An RA-deficient diet resulted in dorsal choroidal hypoplasia, and simple RA treatment of Aldh1a1 -/- pregnant females suppressed choroid hypoplasia in their offspring. We also found downregulation of Sox9 in the dorsal neural retina and RPE of Aldh1a1 -/- mice and RPE-specific disruption of Sox9 phenocopied Aldh1a1 -/- choroidal development. These results suggest that RAs produced by Aldh1a1 in the neural retina directs dorsal choroidal vascular development via Sox9 upregulation in the dorsal RPE cells to enhance RPE-derived VEGF secretion. © 2018, Goto et al.

  1. 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.

  2. MAOA-uVNTR polymorphism may modify the protective effect of ALDH2 gene against alcohol dependence in antisocial personality disorder.

    PubMed

    Lee, Sheng-Yu; Hahn, Cheng-Yi; Lee, Jia-Fu; Chen, Shiou-Lan; Chen, Shih-Heng; Yeh, Tzung Lieh; Kuo, Po-Hsiu; Lee, I Hui; Yang, Yen Kuang; Huang, San-Yuan; Ko, Huei-Chen; Lu, Ru-Band

    2009-06-01

    Antisocial alcoholism is related to dopamine and serotonin which are catalyzed by monoamine oxidase A (MAOA) and acetaldehyde dehydrogenase 2 (ALDH2). The objective of this study is to determine whether the interaction between the MAOA and the ALDH2 genes is associated with subjects with antisocial personality disorder (ASPD) having alcoholism. A total of 294 Han Chinese men in Taiwan including 132 ASPD with alcoholism (Antisocial ALC) and 162 without alcoholism (Antisocial Non-ALC) were recruited in this study. Alcohol dependence and ASPD were diagnosed according to DSM-IV criteria. Genotypes of ALDH2 and MAOA-uVNTR were determined using PCR-RFLP. A significant difference of ALDH2 polymorphisms (p = 3.39E-05), but not of MAOA, was found among the 2 study groups. However, only after the stratification of the MAOA-uVNTR (variable number of tandem repeat located upstream) 3-repeat, a significant association between Antisocial Non-ALC and ALDH2*1/*2 or *2/*2 genotypes was shown (p = 1.46E-05; odds ratio = 3.913); whereas stratification of MAOA-uVNTR 4-repeat revealed no association. Multiple logistic regression analysis further revealed significant interaction of MAOA and ALDH2 gene in antisocial ALC (odds ratio = 2.927; p = 0.032). The possible interaction of MAOA and ALDH2 gene is associated with Antisocial ALC in Han Chinese males in Taiwan. However, the protective effects of the ALDH2*2 allele against alcoholism might disappear in subjects with ASPD and carrying MAOA-uVNTR 4-repeat allele in the Han Chinese male population.

  3. 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

  4. Neural retina-specific Aldh1a1 controls dorsal choroidal vascular development via Sox9 expression in retinal pigment epithelial cells

    PubMed Central

    Goto, So; Misaki, Kazuyo; Yonemura, Shigenobu; Sugita, Sunao; Ito, Hiromi; Ohigashi, Yoko; Ema, Masatsugu; Sakaguchi, Hirokazu; Nishida, Kohji; Takahashi, Masayo

    2018-01-01

    VEGF secreted from retinal pigment epithelial (RPE) cells is responsible for the choroidal vascular development; however, the molecular regulatory mechanism is unclear. We found that Aldh1a1–/– mice showed choroidal hypoplasia with insufficient vascularization in the dorsal region, although Aldh1a1, an enzyme that synthesizes retinoic acids (RAs), is expressed in the dorsal neural retina, not in the RPE/choroid complex. The level of VEGF in the RPE/choroid was significantly decreased in Aldh1a1–/– mice, and RA-dependent enhancement of VEGF was observed in primary RPE cells. An RA-deficient diet resulted in dorsal choroidal hypoplasia, and simple RA treatment of Aldh1a1–/– pregnant females suppressed choroid hypoplasia in their offspring. We also found downregulation of Sox9 in the dorsal neural retina and RPE of Aldh1a1–/– mice and RPE-specific disruption of Sox9 phenocopied Aldh1a1–/– choroidal development. These results suggest that RAs produced by Aldh1a1 in the neural retina directs dorsal choroidal vascular development via Sox9 upregulation in the dorsal RPE cells to enhance RPE-derived VEGF secretion. PMID:29609731

  5. Study on beta-galactosidase enzymatic activity of herbal yogurt.

    PubMed

    Chowdhury, Banani Ray; Chakraborty, Runu; Raychaudhuri, Utpal

    2008-03-01

    Different types of herbal yogurts were developed by mixing standardized milk with pretreated herbs, namely tulsi leaf (Ocimum sanctum), pudina leaf (Mentha arvensis) and coriander leaf (Coriandrum sativum), with leaves separately and a 1:1 (v/v) mixture of the strains of lactic starter cultures---Lactobacillus acidophilus (NCIM 2903) and Lactobacillus plantarum (NCIM 2083)-followed by incubation at 40 degrees C for 6 h. The beta-galactosidase enzymatic activity of the abovementioned herbal yogurts was determined and interestingly noted to exhibit higher enzymatic activity compared with the control yogurt (without any herbs). Among all herbal yogurts, tulsi yogurt had the maximum beta-galactosidase activity.

  6. CD44 and ALDH1 immunoexpression as prognostic indicators of invasion and metastasis in oral squamous cell carcinoma.

    PubMed

    Ortiz, Rafael Carneiro; Lopes, Nathália Martins; Amôr, Nadia Ghinelli; Ponce, José Burgos; Schmerling, Cláudia Kliemann; Lara, Vanessa Soares; Moyses, Raquel Ajub; Rodini, Camila Oliveira

    2018-05-23

    Tumour metastasis has been associated with cancer stem cells, a small population with stem-like cells properties, higher rate of migration and metastatic potential compared to cells from the tumour bulk. Our aim was to evaluate the immunoexpression of the putative cancer stem cell biomarkers ALDH1 and CD44 in primary tumour and corresponding metastatic lymph nodes. Tumour tissue specimens (n=50) and corresponding metastatic lymph nodes (n=25) were surgically obtained from 50 patients with oral squamous cell carcinoma and submitted to immunohistochemistry. CD44 and ALDH1 were semi-quantitatively scored according to the proportion and intensity of positive cells within the invasive front and metastatic lymph nodes as a whole. A combined score was obtained by multiplying both parameters and later dichotomized into a final score classified as low (≤ 2) or high (> 2) immunoexpression. ALDH1 and CD44 immunoexpression was detected in both tumour sites, although the means of ALDH1 (P = 0.0985) and CD44 (P = 0.4220) cells were higher in metastasis compared to primary tumours. ALDH1 high was positively associated (P = 0.0184) with angiolymphatic invasion, while CD44 high was positively associated (P = 0.0181) with metastasis (N+). At multivariate analysis, CD44 significantly increased the odds of lymph node metastasis, regardless of T stage (OR=8,24; 1,64-65,64, p=0,0088). CD44 immunoexpression was a significant predictor of lymph node metastasis, while ALDH1 high immunostaining was associated with angiolymphatic invasion. Altogether, it suggests that immunoexpression of CD44 and ALDH1 links the cancer stem cell phenotype with oral squamous cell carcinoma invasion and metastasis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  7. Crystal structure of human aldehyde dehydrogenase 1A3 complexed with NAD+ and retinoic acid

    PubMed Central

    Moretti, Andrea; Li, Jianfeng; Donini, Stefano; Sobol, Robert W.; Rizzi, Menico; Garavaglia, Silvia

    2016-01-01

    The aldehyde dehydrogenase family 1 member A3 (ALDH1A3) catalyzes the oxidation of retinal to the pleiotropic factor retinoic acid using NAD+. The level of ALDHs enzymatic activity has been used as a cancer stem cell marker and seems to correlate with tumour aggressiveness. Elevated ALDH1A3 expression in mesenchymal glioma stem cells highlights the potential of this isozyme as a prognosis marker and drug target. Here we report the first crystal structure of human ALDH1A3 complexed with NAD+ and the product all-trans retinoic acid (REA). The tetrameric ALDH1A3 folds into a three domain-based architecture highly conserved along the ALDHs family. The structural analysis revealed two different and coupled conformations for NAD+ and REA that we propose to represent two snapshots along the catalytic cycle. Indeed, the isoprenic moiety of REA points either toward the active site cysteine, or moves away adopting the product release conformation. Although ALDH1A3 shares high sequence identity with other members of the ALDH1A family, our structural analysis revealed few peculiar residues in the 1A3 isozyme active site. Our data provide information into the ALDH1As catalytic process and can be used for the structure-based design of selective inhibitors of potential medical interest. PMID:27759097

  8. Single nucleotide polymorphism detection in aldehyde dehydrogenase 2 (ALDH2) gene using bacterial magnetic particles based on dissociation curve analysis.

    PubMed

    Maruyama, Kohei; Takeyama, Haruko; Nemoto, Etsuo; Tanaka, Tsuyoshi; Yoda, Kiyoshi; Matsunaga, Tadashi

    2004-09-20

    Single nucleotide polymorphism (SNP) detection for aldehyde dehydrogenase 2 (ALDH2) gene based on DNA thermal dissociation curve analysis was successfully demonstrated using an automated system with bacterial magnetic particles (BMPs) by developing a new method for avoiding light scattering caused by nanometer-size particles when using commercially available fluorescent dyes such as FITC, Cy3, and Cy5 as labeling chromophores. Biotin-labeled PCR products in ALDH2, two allele-specific probes (Cy3-labeled detection probe for ALDH2*1 and Cy5-labeled detection probe for ALDH2*2), streptavidin-immobilized BMPs (SA-BMPs) were simultaneously mixed. The mixture was denatured at 70 degrees C for 3 min, cooled slowly to 25 degrees C, and incubated for 10 min, allowing the DNA duplex to form between Cy3- or Cy5-labeled detection probes and biotin-labeled PCR products on SA-BMPs. Then duplex DNA-BMP complex was heated to 58 degrees C, a temperature determined by dissociation curve analysis and a dissociated single-base mismatched detection probe was removed at the same temperature under precise control. Furthermore, fluorescence signal from the detection probe was liberated into the supernatant from completely matched duplex DNA-BMP complex by heating to 80 degrees C and measured. In the homozygote target DNA (ALDH2*1/*1 and ALDH2*2/*2), the fluorescence signals from single-base mismatched were decreased to background level, indicating that mismatched hybridization was efficiently removed by the washing process. In the heterozygote target DNA (ALDH2*1/*2), each fluorescence signals was at a similar level. Therefore, three genotypes of SNP in ALDH2 gene were detected using the automated detection system with BMPs. Copyright 2004 Wiley Periodicals, Inc.

  9. Enzymatic Activity of Free-Prostate-Specific Antigen (f-PSA) Is Not Required for Some of its Physiological Activities

    PubMed Central

    Chadha, Kailash C.; Nair, Bindukumar B.; Chakravarthi, Srikant; Zhou, Rita; Godoy, Alejandro; Mohler, James L.; Aalinkeel, Ravikumar; Schwartz, Stanley A.; Smith, Gary J.

    2015-01-01

    BACKGROUND Prostate specific antigen (PSA) is a well known biomarker for early diagnosis and management of prostate cancer. Furthermore, PSA has been documented to have anti-angiogenic and anti-tumorigenic activities in both in vitro and in vivo studies. However, little is known about the molecular mechanism(s) involved in regulation of these processes, in particular the role of the serine-protease enzymatic activity of PSA. METHODS Enzymatic activity of PSA isolated directly from seminal plasma was inhibited specifically (>95%) by incubation with zinc2+. Human umbilical vein endothelial cells (HUVEC) were utilized to compare/contrast the physiological effects of enzymatically active versus inactive PSA. RESULTS Equimolar concentrations of enzymatically active PSA and PSA enzymatically inactivated by incubation with Zn2+ had similar physiological effects on HUVEC, including inhibiting the gene expression of pro-angiogenic growth factors, like VEGF and bFGF, and up-regulation of expression of the anti-angiogenic growth factor IFN-γ; suppression of mRNA expression for markers of blood vessel development, like FAK, FLT, KDR, TWIST-1; P-38; inhibition of endothelial tube formation in the in vitro Matrigel Tube Formation Assay; and inhibition of endothelial cell invasion and migration properties. DISCUSSION Our data provides compelling evidence that the transcriptional regulatory and the anti-angiogenic activities of human PSA are independent of the innate enzymatic activity PMID:21446007

  10. Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells

    PubMed Central

    Liu, P; Brown, S; Goktug, T; Channathodiyil, P; Kannappan, V; Hugnot, J-P; Guichet, P-O; Bian, X; Armesilla, A L; Darling, J L; Wang, W

    2012-01-01

    Background: Glioblastoma multiforme (GBM) cells are resistant to anticancer drugs. Cancer stem cells (CSCs) are a key mediator of chemoresistance. We have reported that disulfiram (DS), an aldehyde dehydrogenase (ALDH) inhibitor, targets breast CSC-like cells. In this study, the effect of DS and combination of DS and gemcitabine (dFdC) on GBM cells and GBM stem-like cells was investigated. Methods: 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), combination index (CI)-isobologram, western blot, luciferase reporter gene assay, electrophoretic mobility-shift assay and ALDH analysis were used in this study. Results: Disulfiram is cytotoxic in GBM cell lines in a copper (Cu)-dependent manner. Disulfiram/copper enhances the cytotoxicity of dFdC. Combination index-isobologram analysis indicates a synergistic effect between DS/Cu and dFdC. Disulfiram/copper induces reactive oxygen species (ROS), activates JNK and p38 pathways and inhibits nuclear factor-kappa B activity in GBM cell lines. Disulfiram/copper may trigger intrinsic apoptotic pathway via modulation of the Bcl2 family. Disulfiram/copper abolishes stem-like cell population in GBM cell lines. Conclusion: Our findings indicate that the cytotoxicity of DS/Cu and the enhancing effect of DS/Cu on the cytotoxicity of dFdC in GBM stem-like cells may be caused by induction of ROS and inhibition of both ALDH and the NFkB pathway. Both DS and dFdC can traverse the blood–brain barrier. Further study may lead them into GBM chemotherapy. PMID:23033007

  11. Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces

    PubMed Central

    2015-01-01

    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. PMID:25420087

  12. Digestive enzymatic activity on tropical gar (Atractosteus tropicus) larvae fed different diets.

    PubMed

    Aguilera, Carlos; Mendoza, Roberto; Iracheta, Israel; Marquez, Gabriel

    2012-06-01

    Digestive enzymatic activity and growth performance on tropical gar (Atractosteus tropicus) larvae fed Artemia nauplii (LF), frozen adult Artemia (AB), an artificial diet (AF) with 46% protein and 16% lipids and a starvation group (SG) from first feeding (5 days after hatching-5 DAH) to 34 DAH were studied. All larvae under starvation (SG) died at 15 DAH. By the end of the experimental period, morphological variables (total length, wet weight and specific growth rate) were significant in larvae fed AF compared to LF and AB. All enzymes studied in the experiment were present since the start of exogenous feeding (including pepsin) and the enzymatic activity varied with the diets. Low levels of enzymatic activity were observed until the 29 DAH; however, after this moment, there was a significant increase (eightfold), particularly for the AF treatment. In vitro protein digestibility tests performed with enzymatic extracts showed that artificial diets with 52% protein and 14% lipids were better digested by larvae before 30 DAH, while diets with 45% protein and 11% lipids were better digested after this age. Taking into account the better growth performance, higher enzymatic activity and better protein digestibility obtained, artificial diets can be used since the start of exogenous feeding on tropical gar larvae, as in other lepisosteids.

  13. Integrating biological and behavioral factors in alcohol use risk: the role of ALDH2 status and alcohol expectancies in a sample of Asian Americans.

    PubMed

    McCarthy, D M; Wall, T L; Brown, S A; Carr, L G

    2000-05-01

    Prior studies have shown that the ALDH2*2 genetic variant, most common in individuals of Asian descent, is related to heightened sensitivity to alcohol and can serve as a protective factor against alcohol problems. This study explored the effect of this factor on alcohol expectancies. It was hypothesized that (a) individuals with ALDH2*2 alleles would have lower positive expectancies and higher negative expectancies, (b) expectancies would mediate the ALDH2-drinking relation, and (c) ALDH2 status would moderate the expectancy-drinking relation. Data were collected from 171 Asian American university students. Positive expectancy and ALDH2 status were correlated with alcohol use. Mediation and moderation hypotheses were supported only in the female sample. Results were not significant for negative expectancies. These results indicate that ALDH2 status may protect against drinking by lowering positive expectancies and reducing the expectancy-drinking relationship.

  14. Enzymatic Activation of the Emerging Drug Resveratrol.

    PubMed

    Koyani, Rina D; Vazquez-Duhalt, Rafael

    2018-05-01

    The plant originated stilbene "resveratrol" (3,4',5-trans-trihydroxystilbene) is well known for its diverse health benefits including anti-tumor, anti-inflammatory, anti-microbial, and anti-oxidant properties. Besides a significant amount of reports on different aspects of its application as prodrug in the last 50 years, still, a strategy leading to the production of the active drug is missing. The aim of this work was to evaluate the enzymatic activation of prodrug resveratrol to the effective drug piceatannol, without engaging expensive cofactors. Five different heme proteins were analyzed for the transformation of resveratrol. Kinetic parameters of resveratrol transformation and analysis of the transformed products were conducted through HPLC and GC-MS. Effect of pH and organic solvent on the transformation process had also been evaluated. Among all tested heme proteins, only a variant of cytochrome P450 BM3 from Bacillus megaterium (CYP BM3 F87A) was found suitable for piceatannol production. The most suitable pH for the reaction conditions was 8.5, while organic solvents did not show any effect on transformation. For resveratrol transformation, the turnover rate (k cat ) was 21.7 (± 0.6) min -1 , the affinity constant (K M ) showed a value of 55.7 (± 16.7) μM for a catalytic efficiency (k cat /K M ) of 389 min -1  mM -1 . GC-MS analysis showed that the only product from resveratrol transformation by cytochrome P450 BM3 is the biologically active piceatannol. The enzymatic transformation of resveratrol, an emerging compound with medical interest, to active product piceatannol by a variant of cytochrome P450 BM3 in the absence of expensive NADPH cofactor is demonstrated. This enzymatic process is economically attractive and can be scaled up to cover the increasing medical demand for piceatannol.

  15. Antimicrobial and enzymatic activity of anemophilous fungi of a public university in Brazil.

    PubMed

    Sobral, Laureana V; Melo, Kelly N; Souza, Cleciana M; Silva, Sílvio F; Silva, Gilvania L R; Silva, Andressa L F; Wanderley, Katharine A A; Oliveira, Idjane S; Cruz, Roberta

    2017-01-01

    To the fungal microbiota the UFPE and biotechnological potential enzymatic and antimicrobial production. Air conditioned environments were sampled using a passive sedimentation technique, the air I ratio and the presence of aflatoxigenic strains evaluated for ANVISA. Icelles were to determine the enzymatic activity of lipase, amylase and protease metabolic liquids to determine antimicrobial activity. Diversity was observed in all CAV environments, CFU/m3 ranged from 14 to 290 and I/E ratio from 0.1 to 1.5. The of the fungal genera were: Aspergillus (50%), Penicillium (21%), Talaromyces (14%), Curvularia and Paecilomyces (7% each). Aspergillus sydowii (Bainier & Sartory) Thom & Church presented enzymatic activity and the Talaromyces purpureogenus Samson, Yilmaz, Houbraken, Spierenb., Seifert, Peterson, Varga & Frisvad presented antibacterial activity against all bacteria that all environments present fungal species biodiversity no toxigenic or pathogenic fungi were found, according to ANVISA legislation for conditioned environments and airborne filamentous fungi present potential for enzymatic and antimicrobial activity.

  16. Mutations in ALDH1A3 represent a frequent cause of microphthalmia/anophthalmia in consanguineous families.

    PubMed

    Abouzeid, Hana; Favez, Tatiana; Schmid, Angélique; Agosti, Céline; Youssef, Mohammed; Marzouk, Iman; El Shakankiry, Nihal; Bayoumi, Nader; Munier, Francis L; Schorderet, Daniel F

    2014-08-01

    Anophthalmia or microphthalmia (A/M), characterized by absent or small eye, can be unilateral or bilateral and represent developmental anomalies due to the mutations in several genes. Recently, mutations in aldehyde dehydrogenase family 1, member A3 (ALDH1A3) also known as retinaldehyde dehydrogenase 3, have been reported to cause A/M. Here, we screened a cohort of 75 patients with A/M and showed that mutations in ALDH1A3 occurred in six families. Based on this series, we estimate that mutations in ALDH1A3 represent a major cause of A/M in consanguineous families, and may be responsible for approximately 10% of the cases. Screening of this gene should be performed in a first line of investigation, together with SOX2. © 2014 WILEY PERIODICALS, INC.

  17. ALDH2 polymorphism, associated with attenuating negative symptoms in patients with schizophrenia treated with add-on dextromethorphan.

    PubMed

    Lee, Sheng-Yu; Chen, Shiou-Lan; Chang, Yun-Hsuan; Chen, Po-See; Huang, San-Yuan; Tzeng, Nian-Sheng; Wang, Liang-Jen; Lee, I-Hui; Wang, Tzu-Yun; Chen, Kao-Chin; Yang, Yen-Kuang; Hong, Jau-Shyong; Lu, Ru-Band

    2015-10-01

    Increasing the evidence of inflammation's contribution to schizophrenia; using anti-inflammatory or neurotrophic therapeutic agents to see whether they improve schizophrenia treatment. Dextromethorphan (DM), a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, might protect monoamine neurons. Whether treating schizophrenia with risperidone plus add-on DM is more effective than risperidone (RISP) alone, and the association between the ALDH2 polymorphism and treatment response were investigated. A double-blind study in which patients with schizophrenia were randomly assigned to the RISP + DM (60 mg/day; n = 74) or the RISP + Placebo (n = 75) group. The Positive and Negative Syndrome Scale (PANSS) and the Scale for the Assessment of Negative Symptoms (SANS) scores were used to evaluate clinical response during weeks 0, 1, 2, 4, 6, 8, and 11. The genotypes of the ALDH2 polymorphism were determined using polymerase chain reactions plus restriction fragment length polymorphism analysis. A generalized estimating equation was used to analyze the effects of ALDH2 polymorphism on the clinical performance of DM. PANSS and SANS scores were significantly lower in both groups after 11 weeks of treatment. SANS total scores were significantly lower in the RISP + DM group in patients with the ALDH2*2*2 genotype. RISP plus add-on DM treatment reduced negative schizophrenia symptoms in patients with the ALDH2 polymorphism. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Meta-analysis of ADH1B and ALDH2 polymorphisms and esophageal cancer risk in China.

    PubMed

    Zhang, Guo-Hong; Mai, Rui-Qin; Huang, Bo

    2010-12-21

    To evaluate whether alcohol dehydrogenase-1B (ADH1B) His47Arg and aldehyde dehydrogenase-2 (ALDH2) Glu487Lys polymorphism is involved in the esophageal squamous cell carcinoma (ESCC) risk in Chinese Han population. Seven studies of ADH1B and ALDH2 genotypes in Chinese Han population in 1450 cases and 2459 controls were included for meta-analysis. Stratified analyses were carried out to determine the gene-alcohol and gene-gene interaction with ESCC risk. Potential sources of heterogeneity between studies were explored, and publication bias was also evaluated. Individuals with ADH1B arginine (Arg)/Arg genotype showed 3.95-fold increased ESCC risk in the recessive genetic model [Arg/Arg vs Arg/histidine (His) + His/His: odds ratio (OR) = 3.95, 95% confidence interval (CI): 2.76-5.67]. Significant association was found in the dominant model for ALDH2 lysine (Lys) allele [glutamate (Glu)/Lys + Lys/Lys vs Glu/Glu: OR = 2.00, 95% CI: 1.54-2.61]. Compared with the non-alcoholics, Arg/Arg (OR = 25.20, 95% CI: 10.87-53.44) and Glu/Lys + Lys/Lys (OR = 21.47, 95% CI: 6.44-71.59) were found to interact with alcohol drinking to increase the ESCC risk. ADH1B Arg+ and ALDH2 Lys+ had a higher risk for ESCC (OR = 7.09, 95% CI: 2.16-23.33). The genetic variations of ADH1B His47Arg and ALDH2 Glu487Lys are susceptible loci for ESCC in Chinese Han population and interact substantially with alcohol consumption. The individuals carrying both risky genotypes have a higher baseline risk of ESCC.

  19. Association between DRD2, 5-HTTLPR, and ALDH2 genes and specific personality traits in alcohol- and opiate-dependent patients.

    PubMed

    Wang, Tzu-Yun; Lee, Sheng-Yu; Chen, Shiou-Lan; Huang, San-Yuan; Chang, Yun-Hsuan; Tzeng, Nian-Sheng; Wang, Chen-Lin; Hui Lee, I; Yeh, Tzung Lieh; Yang, Yen Kuang; Lu, Ru-Band

    2013-08-01

    The vulnerability of developing addictions is associated with genetic factors and personality traits. The predisposing genetic variants and personality traits may be common to all addictions or specific to a particular class of addiction. To investigate the relationship between genetic variances, personality traits, and their interactions in addiction are important. We recruited 175 opiate-dependent patients, 102 alcohol-dependent patients, and 111 healthy controls. All participants were diagnosed using DSM-IV criteria and assessed with Tridimensional Personality Questionnaire (TPQ). The dopamine D2 receptor (DRD2), 5-HTT-linked promoter region (5-HTTLPR), and aldehyde dehydrogenase 2 (ALDH2) genes were genotyped using PCR. The genotype frequency of the 5-HTTLPR and ALDH2 was significantly different between the patients and controls (P=0.013, P<0.001, respectively), and borderline significant (P=0.05) for DRD2 polymorphism. Both Novelty Seeking (NS) and Harm Avoidance (HA) scores were higher for patients (P<0.001). After stratification by candidate genes, addicts with ALDH2 *1/*1 interacting with the low-functional group of DRD2 and 5-HTTLPR genes have higher HA traits, whereas addicts with ALDH2 *1/*2 or *2/*2 and low-functional group of DRD2 and 5-HTTLPR genes have higher NS traits. We concluded that addicts, both alcohol- and opiate-dependent patients, have common genetic variants in DRD2 and 5-HTTLPR but specific for ALDH2. Higher NS and HA traits were found in both patient groups with the interaction with DRD2, 5-HTTLPR, and ALDH2 genes. The ALDH2 gene variants had different effect in the NS and HA dimension while the DRD2 and 5-HTTLPR genes did not. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. 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.

  1. 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

  2. The effects of xylitol and sorbitol on lysozyme- and peroxidase-related enzymatic and candidacidal activities.

    PubMed

    Kim, Bum-Soo; Chang, Ji-Youn; Kim, Yoon-Young; Kho, Hong-Seop

    2015-07-01

    To investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase system. Xylitol and sorbitol were added to hen egg-white lysozyme, bovine lactoperoxidase, glucose oxidase-mediated peroxidase, and whole saliva in solution and on hydroxyapatite surfaces. The enzymatic activities of lysozyme, peroxidase, and glucose oxidase-mediated peroxidase were determined by the turbidimetric method, the NbsSCN assay, and production of oxidized o-dianisidine, respectively. Candidacidal activities were determined by comparing colony forming units using Candida albicans ATCC strains 10231, 11006, and 18804. While xylitol and sorbitol did not affect the enzymatic activity of hen egg-white lysozyme both in solution and on hydroxyapatite surfaces, they did inhibit the enzymatic activity of salivary lysozyme significantly in solution, but not on the surfaces. Xylitol and sorbitol enhanced the enzymatic activities of both bovine lactoperoxidase and salivary peroxidase significantly in a dose-dependent manner in solution, but not on the surfaces. Sorbitol, but not xylitol, inhibited the enzymatic activity of glucose oxidase-mediated peroxidase significantly. Both xylitol and sorbitol did not affect candidacidal activities of hen egg-white lysozyme, the bovine lactoperoxidase system, or the glucose oxidase-mediated bovine lactoperoxidase system. Xylitol and sorbitol inhibited salivary lysozyme activity, but enhanced both bovine lactoperoxidase and salivary peroxidase activities significantly in solution. Xylitol and sorbitol did not augment lysozyme- and peroxidase-related candidacidal activities. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. ALDH1 is an immunohistochemical diagnostic marker for solitary fibrous tumours and haemangiopericytomas of the meninges emerging from gene profiling study

    PubMed Central

    2013-01-01

    Background Solitary Fibrous Tumours (SFT) and haemangiopericytomas (HPC) are rare meningeal tumours that have to be distinguished from meningiomas and more rarely from synovial sarcomas. We recently found that ALDH1A1 was overexpressed in SFT and HPC as compared to soft tissue sarcomas. Using whole-genome DNA microarrays, we defined the gene expression profiles of 16 SFT/HPC (9 HPC and 7 SFT). Expression profiles were compared to publicly available expression profiles of additional SFT or HPC, meningiomas and synovial sarcomas. We also performed an immunohistochemical (IHC) study with anti-ALDH1 and anti-CD34 antibodies on Tissue Micro-Arrays including 38 SFT (25 meningeal and 13 extrameningeal), 55 meningeal haemangiopericytomas (24 grade II, 31 grade III), 163 meningiomas (86 grade I, 62 grade II, 15 grade III) and 98 genetically confirmed synovial sarcomas. Results ALDH1A1 gene was overexpressed in SFT/HPC, as compared to meningiomas and synovial sarcomas. These findings were confirmed at the protein level. 84% of the SFT and 85.4% of the HPC were positive with anti-ALDH1 antibody, while only 7.1% of synovial sarcomas and 1.2% of meningiomas showed consistent expression. Positivity was usually more diffuse in SFT/HPC compared to other tumours with more than 50% of tumour cells immunostained in 32% of SFT and 50.8% of HPC. ALDH1 was a sensitive and specific marker for the diagnosis of SFT (SE = 84%, SP = 98.8%) and HPC (SE = 84.5%, SP = 98.7%) of the meninges. In association with CD34, ALDH1 expression had a specificity and positive predictive value of 100%. Conclusion We show that ALDH1, a stem cell marker, is an accurate diagnostic marker for SFT and HPC, which improves the diagnostic value of CD34. ALDH1 could also be a new therapeutic target for these tumours which are not sensitive to conventional chemotherapy. PMID:24252471

  4. Combination of ALDH2 and ADH1B polymorphisms is associated with smoking initiation: A large-scale cross-sectional study in a Japanese population.

    PubMed

    Masaoka, Hiroyuki; Ito, Hidemi; Gallus, Silvano; Watanabe, Miki; Yokomizo, Akira; Eto, Masatoshi; Matsuo, Keitaro

    2017-04-01

    Aldehyde dehydrogenase 2 (ALDH2; rs671, Glu504Lys) and alcohol dehydrogenase 1B (ADH1B; rs1229984, His47Arg) polymorphisms are known to strongly influence alcohol drinking behavior. Given evidence of an association between smoking and drinking behaviors, we hypothesized that ALDH2/ADH1B polymorphisms might also be associated with smoking initiation, and conducted a cross-sectional study to examine this hypothesis. Study subjects were first-visit outpatients diagnosed not to have cancer at Aichi Cancer Center Hospital between 2001 and 2005, including 4141 never smokers and 2912 ever smokers. Unconditional logistic regression models were applied to estimate odds ratios (OR) and 95% confidence intervals (CI) for smoking initiation by comparing ever smokers with never smokers. Excessive alcohol drinking was associated with a higher likelihood of ever smoking. After adjustment for drinking behaviors, compared to individuals with ALDH2 Glu/Glu, the ORs of ever smoking were 1.71 (95% CI, 1.49-1.95) and 2.28 (1.81-2.87) among those with ALDH2 Glu/Lys and Lys/Lys, respectively. Combination of ALDH2 Lys/Lys and ADH1B His/His (i.e., the most alcohol-intolerant subpopulation) showed the highest OR [2.44 (1.84-3.23)], whereas combination of ALDH2 Glu/Glu and ADH1B Arg/Arg (i.e., the most alcohol-tolerant subpopulation) showed the lowest OR [0.83 (0.57-1.21)] compared with ALDH2 Glu/Glu and ADH1B His/His. Besides the amount and frequency of alcohol drinking, the combination of ALDH2 and ADH1B polymorphisms predicts smoking initiation. This study suggests that alcohol tolerance regulated by ALDH2 and ADH1B polymorphisms is associated with smoking initiation, and facilitates the development of targeted interventions to reduce smoking prevalence. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Increased Expression of ALDH1A1 in Prostate Cancer is Correlated With Tumor Aggressiveness: A Tissue Microarray Study of Iranian Patients.

    PubMed

    Kalantari, Elham; Saadi, Faezeh H; Asgari, Mojgan; Shariftabrizi, Ahmad; Roudi, Raheleh; Madjd, Zahra

    2017-09-01

    Subpopulations of prostate cancer (PCa) cells expressing putative stem cell markers possess the ability to promote tumor growth, maintenance, and progression. This study aimed to evaluate the expression patterns and clinical significance of putative stem cell marker aldehyde dehydrogenase 1 A1 (ALDH1A1) in prostate tumor tissues. ALDH1A1 expression was examined in a well-defined series of prostate tissues, including 105 (68%) samples of PCa, 21 (13%) samples of high-grade prostatic intraepithelial neoplasia, and 31 (19%) samples of benign prostate hyperplasia, which were embedded in tissue microarray blocks. The correlation of ALDH1A1 expression with clinicopathologic parameters was also assessed. There was a significant difference between the expression level of ALDH1A1 in PCa compared with the high-grade prostatic intraepithelial neoplasia and benign prostate hyperplasia samples (P<0.001). PCa cells expressing ALDH1A1 were more often seen in samples with advanced Gleason score (P=0.05) and high serum prostate specific antigen level (P=0.02). In addition, a positive correlation was found between ALDH1A1 expression and primary tumor stage and regional lymph node involvement (P=0.04 and 0.03, respectively). The significant association between ALDH1A1 expressions with Gleason score indicates the potential role of this protein in PCa tumorigenesis and aggressive behavior; therefore, this cancer stem cell marker can be used as a promising candidate for targeted therapy of PCa, especially those with high Gleason score.

  6. DOXIL when combined with Withaferin A (WFA) targets ALDH1 positive cancer stem cells in ovarian cancer.

    PubMed

    Kakar, Sham S; Worth, Christopher A; Wang, Zhenglong; Carter, Kelsey; Ratajczak, Mariusz; Gunjal, Pranesh

    Ovarian cancer is a highly aggressive and deadly disease. Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, mainly cisplatin or carboplatin combined with paclitaxel. Although this regimen is initially effective in a high percentage of cases, unfortunately, after few months of initial treatment, tumor relapse occurs due to platinum-resistance. DOXIL (liposomal preparation of doxorubicin) is a choice of drug for recurrent ovarian cancer. However, its response rate is very low and is accompanied by myocardial toxicity. Resistance to chemotherapy and recurrence of cancer is primarily attributed to the presence of cancer stem cells (CSCs), a small population of cells present in cancer. Effect of DOXIL and withaferin A (WFA), both alone and in combination, was investigated on cell proliferation of ovarian cancer cell line A2780 and tumor growth in SCID mice bearing i.p. ovarian tumors. ALDH1 cells were isolated from A2780 using cell sorter, and effect of DOXIL and WFA both alone and in combination on tumorigenic function of ALDH1 was studied using spheroids formation assays in vitro. Western blots were performed to examine the expression of ALDH1 and Notch 1 genes. In our studies, we showed, for the first time, that DOXIL when combined with withaferin A (WFA) elicits synergistic effect on inhibition of cell proliferation of ovarian cancer cells and inhibits the expression of ALDH1 protein, a marker for ALDH1 positive cancer stem cells (CSCs), and Notch1, a signaling pathway gene required for self-renewal of CSCs. Inhibition of expression of both ALDH1 and Notch1 genes by WFA was found to be dose dependent, whereas DOXIL (200 nM) was found to be ineffective. SCID mice, bearing i.p. ovarian tumors, were treated with a small dose of DOXIL (2 mg/kg) in combination with a sub-optimal dose of WFA (2 mg/kg) which resulted in a highly significant (60% to 70%) reduction in tumor growth, and complete inhibition of metastasis

  7. 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.

  8. PKCalpha regulates phosphorylation and enzymatic activity of cPLA2 in vitro and in activated human monocytes.

    PubMed

    Li, Qing; Subbulakshmi, Venkita; Oldfield, Claudine M; Aamir, Rozina; Weyman, Crystal M; Wolfman, Alan; Cathcart, Martha K

    2007-02-01

    Phospholipases A(2) (PLA(2)) are potent regulators of the inflammatory response. We have observed that Group IV cPLA(2) activity is required for the production of superoxide anion (O(2)(-)) in human monocytes [Li Q., Cathcart M.K. J. Biol. Chem. 272 (4) (1997) 2404-2411.]. We have previously identified PKCalpha as a kinase pathway required for monocyte O(2)(-) production [Li Q., Cathcart M.K. J. Biol. Chem. 269 (26) (1994) 17508-17515.]. We therefore investigated the potential interaction between PKCalpha and cPLA(2) by evaluating the requirement for specific PKC isoenzymes in the process of activating cPLA(2) enzymatic activity and protein phosphorylation upon monocyte activation. We first showed that general PKC inhibitors and antisense oligodeoxyribonucleotides (ODN) to the cPKC group of PKC enzymes inhibited cPLA(2) activity. To distinguish between PKCalpha and PKCbeta isoenzymes in regulating cPLA(2) protein phosphorylation and enzymatic activity, we employed our previously characterized PKCalpha or PKCbeta isoenzyme-specific antisense ODN [Li Q., Subbulakshmi V., Fields A.P., Murray, N.R., Cathcart M.K., J. Biol. Chem. 274 (6) (1999) 3764-3771]. Suppression of PKCalpha expression, but not PKCbeta expression, inhibited cPLA(2) protein phosphorylation and enzymatic activity. Additional studies ruled out a contribution by Erk1/2 to cPLA(2) phosphorylation and activation. We also found that cPLA(2) co-immunoprecipitated with PKCalpha and vice versa. In vitro studies demonstrated that PKCalpha could directly phosphorylate cPLA(2).and enhance enzymatic activity. Finally, we showed that addition of arachidonic acid restored the production of O(2)(-) in monocytes defective in either PKCalpha or cPLA(2) expression. Taken together, our data suggest that PKCalpha, but not PKCbeta, is the predominant cPKC isoenzyme required for cPLA(2) protein phosphorylation and maximal induction of cPLA(2) enzymatic activity upon activation of human monocytes. Our data also support the

  9. Novel splice-site and missense mutations in the ALDH1A3 gene underlying autosomal recessive anophthalmia/microphthalmia.

    PubMed

    Semerci, C Nur; Kalay, Ersan; Yıldırım, Cem; Dinçer, Tuba; Olmez, Akgün; Toraman, Bayram; Koçyiğit, Ali; Bulgu, Yunus; Okur, Volkan; Satıroğlu-Tufan, Lale; Akarsu, Nurten A

    2014-06-01

    This study aimed to identify the underlying genetic defect responsible for anophthalmia/microphthalmia. In total, two Turkish families with a total of nine affected individuals were included in the study. Affymetrix 250 K single nucleotide polymorphism genotyping and homozygosity mapping were used to identify the localisation of the genetic defect in question. Coding region of the ALDH1A3 gene was screened via direct sequencing. cDNA samples were generated from primary fibroblast cell cultures for expression analysis. Reverse transcriptase PCR (RT-PCR) analysis was performed using direct sequencing of the obtained fragments. The causative genetic defect was mapped to chromosome 15q26.3. A homozygous G>A substitution (c.666G>A) at the last nucleotide of exon 6 in the ALDH1A3 gene was identified in the first family. Further cDNA sequencing of ALDH1A3 showed that the c.666G>A mutation caused skipping of exon 6, which predicted in-frame loss of 43 amino acids (p.Trp180_Glu222del). A novel missense c.1398C>A mutation in exon 12 of ALDH1A3 that causes the substitution of a conserved asparagine by lysine at amino acid position 466 (p.Asn466Lys) was observed in the second family. No extraocular findings-except for nevus flammeus in one affected individual and a variant of Dandy-Walker malformation in another affected individual-were observed. Autistic-like behaviour and mental retardation were observed in three cases. In conclusion, novel ALDH1A3 mutations identified in the present study confirm the pivotal role of ALDH1A3 in human eye development. Autistic features, previously reported as an associated finding, were considered to be the result of social deprivation and inadequate parenting during early infancy in the presented families. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  10. Association between ADH1C and ALDH2 polymorphisms and alcoholism in a Turkish sample.

    PubMed

    Ayhan, Yavuz; Gürel, Şeref Can; Karaca, Özgür; Zoto, Teuta; Hayran, Mutlu; Babaoğlu, Melih; Yaşar, Ümit; Bozkurt, Atilla; Dilbaz, Nesrin; Uluğ, Berna Diclenur; Demir, Başaran

    2015-04-01

    Polymorphisms in the genes encoding alcohol metabolizing enzymes are associated with alcohol dependence. To evaluate the association between the alcohol dehydrogenase 1C (ADH1C) Ile350Val and aldehyde dehydrogenase 2 (ALDH2) Glu504Lys polymorphisms and alcohol dependence in a Turkish sample. 235 individuals (115 alcohol-dependent patients and 120 controls) were genotyped for ADH1C and ALDH2 with PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). Association between the polymorphisms and family history, daily and maximum amount of alcohol consumed was investigated. The associations between alcohol dependence, severity of consumption and family history and the polymorphisms were analyzed by chi-square or Fisher's exact test where necessary. Relationship between genotypes and dependence related features was evaluated using analysis of variance (ANOVA). The -350Val allele for ADH1C (ADH1C*2) was increased in alcohol-dependent patients (P = 0.05). In individuals with a positive family history, the genotype distribution differed significantly (P = 0.031) and more patients carried the Val allele compared with controls (P = 0.025). Genotyping of 162 participants did not reveal the -504Lys allele in ALDH2. These findings suggest that ADH1C*2 is associated with alcohol dependence in the Turkish population displaying a dominant inheritance model. ADH1C*2 allele may contribute to the variance in heritability of alcohol dependence. The ALDH2 -504Lys/Lys or Glu/Lys genotypes were not present in alcohol-dependent patients, similar to that seen in European populations and in contrast to the findings in the Asian populations.

  11. The prognostic impact of cancer stem-like cell biomarker aldehyde dehydrogenase-1 (ALDH1) in ovarian cancer: A meta-analysis.

    PubMed

    Ruscito, Ilary; Darb-Esfahani, Silvia; Kulbe, Hagen; Bellati, Filippo; Zizzari, Ilaria Grazia; Rahimi Koshkaki, Hassan; Napoletano, Chiara; Caserta, Donatella; Rughetti, Aurelia; Kessler, Mirjana; Sehouli, Jalid; Nuti, Marianna; Braicu, Elena Ioana

    2018-05-10

    To investigate the association of cancer stem cell biomarker aldehyde dehydrogenase-1 (ALDH1) with ovarian cancer patients' prognosis and clinico-pathological characteristics. The electronic searches were performed in January 2018 through the databases PubMed, MEDLINE and Scopus by searching the terms: "ovarian cancer" AND "immunohistochemistry" AND ["aldehyde dehydrogenase-1" OR "ALDH1" OR "cancer stem cell"]. Studies evaluating the impact of ALDH1 expression on ovarian cancer survival and clinico-pathological variables were selected. 233 studies were retrieved. Thirteen studies including 1885 patients met all selection criteria. ALDH1-high expression was found to be significantly associated with poor 5-year OS (OR = 3.46; 95% CI: 1.61-7.42; P = 0.001, random effects model) and 5-year PFS (OR = 2.14; 95% CI: 1.11-4.13; P = 0.02, random effects model) in ovarian cancer patients. No correlation between ALDH1 expression and tumor histology (OR = 0.60; 95% CI: 0.36-1.02; P = 0.06, random effects model), FIGO Stage (OR = 0.65; 95% CI: 0.33-1.30; P = 0.22, random effects model), tumor grading (OR = 0.76; 95% CI: 0.40-1.45; P = 0.41, random effects model) lymph nodal status (OR = 2.05; 95% CI: 0.81-5.18; P = 0.13, random effects model) or patients' age at diagnosis (OR = 0.83; 95% CI: 0.54-1.29; P = 0.41, fixed effects model) was identified. Basing on the available evidence, this meta-analysis showed that high levels of ALDH1 expression correlate with worse OS and PFS in ovarian cancer patients. Copyright © 2018. Published by Elsevier Inc.

  12. CD10-/ALDH- cells are the sole cisplatin-resistant component of a novel ovarian cancer stem cell hierarchy.

    PubMed

    Ffrench, Brendan; Gasch, Claudia; Hokamp, Karsten; Spillane, Cathy; Blackshields, Gordon; Mahgoub, Thamir Mahmoud; Bates, Mark; Kehoe, Louise; Mooney, Aoibhinn; Doyle, Ronan; Doyle, Brendan; O'Donnell, Dearbhaile; Gleeson, Noreen; Hennessy, Bryan T; Stordal, Britta; O'Riain, Ciaran; Lambkin, Helen; O'Toole, Sharon; O'Leary, John J; Gallagher, Michael F

    2017-10-19

    It is long established that tumour-initiating cancer stem cells (CSCs) possess chemoresistant properties. However, little is known of the mechanisms involved, particularly with respect to the organisation of CSCs as stem-progenitor-differentiated cell hierarchies. Here we aimed to elucidate the relationship between CSC hierarchies and chemoresistance in an ovarian cancer model. Using a single cell-based approach to CSC discovery and validation, we report a novel, four-component CSC hierarchy based around the markers cluster of differentiation 10 (CD10) and aldehyde dehydrogenase (ALDH). In a change to our understanding of CSC biology, resistance to chemotherapy drug cisplatin was found to be the sole property of CD10 - /ALDH - CSCs, while all four CSC types were sensitive to chemotherapy drug paclitaxel. Cisplatin treatment quickly altered the hierarchy, resulting in a three-component hierarchy dominated by the cisplatin-resistant CD10 - /ALDH - CSC. This organisation was found to be hard-wired in a long-term cisplatin-adapted model, where again CD10 - /ALDH - CSCs were the sole cisplatin-resistant component, and all CSC types remained paclitaxel-sensitive. Molecular analysis indicated that cisplatin resistance is associated with inherent- and adaptive-specific drug efflux and DNA-damage repair mechanisms. Clinically, low CD10 expression was consistent with a specific set of ovarian cancer patient samples. Collectively, these data advance our understanding of the relationship between CSC hierarchies and chemoresistance, which was shown to be CSC- and drug-type specific, and facilitated by specific and synergistic inherent and adaptive mechanisms. Furthermore, our data indicate that primary stage targeting of CD10 - /ALDH - CSCs in specific ovarian cancer patients in future may facilitate targeting of recurrent disease, before it ever develops.

  13. ALDH1A3 loss of function causes bilateral anophthalmia/microphthalmia and hypoplasia of the optic nerve and optic chiasm.

    PubMed

    Yahyavi, Mani; Abouzeid, Hana; Gawdat, Ghada; de Preux, Anne-Sophie; Xiao, Tong; Bardakjian, Tanya; Schneider, Adele; Choi, Alex; Jorgenson, Eric; Baier, Herwig; El Sada, Mohamad; Schorderet, Daniel F; Slavotinek, Anne M

    2013-08-15

    The major active retinoid, all-trans retinoic acid, has long been recognized as critical for the development of several organs, including the eye. Mutations in STRA6, the gene encoding the cellular receptor for vitamin A, in patients with Matthew-Wood syndrome and anophthalmia/microphthalmia (A/M), have previously demonstrated the importance of retinol metabolism in human eye disease. We used homozygosity mapping combined with next-generation sequencing to interrogate patients with anophthalmia and microphthalmia for new causative genes. We used whole-exome and whole-genome sequencing to study a family with two affected brothers with bilateral A/M and a simplex case with bilateral anophthalmia and hypoplasia of the optic nerve and optic chiasm. Analysis of novel sequence variants revealed homozygosity for two nonsense mutations in ALDH1A3, c.568A>G, predicting p.Lys190*, in the familial cases, and c.1165A>T, predicting p.Lys389*, in the simplex case. Both mutations predict nonsense-mediated decay and complete loss of function. We performed antisense morpholino (MO) studies in Danio rerio to characterize the developmental effects of loss of Aldh1a3 function. MO-injected larvae showed a significant reduction in eye size, and aberrant axonal projections to the tectum were noted. We conclude that ALDH1A3 loss of function causes anophthalmia and aberrant eye development in humans and in animal model systems.

  14. Inhibition of Aldehyde Dehydrogenase-Activity Expands Multipotent Myeloid Progenitor Cells with Vascular Regenerative Function.

    PubMed

    Cooper, Tyler T; Sherman, Stephen E; Kuljanin, Miljan; Bell, Gillian I; Lajoie, Gilles A; Hess, David A

    2018-05-01

    Blood-derived progenitor cell transplantation holds potential for the treatment of severe vascular diseases. Human umbilical cord blood (UCB)-derived hematopoietic progenitor cells purified using high aldehyde dehydrogenase (ALDH hi ) activity demonstrate pro-angiogenic functions following intramuscular (i.m.) transplantation into immunodeficient mice with hind-limb ischemia. Unfortunately, UCB ALDH hi cells are rare and prolonged ex vivo expansion leads to loss of high ALDH-activity and diminished vascular regenerative function. ALDH-activity generates retinoic acid, a potent driver of hematopoietic differentiation, creating a paradoxical challenge to expand UCB ALDH hi cells while limiting differentiation and retaining pro-angiogenic functions. We investigated whether inhibition of ALDH-activity during ex vivo expansion of UCB ALDH hi cells would prevent differentiation and expand progeny that retained pro-angiogenic functions after transplantation into non-obese diabetic/severe combined immunodeficient mice with femoral artery ligation-induced unilateral hind-limb ischemia. Human UCB ALDH hi cells were cultured under serum-free conditions for 9 days, with or without the reversible ALDH-inhibitor, diethylaminobenzaldehyde (DEAB). Although total cell numbers were increased >70-fold, the frequency of cells that retained ALDH hi /CD34+ phenotype was significantly diminished under basal conditions. In contrast, DEAB-inhibition increased total ALDH hi /CD34+ cell number by ≥10-fold, reduced differentiation marker (CD38) expression, and enhanced the retention of multipotent colony-forming cells in vitro. Proteomic analysis revealed that DEAB-treated cells upregulated anti-apoptotic protein expression and diminished production of proteins implicated with megakaryocyte differentiation. The i.m. transplantation of DEAB-treated cells into mice with hind-limb ischemia stimulated endothelial cell proliferation and augmented recovery of hind-limb perfusion. DEAB

  15. LIPID ABNORMALITIES IN SUCCINATE SEMIALDEHYDE DEHYDROGENASE (Aldh5a1−/−) DEFICIENT MOUSE BRAIN PROVIDE ADDITIONAL EVIDENCE FOR MYELIN ALTERATIONS

    PubMed Central

    Barcelo-Coblijn, G.; Murphy, E. J.; Mills, K.; Winchester, B.; Jakobs, C.; Snead, O.C.; Gibson, KM

    2007-01-01

    Earlier work from our laboratory provided evidence for myelin abnormalities (decreased quantities of proteins associated with myelin compaction, decreased sheath thickness) in cortex and hippocampus of Aldh5a1−/− mice, which have a complete ablation of the succinate semialdehyde dehydrogenase protein [1]. In the current report, we have extended these findings via comprehensive analysis of brain phospholipid fractions, including quantitation of fatty acids in individual phospholipid subclasses and estimation of hexose-ceramide in Aldh5a1−/− brain. In comparison to wild-type littermates (Aldh5a1+/+), we detected a 20% reduction in the ethanolamine glycerophospholipid content of Aldh5a1−/− mice, while other brain phospholipids (choline glycerophospholipid, phosphatidylserine and phosphatidylinositol) were within normal limits. Analysis of individual fatty acids in each of these fractions revealed consistent alterations in n-3 fatty acids, primarily increased 22:6n-3 levels (docosahexaenoic acid; DHA). In the phosphatidyl serine fraction there were marked increases in the proportions of polyunsaturated fatty acids with corresponding decreases of monounsaturated fatty acids. Interestingly, the levels of hexose-ceramide (glucosyl- and galactosylceramide, principal myelin cerebrosides) were decreased in Aldh5a1−/− brain tissue (one-tailed t test, p=0.0449). The current results suggest that lipid and myelin abnormalities in this animal may contribute to the pathophysiology. PMID:17300923

  16. 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.

  17. 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. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. 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.

  19. In phyllodes tumour of the breast expression of c-kit but not of ALDH1A1 is associated with adverse clinico-pathological features.

    PubMed

    Chougule, Abhijit; Bal, Amanjit; Das, Ashim; Kohli, Pavneet Singh; Singh, Gurpreet

    2016-12-01

    Attempts at identification of an ideal prognostic/predictive biomarker in phyllodes tumour (PT) have not been fruitful so far. Studies evaluating c-kit expression in PT have shown contradictory results. Recently aldehyde dehydrogenase 1A1 (ALDH1A1) was proposed as a stem cell marker for malignant PT but its expression has not been studied in benign and borderline tumours. We aimed to evaluate expression and prognostic significance of c-kit and ALDH1A1 in different grades of PT. Epithelial and stromal c-kit and ALDH1A1 expression were studied in 104 PT cases (86 primary and 18 recurrent tumours) and compared with different clinico-pathological features and recurrence rates. Stromal c-kit expression at 1 % cutoff correlated with increasing tumour grade, larger tumour size, hypercellularity, nuclear atypia, stromal overgrowth, infiltrative margins and mitotic count. These associations, however, were lost with higher (5 or 10 %) cutoffs. Conversely, decreased c-kit expression in the epithelial component correlated with increasing tumour grade, regardless of the cutoffs used. Stromal ALDH1A1 expression did not have significant associations with tumour grade or other adverse clinico-pathological features, regardless of different cutoffs. None of the cases showed significant epithelial ALDH1A1 expression. Expression of c-kit was associated with poorer overall survival (p = 0.011), while ALDH1A1 expression was associated with shorter recurrence-free survival (p = 0.036). In conclusion, c-kit expression was associated with higher tumour grade and adverse clinico-pathological features. However, these associations are cutoff dependent, partly explaining the variability in previously reported studies. ALDH1A1 expression did not have significant correlations with tumour grade and adverse clinico-pathological variables.

  20. Whole-Exome Sequencing in a South American Cohort Links ALDH1A3, FOXN1 and Retinoic Acid Regulation Pathways to Autism Spectrum Disorders.

    PubMed

    Moreno-Ramos, Oscar A; Olivares, Ana María; Haider, Neena B; de Autismo, Liga Colombiana; Lattig, María Claudia

    2015-01-01

    Autism spectrum disorders (ASDs) are a range of complex neurodevelopmental conditions principally characterized by dysfunctions linked to mental development. Previous studies have shown that there are more than 1000 genes likely involved in ASD, expressed mainly in brain and highly interconnected among them. We applied whole exome sequencing in Colombian-South American trios. Two missense novel SNVs were found in the same child: ALDH1A3 (RefSeq NM_000693: c.1514T>C (p.I505T)) and FOXN1 (RefSeq NM_003593: c.146C>T (p.S49L)). Gene expression studies reveal that Aldh1a3 and Foxn1 are expressed in ~E13.5 mouse embryonic brain, as well as in adult piriform cortex (PC; ~P30). Conserved Retinoic Acid Response Elements (RAREs) upstream of human ALDH1A3 and FOXN1 and in mouse Aldh1a3 and Foxn1 genes were revealed using bioinformatic approximation. Chromatin immunoprecipitation (ChIP) assay using Retinoid Acid Receptor B (Rarb) as the immunoprecipitation target suggests RA regulation of Aldh1a3 and Foxn1 in mice. Our results frame a possible link of RA regulation in brain to ASD etiology, and a feasible non-additive effect of two apparently unrelated variants in ALDH1A3 and FOXN1 recognizing that every result given by next generation sequencing should be cautiously analyzed, as it might be an incidental finding.

  1. Whole-Exome Sequencing in a South American Cohort Links ALDH1A3, FOXN1 and Retinoic Acid Regulation Pathways to Autism Spectrum Disorders

    PubMed Central

    Moreno-Ramos, Oscar A.; Olivares, Ana María; Haider, Neena B.; de Autismo, Liga Colombiana; Lattig, María Claudia

    2015-01-01

    Autism spectrum disorders (ASDs) are a range of complex neurodevelopmental conditions principally characterized by dysfunctions linked to mental development. Previous studies have shown that there are more than 1000 genes likely involved in ASD, expressed mainly in brain and highly interconnected among them. We applied whole exome sequencing in Colombian—South American trios. Two missense novel SNVs were found in the same child: ALDH1A3 (RefSeq NM_000693: c.1514T>C (p.I505T)) and FOXN1 (RefSeq NM_003593: c.146C>T (p.S49L)). Gene expression studies reveal that Aldh1a3 and Foxn1 are expressed in ~E13.5 mouse embryonic brain, as well as in adult piriform cortex (PC; ~P30). Conserved Retinoic Acid Response Elements (RAREs) upstream of human ALDH1A3 and FOXN1 and in mouse Aldh1a3 and Foxn1 genes were revealed using bioinformatic approximation. Chromatin immunoprecipitation (ChIP) assay using Retinoid Acid Receptor B (Rarb) as the immunoprecipitation target suggests RA regulation of Aldh1a3 and Foxn1 in mice. Our results frame a possible link of RA regulation in brain to ASD etiology, and a feasible non-additive effect of two apparently unrelated variants in ALDH1A3 and FOXN1 recognizing that every result given by next generation sequencing should be cautiously analyzed, as it might be an incidental finding. PMID:26352270

  2. ALDH1A3 loss of function causes bilateral anophthalmia/microphthalmia and hypoplasia of the optic nerve and optic chiasm

    PubMed Central

    Yahyavi, Mani; Abouzeid, Hana; Gawdat, Ghada; de Preux, Anne-Sophie; Xiao, Tong; Bardakjian, Tanya; Schneider, Adele; Choi, Alex; Jorgenson, Eric; Baier, Herwig; El Sada, Mohamad; Schorderet, Daniel F.; Slavotinek, Anne M.

    2013-01-01

    The major active retinoid, all-trans retinoic acid, has long been recognized as critical for the development of several organs, including the eye. Mutations in STRA6, the gene encoding the cellular receptor for vitamin A, in patients with Matthew–Wood syndrome and anophthalmia/microphthalmia (A/M), have previously demonstrated the importance of retinol metabolism in human eye disease. We used homozygosity mapping combined with next-generation sequencing to interrogate patients with anophthalmia and microphthalmia for new causative genes. We used whole-exome and whole-genome sequencing to study a family with two affected brothers with bilateral A/M and a simplex case with bilateral anophthalmia and hypoplasia of the optic nerve and optic chiasm. Analysis of novel sequence variants revealed homozygosity for two nonsense mutations in ALDH1A3, c.568A>G, predicting p.Lys190*, in the familial cases, and c.1165A>T, predicting p.Lys389*, in the simplex case. Both mutations predict nonsense-mediated decay and complete loss of function. We performed antisense morpholino (MO) studies in Danio rerio to characterize the developmental effects of loss of Aldh1a3 function. MO-injected larvae showed a significant reduction in eye size, and aberrant axonal projections to the tectum were noted. We conclude that ALDH1A3 loss of function causes anophthalmia and aberrant eye development in humans and in animal model systems. PMID:23591992

  3. Enzymatically Active Microgels from Self-Assembling Protein Nanofibrils for Microflow Chemistry.

    PubMed

    Zhou, Xiao-Ming; Shimanovich, Ulyana; Herling, Therese W; Wu, Si; Dobson, Christopher M; Knowles, Tuomas P J; Perrett, Sarah

    2015-06-23

    Amyloid fibrils represent a generic class of protein structure associated with both pathological states and with naturally occurring functional materials. This class of protein nanostructure has recently also emerged as an excellent foundation for sophisticated functional biocompatible materials including scaffolds and carriers for biologically active molecules. Protein-based materials offer the potential advantage that additional functions can be directly incorporated via gene fusion producing a single chimeric polypeptide that will both self-assemble and display the desired activity. To succeed, a chimeric protein system must self-assemble without the need for harsh triggering conditions which would damage the appended functional protein molecule. However, the micrometer to nanoscale patterning and morphological control of protein-based nanomaterials has remained challenging. This study demonstrates a general approach for overcoming these limitations through the microfluidic generation of enzymatically active microgels that are stabilized by amyloid nanofibrils. The use of scaffolds formed from biomaterials that self-assemble under mild conditions enables the formation of catalytic microgels while maintaining the integrity of the encapsulated enzyme. The enzymatically active microgel particles show robust material properties and their porous architecture allows diffusion in and out of reactants and products. In combination with microfluidic droplet trapping approaches, enzymatically active microgels illustrate the potential of self-assembling materials for enzyme immobilization and recycling, and for biological flow-chemistry. These design principles can be adopted to create countless other bioactive amyloid-based materials with diverse functions.

  4. Enzymatically Active Microgels from Self-Assembling Protein Nanofibrils for Microflow Chemistry

    PubMed Central

    2015-01-01

    Amyloid fibrils represent a generic class of protein structure associated with both pathological states and with naturally occurring functional materials. This class of protein nanostructure has recently also emerged as an excellent foundation for sophisticated functional biocompatible materials including scaffolds and carriers for biologically active molecules. Protein-based materials offer the potential advantage that additional functions can be directly incorporated via gene fusion producing a single chimeric polypeptide that will both self-assemble and display the desired activity. To succeed, a chimeric protein system must self-assemble without the need for harsh triggering conditions which would damage the appended functional protein molecule. However, the micrometer to nanoscale patterning and morphological control of protein-based nanomaterials has remained challenging. This study demonstrates a general approach for overcoming these limitations through the microfluidic generation of enzymatically active microgels that are stabilized by amyloid nanofibrils. The use of scaffolds formed from biomaterials that self-assemble under mild conditions enables the formation of catalytic microgels while maintaining the integrity of the encapsulated enzyme. The enzymatically active microgel particles show robust material properties and their porous architecture allows diffusion in and out of reactants and products. In combination with microfluidic droplet trapping approaches, enzymatically active microgels illustrate the potential of self-assembling materials for enzyme immobilization and recycling, and for biological flow-chemistry. These design principles can be adopted to create countless other bioactive amyloid-based materials with diverse functions. PMID:26030507

  5. Tamoxifen enhances stemness and promotes metastasis of ERα36+ breast cancer by upregulating ALDH1A1 in cancer cells

    PubMed Central

    Wang, Qiang; Jiang, Jun; Ying, Guoguang; Xie, Xiao-Qing; Zhang, Xia; Xu, Wei; Zhang, Xuemin; Song, Erwei; Bu, Hong; Ping, Yi-Fang; Yao, Xiao-Hong; Wang, Bin; Xu, Shilei; Yan, Ze-Xuan; Tai, Yanhong; Hu, Baoquan; Qi, Xiaowei; Wang, Yan-Xia; He, Zhi-Cheng; Wang, Yan; Wang, Ji Ming; Cui, You-Hong; Chen, Feng; Meng, Kun; Wang, Zhaoyi; Bian, Xiu-Wu

    2018-01-01

    The 66 kDa estrogen receptor alpha (ERα66) is the main molecular target for endocrine therapy such as tamoxifen treatment. However, many patients develop resistance with unclear mechanisms. In a large cohort study of breast cancer patients who underwent surgery followed by tamoxifen treatment, we demonstrate that ERα36, a variant of ERα66, correlates with poor prognosis. Mechanistically, tamoxifen directly binds and activates ERα36 to enhance the stemness and metastasis of breast cancer cells via transcriptional stimulation of aldehyde dehydrogenase 1A1 (ALDH1A1). Consistently, the tamoxifen-induced stemness and metastasis can be attenuated by either ALDH1 inhibitors or a specific ERα36 antibody. Thus, tamoxifen acts as an agonist on ERα36 in breast cancer cells, which accounts for hormone therapy resistance and metastasis of breast cancer. Our study not only reveals ERα36 as a stratifying marker for endocrine therapy but also provides a promising therapeutic avenue for tamoxifen-resistant breast cancer. PMID:29393296

  6. Interaction of the SPG21 protein ACP33/maspardin with the aldehyde dehydrogenase ALDH16A1

    PubMed Central

    2017-01-01

    Mast syndrome (SPG21) is an autosomal-recessive complicated form of hereditary spastic paraplegia characterized by dementia, thin corpus callosum, white matter abnormalities, and cerebellar and extrapyramidal signs in addition to spastic paraparesis. A nucleotide insertion resulting in premature truncation of the SPG21 gene product acidic cluster protein 33 (ACP33)/maspardin underlies this disorder, likely causing loss of protein function. However, little is known about the function of maspardin. Here, we report that maspardin localizes prominently to cytoplasm as well as to membranes, possibly at trans-Golgi network/late endosomal compartments. Immunoprecipitation of maspardin with identification of coprecipitating proteins by mass spectrometry revealed the aldehyde dehydrogenase ALDH16A1 as an interacting protein. This interaction was confirmed using overexpressed proteins as well as by fusion protein pull down experiments, and these proteins colocalized in cells. Further studies of the function of ALDH16A1 and the role of the maspardin–ALDH16A1 interaction in neuronal cells may clarify the cellular pathogenesis of Mast syndrome. PMID:19184135

  7. Comparative Proteomics Analysis Reveals L-Arginine Activates Ethanol Degradation Pathways in HepG2 Cells.

    PubMed

    Yan, Guokai; Lestari, Retno; Long, Baisheng; Fan, Qiwen; Wang, Zhichang; Guo, Xiaozhen; Yu, Jie; Hu, Jun; Yang, Xingya; Chen, Changqing; Liu, Lu; Li, Xiuzhi; Purnomoadi, Agung; Achmadi, Joelal; Yan, Xianghua

    2016-03-17

    L-Arginine (Arg) is a versatile amino acid that plays crucial roles in a wide range of physiological and pathological processes. In this study, to investigate the alteration induced by Arg supplementation in proteome scale, isobaric tags for relative and absolute quantification (iTRAQ) based proteomic approach was employed to comparatively characterize the differentially expressed proteins between Arg deprivation (Ctrl) and Arg supplementation (+Arg) treated human liver hepatocellular carcinoma (HepG2) cells. A total of 21 proteins were identified as differentially expressed proteins and these 21 proteins were all up-regulated by Arg supplementation. Six amino acid metabolism-related proteins, mostly metabolic enzymes, showed differential expressions. Intriguingly, Ingenuity Pathway Analysis (IPA) based pathway analysis suggested that the three ethanol degradation pathways were significantly altered between Ctrl and +Arg. Western blotting and enzymatic activity assays validated that the key enzymes ADH1C, ALDH1A1, and ALDH2, which are mainly involved in ethanol degradation pathways, were highly differentially expressed, and activated between Ctrl and +Arg in HepG2 cells. Furthermore, 10 mM Arg significantly attenuated the cytotoxicity induced by 100 mM ethanol treatment (P < 0.0001). This study is the first time to reveal that Arg activates ethanol degradation pathways in HepG2 cells.

  8. 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.

  9. Novel therapy for pyridoxine dependent epilepsy due to ALDH7A1 genetic defect: L-arginine supplementation alternative to lysine-restricted diet.

    PubMed

    Mercimek-Mahmutoglu, Saadet; Cordeiro, Dawn; Cruz, Vivian; Hyland, Keith; Struys, Eduard A; Kyriakopoulou, Lianna; Mamak, Eva

    2014-11-01

    Pyridoxine dependent epilepsy (PDE) due to mutations in the ALDH7A1 gene (PDE-ALDH7A1) is caused by α-aminoadipic-semialdehyde-dehydrogenase enzyme deficiency in the lysine pathway resulting in the accumulation of α-aminoadipic acid semialdehyde (α-AASA). Classical presentation is neonatal intractable seizures with a dramatic response to pyridoxine. Pyridoxine therapy does not prevent developmental delays in the majority of the patients. We hypothesized that L-arginine supplementation will decrease accumulation of α-AASA by competitive inhibition of lysine transport into the central nervous system and improve neurodevelopmental and neurocognitive functions in PDE-ALDH7A1. A 12-year-old male with PDE-ALDH7A1 was treated with l-arginine supplementation as an innovative therapy. Treatment outcome was monitored by cerebral-spinal-fluid (CSF) α-AASA measurements at baseline, 6th and 12th months of therapy. Neuropsychological assessments were performed at baseline and 12th months of therapy. L-arginine therapy was well tolerated without side effects. CSF α-AASA was decreased 57% at 12th months of therapy. Neuropsychological assessments revealed improvements in general abilities index from 108 to 116 and improvements in verbal and motor functioning at 12th months of therapy. The short-term treatment outcome of this novel L-arginine supplementation therapy for PDE-ALDH7A1 was successful for biochemical and neurocognitive improvements. Copyright © 2014 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

  10. Characterization of selected cellulolytic activities of multi-enzymatic complex system from Penicillium funiculosum.

    PubMed

    Karboune, Salwa; Geraert, Pierre-André; Kermasha, Selim

    2008-02-13

    The presence of endo-1,4-beta-D-glucanase, cellobiohydrolase, and beta-glucosidase activities in a multi-enzymatic complex system from Penicillium funiculosum was investigated. The interesting feature of these enzymes is their synergistic action for the hydrolysis of the native cellulose into glucose units. Both endo-1,4-beta-D-glucanase and cellobiohydrolase showed broader pH activity profiles, with pH optima of 4.0 and 4.0-5.0, respectively. However, beta-glucosidase activity showed a narrow pH-activity profile, with an optimum pH of 4.5. The different cellulolytic activities were stable in the acidic pH range of 2.5-6.0 and showed a similar optimal temperature of 60 degrees C. Although beta-glucosidase has shown a close catalytic efficiency as that of endo-1,4-beta-D-glucanase, its thermal stability was lower. However, the thermal stability profile of cellobiohydrolase was close to that of endo-1,4-beta-D-glucanase. The results also revealed the presence of high levels of endo-1,3-1,4-beta-D-glucanase, endo-1,3-beta- d-glucanase, and pectinase activities in the multi-enzymatic cellulolytic complex system. Moreover, the investigated multi-enzymatic complex system was effective in degrading the nonstarch polysaccharides of soybean meal.

  11. Host-parasite co-metabolic activation of antitrypanosomal aminomethyl-benzoxaboroles

    PubMed Central

    Scullion, Paul; del Pino, Ricardo C.; Vincent, Isabel M.; Zhang, Yong-Kang; Alley, Michael R. K.; Jacobs, Robert T.; Read, Kevin D.

    2018-01-01

    Recent development of benzoxaborole-based chemistry gave rise to a collection of compounds with great potential in targeting diverse infectious diseases, including human African Trypanosomiasis (HAT), a devastating neglected tropical disease. However, further medicinal development is largely restricted by a lack of insight into mechanism of action (MoA) in pathogenic kinetoplastids. We adopted a multidisciplinary approach, combining a high-throughput forward genetic screen with functional group focused chemical biological, structural biology and biochemical analyses, to tackle the complex MoAs of benzoxaboroles in Trypanosoma brucei. We describe an oxidative enzymatic pathway composed of host semicarbazide-sensitive amine oxidase and a trypanosomal aldehyde dehydrogenase TbALDH3. Two sequential reactions through this pathway serve as the key underlying mechanism for activating a series of 4-aminomethylphenoxy-benzoxaboroles as potent trypanocides; the methylamine parental compounds as pro-drugs are transformed first into intermediate aldehyde metabolites, and further into the carboxylate metabolites as effective forms. Moreover, comparative biochemical and crystallographic analyses elucidated the catalytic specificity of TbALDH3 towards the benzaldehyde benzoxaborole metabolites as xenogeneic substrates. Overall, this work proposes a novel drug activation mechanism dependent on both host and parasite metabolism of primary amine containing molecules, which contributes a new perspective to our understanding of the benzoxaborole MoA, and could be further exploited to improve the therapeutic index of antimicrobial compounds. PMID:29425238

  12. Enzymatic Activity of Candida spp. from Oral Cavity and Urine in Children with Nephrotic Syndrome.

    PubMed

    Olczak-Kowalczyk, Dorota; Roszkowska-Blaim, Maria; Dąbkowska, Maria; Swoboda-Kopeć, Ewa; Gozdowski, Dariusz; Mizerska-Wasiak, Małgorzata; Demkow, Urszula; Pańczyk-Tomaszewska, Małgorzata

    2017-01-01

    Oral colonization with Candida spp. is not synonymous with a systemic active infection. The aim of the study was to evaluate enzymatic activity of Candida strains isolated from the oral cavity in patients with nephrotic syndrome (NS) and to compare it with the activity determined in urine. We studied 32 children with NS and 26 control healthy children. Children with NS were treated with glucocorticosteroids, cyclosporin A, mycophenolate mofetil or azathioprine. In all children, API-ZYM enzymatic tests were performed to evaluate hydrolytic enzymes of Candida isolated from the oral cavity and in urine. Candida spp. were isolated from the oral cavity in 11 patients with NS (34.4%), all receiving immunosuppressive treatment. All strains produced valine arylamidase, 9 alpha-glucosidase (E16), and 9 N-acetyl-beta-glucosaminidase (E18). A positive correlation between the presence of Candida in the oral cavity and E16 and E18 enzymatic activity in both oral cavity and urine was found. A dose of cyclosporin A had an effect on the enzymatic activity (p < 0.05). We conclude that immunosuppressive treatment of NS in children may predispose to systemic Candida invasion. The results of this study suggest that oral candida infection should be monitored in children with nephrotic syndrome, particularly those treated with immunosuppressive agents.

  13. Genetic polymorphisms of ADH1B, ADH1C and ALDH2 in Turkish alcoholics: lack of association with alcoholism and alcoholic cirrhosis.

    PubMed

    Vatansever, Sezgin; Tekin, Fatih; Salman, Esin; Altintoprak, Ender; Coskunol, Hakan; Akarca, Ulus Salih

    2015-05-17

    No data exists regarding the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) gene polymorphisms in Turkish alcoholic cirrhotics. We studied the polymorphisms of ADH1B, ADH1C and ALDH2 genes in alcoholic cirrhotics and compared the results with non-cirrhotic alcoholics and healthy volunteers. Overall, 237 subjects were included for the study: 156 alcoholic patients (78 cirrhotics, 78 non-cirrhotic alcoholics) and 81 healthy volunteers. Three different single-nucleotide-polymorphism genotyping methods were used. ADH1C genotyping was performed using a polymerase chain reaction-restriction fragment length polymorphism method. The identified ADH1C genotypes were named according to the presence or absence of the enzyme restriction sites. ADH1B (Arg47Hys) genotyping was performed using the allele specific primer extension method, and ALDH2 (Glu487Lys) genotyping was performed by a multiplex polymerase chain reaction using two allele-specific primer pairs. For ADH1B, the frequency of allele *1 in the cirrhotics, non-cirrhotic alcoholics and healthy volunteers was 97.4%, 94.9% and 99.4%, respectively. For ADH1C, the frequency of allele *1 in the cirrhotics, non-cirrhotic alcoholics and healthy volunteers was 47%, 36.3% and 45%, respectively. There was no statistical difference between the groups for ADH1B and ADH1C (p>0.05). All alcoholic and non-alcoholic subjects (100%) had the allele *1 for ALDH2. The obtained results for ADH1B, ADH1C, and ALDH gene polymorphisms in the present study are similar to the results of Caucasian studies. ADH1B and ADH1C genetic variations are not related to the development of alcoholism or susceptibility to alcoholic cirrhosis. ALDH2 gene has no genetic variation in the Turkish population.

  14. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  16. 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.

  17. High Aldehyde Dehydrogenase Activity Identifies a Subset of Human Mesenchymal Stromal Cells with Vascular Regenerative Potential.

    PubMed

    Sherman, Stephen E; Kuljanin, Miljan; Cooper, Tyler T; Putman, David M; Lajoie, Gilles A; Hess, David A

    2017-06-01

    During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDH hi ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDH l ° and ALDH hi MSC subsets demonstrated similar expression of stromal cell (>95% CD73 + , CD90 + , CD105 + ) and pericyte (>95% CD146 + ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDH hi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDH hi MSC or CDM produced by ALDH hi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDH l ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDH hi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix

  18. Genetic polymorphisms in ALDH2 are associated with drug addiction in a Chinese Han population

    PubMed Central

    Zhang, Chan; Ding, Heng; Cheng, Yujing; Chen, Wanlu; Li, Qi; Li, Qing; Dai, Run; Luo, Manlin

    2017-01-01

    We investigated the association between single nucleotide polymorphisms (SNPs) in ALDH2, which has been associated with alcohol dependence and several types of diseases, and the risk of drug addiction in a Chinese Han population. In a case-control study that included 692 cases and 700 healthy controls, eight SNPs in ALDH2 were selected and genotyped using the Sequenom MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression after adjusting for age and gender. We determined that rs671 is significantly associated with a 1.551-fold increased drug addiction risk (95% CI = 1.263-1.903; p < 0.001). In the genetic model analysis, we found that rs671 is associated with an increased risk of drug addiction under additive, dominant and recessive models (p < 0.001), while rs886205, rs441 and rs4646778 displayed a decreased drug addiction risk under additive and recessive model, respectively (p < 0.05). SNP rs671 remained significant after Bonferroni correction (p<0.00125). Additionally, we observed that haplotype “GTCAC” was associated with increased drug addiction risk (OR = 1.668; 95% CI, 1.328–2.094, p < 0.001); in contrast, “ATCGC” was a protective haplotype for drug addiction risk (OR = 0.444; 95% CI, 0.281–0.704, p < 0.001). Our findings showed that ALDH2 polymorphisms are significantly associated with the risk of drug addiction in the Chinese Han population. PMID:28052001

  19. Genetic polymorphisms in ALDH2 are associated with drug addiction in a Chinese Han population.

    PubMed

    Zhang, Chan; Ding, Heng; Cheng, Yujing; Chen, Wanlu; Li, Qi; Li, Qing; Dai, Run; Luo, Manlin

    2017-01-31

    We investigated the association between single nucleotide polymorphisms (SNPs) in ALDH2, which has been associated with alcohol dependence and several types of diseases, and the risk of drug addiction in a Chinese Han population. In a case-control study that included 692 cases and 700 healthy controls, eight SNPs in ALDH2 were selected and genotyped using the Sequenom MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression after adjusting for age and gender. We determined that rs671 is significantly associated with a 1.551-fold increased drug addiction risk (95% CI = 1.263-1.903; p < 0.001). In the genetic model analysis, we found that rs671 is associated with an increased risk of drug addiction under additive, dominant and recessive models (p < 0.001), while rs886205, rs441 and rs4646778 displayed a decreased drug addiction risk under additive and recessive model, respectively (p < 0.05). SNP rs671 remained significant after Bonferroni correction (p<0.00125). Additionally, we observed that haplotype "GTCAC" was associated with increased drug addiction risk (OR = 1.668; 95% CI, 1.328-2.094, p < 0.001); in contrast, "ATCGC" was a protective haplotype for drug addiction risk (OR = 0.444; 95% CI, 0.281-0.704, p < 0.001). Our findings showed that ALDH2 polymorphisms are significantly associated with the risk of drug addiction in the Chinese Han population.

  20. The Impact of Marine Enzymatic Activity on Sea Spray Aerosol Properties

    NASA Astrophysics Data System (ADS)

    Ryder, O. S.; Michaud, J. M.; Sauer, J. S.; Lee, C.; Förster, J. D.; Pöhlker, C.; Andreae, M. O.; Prather, K. A.

    2016-12-01

    The composition of sea spray aerosol (SSA) and the relationship between its organic fraction and biological ocean conditions is not well understood, resulting in considerable disagreement in the literature linking biological markers to SSA chemical composition. Recent work suggests that enzymatic activity in seawater may play a key role in dictating aerosol composition by changing the organic pool from which SSA is formed. Here we investigate the role of enzymatic activity on SSA spatial chemical composition, aerosol phase and morphological microstructure. In these experiments, SSA was generated using a novel mini-Marine Aerosol Reference Tank system. SSA collected onto substrates was generated from artificial salt water that had been doped with either 1) unsaturated triglycerides or 2) diatom cellular lysate, both followed by lipase. Results from analysis including morphological studies via atomic force microscopy, and chemical composition investigations both under dry and RH conditions via STXM-NEXAFS are presented.

  1. Salivary aldehyde dehydrogenase - temporal and population variability, correlations with drinking and smoking habits and activity towards aldehydes contained in food.

    PubMed

    Giebułtowicz, Joanna; Dziadek, Marta; Wroczyński, Piotr; Woźnicka, Katarzyna; Wojno, Barbara; Pietrzak, Monika; Wierzchowski, Jacek

    2010-01-01

    Fluorimetric method based on oxidation of the fluorogenic 6-methoxy-2-naphthaldehyde was applied to evaluate temporal and population variability of the specific activity of salivary aldehyde dehydrogenase (ALDH) and the degree of its inactivation in healthy human population. Analyzed was also its dependence on drinking and smoking habits, coffee consumption, and its sensitivity to N-acetylcysteine. Both the specific activity of salivary ALDH and the degree of its inactivation were highly variable during the day, with the highest activities recorded in the morning hours. The activities were also highly variable both intra- and interpersonally, and negatively correlated with age, and this correlation was stronger for the subgroup of volunteers declaring abstinence from alcohol and tobacco. Moderately positive correlations of salivary ALDH specific activity with alcohol consumption and tobacco smoking were also recorded (r(s) ~0.27; p=0.004 and r(s) =0.30; p=0.001, respectively). Moderate coffee consumption correlated positively with the inactivation of salivary ALDH, particularly in the subgroup of non-drinking and non-smoking volunteers. It was found that mechanical stimulation of the saliva flow increases the specific activity of salivary ALDH. The specific activity of the salivary ALDH was strongly and positively correlated with that of superoxide dismutase, and somewhat less with salivary peroxidase. The antioxidant-containing drug N-acetylcysteine increased activity of salivary ALDH presumably by preventing its inactivation in the oral cavity. Some food-related aldehydes, mainly cinnamic aldehyde and anisaldehyde, were excellent substrates of the salivary ALDH3A1 enzyme, while alkenals, particularly those with short chain, were characterized by lower affinity towards this enzyme but high catalytic constants. The protective role of salivary ALDH against aldehydes in food and those found in the cigarette smoke is discussed, as well as its participation in

  2. 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

  3. MiR-23b controls ALDH1A1 expression in cervical cancer stem cells.

    PubMed

    Wang, Weiwen; Li, Yang; Liu, Na; Gao, Yu; Li, Long

    2017-04-27

    Cancer stem cells has been widely investigated due to its essential role in cancer progression and drug resistance. Here, we try to find a new therapeutic target for cervical cancer stem cells. We detected ALDH1A1-associated miRNAs expression in our isolated tumorspheres and their corresponding parental cells. Sphere formation assay was also used to determine stemness after cells were manipulated with miR-23b plasmid or miR-23b inhibitor. We found that miR-23b was under-expressed in cervical cancer stem cells to maintain high levels of ALDH1A1. Introduction of miR-23b into cervical cancer cells could alter stemness and cisplatin sensitivity. miR-23b plays key role in maintaining stemness of cervical cancer stem cells and can be developed as therapeutic target to better fight against cervical cancer.

  4. ALDH2 protects against high fat diet-induced obesity cardiomyopathy and defective autophagy: role of CaM kinase II, histone H3K9 methyltransferase SUV39H, Sirt1, and PGC-1α deacetylation.

    PubMed

    Wang, Shuyi; Wang, Cong; Turdi, Subat; Richmond, Kacy L; Zhang, Yingmei; Ren, Jun

    2018-06-01

    Uncorrected obesity contributes to cardiac remodeling and contractile dysfunction although the underlying mechanism remains poorly understood. Mitochondrial aldehyde dehydrogenase (ALDH2) is a mitochondrial enzyme with some promises in a number of cardiovascular diseases. This study was designed to evaluate the impact of ALDH2 on cardiac remodeling and contractile property in high fat diet-induced obesity. Wild-type (WT) and ALDH2 transgenic mice were fed low (10% calorie from fat) or high (45% calorie from fat) fat diet for 5 months prior to the assessment of cardiac geometry and function using echocardiography, IonOptix system, Lectin, and Masson Trichrome staining. Western blot analysis was employed to evaluate autophagy, CaM kinase II, PGC-1α, histone H3K9 methyltransferase SUV39H, and Sirt-1. Our data revealed that high fat diet intake promoted weight gain, cardiac remodeling (hypertrophy and interstitial fibrosis, p < 0.0001) and contractile dysfunction (reduced fractional shortening (p < 0.0001), cardiomyocyte function (p < 0.0001), and intracellular Ca 2+ handling (p = 0.0346)), mitochondrial injury (elevated O 2 - levels, suppressed PGC-1α, and enhanced PGC-1α acetylation, p < 0.0001), elevated SUV39H, suppressed Sirt1, autophagy and phosphorylation of AMPK and CaM kinase II, the effects of which were negated by ALDH2 (p ≤ 0.0162). In vitro incubation of the ALDH2 activator Alda-1 rescued against palmitic acid-induced changes in cardiomyocyte function, the effect of which was nullified by the Sirt-1 inhibitor nicotinamide and the CaM kinase II inhibitor KN-93 (p < 0.0001). The SUV39H inhibitor chaetocin mimicked Alda-1-induced protection again palmitic acid (p < 0.0001). Examination in overweight human revealed an inverse correlation between diastolic cardiac function and ALDH2 gene mutation (p < 0.05). Taken together, these data suggest that ALDH2 serves as an indispensable factor against cardiac anomalies in diet

  5. Show Yourself, Asparaginase: An Enzymatic Reaction Explained through a Hands-On Interactive Activity

    PubMed Central

    2017-01-01

    Determining the catalytic activity of an enzyme can be the perfect method for its identification, for example during purification procedures or for isolation purposes. Herein, we used a pharmaceutically relevant protein to bring the concept of enzymatic activity to the classroom. We designed a hands-on interactive activity in which a medically relevant enzyme, asparaginase, was distinguished from a nonenzymatic protein based on its specific enzymatic activity. The experiment was carried out in the classroom, designed to impact different educational levels from elementary to high school. Our main purposes were to promote the emerging field of protein-based drugs as a source of scientific careers in bionanotechnology and to show the students an image of a “scientist” as that of a common and educated person working in an exciting profession. In addition of being inexpensive, this activity proved to be adaptable for various educational levels and can be easily implemented in different scenarios, for example, scientific fairs, some schools, and so forth. PMID:29599566

  6. Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

    PubMed Central

    SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

    2016-01-01

    SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

  7. Allelic variants of ADH, ALDH and the five factor model of personality in alcohol dependence syndrome

    PubMed Central

    Salujha, S. K.; Chaudhury, S.; Menon, P. K.; Srivastava, K.; Gupta, A.

    2014-01-01

    Background: The etiology of alcohol dependence is a complex interplay of biopsychosocial factors. The genes for alcohol-metabolizing enzymes: Alcohol dehydrogenase (ADH2 and ADH3) and aldehyde dehydrogenase (ALDH2) exhibit functional polymorphisms. Vulnerability of alcohol dependence may also be in part due to heritable personality traits. Aim: To determine whether any association exists between polymorphisms of ADH2, ADH3 and ALDH2 and alcohol dependence syndrome in a group of Asian Indians. In addition, the personality of these patients was assessed to identify traits predisposing to alcoholism. Materials and Methods: In this study, 100 consecutive males with alcohol dependence syndrome attending the psychiatric outpatient department of a tertiary care service hospital and an equal number of matched healthy controls were included with their consent. Blood samples of all the study cases and controls were collected and genotyped for the ADH2, ADH3 and ALDH2 loci. Personality was evaluated using the neuroticism, extraversion, openness (NEO) personality inventory and sensation seeking scale. Results: Allele frequencies of ADH2*2 (0.50), ADH3*1 (0.67) and ALSH2*2 (0.09) were significantly low in the alcohol dependent subjects. Personality traits of NEO personality inventory and sensation seeking were significantly higher when compared to controls. Conclusions: The functional polymorphisms of genes coding for alcohol metabolizing enzymes and personality traits of NEO and sensation seeking may affect the propensity to develop dependence. PMID:25535445

  8. Constraints imposed by transmembrane domains affect enzymatic activity of membrane-associated human CD39/NTPDase1 mutants.

    PubMed

    Musi, Elgilda; Islam, Naziba; Drosopoulos, Joan H F

    2007-05-01

    Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.

  9. Interaction between ALDH2*1*1 and DRD2/ANKK1 TaqI A1A1 genes may be associated with antisocial personality disorder not co-morbid with alcoholism.

    PubMed

    Lu, Ru-Band; Lee, Jia-Fu; Huang, San-Yuan; Lee, Sheng-Yu; Chang, Yun-Hsuan; Kuo, Po-Hsiu; Chen, Shiou-Lan; Chen, Shih-Heng; Chu, Chun-Hsien; Lin, Wei-Wen; Wu, Pei-Lin; Ko, Huei-Chen

    2012-09-01

    Previous studies on acetaldehyde dehydrogenase 2 (ALDH2) focused on drinking behavior or alcoholism because the ALDH2*2 allele protects against the risk of developing alcoholism. The mechanism provides that the ALDH2 gene's protective effect is also involved in dopamine metabolism. The interaction of the ALDH2 gene with neurotransmitters, such as dopamine, is suggested to be related to alcoholism. Because alcoholism is often co-morbid with antisocial personality disorder (ASPD), previous association studies on antisocial alcoholism cannot differentiate whether those genes relate to ASPD with alcoholism or ASPD only. This study examined the influence of the interaction effect of the ALDH2*1*1, *1*2 or *2*2 polymorphisms with the dopamine 2 receptor (DRD2) Taq I polymorphism on ASPD. Our 541 Han Chinese male participants were classified into three groups: antisocial alcoholism (ASPD co-morbid with alcohol dependence, antisocial ALC; n = 133), ASPD without alcoholism (ASPD not co-morbid with alcohol dependence, antisocial non-ALC; n = 164) and community controls (healthy volunteers from the community; n = 244). Compared with healthy controls, individuals with the DRD2 A1/A1 and the ALDH2*1/*1 genotypes were at a 5.39 times greater risk for antisocial non-ALC than were those with other genotypes. Our results suggest that the DRD2/ANKK1 and ALDH2 genes interacted in the antisocial non-ALC group; a connection neglected in previous studies caused by not separating antisocial ALC from ASPD. Our study made this distinction and showed that these two genes may be associated ASPD without co-morbid alcoholism. © 2010 The Authors, Addiction Biology © 2010 Society for the Study of Addiction.

  10. Genome-wide DNA methylation profiling identifies ALDH1A3 promoter methylation as a prognostic predictor in G-CIMP- primary glioblastoma.

    PubMed

    Zhang, Wei; Yan, Wei; You, Gan; Bao, Zhaoshi; Wang, Yongzhi; Liu, Yanwei; You, Yongping; Jiang, Tao

    2013-01-01

    To date, the aberrations in the DNA methylation patterns that are associated with different prognoses of G-CIMP- primary GBMs remain to be elucidated. Here, DNA methylation profiling of primary GBM tissues from 13 long-term survivors (LTS; overall survival ⩾18months) and 20 short-term survivors (STS; overall survival ⩽9months) was performed. Then G-CIMP+ samples were excluded. The differentially expressed CpG loci were identified between residual 18 STS and 9 LTS G-CIMP- samples. Methylation levels of 11 CpG loci (10genes) were statistically significantly lower, and 43 CpG loci (40genes) were statistically significantly higher in the tumor tissues of LTS than those of STS G-CIMP- samples (P<0.01). Of the 43 CpG loci that were hypermethylated in LTS G-CIMP- samples, 3 CpG loci localized in the promoter of ALDH1A3. Furthermore, using an independent validation cohort containing 37 primary GBM samples without IDH1 mutation and MGMT promoter methylation, the hypermethylation status of ALDH1A3 promoter predicted a better prognosis with an accompanied low expression of ALDH1A3 protein. Taken together, our results defined prognosis-related methylation signatures systematically for the first time in G-CIMP- primary GBMs. ALDH1A3 promoter methylation conferred a favorable prognosis in G-CIMP- primary GBMs. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  11. Galectin-9 Produced by Intestinal Epithelial Cells Enhances Aldehyde Dehydrogenase Activity in Dendritic Cells in a PI3K- and p38-Dependent Manner.

    PubMed

    de Kivit, Sander; Kostadinova, Atanaska I; Kerperien, JoAnn; Ayechu Muruzabal, Veronica; Morgan, Mary E; Knippels, Leon M J; Kraneveld, Aletta D; Garssen, Johan; Willemsen, Linette E M

    2017-01-01

    Intestinal epithelial cells (IEC) drive regulatory T cell (Treg) responses by promoting the differentiation of aldehyde dehydrogenase (ALDH)-expressing CD103+ dendritic cells (DC). Apical stimulation of TLR9 by CpG DNA on IEC supports galectin-9 expression by IEC, which is promoted by short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides (GF). While galectin-9 can induce the maturation of monocyte-derived DC (moDC), the contribution of galectin-9 on the induction of ALDH activity in DC is not known. To this end, DC were stimulated with galectin-9, and ALDH activity and the expression of CD103 were assessed. ALDH activity was increased by moDC exposed to galectin-9, while the expression of CD103 remained unaltered. Galectin-9 secreted by IEC apically exposed to CpG DNA and GF enhanced ALDH activity, but not CD103 expression by moDC, which was abrogated upon galectin-9 neutralization. Similar observations were found in murine GM-CSF-cultured bone marrow-derived DC (BMDC). Using Flt3L-cultured BMDC and ex vivo murine splenic DC, it was observed that galectin-9 only enhanced ALDH activity in the presence of GM-CSF in CD103- cells. The induction of ALDH activity in BMDC was dependent on p38 and PI3K signaling. These data indicate a novel role for galectin-9 in modulating innate immunity by inducing ALDH activity in DC. © 2017 S. Karger AG, Basel.

  12. Meta-Analyses of ALDH2 and ADH1B with Alcohol Dependence in Asians

    ERIC Educational Resources Information Center

    Luczak, Susan E.; Glatt, Stephen J.; Wall, Tamara J.

    2006-01-01

    Meta-analyses were conducted to determine the magnitude of relationships between polymorphisms in 2 genes, ALDH2 and ADH1B, with alcohol dependence in Asians. For each gene, possession of 1 variant [asterisk]2 allele was protective against alcohol dependence, and possession of a 2nd [asterisk]2 allele did not offer significant additional…

  13. Reduced adiponectin expression after high-fat diet is associated with selective up-regulation of ALDH1A1 and further retinoic acid receptor signaling in adipose tissue

    PubMed Central

    Landrier, Jean-Francois; Kasiri, Elnaz; Karkeni, Esma; Mihály, Johanna; Béke, Gabriella; Weiss, Kathrin; Lucas, Renata; Aydemir, Gamze; Salles, Jérome; Walrand, Stéphane; de Lera, Angel R.; Rühl, Ralph

    2017-01-01

    Adiponectin is an adipocyte-derived adipokine with potent antidiabetic, anti-inflammatory, and antiatherogenic activity. Long-term, high-fat diet results in gain of body weight, adiposity, further inflammatory-based cardiovascular diseases, and reduced adiponectin secretion. Vitamin A derivatives/retinoids are involved in several of these processes, which mainly take place in white adipose tissue (WAT). In this study, we examined adiponectin expression as a function of dietary high-fat and high–vitamin A conditions in mice. A decrease of adiponectin expression in addition to an up-regulation of aldehyde dehydrogenase A1 (ALDH1A1), retinoid signaling, and retinoic acid response element signaling was selectively observed in WAT of mice fed a normal–vitamin A, high-fat diet. Reduced adiponectin expression in WAT was also observed in mice fed a high–vitamin A diet. Adipocyte cell culture revealed that endogenous and synthetic retinoic acid receptor (RAR)α- and RARγ-selective agonists, as well as a synthetic retinoid X receptor agonist, efficiently reduced adiponectin expression, whereas ALDH1A1 expression only increased with RAR agonists. We conclude that reduced adiponectin expression under high-fat dietary conditions is dependent on 1) increased ALDH1A1 expression in adipocytes, which does not increase all-trans-retinoic acid levels; 2) further RAR ligand–induced, WAT-selective, increased retinoic acid response element–mediated signaling; and 3) RAR ligand–dependent reduction of adiponectin expression.—Landrier, J.-F., Kasiri, E., Karkeni, E., Mihály, J., Béke, G., Weiss, K., Lucas, R., Aydemir, G., Salles, J., Walrand, S., de Lera, A. R., Rühl, R. Reduced adiponectin expression after high-fat diet is associated with selective up-regulation of ALDH1A1 and further retinoic acid receptor signaling in adipose tissue. PMID:27729412

  14. A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

    PubMed Central

    Guo, Hou-Fu; Cho, Eun Jeong; Devkota, Ashwini K.; Chen, Yulong; Russell, William; Phillips, George N.; Yamauchi, Mitsuo; Dalby, Kevin; Kurie, Jonathan M.

    2017-01-01

    Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated previously using E coli– or insect-based expression systems was either insoluble or enzymatically unstable, and LH2 enzymatic activity assays have measured radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems. PMID:28216326

  15. Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes

    PubMed Central

    Covian, Raul; Chess, David; Balaban, Robert S.

    2012-01-01

    Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction media recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase where catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. PMID:22975200

  16. Continuous monitoring of enzymatic activity within native electrophoresis gels: application to mitochondrial oxidative phosphorylation complexes.

    PubMed

    Covian, Raul; Chess, David; Balaban, Robert S

    2012-12-01

    Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light-scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze the enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction medium recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high-resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase in which catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. Published by Elsevier Inc.

  17. 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.

  18. Respiration and enzymatic activities as indicators of stabilization of sewage sludge composting.

    PubMed

    Nikaeen, Mahnaz; Nafez, Amir Hossein; Bina, Bijan; Nabavi, BiBi Fatemeh; Hassanzadeh, Akbar

    2015-05-01

    The objective of this work was to study the evolution of physico-chemical and microbial parameters in the composting process of sewage sludge (SS) with pruning wastes (PW) in order to compare these parameters with respect to their applicability in the evaluation of organic matter (OM) stabilization. To evaluate the composting process and organic matter stability, different microbial activities were compared during composting of anaerobically digested SS with two volumetric ratios, 1:1 and 3:1 of PW:SS and two aeration techniques including aerated static piles (ASP) and turned windrows (TW). Dehydrogenase activity, fluorescein diacetate hydrolysis, and specific oxygen uptake rate (SOUR) were used as microbial activity indices. These indices were compared with traditional parameters, including temperature, pH, moisture content, organic matter, and C/N ratio. The results showed that the TW method and 3:1 (PW:SS) proportion was superior to the ASP method and 1:1 proportion, since the former accelerate the composting process by catalyzing the OM stabilization. Enzymatic activities and SOUR, which reflect microbial activity, correlated well with temperature fluctuations. Based on these results it appears that SOUR and the enzymatic activities are useful parameters to monitor the stabilization of SS compost. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. 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

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

    2017-02-17

    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.

  20. Yeasts from sub-Antarctic region: biodiversity, enzymatic activities and their potential as oleaginous microorganisms.

    PubMed

    Martinez, A; Cavello, I; Garmendia, G; Rufo, C; Cavalitto, S; Vero, S

    2016-09-01

    Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. Sixty-one yeasts strains were isolated from King George Island, Antarctica which were characterized physiologically and identified at the molecular level using the D1/D2 region of rDNA. Fifty-eight yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Rhodotorula, Guehomyces, Candida, Metschnikowia and Debaryomyces) were screened for extracellular amylolytic, proteolytic, esterasic, pectinolytic, inulolytic xylanolytic and cellulolytic activities at low and moderate temperatures. Esterase activity was the most common enzymatic activity expressed by the yeast isolates regardless the assay temperature and inulinase was the second most common enzymatic activity. No cellulolytic activity was detected. One yeast identified as Guehomyces pullulans (8E) showed significant activity across six of seven enzymes types tested. Twenty-eight yeast isolates were classified as oleaginous, being the isolate 8E the strain that accumulated the highest levels of saponifiable lipids (42 %).

  1. Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.

    PubMed

    Santini, Roberta; Vinci, Maria C; Pandolfi, Silvia; Penachioni, Junia Y; Montagnani, Valentina; Olivito, Biagio; Gattai, Riccardo; Pimpinelli, Nicola; Gerlini, Gianni; Borgognoni, Lorenzo; Stecca, Barbara

    2012-09-01

    The question of whether cancer stem/tumor-initiating cells (CSC/TIC) exist in human melanomas has arisen in the last few years. Here, we have used nonadherent spheres and the aldehyde dehydrogenase (ALDH) enzymatic activity to enrich for CSC/TIC in a collection of human melanomas obtained from a broad spectrum of sites and stages. We find that melanomaspheres display extensive in vitro self-renewal ability and sustain tumor growth in vivo, generating human melanoma xenografts that recapitulate the phenotypic composition of the parental tumor. Melanomaspheres express high levels of Hedgehog (HH) pathway components and of embryonic pluripotent stem cell factors SOX2, NANOG, OCT4, and KLF4. We show that human melanomas contain a subset of cells expressing high ALDH activity (ALDH(high)), which is endowed with higher self-renewal and tumorigenic abilities than the ALDH(low) population. A good correlation between the number of ALDH(high) cells and sphere formation efficiency was observed. Notably, both pharmacological inhibition of HH signaling by the SMOOTHENED (SMO) antagonist cyclopamine and GLI antagonist GANT61 and stable expression of shRNA targeting either SMO or GLI1 result in a significant decrease in melanoma stem cell self-renewal in vitro and a reduction in the number of ALDH(high) melanoma stem cells. Finally, we show that interference with the HH-GLI pathway through lentiviral-mediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDH(high) melanoma stem cells. In conclusion, our data indicate an essential role of the HH-GLI1 signaling in controlling self-renewal and tumor initiation of melanoma CSC/TIC. Targeting HH-GLI1 is thus predicted to reduce the melanoma stem cell compartment. Copyright © 2012 AlphaMed Press.

  2. In situ nanoplasmonic probing of enzymatic activity of monolayer-confined glucose oxidase on colloidal nanoparticles.

    PubMed

    He, Haili; Xu, Xiaolong; Wu, Haoxi; Zhai, Yujuan; Jin, Yongdong

    2013-05-07

    In situ probing protein-particle interactions and activities of proteins on colloidal nanoparticle (NP) surfaces is a long-standing key challenge in understanding the nanobio interfaces and virtually important for a variety of biological and biomedical applications. The interactions of NPs with proteins, for instance, are known to form NP bioconjugates or protein coronas; protein surface immobilization and molecular layer-by-layer deposition techniques are widely used, but a clear understanding of the confinement effect on protein activity by molecular coating, at the monolayer level, remains poorly understood. We explore here a novel approach, using colloidal plasmonic nanocomplexes coated with glucose oxidase (GOx) as self-sensing nanoprobes for in situ optical probing of surface-confined enzymatic activity, which is at least 1-2 orders of magnitude more sensitive than standard colorimetric assays for detecting GOx activity. We found that enzymatic activity of monolayer-confined GOx on colloidal NPs was significantly enhanced as compared with free GOx (also proved by conformational changes from circular dichroism studies), with a low apparent Michaelis-Menten constant Km of ~0.115 mM and high turnover kcat/Km of ~8394 M(-1)·s(-1); compared with the "anchored-type" suspending GOx, the outmost polyelectrolyte monolayer-protected "sandwiched-type" GOx exhibits significantly improved enzymatic activities toward higher temperatures and wider pH range. This finding is of fundamental important and instructive for safe use of such nanomaterials for bioapplications.

  3. Ship-borne measurements of microbial enzymatic activity: A rapid biochemical indicator for microbial water quality monitoring

    NASA Astrophysics Data System (ADS)

    Stadler, Philipp; Loken, Luke; Crawford, John; Schramm, Paul; Sorsa, Kirsti; Kuhn, Catherine; Savio, Domenico; Striegl, Rob; Butman, David; Stanley, Emily; Farnleitner, Andreas H.; Zessner, Matthias

    2017-04-01

    Contamination of aquatic ecosystems by human and animal wastes is a global concern for water quality. Disclosing fate and transport processes of fecal indicator organism (FIO) in large water bodies is a big challenge due to material intensive and time consuming methods used in microbiological water quality monitoring. In respect of utilization of large surface water resources there is a dearth of rapid microbiological methods that allow a near-real time health related water quality monitoring to be implemented into early warning systems. The detection of enzymatic activities has been proposed as a rapid surrogate for microbiological pollution monitoring of water and water resources (Cabral, 2010; Farnleitner et al., 2001, 2002). Methods such as the beta-D-Glucuronidase assay (GLUC), targeting FIO such as E. coli, were established. New automated enzymatic assays have been implemented during the last years into on-site monitoring stations, ranging from ground- to surface waters (Ryzinska-Paier et al., 2014; Stadler et al., 2017, 2016). While these automated enzymatic methods cannot completely replace assays for culture-based FIO enumeration, they yielded significant information on pollution events and temporal dynamics on a catchment specific basis, but were restricted to stationary measurements. For the first time we conducted ship-borne and automated measurements of enzymatic GLUC activity on large fresh water bodies, including the Columbia River, the Mississippi River and Lake Mendota. Not only are automated enzymatic assays technically feasible from a mobile vessel, but also can be used to localize point sources of potential microbial fecal contamination, such as tributaries or storm drainages. Spatial and temporal patterns of enzymatic activity were disclosed and the habitat specific correlation with microbiological standard assays for FIO determined due to reference samples. The integration of rapid and automated enzymatic assays into well-established systems

  4. 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.

  5. 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

  6. 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.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 ofmore » 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

  8. Ethanol- and acetaldehyde-induced cholinergic imbalance in the hippocampus of Aldh2-knockout mice does not affect nerve growth factor or brain-derived neurotrophic factor.

    PubMed

    Jamal, Mostofa; Ameno, Kiyoshi; Ruby, Mostofa; Miki, Takanori; Tanaka, Naoko; Nakamura, Yu; Kinoshita, Hiroshi

    2013-11-20

    Neurotrophins, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), play an important role in the maintenance of cholinergic-neuron function. The objective of this study was to investigate whether ethanol (EtOH)- and acetaldehyde (AcH)- induced cholinergic effects would cause neurotrophic alterations in the hippocampus of mice. We used Aldh2 knockout (Aldh2-KO) mice, a model of aldehyde dehydrogenase 2 (ALDH2)-deficiency in humans, to examine the effects of acute administration of EtOH and the role of AcH. Hippocampal slices were collected and the mRNA and protein levels of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), NGF and BDNF were analyzed 30 min after the i.p. administration of EtOH (0.5, 1.0, or 2.0 g/kg). We show that treatment with 2.0 g/kg of EtOH decreased ChAT mRNA and protein levels in Aldh2-KO mice but not in wild-type (WT) mice, which suggests a role for AcH in the mechanism of action of EtOH. The administration of 2.0 g/kg of EtOH increased AChE mRNA in both strains of mice. EtOH failed to change the levels of NGF or BDNF at any dose. Aldh2-KO mice exhibited a distinctly lower expression of ChAT and a higher expression of NGF both at mRNA and protein levels in the hippocampus compared with WT mice. Our observations suggest that administration of EtOH and elevated AcH can alter cholinergic markers in the hippocampus of mice, and this effect did not change the levels of NGF or BDNF. © 2013 Elsevier B.V. All rights reserved.

  9. Hypoglycemic drugs induce antioxidant aldehyde dehydrogenase activity and remain high in patients with glycemic control in type 2 diabetes.

    PubMed

    Picazo, Alejandra; Jiménez-Osorio, Angélica S; Zúñiga-Mejía, Porfirio; Pedraza-Chaverri, José; Monroy, Adriana; Rodríguez-Arellano, M Eunice; Barrera-Oviedo, Diana

    2017-04-05

    The antioxidant system results essential to control and prevent lipid peroxidation due to stress damage in type 2 diabetes. An example is aldehyde dehydrogenase (ALDH), an enzyme that is involved in the detoxification of aldehydes formed during lipid peroxidation. This study was conducted to evaluate ALDH activity and to determine their association with hypoglycemic treatment in type 2 diabetes patients. The study population consisted of 422 Mexican subjects: a control group and type 2 diabetes patients. Type 2 diabetes patients were re-classified as those with or without hypoglycemic treatment and those with or without glycemic control (according to glycated hemoglobin (HbA1c)). Clinical parameters, antioxidant enzyme activities (ALDH, superoxide dismutase (SOD), catalase and glutathione peroxidase) and oxidative markers (reactive oxygen species and thiobarbituric acid reactive substances (TBARS)) were evaluated. The activity of antioxidant enzymes and oxidative stress markers were higher in type 2 diabetes patients with hypoglycemic treatment and without glycemic control than control group. The activity of ALDH and SOD remained high in type 2 diabetes patients with moderate glycemic control while only ALDH's remained high in type 2 diabetes patients with tight glycemic control. Increased ALDH and SOD activities were associated with hypoglycemic therapy. TBARS levels were associated with glycemic control. The persistence of high ALDH and SOD activities in type 2 diabetes patients with glycemic control may be to avoid a significant damage due to the increase in reactive oxygen species and TBARS. It is possible that this new oxidative status prevented the development the classical complications of diabetes. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Blockade of Notch3 inhibits the stem-like property and is associated with ALDH1A1 and CD44 via autophagy in non-small lung cancer.

    PubMed

    Ma, Yuanyuan; Li, Mingzhen; Si, Jiahui; Xiong, Ying; Lu, Fangliang; Zhang, Jianzhi; Zhang, Liyi; Zhang, Panpan; Yang, Yue

    2016-06-01

    Acquired resistance to standard chemotherapy causes treatment failure in patients with local advanced and advanced non-small lung cancer (NSCLC). Cancer stem cells (CSCs) are a small subpopulation within cancer that is thought to be resistant to conventional chemotherapy. The Notch pathway is one of the most intensively studied for putative therapeutic targets of CSCs in solid tumors. In our study, suppression of Notch3 decreased colony and sphere formation of stem-like property in lung cancer cells. In addition, Notch3 expression was demonstrated to be upregulated in the patients with chemoresistance and related to poor prognosis of NSCLC patients. Our results also showed that CSC markers ALDH1A1 and CD44 were highly expressed in NSCLC patients with chemoresistance and these two markers were positively correlated with Notch3 expression in lung cancer specimens from TCGA database. Furthermore, the lung cancer cells with drug resistance were shown to be associated with activation of autophagy. All the data support a crucial role of Notch3 in the increase of stem-like property in NSCLC cells that might be associated with upregulation of ALDH1A1 and CD44 and activation of autophagy.

  11. Membrane Phospholipid Augments Cytochrome P4501a Enzymatic Activity by Modulating Structural Conformation during Detoxification of Xenobiotics

    PubMed Central

    Ghosh, Manik C.; Ray, Arun K.

    2013-01-01

    Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment. PMID:23469105

  12. Membrane phospholipid augments cytochrome P4501a enzymatic activity by modulating structural conformation during detoxification of xenobiotics.

    PubMed

    Ghosh, Manik C; Ray, Arun K

    2013-01-01

    Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

  13. Effect of wheat and Miscanthus straw biochars on soil enzymatic activity, ecotoxicity, and plant yield

    NASA Astrophysics Data System (ADS)

    Mierzwa-Hersztek, Monika; Gondek, Krzysztof; Klimkowicz-Pawlas, Agnieszka; Baran, Agnieszka

    2017-07-01

    The variety of technological conditions and raw materials from which biochar is produced is the reason why its soil application may have different effects on soil properties and plant growth. The aim of this study was to evaluate the effect of the addition of wheat straw and Miscanthus giganteus straw (5 t DM ha-1) and biochar obtained from this materials in doses of 2.25 and 5 t DM ha-1 on soil enzymatic activity, soil ecotoxicity, and plant yield (perennial grass mixture with red clover). The research was carried out under field conditions on soil with the granulometric composition of loamy sand. No significant effect of biochar amendment on soil enzymatic activity was observed. The biochar-amended soil was toxic to Vibrio fischeri and exhibited low toxicity to Heterocypris incongruens. Application of wheat straw biochar and M. giganteus straw biochar in a dose of 5 t DM ha-1 contributed to an increase in plant biomass production by 2 and 14%, respectively, compared to the soil with mineral fertilisation. Biochars had a more adverse effect on soil enzymatic activity and soil ecotoxicity to H. incongruens and V. fischeri than non-converted wheat straw and M. giganteus straw, but significantly increased the grass crop yield.

  14. Antioxidant and Cytoprotective Activities of Enzymatic Extracts from Rhizoid of Laminaria japonica

    PubMed Central

    Je, Jae-Young; Park, Soo Yeon; Ahn, Chang-Bum

    2017-01-01

    Rhizoid of Laminaria japonica was hydrolyzed with proteases and carbohydrases to obtain antioxidant materials. Oxygen radical absorbance capacity (ORAC) of the enzymatic extracts was evaluated and the Protamex extract (PE) exhibited the highest ORAC value. PE also potently scavenged 2,2-diphenyl-1-picrylhydrazyl radical, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic) acid cation radical, and hydrogen peroxide (H2O2) and had good reducing power. PE inhibited hydroxyl radical-induced DNA scission by measuring the conversion of supercoiled pBR322 plasmid DNA to the open circular form. The cytoprotective effect of PE against H2O2-induced hepatic cell damage was also investigated. PE showed a dose-dependent cytoprotective effect in cultured hepatocytes by inhibiting intracellular reactive oxygen species scavenging activity. In addition, PE up-regulated the expression of heme oxygenase-1, which is a cytoprotective enzyme, by activating translocation of nuclear factor-erythroid 2-related factor 2. Taken together, the enzymatic extract of rhizoid of L. japonica, particularly PE, may be useful for antioxidant additives. PMID:29333384

  15. Unraveling the Enzymatic Activity of Oxygenated Carbon Nanotubes and Their Application in the Treatment of Bacterial Infections.

    PubMed

    Wang, Huan; Li, Penghui; Yu, Dongqin; Zhang, Yan; Wang, Zhenzhen; Liu, Chaoqun; Qiu, Hao; Liu, Zhen; Ren, Jinsong; Qu, Xiaogang

    2018-05-17

    Carbon nanotubes (CNTs) and their derivatives have emerged as a series of efficient biocatalysts to mimic the function of natural enzymes in recent years. However, the unsatisfiable enzymatic efficiency usually limits their practical usage ranging from materials science to biotechnology. Here, for the first time, we present the synthesis of several oxygenated-group-enriched carbon nanotubes (o-CNTs) via a facile but green approach, as well as their usage as high-performance peroxidase mimics for biocatalytic reaction. Exhaustive characterizations of the enzymatic activity of o-CNTs have been provided by exploring the accurate effect of various oxygenated groups on their surface including carbonyl, carboxyl, and hydroxyl groups. Because of the "competitive inhibition" effect among all of these oxygenated groups, the catalytic efficiency of o-CNTs is significantly enhanced by weakening the presence of noncatalytic sites. Furthermore, the admirable enzymatic activity of these o-CNTs has been successfully applied in the treatment of bacterial infections, and the results of both in vitro and in vivo nanozyme-mediated bacterial clearance clearly demonstrate the feasibility of o-CNTs as robust peroxidase mimics to effectively decrease the bacterial viability under physiological conditions. We believe that the present study will not only facilitate the construction of novel efficient nanozymes by rationally adjusting the degree of the "competitive inhibition" effect, but also broaden the biological usage of o-CNT-based nanomaterials via their satisfactory enzymatic activity.

  16. The activity of salivary aldehyde dehydrogenase during the menstrual cycle and pregnancy.

    PubMed

    Giebułtowicz, Joanna; Wroczyński, Piotr; Kosiński, Przemysław; Pietrzak, Bronisława

    2013-03-01

    The aim of the present study was to describe the changes in the activity of ALDH3A1 in saliva in relation to the menstrual cycle and pregnancy. We also measured major salivary antioxidants, salivary peroxidase (SPO) activity and uric acid (UA) concentration. Fasting saliva samples were collected from 63 women with uncomplicated pregnancies and from 39 healthy women of reproductive age, but not pregnant. Saliva samples were also collected from 10 healthy women with regular menstrual cycles in the early follicular, the mid-cycle and the mid-luteal phase during one menstrual cycle. SPO and ALDH3A1 activity was determined fluorimetrically, whereas UA concentration photometrically. The ALDH3A1 did not vary significantly among phases of menstrual cycle. However, the enzyme activity decreased with the length of pregnancy and in the third trimester is significantly lower than that in the saliva of non-pregnant women. Lower concentration of UA and in the third trimester the activity of ALDH3A1 in saliva of pregnant women could be a risk factor of, e.g. oral pathologies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Changes in the amino acid profiles and free radical scavenging activities of Tenebrio molitor larvae following enzymatic hydrolysis.

    PubMed

    Tang, Yujiao; Debnath, Trishna; Choi, Eun-Ju; Kim, Young Wook; Ryu, Jung Pyo; Jang, Sejin; Chung, Sang Uk; Choi, Young-Jin; Kim, Eun-Kyung

    2018-01-01

    Tenebrio molitor (T. molitor) larvae provide food at low environmental cost and contribute positively to livelihoods. In this research, we compared the amino acids compositions and antioxidant activities of various extracts of T. molitor to enhance their quality as food. For the comparison, distilled water extracts, enzymatic hydrolysates, and condensed enzymatic hydrolysates of T. molitor larvae were prepared. Their amino acids (AAs) profiles and antioxidant activities, including ferric-reducing antioxidant power, oxygen radical absorption capacity, and DPPH, hydroxyl radical, and hydrogen peroxide radical scavenging properties assay were analyzed. DW extracts had the lowest AAs contents and antioxidant activity compared with enzymatic extracts. Condensed hydrolysates with a combination of alcalase and flavourzyme (C-A+F) exhibited the highest levels of total free AAs (11.1759 g/100 g). C-A+F produced higher total hydrolyzed AAs (32.5292 g/100 g) compared with the other groups. The C-A+F possessed the strongest antioxidant activity. Notably, the antioxidant activities of the hydrolysates and the total hydrolyzed AAs amount were correlated. Taken together, our findings showed that C-A+F was a promising technique for obtaining extracts of T. molitor larvae with antioxidant activity as potential nutritious functional food.

  18. 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.

  19. Ultra-high-pressure processing improves proteolysis and release of bioactive peptides with activation activities on alcohol metabolic enzymes in vitro from mushroom foot protein.

    PubMed

    Zhao, Rui-Jie; Huo, Chun-Yan; Qian, Yang; Ren, Di-Feng; Lu, Jun

    2017-09-15

    This study was to find an effective process to extract bioactive peptides from mushroom foot and determine their effects on activation of alcohol metabolic enzymes in vitro. The optimum extraction assisted by ultra-high-pressure processing of mushroom foot peptides was obtained with a pressure of 400MPa and a processing time of 10min. After ultrafiltration, peptides with molecular weight of 0-3kDa had the highest activity to activate alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) by 70.79% and 71.35%, respectively. Following dextran gel chromatography, two peaks (p-I and p-II) appeared and the activation activities on ADH and ALDH of p-I were 72.00% and 73.43%, both higher than p-II. Nine peptides were found in p-I as determined by LC-MS/MS, and two of them (IPLH and IPIVLL) were synthesized. IPLH activated ADH and ALDH by 42.7% and 29.2% respectively, which were higher than IPIVLL. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. 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.

  1. Controlling enzymatic activity by immobilization on graphene oxide

    NASA Astrophysics Data System (ADS)

    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.

  2. Protective effect of ALDH2 against cyclophosphamide-induced acute hepatotoxicity via attenuating oxidative stress and reactive aldehydes.

    PubMed

    Zhai, Xiaoxuan; Zhang, Zhenxiao; Liu, Wenwen; Liu, Baoshan; Zhang, Rui; Wang, Wenjun; Zheng, Wen; Xu, Feng; Wang, Jiali; Chen, Yuguo

    2018-04-30

    Cyclophosphamide (CY) is a widely used chemotherapeutic agent that is associated with severe side effects, such as hepatotoxicity and nephrotoxicity. However, the extent, mechanisms and potential prevention and treatment strategies of CY-induced acute hepatotoxicity and nephrotoxicity are largely unknown. In this study, we determined the existence and extent of CY-induced acute hepatotoxicity and nephrotoxicity, and demonstrated the effect of ALDH2 on CY-induced acute tissue toxicity and related mechanisms. Adult male C57BL/6J (wide-type, WT) and ALDH2 -/- (KO) mice were divided into four groups: WT, WT + CY, KO + CY and WT + CY + Alda-1. Biochemical analysis showed that plasma ALT was increased by 35.8% in KO + CY group and decreased by 21.1% in WT + CY + Alda-1 group compared to WT + CY group (P < 0.05, respectively). However, there was no significant difference among WT, WT + CY and KO + CY groups regarding plasma renal marker enzymes, including blood urea nitrogen (BUN), creatinine and cystatin C (CysC). Levels of reactive oxygen species (ROS) and toxic aldehydes (acrolein, 4-hydroxynonenol and malondialdehyde) were increased significantly in KO + CY group and decreased significantly in WT + CY + Alda-1 group compared to WT + CY group (P < 0.05, respectively). These findings demonstrate that CY could induce acute hepatotoxicity without nephrotoxicity, and ALDH2 plays a protective role in CY-induced acute hepatotoxicity. The underlying mechanisms are associated with attenuating oxidative stress and detoxifying reactive aldehydes. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. STAT3 as a promising chemoresistance biomarker associated with the CD44+/high/CD24-/low/ALDH+ BCSCs-like subset of the triple-negative breast cancer (TNBC) cell line.

    PubMed

    Moreira, Milene Pereira; da Conceição Braga, Letícia; Cassali, Geovanni Dantas; Silva, Luciana Maria

    2018-02-15

    The cancer stem cell (CSC) concept is currently employed to explain the mechanism of multidrug resistance that is implicated in the reduced efficacy of many chemotherapeutic agents, consequently leading to metastatic spread and disease relapse. We searched for potential predictive markers of doxorubicin (DOX) resistance in breast cancer stem cells (BCSCs) of the BT-549 human triple-negative breast cancer (TNBC) cell line classified as a claudin-low subtype. In this study, we show that BT-549 presents a BCSCs-like subset determined by a CD44 +/high /CD24 -/low /ALDH1 + phenotype. The CD44 +/high /CD24 -/low /ALDH + BCSCs-like subset presented the downregulation of a majority of the genes analyzed (64 genes), and only 3 genes were upregulated after DOX treatment. Among the upregulated genes, MAPK3, PRKCZ and STAT3, STAT3 presented a higher level of upregulation in the DOX-treated CD44 +/high /CD24 -/low /ALDH + BCSCs-like subset. The identification of biomarkers that predict antitumor responses is at the top of cancer research priorities. STAT3 was highlighted as a molecular signature in the CD44 +/high /CD24 -/low /ALDH1 + BCSCs-like subset obtained from the TNBC BT-549 cell line related to DOX resistance. A majority of the evaluated genes in the EGF pathway appear to be not associated with DOX resistance, as observed in the CD44 +/high /CD24 -/low /ALDH1 + BCSCs-like subset. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines.

    PubMed

    Hanavan, Paul D; Borges, Chad R; Katchman, Benjamin A; Faigel, Douglas O; Ho, Thai H; Ma, Chen-Ting; Sergienko, Eduard A; Meurice, Nathalie; Petit, Joachim L; Lake, Douglas F

    2015-07-30

    Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a "proof-of-principle" that enzymatic inhibition of QSOX1 may have clinical relevancy.

  6. Ethanol disrupts chondrification of the neurocranial cartilages in medaka embryos without affecting aldehyde dehydrogenase 1A2 (Aldh1A2) promoter methylation

    PubMed Central

    Hu, Yuhui; Willett, Kristine L.; Khan, Ikhlas A.; Scheffler, Brian E.; Dasmahapatra, Asok K.

    2009-01-01

    Medaka (Oryzias latipes) embryos at different developmental stages were exposed to ethanol for 48 h, then allowed to hatch. Teratogenic effects were evaluated in hatchlings after examining chondrocranial cartilage deformities. Ethanol disrupted cartilage development in medaka in a dose and developmental stage-specific manner. Compared to controls, the linear length of the neurocranium and other cartilages were reduced in ethanol-treated groups. Moreover, the chondrification in cartilages, specifically trabeculae and polar cartilages, were inhibited by ethanol. To understand the mechanism of ethanol teratogenesis, NAD+: NADH status during embryogenesis and the methylation pattern of Aldh1A2 promoter in whole embryos and adult tissues (brain, eye, heart and liver) were analyzed. Embryos 6 dpf had higher NAD+ than embryos 0 or 2 dpf. Ethanol (200 or 400 mM) was able to reduce NAD+ content in 2 and 6 dpf embryos. However, in both cases reductions were not significantly different from the controls. Moreover, no significant difference in either NADH content or in NAD+: NADH status of the ethanol-treated embryos, with regard to controls, was observed. The promoter of Aldh1A2 contains 31 CpG dinucleotides (-705 to +154, ATG = +1); none of which were methylated. Compared to controls, embryonic ethanol exposure (100 and 400 mM) was unable to alter Aldh1A2 promoter methylation in embryos or in the tissues of adults (breeding) developmentally exposed to ethanol (300 mM, 48 hpf). From these data we conclude that ethanol teratogenesis in medaka does not induce alteration in the methylation pattern of Aldh1A2 promoter, but does change cartilage development. PMID:19651241

  7. Co-transforming bar and CsALDH Genes Enhanced Resistance to Herbicide and Drought and Salt Stress in Transgenic Alfalfa (Medicago sativa L.)

    PubMed Central

    Duan, Zhen; Zhang, Daiyu; Zhang, Jianquan; Di, Hongyan; Wu, Fan; Hu, Xiaowen; Meng, Xuanchen; Luo, Kai; Zhang, Jiyu; Wang, Yanrong

    2015-01-01

    Drought and high salinity are two major abiotic factors that restrict the productivity of alfalfa. By application of the Agrobacterium-mediated transformation method, an oxidative responsive gene, CsALDH12A1, from the desert grass Cleistogenes songorica together with the bar gene associated with herbicide resistance, were co-transformed into alfalfa (Medicago sativa L.). From the all 90 transformants, 16 were positive as screened by spraying 1 mL L-1 10% Basta solution and molecularly diagnosis using PCR. Real-time PCR analysis indicated that drought and salt stress induced high CsALDH expression in the leaves of the transgenic plants. The CsALDH expression levels under drought (15 d) and salt stress (200 mM NaCl) were 6.11 and 6.87 times higher than in the control plants, respectively. In comparison to the WT plants, no abnormal phenotypes were observed among the transgenic plants, which showed significant enhancement of tolerance to 15 d of drought and 10 d of salinity treatment. Evaluation of the physiological and biochemical indices during drought and salt stress of the transgenic plants revealed relatively lower Na+ content and higher K+ content in the leaves relative to the WT plants, a reduction of toxic on effects and maintenance of osmotic adjustment. In addition, the transgenic plants could maintain a higher relative water content level, higher shoot biomass, fewer changes in the photosystem, decreased membrane injury, and a lower level of osmotic stress. These results indicate that the co-expression of the introduced bar and CsALDH genes enhanced the herbicide, drought and salt tolerance of alfalfa and therefore can potentially be used as a novel genetic resource for the future breeding programs to develop new cultivars. PMID:26734025

  8. Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins

    PubMed Central

    Dai, Meiling; Guo, Hongbo; Dortmans, Jos C. F. M.; Dekkers, Jojanneke; Nordholm, Johan; Daniels, Robert; van Kuppeveld, Frank J. M.; de Vries, Erik

    2016-01-01

    ABSTRACT Influenza A virus (IAV) attachment to and release from sialoside receptors is determined by the balance between hemagglutinin (HA) and neuraminidase (NA). The molecular determinants that mediate the specificity and activity of NA are still poorly understood. In this study, we aimed to design the optimal recombinant soluble NA protein to identify residues that affect NA enzymatic activity. To this end, recombinant soluble versions of four different NA proteins from H5N1 viruses were compared with their full-length counterparts. The soluble NA ectodomains were fused to three commonly used tetramerization domains. Our results indicate that the particular oligomerization domain used does not affect the Km value but may affect the specific enzymatic activity. This particularly holds true when the stalk domain is included and for NA ectodomains that display a low intrinsic ability to oligomerize. NA ectodomains extended with a Tetrabrachion domain, which forms a nearly parallel four-helix bundle, better mimicked the enzymatic properties of full-length proteins than when other coiled-coil tetramerization domains were used, which probably distort the stalk domain. Comparison of different NA proteins and mutagenic analysis of recombinant soluble versions thereof resulted in the identification of several residues that affected oligomerization of the NA head domain (position 95) and therefore the specific activity or sialic acid binding affinity (Km value; positions 252 and 347). This study demonstrates the potential of using recombinant soluble NA proteins to reveal determinants of NA assembly and enzymatic activity. IMPORTANCE The IAV HA and NA glycoproteins are important determinants of host tropism and pathogenicity. However, NA is relatively understudied compared to HA. Analysis of soluble versions of these glycoproteins is an attractive way to study their activities, as they are easily purified from cell culture media and applied in downstream assays. In the

  9. Enzymatic activity in the surface microlayer and subsurface water in the harbour channel

    NASA Astrophysics Data System (ADS)

    Perliński, Piotr; Mudryk, Zbigniew J.; Antonowicz, Józef

    2017-09-01

    Hydrolytic activity of eight extracellular enzymes was determined spectrofluorimetric method in the surface microlayer and subsurface water in the harbour channel in Ustka. The ranking order of the potential enzyme activity rates in the studied water layers was as follows: lipase > phosphatase > aminopeptidase > β-glucosidase > α-glucosidase > xylanase > cellulase > chitinase. The level of activity of all studied hydrolases was higher in the surface microlayer than subsurface water. No clear gradients in the level of enzymatic activity were determined along the horizontal profile of the studied channel. Activity of extracellular enzymes was strongly influenced by the season.

  10. 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

  11. Ovarian cancer risk, ALDH2 polymorphism and alcohol drinking: Asian data from the Ovarian Cancer Association Consortium.

    PubMed

    Ugai, Tomotaka; Kelemen, Linda E; Mizuno, Mika; Ong, Jue-Sheng; Webb, Penelope M; Chenevix-Trench, Georgia; Wicklund, Kristine G; Doherty, Jennifer Anne; Rossing, Mary Anne; Thompson, Pamela J; Wilkens, Lynne R; Carney, Michael E; Goodman, Marc T; Schildkraut, Joellen M; Berchuck, Andrew; Cramer, Daniel W; Terry, Kathryn L; Cai, Hui; Shu, Xiao-Ou; Gao, Yu-Tang; Xiang, Yong-Bing; Van Den Berg, David; Pike, Malcom C; Wu, Anna H; Pearce, Celeste Leigh; Matsuo, Keitaro

    2018-02-01

    The aldehyde dehydrogenase 2 (ALDH2) polymorphism rs671 (Glu504Lys) causes ALDH2 inactivation and adverse acetaldehyde exposure among Asians, but little is known of the association between alcohol consumption and rs671 and ovarian cancer (OvCa) in Asians. We conducted a pooled analysis of Asian ancestry participants in the Ovarian Cancer Association Consortium. We included seven case-control studies and one cohort study comprising 460 invasive OvCa cases, 37 borderline mucinous OvCa and 1274 controls of Asian descent with information on recent alcohol consumption. Pooled odds ratios (OR) with 95% confidence intervals (CI) for OvCa risk associated with alcohol consumption, rs671 and their interaction were estimated using logistic regression models adjusted for potential confounders. No significant association was observed for daily alcohol intake with invasive OvCa (OR comparing any consumption to none = 0.83; 95% CI = 0.58-1.18) or with individual histotypes. A significant decreased risk was seen for carriers of one or both Lys alleles of rs671 for invasive mucinous OvCa (OR = 0.44; 95% CI = 0.20-0.97) and for invasive and borderline mucinous tumors combined (OR = 0.48; 95% CI = 0.26-0.89). No significant interaction was observed between alcohol consumption and rs671 genotypes. In conclusion, self-reported alcohol consumption at the quantities estimated was not associated with OvCa risk among Asians. Because the rs671 Lys allele causes ALDH2 inactivation leading to increased acetaldehyde exposure, the observed inverse genetic association with mucinous ovarian cancer is inferred to mean that alcohol intake may be a risk factor for this histotype. This association will require replication in a larger sample. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  12. Structure and Activity of a New Low Molecular Weight Heparin Produced by Enzymatic Ultrafiltration

    PubMed Central

    FU, LI; ZHANG, FUMING; LI, GUOYUN; ONISHI, AKIHIRO; BHASKAR, UJJWAL; SUN, PEILONG; LINHARDT, ROBERT J.

    2014-01-01

    The standard process for preparing the low molecular weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield due to 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. PMID:24634007

  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. Characterization of polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 and relationship to the alcoholism in a Colombian population.

    PubMed

    Méndez, Claudia; Rey, Mauricio

    2015-12-30

    Identify and characterize polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 in a Colombian population residing in the city of Bogotá and determine its possible relationship to the alcoholism. ADH2, ADH3, ALDH2, and CYP2E1 genotypes a population of 148 individuals with non-problematic alcohol and 65 individuals with alcoholism were determined with TaqMan probes and PCR-RFLP. DNA was obtained from peripheral blood white cells. Significant difference was found in family history of alcoholism and use of other psychoactive substances to compare alcoholics with controls. When allelic frequencies for each category (gender) were considered, frequency of A2 allele carriers in ADH2 was found higher in male patients than controls. In women, the relative frequency for c1 allele in CYP2E1 was lower in controls than alcoholics. The ALDH2 locus is monomorphic. No significant differences in allele distributions of the loci examined to compare two populations were observed, however when stratifying the same trend was found that these differences tended to be significant. This study allows us to conclude the positive association between family history of alcoholism and alcoholism suggesting that there is a favourable hereditary predisposition. Since substance dependence requires interaction of multiple genes, the combination of genotypes ADH2 * 2, CYP2E1 * 1 combined with genotype homozygous ALDH2 * 1 found in this study could be leading to the population to a potential risk to alcoholism.

  15. Human Salivary Aldehyde Dehydrogenase: Purification, Kinetic Characterization and Effect of Ethanol, Hydrogen Peroxide and Sodium Dodecyl Sulfate on the Activity of the Enzyme.

    PubMed

    Alam, Md Fazle; Laskar, Amaj Ahmed; Choudhary, Hadi Hasan; Younus, Hina

    2016-09-01

    Human salivary aldehyde dehydrogenase (hsALDH) enzyme appears to be the first line of defense in the body against exogenous toxic aldehydes. However till date much work has not been done on this important member of the ALDH family. In this study, we have purified hsALDH to homogeneity by diethylaminoethyl-cellulose (DEAE-cellulose) ion-exchange chromatography in a single step. The molecular mass of the homodimeric enzyme was determined to be approximately 108 kDa. Four aromatic substrates; benzaldehyde, cinnamaldehyde, 2-naphthaldehyde and 6-methoxy-2-naphthaldehyde were used for determining the activity of pure hsALDH. K m values for these substrates were calculated to be 147.7, 5.31, 0.71 and 3.31 μM, respectively. The best substrates were found to be cinnamaldehyde and 2-naphthaldehyde since they exhibited high V max /K m values. 6-methoxy-2-naphthaldehyde substrate was used for further kinetic characterization of pure hsALDH. The pH and temperature optima of hsALDH were measured to be pH 8 and 45 °C, respectively. The pure enzyme is highly unstable at high temperatures. Ethanol, hydrogen peroxide and SDS activate hsALDH, therefore it is safe and beneficial to include them in mouthwashes and toothpastes in low concentrations.

  16. Preventive effects of Chlorella on skeletal muscle atrophy in muscle-specific mitochondrial aldehyde dehydrogenase 2 activity-deficient mice.

    PubMed

    Nakashima, Yuya; Ohsawa, Ikuroh; Nishimaki, Kiyomi; Kumamoto, Shoichiro; Maruyama, Isao; Suzuki, Yoshihiko; Ohta, Shigeo

    2014-10-11

    Oxidative stress is involved in age-related muscle atrophy, such as sarcopenia. Since Chlorella, a unicellular green alga, contains various antioxidant substances, we used a mouse model of enhanced oxidative stress to investigate whether Chlorella could prevent muscle atrophy. Aldehyde dehydrogenase 2 (ALDH2) is an anti-oxidative enzyme that detoxifies reactive aldehydes derived from lipid peroxides such as 4-hydroxy-2-nonenal (4-HNE). We therefore used transgenic mice expressing a dominant-negative form of ALDH2 (ALDH2*2 Tg mice) to selectively decrease ALDH2 activity in the muscles. To evaluate the effect of Chlorella, the mice were fed a Chlorella-supplemented diet (CSD) for 6 months. ALDH2*2 Tg mice exhibited small body size, muscle atrophy, decreased fat content, osteopenia, and kyphosis, accompanied by increased muscular 4-HNE levels. The CSD helped in recovery of body weight, enhanced oxidative stress, and increased levels of a muscle impairment marker, creatine phosphokinase (CPK) induced by ALDH2*2. Furthermore, histological and histochemical analyses revealed that the consumption of the CSD improved skeletal muscle atrophy and the activity of the mitochondrial cytochrome c oxidase. This study suggests that long-term consumption of Chlorella has the potential to prevent age-related muscle atrophy.

  17. High aldehyde dehydrogenase activity identifies cancer stem cells in human cervical cancer

    PubMed Central

    Liu, Shu-Yan; Zheng, Peng-Sheng

    2013-01-01

    High aldehyde dehydrogenase (ALDH) activity characterizes a subpopulation of cells with cancer stem cell (CSC) properties in several malignancies. To clarify whether ALDH can be used as a marker of cervical cancer stem cells (CCSCs), ALDHhigh and ALDHlow cells were sorted from 4 cervical cancer cell lines and 5 primary tumor xenografts and examined for CSC characteristics. Here, we demonstrate that cervical cancer cells with high ALDH activity fulfill the functional criteria for CSCs: (1) ALDHhigh cells, unlike ALDHlow cells, are highly tumorigenic in vivo; (2) ALDHhigh cells can give rise to both ALDHhigh and ALDHlow cells in vitro and in vivo, thereby establishing a cellular hierarchy; and (3) ALDHhigh cells have enhanced self-renewal and differentiation potentials. Additionally, ALDHhigh cervical cancer cells are more resistant to cisplatin treatment than ALDHlow cells. Finally, expression of the stem cell self-renewal-associated transcription factors OCT4, NANOG, KLF4 and BMI1 is elevated in ALDHhigh cervical cancer cells. Taken together, our data indicated that high ALDH activity may represent both a functional marker for CCSCs and a target for novel cervical cancer therapies. PMID:24318570

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

    NASA Astrophysics Data System (ADS)

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

    2017-04-01

    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.

  19. Lipoic acid protects gastric mucosa from ethanol-induced injury in rat through a mechanism involving aldehyde dehydrogenase 2 activation.

    PubMed

    Li, Jia-Hui; Ju, Gui-Xia; Jiang, Jun-Lin; Li, Nian-Sheng; Peng, Jun; Luo, Xiu-Ju

    2016-11-01

    Numerous studies demonstrate that reactive aldehydes are highly toxic and aldehyde dehydrogenase 2 (ALDH2)-mediated detoxification of reactive aldehydes is thought as an endogenous protective mechanism against reactive aldehydes-induced cell injury. This study aims to explore whether lipoic acid, a potential ALDH2 activator, is able to protect gastric mucosa from ethanol-induced injury through a mechanism involving clearance of reactive aldehydes. The rats received 60% of acidified ethanol through intragastric administration and held for 1 h to establish a mucosal injury model. Lipoic acid (10 or 30 mg/kg) or Alda-1 (a positive control, 10 mg/kg) was given 45 min before the ethanol treatment. The gastric tissues were collected for analysis of gastric ulcer index, cellular apoptosis, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) contents, and ALDH2 activity. The results showed that acute administration of ethanol led to an increase in gastric ulcer index, cellular apoptosis, 4-HNE and MDA contents concomitant with a decrease in ALDH2 activity; these phenomena were reversed by lipoic acid or Alda-1. The gastric protection of lipoic acid was attenuated in the presence of ALDH2 inhibitor. Based on these observations, we conclude that lipoic acid exerts the beneficial effects on ethanol-induced injury through a mechanism involving, at least in part, ALDH2 activation. As a dietary supplement or a medicine already in some countries, lipoic acid can be used to treat the ethanol - induced gastric mucosal injury. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Decomposition rate and enzymatic activity of composted municipal waste and poultry manure in the soil in a biofuel crops field.

    PubMed

    Cordovil, Cláudia Marques-Dos-Santos; de Varennes, Amarilis; Pinto, Renata Machado Dos Santos; Alves, Tiago Filipe; Mendes, Pedro; Sampaio, Sílvio César

    2017-05-01

    Biofuel crops are gaining importance because of the need to replace non-renewable sources. Also, due to the increasing amounts of wastes generated, there is the need to recycle them to the soil, both to fertilize crops and to improve soil physical properties through organic matter increase and microbiological changes in the rhizosphere. We therefore studied the influence of six biofuel crops (elephant grass, giant cane, sugarcane, blue gum, black cottonwood, willow) on the decomposition rate and enzymatic activity of composted municipal solid waste and poultry manure. Organic amendments were incubated in the field (litterbag method), buried near each plant or bare soil. Biomass decrease and dehydrogenase, urease and acid phosphatase level in amendments was monitored over a 180-day period. Soil under the litterbags was analysed for the same enzymatic activity and organic matter fractions (last sampling). After 365 days, a fractionation of organic matter was carried out in both amendments and soil under the litterbags. For compost, willow and sugarcane generally led to the greatest enzymatic activity, at the end of the experiment. For manure, dehydrogenase activity decreased sharply with time, the smallest value near sugarcane, while phosphatase and urease generally presented the highest values, at the beginning or after 90 days' incubation. Clustering showed that plant species could be grouped based on biomass and enzymes measured over time. Plant species influenced the decomposition rate and enzymatic activities of the organic amendments. Overall, mineralization of both amendments was associated with a greater urease activity in soils. Dehydrogenase activity in manure was closely associated with urease activity. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  2. 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. Copyright © 2013 American Dairy Science Association

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

    DOEpatents

    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.

  4. Effects of lead contamination on soil enzymatic activities, microbial biomass, and rice physiological indices in soil-lead-rice (Oryza sativa L.) system.

    PubMed

    Zeng, Lu S; Liao, Min; Chen, Cheng L; Huang, Chang Y

    2007-05-01

    The effect of lead (Pb) treatment on the soil enzymatic activities, soil microbial biomass, rice physiological indices and rice biomass were studied in a greenhouse pot experiment. Six levels of Pb viz. 0(CK), 100, 300, 500, 700, 900 mg/kg soil were applied in two types of paddy soils. The results showed that Pb treatment had a stimulating effect on soil enzymatic activities and microbial biomass carbon (Cmic) at low concentration and an inhibitory influence at higher concentration. The degree of influence on enzymatic activities and Cmic by Pb was related to the clay and organic matter contents of the soils. When the Pb treatment was raised to the level of 500 mg/kg, ecological risk appeared both to soil microorganisms and plants. The results also revealed a consistent trend of increased chlorophyll contents and rice biomass initially, maximum at a certain Pb treatment, and then decreased gradually with the increase in Pb concentration. Pb was effective in inducing proline accumulation and its toxicity causes oxidative stress in rice plants. Therefore, it was concluded that soil enzymatic activities, Cmic and rice physiological indices, could be sensitive indicators to reflect environmental stress in soil-lead-rice system.

  5. 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. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  6. Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry

    PubMed Central

    Idewaki, Yasuhiro; Iwase, Masanori; Fujii, Hiroki; Ohkuma, Toshiaki; Ide, Hitoshi; Kaizu, Shinako; Jodai, Tamaki; Kikuchi, Yohei; Hirano, Atsushi; Nakamura, Udai; Kubo, Michiaki; Kitazono, Takanari

    2015-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671) was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity) and drinking habits (lifetime abstainers vs. former or current drinkers) was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2). The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI)]: *1/*1 abstainers as the referent, 0.94 [0.76–1.16] in abstainers with *2, 1.00 [0.80–1.26] in *1/*1 drinkers, 0.71 [0.54–0.93] in drinkers with *2). Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28–6.13] in abstainers with *2, 1.89 [0.89–4.51] in *1/*1 drinkers, 2.35 [1.06–5.79] in drinkers with *2). In summary, patients with type 2 diabetes and ALDH2 *2

  7. Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry.

    PubMed

    Idewaki, Yasuhiro; Iwase, Masanori; Fujii, Hiroki; Ohkuma, Toshiaki; Ide, Hitoshi; Kaizu, Shinako; Jodai, Tamaki; Kikuchi, Yohei; Hirano, Atsushi; Nakamura, Udai; Kubo, Michiaki; Kitazono, Takanari

    2015-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671) was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity) and drinking habits (lifetime abstainers vs. former or current drinkers) was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2). The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI)]: *1/*1 abstainers as the referent, 0.94 [0.76-1.16] in abstainers with *2, 1.00 [0.80-1.26] in *1/*1 drinkers, 0.71 [0.54-0.93] in drinkers with *2). Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28-6.13] in abstainers with *2, 1.89 [0.89-4.51] in *1/*1 drinkers, 2.35 [1.06-5.79] in drinkers with *2). In summary, patients with type 2 diabetes and ALDH2 *2 displayed a

  8. Delta-Secretase Phosphorylation by SRPK2 Enhances Its Enzymatic Activity, Provoking Pathogenesis in Alzheimer's Disease.

    PubMed

    Wang, Zhi-Hao; Liu, Pai; Liu, Xia; Manfredsson, Fredric P; Sandoval, Ivette M; Yu, Shan Ping; Wang, Jian-Zhi; Ye, Keqiang

    2017-09-07

    Delta-secretase, a lysosomal asparagine endopeptidase (AEP), simultaneously cleaves both APP and tau, controlling the onset of pathogenesis of Alzheimer's disease (AD). However, how this protease is post-translationally regulated remains unclear. Here we report that serine-arginine protein kinase 2 (SRPK2) phosphorylates delta-secretase and enhances its enzymatic activity. SRPK2 phosphorylates serine 226 on delta-secretase and accelerates its autocatalytic cleavage, leading to its cytoplasmic translocation and escalated enzymatic activities. Delta-secretase is highly phosphorylated in human AD brains, tightly correlated with SRPK2 activity. Overexpression of a phosphorylation mimetic (S226D) in young 3xTg mice strongly promotes APP and tau fragmentation and facilitates amyloid plaque deposits and neurofibrillary tangle (NFT) formation, resulting in cognitive impairment. Conversely, viral injection of the non-phosphorylatable mutant (S226A) into 5XFAD mice decreases APP and tau proteolytic cleavage, attenuates AD pathologies, and reverses cognitive defects. Our findings support that delta-secretase phosphorylation by SRPK2 plays a critical role in aggravating AD pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. 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, v max decreased significantly (P < 0.05) and K M 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

  10. Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

    PubMed

    Kalb, Suzanne R; Baudys, Jakub; Smith, Theresa J; Smith, Leonard A; Barr, John R

    2014-04-01

    Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.

  11. An association between Schistosoma mansoni worms and an enzymatically-active protease/peptidase in mouse blood.

    PubMed

    Darani, H Y; Doenhoff, M J

    2008-04-01

    An enzyme found previously in extracts of adult Schistosoma mansoni worms, that hydrolysed the chromogenic substrate N-acetyl-DL-phenylalanine beta-naphthyl-ester, has here been further investigated and characterized. Evidence that the molecule found in the parasite was antigenically and enzymatically homologous with a constituent of normal mouse plasma has been consolidated using a monospecific serum in immunoelectrophoresis and Western immunoblotting. The molecular size of the enzyme was found to be approximately 70 kDa and it was inhibited by a serine protease inhibitor, but not by inhibitors of other classes of protease. The enzymatic activity found in normal mouse serum was also found in normal rat serum, but not in sera from several other mammalian species.

  12. Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C.

    PubMed

    Faustini, Massimo; Torre, Maria Luisa; Stacchezzini, Simona; Norberti, Roberta; Consiglio, Anna Lange; Porcelli, Franca; Conte, Ubaldo; Munari, Eleonora; Russo, Vincenzo; Vigo, Daniele

    2004-01-01

    The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.

  13. First description and evaluation of SNPs in the ADH and ALDH genes in a population of alcoholics in Central-West Brazil.

    PubMed

    Teixeira, Thallita Monteiro; da Silva, Hugo Delleon; Goveia, Rebeca Mota; Ribolla, Paulo Eduardo Martins; Alonso, Diego Peres; Alves, Alessandro Arruda; Melo E Silva, Daniela; Collevatti, Rosane Garcia; Bicudo, Lucilene Arilho; Bérgamo, Nádia Aparecida; de Paula Silveira-Lacerda, Elisângela

    2017-12-01

    Worldwide, different studies have reported an association of alcohol-use disorder (AUD) with different types of Single Nucleotide Polymorphisms (SNPs) in the genes for aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH). In Brazil, there is little information about the occurrence of these SNPs in the AUD population and an absence of studies characterizing the population in the Central-West Region of Brazil. Actually, in Brazil, there are more than 4 million people with AUD. Despite the major health hazards of AUD, information on alcohol consumption and its consequences are not well understood. Therefore, it is extremely important to characterize these SNPs for the better understanding of AUD as a genetic disease in the Brazilian population. The present study, unlike other studies in other countries, is done with a subject population that shows a significant amount of racial homogenization. We evaluated the presence of SNPs in the ADH (ADH1B, ADH1C, and ADH4) and ALDH (ALDH2) genes in alcohol users of Goiânia, State of Goiás - Brazil, and then we established a possible relationship with AUD by allelic and genotypic study. This study was conducted with a population of people with AUD (n = 99) from Goiás Alcohol Dependence Recovery Center (GO CEREA) and Psychosocial Care Center for Alcohol and Drugs (CAPS AD), and with a population of people without AUD as controls (n = 100). DNA was extracted from whole-blood samples and the genotyping was performed using TaqMan ® SNP genotyping assays. For characterization and evaluation of SNPs in the population, genotype frequency, allele frequency, haplotype frequency, Hardy-Weinberg equilibrium, and linkage disequilibrium were analyzed. Statistical analyses were calculated by GENEPOP 4.5 and Haploview software. The allele 1 was considered as "wild" (or *1) and allele 2 as mutant (or *2). Significant differences were found for ADH1B*, ADH4*2, and ALDH2*2 SNPs when the genotype and allele frequencies were

  14. Enzymatic digestive activity and absorption efficiency in Tagelus dombeii upon Alexandrium catenella exposure

    NASA Astrophysics Data System (ADS)

    Fernández-Reiriz, M. J.; Navarro, J. M.; Cisternas, B. A.; Babarro, J. M. F.; Labarta, U.

    2013-12-01

    We analyzed absorption efficiency (AE) and digestive enzyme activity (amylase, cellulase complex, and laminarinase) of the infaunal bivalve Tagelus dombeii originating from two geographic sites, Corral-Valdivia and Melinka-Aysén, which have different long-term paralytic shellfish poisoning (PSP) exposure rates. We report the effects of past feeding history (origin) on T. dombeii exposed to a mixed diet containing the toxic dinoflagellate Alexandrium catenella and another dinoflagellate-free control diet over a 12-day period in the laboratory. Absorption efficiency values of T. dombeii individuals that experienced PSP exposure in their habitat (Melinka-Aysén) remained unchanged during exposure to toxic food in the laboratory. In contrast, T. dombeii from a non-PSP exposure field site (Corral-Valdivia) showed a significant reduction in AE with toxic exposure time. This study established that the amylase and cellulase complexes were the most important enzymes in the digestive glands of Tagelus from both sites. The temporal evolution of enzymatic activity under toxic diet was fitted to exponential (amylase and cellulase) and to a logarithmic (laminarinase) models. In all fits, we found significant effect of origin in the model parameters. At the beginning of the experiment, higher enzymatic activity was observed for clams from Corral-Valdivia. The amylase activity decreased with time exposure for individuals from Corral and increased for individuals from Melinka. Cellulase activity did not vary over time for clams from Corral, but increased for individuals from Melinka and laminarinase activity decreased over time for individuals from Corral and remained unchanged over time for Melinka. A feeding history of exposure to the dinoflagellate A. catenella was reflected in the digestive responses of both T. dombeii populations.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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, suggestingmore » 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.« less

  16. 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.

  17. Enzymatic activities produced by mixed Saccharomyces and non-Saccharomyces cultures: relationship with wine volatile composition.

    PubMed

    Maturano, Yolanda Paola; Assof, Mariela; Fabani, María Paula; Nally, María Cristina; Jofré, Viviana; Rodríguez Assaf, Leticia Anahí; Toro, María Eugenia; Castellanos de Figueroa, Lucía Inés; Vazquez, Fabio

    2015-11-01

    During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as β-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. β-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of β-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.

  18. Sirtuin 1 Enzymatic Activity Is Required for Cartilage Homeostasis In Vivo in a Mouse Model

    PubMed Central

    Gabay, Odile; Sanchez, Christelle; Dvir-Ginzberg, Mona; Gagarina, Viktoria; Zaal, Kristien J.; Song, Yingjie; He, Xiao Hong; McBurney, Michael W.

    2014-01-01

    Objective We and others previously demonstrated that sirtuin 1 (SIRT-1) regulates apoptosis and cartilage-specific gene expression in human chondrocytes and mouse models. This study was undertaken to determine if SIRT-1 enzymatic activity plays a protective role in cartilage homeostasis in vivo, by investigating mice with SIRT-1 mutations to characterize their cartilage. Methods Articular cartilage was harvested from the paws and knees of 5- and 6-month-old wild-type (WT) mice and mice homozygous for SIRT-1tm2.1Mcby (SIRT-1y/y), an allele carrying a point mutation that encodes a SIRT-1 protein with no enzymatic activity (y/y mice). Mice ages 2 days old and 6–7 days old were also examined. Mouse joint cartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures. Results We found that articular cartilage tissue sections from y/y mice of up to 6 months of age contained reduced levels of type II collagen, aggrecan, and glycosaminoglycan compared to sections from WT mice. In contrast, protein levels of matrix metalloproteinase 8 (MMP-8), MMP-9, and MMP-13 were elevated in the cartilage of y/y mice. In addition, chondrocyte apoptosis was elevated in SIRT-1 mutant mice as compared to their WT littermates. Consistent with these observations, protein tyrosine phosphatase 1b was elevated in the y/y mice. Conclusion Our in vivo findings in this animal model demonstrate that mice with defective SIRT-1 also have defective cartilage, with elevated rates of cartilage degradation with age. Hence, normal cartilage homeostasis requires enzymatically active SIRT-1 protein. PMID:23124828

  19. Pharmacological activation of aldehyde dehydrogenase 2 promotes osteoblast differentiation via bone morphogenetic protein-2 and induces bone anabolic effect

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mittal, Monika; Pal, Subhashis; China, Shyamsundar

    Aldehyde dehydrogenases (ALDHs) are a family of enzymes involved in detoxifying aldehydes. Previously, we reported that an ALDH inhibitor, disulfiram caused bone loss in rats and among ALDHs, osteoblast expressed only ALDH2. Loss-of-function mutation in ALDH2 gene is reported to cause bone loss in humans which suggested its importance in skeletal homeostasis. We thus studied whether activating ALDH2 by N-(1, 3-benzodioxol-5-ylmethyl)-2, 6-dichlorobenzamide (alda-1) had osteogenic effect. We found that alda-1 increased and acetaldehyde decreased the differentiation of rat primary osteoblasts and expressions of ALDH2 and bone morphogenetic protein-2 (BMP-2). Silencing ALDH2 in osteoblasts abolished the alda-1 effects. Further, alda-1 attenuatedmore » the acetaldehyde-induced lipid-peroxidation and oxidative stress. BMP-2 is essential for bone regeneration and alda-1 increased its expression in osteoblasts. We then showed that alda-1 (40 mg/kg dose) augmented bone regeneration at the fracture site with concomitant increase in BMP-2 protein compared with control. The osteogenic dose (40 mg/kg) of alda-1 attained a bone marrow concentration that was stimulatory for osteoblast differentiation, suggesting that the tissue concentration of alda-1 matched its pharmacologic effect. In addition, alda-1 promoted modeling-directed bone growth and peak bone mass achievement, and increased bone mass in adult rats which reiterated its osteogenic effect. In osteopenic ovariectomized (OVX) rats, alda-1 reversed trabecular osteopenia with attendant increase in serum osteogenic marker (procollagen type I N-terminal peptide) and decrease in oxidative stress. Alda-1 has no effect on liver and kidney function. We conclude that activating ALDH2 by alda-1 had an osteoanabolic effect involving increased osteoblastic BMP-2 production and decreased OVX-induced oxidative stress. - Highlights: • Alda-1 induced osteoblast differentiation that involved upregulation of ALDH2 and BMP-2 • Alda

  20. Inhibition of Wnt/beta-catenin signaling downregulates expression of aldehyde dehydrogenase isoform 3A1 (ALDH3A1) to reduce resistance against temozolomide in glioblastoma in vitro.

    PubMed

    Suwala, Abigail Kora; Koch, Katharina; Rios, Dayana Herrera; Aretz, Philippe; Uhlmann, Constanze; Ogorek, Isabella; Felsberg, Jörg; Reifenberger, Guido; Köhrer, Karl; Deenen, René; Steiger, Hans-Jakob; Kahlert, Ulf D; Maciaczyk, Jaroslaw

    2018-04-27

    Glioblastoma is the most aggressive type of glioma. The Wingless (Wnt) signaling pathway has been shown to promote stem cell properties and resistance to radio- and chemotherapy in glioblastoma. Here, we demonstrate that pharmacological Wnt pathway inhibition using the porcupine inhibitor LGK974 acts synergistically with temozolomide (TMZ), the chemotherapeutic drug currently used as standard treatment for glioblastoma, to suppress in vitro growth of glioma cells. Synergistic growth inhibition was independent of the O 6 -alkylguanine DNA alkyltransferase ( MGMT ) promoter methylation status. Transcriptomic analysis revealed that expression of aldehyde dehydrogenase 3A1 ( ALDH3A1 ) was significantly down-regulated when cells were treated with LGK974 and TMZ. Suppressing ALDH3A1 expression increased the efficacy of TMZ and reduced clonogenic potential accompanied by decreased expression of stem cell markers CD133, Nestin and Sox2. Taken together, our study suggests that previous observations concerning Wnt signaling blockade to reduce chemoresistance in glioblastoma is at least in part mediated by inhibition of ALDH3A1.

  1. Effects of molecular weight of hyaluronic acid on its viscosity and enzymatic activities of lysozyme and peroxidase.

    PubMed

    Kim, Jihoon; Chang, Ji-Youn; Kim, Yoon-Young; Kim, Moon-Jong; Kho, Hong-Seop

    2018-05-01

    To investigate the effects of the molecular weight of hyaluronic acid on its viscosity and enzymatic activities of lysozyme and peroxidase in solution and on the hydroxyapatite surface. Hyaluronic acids of four different molecular weights (10 kDa, 100 kDa, 1 MDa, and 2 MDa), hen egg-white lysozyme, bovine lactoperoxidase, and human whole saliva were used. Viscosity values of hyaluronic acids were measured using a cone-and-plate viscometer at six different concentrations (0.1-5.0 mg/mL). Enzymatic activities of lysozyme and peroxidase were examined by hydrolysis of fluorescein-labeled Micrococcus lysodeikticus and oxidation of fluorogenic 2',7'-dichlorofluorescein to fluorescing 2',7'-dichlorofluorescein, respectively. In solution assays, only 2 MDa-hyaluronic acid significantly inhibited lysozyme activities in saliva. In surface assays, hyaluronic acids inhibited lysozyme and peroxidase activities; the inhibitory activities were more apparent with high-molecular-weight ones in saliva than in purified enzymes. The 100 kDa-hyaluronic acid at 5.0 mg/mL, 1 MDa-one at 0.5 mg/mL, and 2 MDa-one at 0.2 mg/mL showed viscosity values similar to those of human whole saliva at a shear rate range required for normal oral functions. The differences among the influences of the three conditions on the enzymatic activities were not statistically significant. High-molecular-weight hyaluronic acids at low concentration and low-molecular-weight ones at high concentration showed viscosity values similar to those of human whole saliva. Inhibitory effects of hyaluronic acids on lysozyme and peroxidase activities were more significant with high-molecular-weight ones on the surface and in saliva compared with in solution and on purified enzymes. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. CWP232228 targets liver cancer stem cells through Wnt/β-catenin signaling: a novel therapeutic approach for liver cancer treatment.

    PubMed

    Kim, Ji-Young; Lee, Hwa-Yong; Park, Kwan-Kyu; Choi, Yang-Kyu; Nam, Jeong-Seok; Hong, In-Sun

    2016-04-12

    Liver cancer stem cells (CSCs) are resistant to conventional chemotherapy and radiation, which may destroy tumor masses, but not all liver CSCs contribute to tumor initiation, metastasis, and relapse. In the present study, we showed that liver CSCs with elevated Wnt/β-catenin signaling possess much greater self-renewal and clonogenic potential. We further documented that the increased clonogenic potential of liver CSCs is highly associated with changes in Wnt/β-catenin signaling and that Wnt/β-catenin signaling activity is positively correlated with CD133 expression and aldehyde dehydrogenase (ALDH) enzymatic activity. Notably, the small molecule inhibitor CWP232228, which antagonizes the binding of β-catenin to TCF in the nucleus, inhibits Wnt/β-catenin signaling and depletes CD133+/ALDH+ liver CSCs, thus ultimately diminishing the self-renewal capacity of CSCs and decreasing tumorigenicity in vitro and in vivo. Taken together, our findings suggest that CWP232228 acts as a candidate therapeutic agent for liver cancer by preferentially targeting liver CSCs.

  3. Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

    PubMed

    Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

    2013-04-10

    Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. 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.

  5. Trends in gastrectomy and ADH1B and ALDH2 genotypes in Japanese alcoholic men and their gene-gastrectomy, gene-gene and gene-age interactions for risk of alcoholism.

    PubMed

    Yokoyama, Akira; Yokoyama, Tetsuji; Matsui, Toshifumi; Mizukami, Takeshi; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

    2013-01-01

    The life-time drinking profiles of Japanese alcoholics have shown that gastrectomy increases susceptibility to alcoholism. We investigated the trends in gastrectomy and alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) genotypes and their interactions in alcoholics. This survey was conducted on 4879 Japanese alcoholic men 40 years of age or older who underwent routine gastrointestinal endoscopic screening during the period 1996-2010. ADH1B/ALDH2 genotyping was performed in 3702 patients. A history of gastrectomy was found in 508 (10.4%) patients. The reason for the gastrectomy was peptic ulcer in 317 patients and gastric cancer in 187 patients. The frequency of gastrectomy had gradually decreased from 13.3% in 1996-2000 to 10.5% in 2001-2005 and to 7.8% in 2006-2010 (P < 0.0001). ADH1B*1/*1 was less frequent in the gastrectomy group than in the non-gastrectomy group (age-adjusted prevalence: 20.4 vs. 27.6%, P = 0.006). ALDH2 genotype distribution did not differ between the two groups. The frequency of inactive ALDH2*1/*2 heterozygotes increased slightly from 13.0% in 1996-2000 to 14.0% in 2001-2005 and to 15.4% in 2006-2010 (P < 0.08). Two alcoholism-susceptibility genotypes, ADH1B*1/*1 and ALDH2*1/*1, modestly but significantly tended not to occur in the same individual (P = 0.026). The frequency of ADH1B*1/*1 decreased with ascending age groups. The high frequency of history of gastrectomy suggested that gastrectomy is still a risk factor for alcoholism, although the percentage decreased during the period. The alcoholism-susceptibility genotype ADH1B*1/*1 was less frequent in the gastrectomy group, suggesting a competitive gene-gastrectomy interaction for alcoholism. A gene-gene interaction and gene-age interactions regarding the ADH1B genotype were observed.

  6. Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

    NASA Astrophysics Data System (ADS)

    Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing

    2014-03-01

    Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

  7. Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes, and correlation with antifungal activity.

    PubMed

    Park, Sang-Wook; Stevens, Noah M; Vivanco, Jorge M

    2002-12-01

    Ribosome-inactivating proteins (RIPs) are enzymes that cleave a specific adenine base from the highly conserved sarcin/ricin (S/R) loop of the large ribosomal RNA, thus arresting protein synthesis at the translocation step. In the present study, we employed three RIPs to dissect the antifungal activity of RIPs as plant defense proteins. We measured the catalytic activity of RAT (the catalytic A-chain of ricin from Ricinus communis L.), saporin-S6 (from Saponaria officinalis L.), and ME (RIP from Mirabilis expansa R&P) against intact ribosomal substrates isolated from various pathogenic fungi. We further determined the enzymatic specificity of these three RIPs against fungal ribosomes, from Rhizoctonia solani Kuhn, Alternaria solani Sorauer, Trichoderma reesei Simmons and Candida albicans Berkhout, and correlated the data with antifungal activity. RAT showed the strongest toxicity against all tested fungal ribosomes, except for the ribosomes isolated from C. albicans, which were most susceptible to saporin. RAT and saporin showed higher enzymatic activity than ME against ribosomes from all of the fungal species assayed, but did not show detectable antifungal activity. In contrast, ME showed substantial inhibitory activity against fungal growth. Using N-hydroxysuccinimide-fluorescein labeling of RIPs and fluorescence microscopy, we determined that ME was targeted to the surface of fungal cells and transferred into the cells. Thus, ME caused ribosome depurination and subsequent fungal mortality. In contrast, saporin did not interact with fungal cells, correlating with its lack of antifungal activity.

  8. 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.

  9. Impact of potato cultivation and cattle farming on physicochemical parameters and enzymatic activities of Neotropical high Andean Páramo ecosystem soils.

    PubMed

    Avellaneda-Torres, Lizeth Manuela; León Sicard, Tomás Enrique; Torres Rojas, Esperanza

    2018-08-01

    The Andean Páramos are high mountain ecosystems whose soils are essential for the management of South American water resources, but research on anthropic impacts to these soils is currently minimal and insufficient. The objective of this study was to evaluate the impacts of potato (Solanum tuberosum) cultivation and livestock on the physicochemical parameters and enzymatic activities that determine the soil quality of the Neotropical high Andean Páramo ecosystem in the Nevados National Natural Park (Nevados NNP) in Colombia. It was hypothesised that sites with potato crops and livestock farming would exhibit significant changes in soil physicochemical parameters and enzymatic activities compared with Páramo sites that have been conserved without agriculture. Samples were collected from soils under potato cultivation, livestock and Páramo (subject to the lowest degree of human intervention possible), on three farms in the El Bosque District at three different altitudes (Buenos Aires, El Edén and La Secreta) during two seasons (dry and rainy). The results showed that none of the physical parameters under study presented statistically significant differences due to the type of use (livestock, potato crop or Páramo), season of sampling (dry or rainy season) or altitude (different farms). The chemical parameters that statistically significantly differed due to land use were organic carbon, cation exchange capacity, calcium, potassium, and ammonium and those that showed statistically significant differences associated with the sampling timing were organic carbon, nitrogen, cation exchange capacity, total carbon, C/N and nitrate. Additionally, there were differences in organic carbon due to the altitude of the farms. With respect to enzymatic activities, those of β-glucosidase, phosphodiesterase and urease significantly decreased in soils under potato cultivation and livestock relative to those of Páramo, but those of acid phosphatase and protease increased

  10. 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.

  11. In vivo antitumor activity of 4-amino 4-methyl 2-pentyne 1-al, an inhibitor of aldehyde dehydrogenase.

    PubMed

    Quemener, V; Quash, G; Moulinoux, J P; Penlap, V; Ripoll, H; Havouis, R; Doutheau, A; Goré, J

    1989-01-01

    4-amino-4-methyl-2-pentyne-1-al (AMPAL), a new irreversible inhibitor of aldehyde dehydrogenase (ALDH) has been assayed for its in vitro and in vivo antitumor activity. In vitro, AMPAL inhibits the proliferation and the ALDH activity of L1210 and RBL5 cell lines. In vivo, AMPAL significantly increases the mean survival time of mice i.p. grafted with leukemia (L1210, P815, MBL2, EL4, RBL5 cell lines) or carcinoma cells (Krebs cell line), without haematopoetic toxicity. No carcinostatic effect was observed against the P388 leukemia and the 3LL Lewis lung carcinoma. A possible relationship between the ALDH isoenzyme activity of the tumor and its sensitivity to AMPAL is discussed in the light of previous reports concerning the role of aldehydes in cell growth control.

  12. Identification of rs671, a common variant of ALDH2, as a gout susceptibility locus.

    PubMed

    Sakiyama, Masayuki; Matsuo, Hirotaka; Nakaoka, Hirofumi; Yamamoto, Ken; Nakayama, Akiyoshi; Nakamura, Takahiro; Kawai, Sayo; Okada, Rieko; Ooyama, Hiroshi; Shimizu, Toru; Shinomiya, Nariyoshi

    2016-05-16

    Gout is a common disease resulting from hyperuricemia. Recently, a genome-wide association study identified an association between gout and a single nucleotide polymorphism (SNP) rs2188380, located on an intergenic region between MYL2 and CUX2 on chromosome 12. However, other genes around rs2188380 could possibly be gout susceptibility genes. Therefore, we performed a fine-mapping study of the MYL2-CUX2 region. From 8,595 SNPs in the MYL2-CUX2 region, 9 tag SNPs were selected, and genotyping of 1,048 male gout patients and 1,334 male controls was performed by TaqMan method. Eight SNPs showed significant associations with gout after Bonferroni correction. rs671 (Glu504Lys) of ALDH2 had the most significant association with gout (P = 1.7 × 10(-18), odds ratio = 0.53). After adjustment for rs671, the other 8 SNPs no longer showed a significant association with gout, while the significant association of rs671 remained. rs671 has been reportedly associated with alcohol drinking behavior, and it is well-known that alcohol drinking elevates serum uric acid levels. These data suggest that rs671, a common functional SNP of ALDH2, is a genuine gout-associated SNP in the MYL2-CUX2 locus and that "A" allele (Lys) of rs671 plays a protective role in the development of gout.

  13. Enzymatic vegetable organic extracts as soil biochemical biostimulants and atrazine extenders.

    PubMed

    García-Martínez, Ana María; Tejada, Manuel; Díaz, Ana Isabel; Rodríguez-Morgado, Bruno; Bautista, Juan; Parrado, Juan

    2010-09-08

    The purpose of this study was to gather information on the potential effects of organic biostimulants on soil activity and atrazine biodegradation. Carob germ enzymatic extract (CGEE) and wheat condensed distiller solubles enzymatic extract (WCDS-EE) have been obtained using an enzymatic process; their main organic components are soluble carbohydrates and proteins in the form of peptides and free amino acids. Their application to soil results in high biostimulation, rapidly increased dehydrogenase, phosphatase and glucosidase activities, and an observed atrazine extender capacity due to inhibition of its mineralization. The extender capacity of both extracts is proportional to the protein/carbohydrate ratio content. As a result, these enzymatic extracts are highly microbially available, leading to two independent phenomena, fertility and an atrazine persistence that is linked to increased soil activity.

  14. Production of 3-hydroxypropionic acid by balancing the pathway enzymes using synthetic cassette architecture.

    PubMed

    Sankaranarayanan, Mugesh; Somasundar, Ashok; Seol, Eunhee; Chauhan, Ashish Singh; Kwon, Seongjin; Jung, Gyoo Yeol; Park, Sunghoon

    2017-10-10

    Biological 3-hydroxypropionic acid (3-HP) production from glycerol is a two-step reaction catalyzed by glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH). Recombinant strains developed for 3-HP production often suffer from the accumulation of a toxic intermediate, 3-hydroxypropionaldehyde (3-HPA). In order to avoid 3-HPA accumulation, balancing of the two enzymatic activities, in the present study, was attempted by employment of synthetic-regulatory cassettes comprising varying-strength promoters and bicistronic ribosome-binding sites (RBSs). When tested in recombinant Escherichia coli, the cassettes could precisely and differentially control the gene expression in transcription, protein expression and enzymatic activity. Five recombinant strains showing different expressions for GDHt were developed and studied for 3-HPA accumulation and 3-HP production. It was found that 3-HPA accumulation could be completely abolished when expressing ALDH at a level approximately 8-fold higher than that of GDHt. One of the strains, SP4, produced 625mM (56.4g/L) of 3-HP in a fed-batch bioreactor, though late-period production was limited by acetate accumulation. Overall, this study demonstrated the importance of pathway balancing in 3-HP production as well as the utility of the synthetic cassette architecture for precise control of bacterial gene expression. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Antioxidative activities of hydrolysates from edible birds nest using enzymatic hydrolysis

    NASA Astrophysics Data System (ADS)

    Muhammad, Nurul Nadia; Babji, Abdul Salam; Ayub, Mohd Khan

    2015-09-01

    Edible bird's nest protein hydrolysates (EBN) were prepared via enzymatic hydrolysis to investigate its antioxidant activity. Two types of enzyme (alcalase and papain) were used in this study and EBN had been hydrolysed with different hydrolysis time (30, 60, 90 and 120 min). Antioxidant activities in EBN protein hydrolysate were measured using DPPH, ABTS+ and Reducing Power Assay. From this study, increased hydrolysis time from 30 min to 120 min contributed to higher DH, as shown by alcalase (40.59%) and papain (24.94%). For antioxidant assay, EBN hydrolysed with papain showed higher scavenging activity and reducing power ability compared to alcalase. The highest antioxidant activity for papain was at 120 min hydrolysis time with ABTS (54.245%), DPPH (49.78%) and Reducing Power (0.0680). Meanwhile for alcalase, the highest antioxidant activity was at 30 min hydrolysis time. Even though scavenging activity for EBN protein hydrolysates were high, the reducing power ability was quite low as compared to BHT and ascorbic Acid. This study showed that EBN protein hydrolysate with alcalase and papain treatments potentially exhibit high antioxidant activity which have not been reported before.

  16. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Solid-State Treatment of Castor Cake Employing the Enzymatic Cocktail Produced from Pleurotus djamor Fungi.

    PubMed

    Sánchez-Cantú, Manuel; Ortiz-Moreno, Liliana; Ramos-Cassellis, María E; Marín-Castro, Marco; De la Cerna-Hernández, C

    2018-06-01

    In this work, the enzymatic cocktail produced by Pleurotus djamor fungi extracted at pH of 4.8 and 5.3 was employed for castor cake solid-state treatment. Proximal, X-ray powder diffraction and scanning electron microscopy analysis of the pristine castor cake were carried out. First, Pleurotus djamor stain was inoculated in castor cake for the enzymatic production and the enzymatic activity was determined. The maximum enzymatic activity was identified at days 14 (65.9 UI/gss) and 11 (140.3 UI/gss) for the enzymatic cocktail obtained at pH 5.3 and 4.8, respectively. Then, the enzymatic cocktail obtained at the highest enzymatic activity days was employed directly over castor cake. Lignin was degraded throughout incubation time achieving a 47 and 45% decrease for the cocktail produced at pH 4.8 and 5.3, correspondingly. These results were corroborated by the SEM and XRD analysis where a higher porosity and xylan degradation were perceived throughout the enzymatic treatment.

  18. Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver.

    PubMed

    Tran, Steven; Nowicki, Magda; Chatterjee, Diptendu; Gerlai, Robert

    2015-01-02

    Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. 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

  20. A redundant role of human thyroid peroxidase propeptide for cellular, enzymatic, and immunological activity.

    PubMed

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

    2014-02-01

    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. 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. 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 simulations were consistent with

  1. Mapping of Functional Domains of the Lipid Kinase Phosphatidylinositol 4-Kinase Type III Alpha Involved in Enzymatic Activity and Hepatitis C Virus Replication

    PubMed Central

    Harak, Christian; Radujkovic, Danijela; Taveneau, Cyntia; Reiss, Simon; Klein, Rahel; Bressanelli, Stéphane

    2014-01-01

    ABSTRACT The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an endoplasmic reticulum (ER)-resident enzyme that synthesizes phosphatidylinositol 4-phosphate (PI4P). PI4KIIIα is an essential host factor for hepatitis C virus (HCV) replication. Interaction with HCV nonstructural protein 5A (NS5A) leads to kinase activation and accumulation of PI4P at intracellular membranes. In this study, we investigated the structural requirements of PI4KIIIα in HCV replication and enzymatic activity. Therefore, we analyzed PI4KIIIα mutants for subcellular localization, reconstitution of HCV replication in PI4KIIIα knockdown cell lines, PI4P induction in HCV-positive cells, and lipid kinase activity in vitro. All mutants still interacted with NS5A and localized in a manner similar to that of the full-length enzyme, suggesting multiple regions of PI4KIIIα are involved in NS5A interaction and subcellular localization. Interestingly, the N-terminal 1,152 amino acids were dispensable for HCV replication, PI4P induction, and enzymatic function, whereas further N-terminal or C-terminal deletions were deleterious, thereby defining the minimal PI4KIIIα core enzyme at a size of ca. 108 kDa. Additional deletion of predicted functional motifs within the C-terminal half of PI4KIIIα also were detrimental for enzymatic activity and for the ability of PI4KIIIα to rescue HCV replication, with the exception of a proposed nuclear localization signal, suggesting that the entire C-terminal half of PI4KIIIα is involved in the formation of a minimal enzymatic core. This view was supported by structural modeling of the PI4KIIIα C terminus, suggesting a catalytic center formed by an N- and C-terminal lobe and an armadillo-fold motif, which is preceded by three distinct alpha-helical domains probably involved in regulation of enzymatic activity. IMPORTANCE The lipid kinase PI4KIIIα is of central importance for cellular phosphatidylinositol metabolism and is a key host cell

  2. Aldehyde dehydrogenase 3A1 activation prevents radiation-induced xerostomia by protecting salivary stem cells from toxic aldehydes

    PubMed Central

    Saiki, Julie P.; Cao, Hongbin; Van Wassenhove, Lauren D.; Viswanathan, Vignesh; Bloomstein, Joshua; Nambiar, Dhanya K.; Mattingly, Aaron J.; Jiang, Dadi; Chen, Che-Hong; Simmons, Amanda L.; Park, Hyun Shin; von Eyben, Rie; Kool, Eric T.; Sirjani, Davud; Knox, Sarah M.; Le, Quynh Thu; Mochly-Rosen, Daria

    2018-01-01

    Xerostomia (dry mouth) is the most common side effect of radiation therapy in patients with head and neck cancer and causes difficulty speaking and swallowing. Since aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in mouse salivary stem/progenitor cells (SSPCs), we sought to determine the role of ALDH3A1 in SSPCs using genetic loss-of-function and pharmacologic gain-of-function studies. Using DarkZone dye to measure intracellular aldehydes, we observed higher aldehyde accumulation in irradiated Aldh3a1−/− adult murine salisphere cells and in situ in whole murine embryonic salivary glands enriched in SSPCs compared with wild-type glands. To identify a safe ALDH3A1 activator for potential clinical testing, we screened a traditional Chinese medicine library and isolated d-limonene, commonly used as a food-flavoring agent, as a single constituent activator. ALDH3A1 activation by d-limonene significantly reduced aldehyde accumulation in SSPCs and whole embryonic glands, increased sphere-forming ability, decreased apoptosis, and improved submandibular gland structure and function in vivo after radiation. A phase 0 study in patients with salivary gland tumors showed effective delivery of d-limonene into human salivary glands following daily oral dosing. Given its safety and bioavailability, d-limonene may be a good clinical candidate for mitigating xerostomia in patients with head and neck cancer receiving radiation therapy. PMID:29794221

  3. 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.

  4. Exploring the clonal evolution of CD133/aldehyde-dehydrogenase-1 (ALDH1)-positive cancer stem-like cells from primary to recurrent high-grade serous ovarian cancer (HGSOC). A study of the Ovarian Cancer Therapy-Innovative Models Prolong Survival (OCTIPS) Consortium.

    PubMed

    Ruscito, Ilary; Cacsire Castillo-Tong, Dan; Vergote, Ignace; Ignat, Iulia; Stanske, Mandy; Vanderstichele, Adriaan; Ganapathi, Ram N; Glajzer, Jacek; Kulbe, Hagen; Trillsch, Fabian; Mustea, Alexander; Kreuzinger, Caroline; Benedetti Panici, Pierluigi; Gourley, Charlie; Gabra, Hani; Kessler, Mirjana; Sehouli, Jalid; Darb-Esfahani, Silvia; Braicu, Elena Ioana

    2017-07-01

    High-grade serous ovarian cancer (HGSOC) causes 80% of all ovarian cancer (OC) deaths. In this setting, the role of cancer stem-like cells (CSCs) is still unclear. In particular, the evolution of CSC biomarkers from primary (pOC) to recurrent (rOC) HGSOCs is unknown. Aim of this study was to investigate changes in CD133 and aldehyde dehydrogenase-1 (ALDH1) CSC biomarker expression in pOC and rOC HGSOCs. Two-hundred and twenty-four pOC and rOC intrapatient paired tissue samples derived from 112 HGSOC patients were evaluated for CD133 and ALDH1 expression using immunohistochemistry (IHC); pOCs and rOCs were compared for CD133 and/or ALDH1 levels. Expression profiles were also correlated with patients' clinicopathological and survival data. Some 49.1% of the patient population (55/112) and 37.5% (42/112) pOCs were CD133+ and ALDH1+ respectively. CD133+ and ALDH1+ samples were detected in 33.9% (38/112) and 36.6% (41/112) rOCs. CD133/ALDH1 coexpression was observed in 23.2% (26/112) and 15.2% (17/112) of pOCs and rOCs respectively. Pairwise analysis showed a significant shift of CD133 staining from higher (pOCs) to lower expression levels (rOCs) (p < 0.0001). Furthermore, all CD133 + pOC patients were International Federation of Gynaecology and Obstetrics (FIGO)-stage III/IV (p < 0.0001) and had significantly worse progression-free interval (PFI) (p = 0.04) and overall survival (OS) (p = 0.02). On multivariate analysis, CD133/ALDH1 coexpression in pOCs was identified as independent prognostic factor for PFI (HR: 1.64; 95% CI: 1.03-2.60; p = 0.036) and OS (HR: 1.71; 95% CI: 1.01-2.88; p = 0.045). Analysis on 52 pts patients with known somatic BRCA status revealed that BRCA mutations did not influence CSC biomarker expression. The study showed that CD133/ALDH1 expression impacts HGSOC patients' survival and first suggests that CSCs might undergo phenotypic change during the disease course similarly to non stem-like cancer cells, providing also a first

  5. The Activity of Class I-IV Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase in Bladder Cancer Cells.

    PubMed

    Orywal, Karolina; Jelski, Wojciech; Werel, Tadeusz; Szmitkowski, Maciej

    2018-01-02

    The aim of this study was to determine the differences in the activity of Alcohol Dehydrogenase (ADH) isoenzymes and Aldehyde Dehydrogenase (ALDH) in normal and cancerous bladder cells. Class III, IV of ADH and total ADH activity were measured by the photometric method and class I, II ADH and ALDH activity by the fluorometric method. Significantly higher total activity of ADH was found in both, low-grade and high-grade bladder cancer, in comparison to healthy tissues. The increased activity of total ADH in bladder cancer cells may be the cause of metabolic disorders in cancer cells, which may intensify carcinogenesis.

  6. A pilot study of the modulation of sirtuins on arylamine N-acetyltransferase 1 and 2 enzymatic activity.

    PubMed

    Turiján-Espinoza, Eneida; Salazar-González, Rául Alejandro; Uresti-Rivera, Edith Elena; Hernández-Hernández, Gloria Estela; Ortega-Juárez, Montserrat; Milán, Rosa; Portales-Pérez, Diana

    2018-03-01

    Arylamine N -acetyltransferase (NAT; E.C. 2.3.1.5) enzymes are responsible for the biotransformation of several arylamine and hydrazine drugs by acetylation. In this process, the acetyl group transferred to the acceptor substrate produces NAT deacetylation and, in consequence, it is susceptible of degradation. Sirtuins are protein deacetylases, dependent on nicotine adenine dinucleotide, which perform post-translational modifications on cytosolic proteins. To explore possible sirtuin participation in the enzymatic activity of arylamine NATs, the expression levels of NAT1, NAT2, SIRT1 and SIRT6 in peripheral blood mononuclear cells (PBMC) from healthy subjects were examined by flow cytometry and Western blot. The in situ activity of the sirtuins on NAT enzymatic activity was analyzed by HPLC, in the presence or absence of an agonist (resveratrol) and inhibitor (nicotinamide) of sirtuins. We detected a higher percentage of positive cells for NAT2 in comparison with NAT1, and higher numbers of SIRT1+ cells compared to SIRT6 in lymphocytes. In situ NAT2 activity in the presence of NAM inhibitors was higher than in the presence of its substrate, but not in the presence of resveratrol. In contrast, the activity of NAT1 was not affected by sirtuins. These results showed that NAT2 activity might be modified by sirtuins.

  7. Effect of pentachlorophenol and 2,6-dichloro-4-nitrophenol on the activity of cDNA-expressed human alcohol and aldehyde dehydrogenases.

    PubMed

    Kollock, Ronny; Rost, Katharina; Batke, Monika; Glatt, Hansruedi

    2009-12-15

    Pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP), potent inhibitors of phenol sulphotransferases, are frequently used in animal studies to elucidate the role of these enzymes in the biotransformation and toxicity of xenobiotics. An unexpected finding with 1-hydroxymethylpyrene--a strong decrease in the excretion of the corresponding carboxylic acid in rats concurrently treated with PCP-led us to suspect that this sulphotransferase inhibitor may also affect alcohol dehydrogenases (ADHs) and/or aldehyde dehydrogenases (ALDHs). Subsequently we investigated the influence of PCP and DCNP on the activity of cDNA-expressed human ADHs and ALDHs. PCP inhibited all four ADHs studied. The inhibition was strong for ADH3 (K(i) 1.4 microM, K(i)' 5.2 microM, mixed-type) and ADH2 (K(i) 3.7 microM, competitive), but moderate for ADH4 (K(i) 81 microM, competitive) and ADH1C (K(i)' 310 microM, uncompetitive). Activities of ALDH2 and ALDH3A1 were unaffected by PCP (used up to a concentration of 1 mM). In contrast, DCNP primarily inhibited ALDH2 (K(i)=K(i)' 7.4 microM, non-competitive), showed moderate competitive inhibition of ADH2 (K(i) 160 microM) and ADH4 (K(i) 710 microM), but did not affect the remaining enzymes (ADH1C, ADH3 and ALDH3A1). The study demonstrates that caution is required when using putative specific enzyme inhibitors in biotransformation studies.

  8. Enzymatic activities and effects of mycovirus infection on the virulence of Metarhizium anisopliae in Rhipicephalus microplus.

    PubMed

    Perinotto, Wendell M S; Golo, Patricia S; Coutinho Rodrigues, Caio J B; Sá, Fillipe A; Santi, Lucélia; Beys da Silva, Walter O; Junges, Angela; Vainstein, Marilene H; Schrank, Augusto; Salles, Cristiane M C; Bittencourt, Vânia R E P

    2014-06-16

    The present study aimed to evaluate the pathogenic potential of different Metarhizium anisopliae s.l. isolates and to determine whether differences in enzymatic activities of proteases, lipases and chitinases and infection with mycoviruses affect the control of Rhipicephalus microplus achieved by these fungal isolates. Engorged female ticks were exposed to fungal suspensions. The lipolytic and proteolytic activities in the isolates were evaluated using chromogenic substrates and the chitinolytic activity was determined using fluorescent substrates. A gel zymography was performed to determine the approximate size of serine proteases released by M. anisopliae isolates. To detect mycoviral infections, dsRNA was digested using both RNAse A and S1 endonuclease; samples were analyzed on an agarose gel. Four of the five isolates tested were infected with mycovirus; however, the level of control of R. microplus ticks achieved with the only isolate free of infection (isolate CG 347) was low. This finding suggests that mycoviral infection does not affect the virulence of fungi against ticks. Although all five isolates were considered pathogenic to R. microplus, the best tick control and the highest levels of enzymatic activity were achieved with the isolates CG 629 and CG 148. The in vitro activities of lipases, proteases and chitinases produced by M. anisopliae s.l. differed among isolates and may be related to their virulence. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. A missense mutation in ALDH18A1, encoding Delta1-pyrroline-5-carboxylate synthase (P5CS), causes an autosomal recessive neurocutaneous syndrome.

    PubMed

    Bicknell, Louise S; Pitt, James; Aftimos, Salim; Ramadas, Ram; Maw, Marion A; Robertson, Stephen P

    2008-10-01

    There are several rare syndromes combining wrinkled, redundant skin and neurological abnormalities. Although phenotypic overlap between conditions has suggested that some might be allelic to one another, the aetiology for many of them remains unknown. A consanguineous New Zealand Maori family has been characterised that segregates an autosomal recessive connective tissue disorder (joint dislocations, lax skin) associated with neurological abnormalities (severe global developmental delay, choreoathetosis) without metabolic abnormalities in four affected children. A genome-screen performed under a hypothesis of homozygosity by descent for an ancestral mutation, identified a locus at 10q23 (Z = 3.63). One gene within the candidate interval, ALDH18A1, encoding Delta1-pyrroline-5-carboxylate synthase (P5CS), was considered a plausible disease gene since a missense mutation had previously been shown to cause progressive neurodegeneration, cataracts, skin laxity, joint dislocations and metabolic derangement in a consanguineous Algerian family. A missense mutation, 2350C>T, was identified in ALDH18A1, which predicts the substitution H784Y. H784 is invariant across all phyla and lies within a previously unrecognised, conserved C-terminal motif in P5CS. In an in vivo assay of flux through this metabolic pathway using dermal fibroblasts obtained from an affected individual, proline and ornithine biosynthetic activity of P5CS was not affected by the H784Y substitution. These data suggest that P5CS may possess additional uncharacterised functions that affect connective tissue and central nervous system function.

  10. Association between enzymatic and non-enzymatic antioxidant defense mechanism with apolipoprotein E genotypes in Alzheimer disease.

    PubMed

    Kharrazi, Hadi; Vaisi-Raygani, Asad; Rahimi, Zohreh; Tavilani, Haidar; Aminian, Mahdi; Pourmotabbed, Tayebeh

    2008-08-01

    There are evidence suggesting that APOE-varepsilon4 allele play an important role in the pathogenesis of Alzheimer's disease (AD) by reducing peripheral levels and activities of a broad spectrum of nonenzymatic and enzymatic antioxidants systems. However, the link between APOE genotype, oxidative stress, and AD has yet to be established. In this study we examined whether antioxidant defense mechanism exacerbates the risk of AD in individual carrying APOE-varepsilon4 allele in a population from Tehran, Iran. We determined the enzymatic activities of the erythrocyte Cu-Zn superoxide dismutase (Cu-Zn SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and serum level of total antioxidant status(TAS) in various APOE genotypes in 91 patients with AD and 91 healthy subjects as control group (age and sex-matched). The results showed that the TAS level and the activities of enzymatic antioxidants CAT and GSH-Px were significantly lower and the SOD activity was significantly higher in AD patients compared to controls. The AD patients with APOE-varepsilon4 allele genotype had significantly lower serum TAS concentration and lower erythrocytes GSH-Px and CAT activities (p=0.001) but significantly higher erythrocytes Cu-Zn SOD activity (p=0.001) than the non-APOE-varepsilon4 carrier AD and the control group. In addition, the association observed between the factors involved in an antioxidant defense mechanism and APOE-varepsilon4 allele in AD increased with age of the subjects. These data indicate that the reduced serum level of TAS and activity of CAT, GSH-Px and increased SOD exacerbate the risk of AD in individuals carrying APOE-varepsilon4 allele. The reduced antioxidants defense in APOE-varepsilon4 allele carrier may contribute to beta-amyloidosis. This effect, however, is more pronounced in the AD patients older than 75 years of age. This suggests that a therapeutic modality should be considered for these subjects.

  11. 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.

  12. Enzymatic Activity Detection via Electrochemistry for Enceladus

    NASA Technical Reports Server (NTRS)

    Studemeister, Lucy; Koehne, Jessica; Quinn, Richard

    2017-01-01

    Electrochemical detection of biological molecules is a pertinent topic and application in many fields such as medicine, environmental spills, and life detection in space. Proteases, a class of molecules of interest in the search for life, catalyze the hydrolysis of peptides. Trypsin, a specific protease, was chosen to investigate an optimized enzyme detection system using electrochemistry. This study aims at providing the ideal functionalization of an electrode that can reliably detect a signal indicative of an enzymatic reaction from an Enceladus sample.

  13. Phenotypic characterization of enzymatic activity of clinical dermatophyte isolates from animals with and without skin lesions and humans.

    PubMed

    Gnat, Sebastian; Łagowski, Dominik; Nowakiewicz, Aneta; Zięba, Przemysław

    2018-05-20

    The pathogenesis of dermatophytoses is associated with the secretion of enzymes degrading the infected tissue components. Although many studies on enzymatic activity of dermatophytes have been conducted over the years, there have been no concrete proposals on construction of the profile of enzymes characteristic of individual species, genus, or ecological types of dermatophytes. The aim of this study was to assess the capability of clinical dermatophyte isolates from both symptomatic and asymptomatic animals and humans to produce different enzymes. Clinical isolates of 234 dermatophyte strains collected during routine examination of animal health were used in this study. The enzymatic production of keratinase, elastase, phospholipase, lipase, protease, DNase, and gelatinase as well as the haemolytic activity were evaluated using specific test media. The overall degree of enzymatic activity of the analysed clinical isolates of the dermatophytes was 67%. All tested clinical isolates of different species of dermatophytes showed keratinase activity and 96% additionally exhibited phospholipase activity. The weakest activity among the tested enzymes was demonstrated for elastase and gelatinase. 83% of the isolates of the dermatophytes showed haemolytic activity. Our data indicate that clinical isolates of dermatophytes from different species produce enzymes with different levels of activities. Profile of enzymes characteristic of individual species, genus, or ecological types of dermatophytes is possibly dependent upon factors related to the host. The relationship between each enzyme and the occurrence of skin lesions in animals and humans or asymptomatic animal carriers varies on whether the infection is caused by T. mentagrophytes, T. verrucosum, or M. canis. Interestingly, only keratinase seems to be correlated with the appearance of dermatophyte infections, irrespective of the pathogen species, and elastase is a characteristic enzyme for dermatophyte strains

  14. New eutectic ionic liquids for lipase activation and enzymatic preparation of biodiesel†

    PubMed Central

    Zhao, Hua; Baker, Gary A.; Holmes, Shaletha

    2012-01-01

    The enzymatic preparation of biodiesel has been hampered by the lack of suitable solvents with desirable properties such as high lipase compatibility, low cost, low viscosity, high biodegradability, and ease of product separation. Recent interest in using ionic liquids (ILs) as advanced reaction media has led to fast reaction rates and high yields in the enzymatic synthesis of biodiesel. However, conventional (i.e., cation–anion paired) ILs based on imidazolium and other quaternary ammonium salts remain too expensive for wide application at industrial scales. In this study, we report on newly-synthesized eutectic ILs derived from choline acetate or choline chloride coupled with biocompatible hydrogen-bond donors, such as glycerol. These eutectic solvents have favorable properties including low viscosity, high biodegradability, and excellent compatibility with Novozym® 435, a commercial immobilized Candida antarctica lipase B. Furthermore, in a model biodiesel synthesis system, we demonstrate high reaction rates for the enzymatic transesterification of Miglyol® oil 812 with methanol, catalyzed by Novozym® 435 in choline acetate/glycerol (1 : 1.5 molar ratio). The high conversion (97%) of the triglyceride obtained within 3 h, under optimal conditions, suggests that these novel eutectic solvents warrant further exploration as potential media in the enzymatic production of biodiesel. PMID:21283901

  15. Real-time ESI-MS of enzymatic conversion: impact of organic solvents and multiplexing.

    PubMed

    Scheerle, Romy K; Grassmann, Johanna; Letzel, Thomas

    2012-01-01

    Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.

  16. Photoelectrochemical enzymatic biosensors.

    PubMed

    Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2017-06-15

    Enzymatic biosensors have been valuable bioanalytical devices for analysis of diverse targets in disease diagnosis, biological and biomedical research, etc. Photoelectrochemical (PEC) bioanalysis is a recently emerged method that promptly becoming a subject of new research interests due to its attractive potential for future bioanalysis with high sensitivity and specificity. PEC enzymatic biosensors integrate the inherent sensitivities of PEC bioanalysis and the selectivity of enzymes and thus share their both advantages. Currently, PEC enzymatic biosensors have become a hot topic of significant research and the recent impetus has grown rapidly as demonstrated by increased research papers. Given the pace of advances in this area, this review will make a thorough discussion and survey on the fundamentals, sensing strategies, applications and the state of the art in PEC enzymatic biosensors, followed by future prospects based on our own opinions. We hope this work could provide an accessible introduction to PEC enzymatic biosensors for any scientist. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Effect of gypsum application on enzymatic browning activity in lettuce.

    PubMed

    Chutichudet, Prasit; Chutichudet, B; Kaewsit, S

    2009-09-15

    A comprehensive study to evaluate calcium, in terms of gypsum (CaSO4.2H2O) by soil dressing application, on enzymatic browning activity of Polyphenol oxidase (PPO) and internal qualities was tested on lettuce var. Grand Rapids under field conditions. A factorial in completely randomized design was arranged with four replications. The results showed that plants-treated with 50 mg kg(-1) gypsum applied at 40 DAP had the maximal fresh weight of 25.83 g plant(-1). The internal qualities of the lettuce at harvest showed that plants treated with 50 mg kg(-1) gypsum had the maximal chlorophyll content (26.80 mg m(-2)), while all gypsum concentrations applied in this study, had less content of ascorbic acid than the control plants. Plants-treated with 100 mg kg(-1) gypsum affected to the lowest level of PPO activity at week 3 after transplanting. Furthermore, gypsum application had no effect to biomass, leaf colour, the contents of phenolic and quinone in lettuce at harvesting stage.

  18. Biocolloids with ordered urease multilayer shells as enzymatic reactors.

    PubMed

    Lvov, Y; Caruso, F

    2001-09-01

    The preparation of biocolloids with organized enzyme-containing multilayer shells for exploitation as colloidal enzymatic nanoreactors is described. Urease multilayers were assembled onto submicrometer-sized polystyrene spheres by the sequential adsorption of urease and polyelectrolyte, in a predetermined order, utilizing electrostatic interactions for layer growth. The catalytic activity of the biocolloids increased proportionally with the number of urease layers deposited on the particles, demonstrating that biocolloid particles with tailored enzymatic activities can be produced. It was further found that precoating the latex spheres with nanoparticles (40-nm silica or 12-nm magnetite) enhanced both the stability (with respect to adsorption) and enzymatic activity of the urease multilayers. The presence of the magnetite nanoparticle coating also provided a magnetic function that allowed the biocolloids to be easily and rapidly separated with a permanent magnet. The fabrication of such colloids opens new avenues for the application of bioparticles and represents a promising route for the creation of complex catalytic particles.

  19. Tissue Factor promotes breast cancer stem cell activity in vitro.

    PubMed

    Shaker, Hudhaifah; Harrison, Hannah; Clarke, Robert; Landberg, Goran; Bundred, Nigel J; Versteeg, Henri H; Kirwan, Cliona C

    2017-04-18

    Cancer stem cells (CSCs) are a subpopulation of cells that can self-renew and initiate tumours. The clotting-initiating protein Tissue Factor (TF) promotes metastasis and may be overexpressed in cancer cells with increased CSC activity. We sought to determine whether TF promotes breast CSC activity in vitro using human breast cancer cell lines. TF expression was compared in anoikis-resistant (CSC-enriched) and unselected cells. In cells sorted into of TF-expressing and TF-negative (FACS), and in cells transfected to knockdown TF (siRNA) and overexpress TF (cDNA), CSC activity was compared by (i) mammosphere forming efficiency (MFE) (ii) holoclone colony formation (Hc) and (iii) ALDH1 activity. TF expression was increased in anoikis-resistant and high ALDH1-activity T47D cells compared to unselected cells. FACS sorted TF-expressing T47Ds and TF-overexpressing MCF7s had increased CSC activity compared to TF-low cells. TF siRNA cells (MDAMB231,T47D) had reduced CSC activity compared to control cells. FVIIa increased MFE and ALDH1 in a dose-dependent manner (MDAMB231, T47D). The effects of FVIIa on MFE were abrogated by TF siRNA (T47D). Breast CSCs (in vitro) demonstrate increased activity when selected for high TF expression, when induced to overexpress TF, and when stimulated (with FVIIa). Targeting the TF pathway in vivo may abrogate CSC activity.

  20. Enzymatic biofuel cell-based self-powered biosensing of protein kinase activity and inhibition via thiophosphorylation-mediated interface engineering.

    PubMed

    Gu, Chengcheng; Gai, Panpan; Han, Lei; Yu, Wen; Liu, Qingyun; Li, Feng

    2018-05-24

    We developed a facile and ultrasensitive enzymatic biofuel cell (EBFC)-based self-powered biosensor of protein kinase A (PKA) activity and inhibition via thiophosphorylation-mediated interface engineering. The detection limit was down to 0.00022 U mL-1 (S/N = 3). In addition, the PKA activities from MCF-7 and A549 cell lysates were analyzed and achieved reliable results.

  1. Herbivore species identity and composition affect soil enzymatic activity through altered plant composition in a coastal tallgrass prairie

    USDA-ARS?s Scientific Manuscript database

    Although single species of herbivores are known to affect soil microbial communities, the effects of herbivore species identity and functional composition on soil microbes is unknown. We tested the effects of single species of orthopterans and multiple species combinations on soil enzymatic activity...

  2. 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.

  3. Self-Assembly of Spider Silk-Fusion Proteins Comprising Enzymatic and Fluorescence Activity.

    PubMed

    Humenik, Martin; Mohrand, Madeleine; Scheibel, Thomas

    2018-04-18

    The recombinant spider silk protein eADF4(C16) was genetically fused either with esterase 2 (EST2) or green fluorescent protein (GFP). The fusions EST-eADF4(C16) and GFP-eADF4(C16) were spectroscopically investigated and showed native structures of EST and GFP. The structural integrity was confirmed by the enzymatic activity of EST and the fluorescence of GFP. The spider silk moiety retained its intrinsically unstructured conformation in solution and the self-assembly into either nanofibrils or nanoparticles could be controlled by the concentration of phosphate. Particles, however, showed significantly lower activity of the EST and GFP domains likely caused by a steric hindrance. However, upon self-assembly of EST-eADF4(C16) and GFP-eADF4(C16) into fibrils the protein activities were retained. In general, the fusion of globular enzymes with the spider silk domain allows the generation of fibrous biomaterials with catalytic or light emitting properties.

  4. Vancomycin analogues containing monosaccharides exhibit improved antibiotic activity: a combined one-pot enzymatic glycosylation and chemical diversification strategy.

    PubMed

    Thayer, Desiree A; Wong, Chi-Huey

    2006-09-18

    Many natural products contain carbohydrate moieties that contribute to their biological activity. Manipulation of the carbohydrate domain of natural products through multiple glycosylations to identify new derivatives with novel biological activities has been a difficult and impractical process. We report a practical one-pot enzymatic approach with regeneration of cosubstrates to synthesize analogues of vancomycin that contain an N-alkyl glucosamine, which exhibited marked improvement in antibiotic activity against a vancomycin-resistant strain of Enterococcus.

  5. Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

    PubMed Central

    Gherib, Rami; Dokainish, Hisham M.; Gauld, James W.

    2014-01-01

    Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM) can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis. PMID:24384841

  6. Improvement of antioxidant and moisture-preserving activities of Sargassum horneri polysaccharide enzymatic hydrolyzates.

    PubMed

    Shao, Ping; Chen, Xiaoxiao; Sun, Peilong

    2015-03-01

    In the previous study, we have found that polysaccharides isolated from Sargassum horneri exhibited bioactivities. The aim of this study was to investigate the antioxidant and moisture-preserving activities of molecular weight alteration of Sargassum horneri polysaccharide in vitro. For this purpose, the homogeneous active polysaccharide SHP was isolated from Sargassum horneri, and response surface methodology was employed to optimize the enzymatic degradation conditions to get SHP-derived fragments with different molecular weight. Results proved that the polysaccharide is capable of scavenging both ABTS and DPPH radicals in vitro. The study revealed that the polysaccharides had strong moisture-absorption and -retention capacities as compared to propanediol and glycerin. Furthermore, these data demonstrated that molecular weight had a certain effect on antioxidant activities and strong moisture-retention capacities of the polysaccharide from Sargassum horneri. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Dissecting the link between the enzymatic activity and the SaPI inducing capacity of the phage 80α dUTPase.

    PubMed

    Alite, Christian; Humphrey, Suzanne; Donderis, Jordi; Maiques, Elisa; Ciges-Tomas, J Rafael; Penadés, José R; Marina, Alberto

    2017-09-11

    The trimeric staphylococcal phage-encoded dUTPases (Duts) are signalling molecules that induce the cycle of some Staphylococcal pathogenicity islands (SaPIs) by binding to the SaPI-encoded Stl repressor. To perform this regulatory role, these Duts require an extra motif VI, as well as the Dut conserved motifs IV and V. While the apo form of Dut is required for the interaction with the Stl repressor, usually only those Duts with normal enzymatic activity can induce the SaPI cycle. To understand the link between the enzymatic activities and inducing capacities of the Dut protein, we analysed the structural, biochemical and physiological characteristics of the Dut80α D95E mutant, which loses the SaPI cycle induction capacity despite retaining enzymatic activity. Asp95 is located at the threefold central channel of the trimeric Dut where it chelates a divalent ion. Here, using state-of-the-art techniques, we demonstrate that D95E mutation has an epistatic effect on the motifs involved in Stl binding. Thus, ion binding in the central channel correlates with the capacity of motif V to twist and order in the SaPI-inducing disposition, while the tip of motif VI is disturbed. These alterations in turn reduce the affinity for the Stl repressor and the capacity to induce the SaPI cycle.

  8. Enzymatic dehalogenation of pentachlorophenol by extracts from Arthrobacter sp. strain ATCC 33790.

    PubMed Central

    Schenk, T; Müller, R; Mörsberger, F; Otto, M K; Lingens, F

    1989-01-01

    Arthrobacter sp. strain ATCC 33790 was grown with pentachlorophenol (PCP) as the sole source of carbon and energy. Crude extracts, which were prepared by disruption of the bacteria with a French pressure cell, showed no dehalogenating activity with PCP as the substrate. After sucrose density ultracentrifugation of the crude extract at 145,000 x g, various layers were found in the gradient. One yellow layer showed enzymatic conversion of PCP. One chloride ion was released per molecule of PCP. The product of the enzymatic conversion was tetrachlorohydroquinone. NADPH and oxygen were essential for this reaction. EDTA stimulated the enzymatic activity by 67%. The optimum pH for the enzyme activity was 7.5, and the temperature optimum was 25 degrees C. Enzymatic activity was also detected with 2,4,5-trichlorophenol, 2,3,4-trichlorophenol, 2,4,6-trichlorophenol, and 2,3,4,5-tetrachlorophenol as substrates, whereas 3,4,5-trichlorophenol, 2,4-dichlorophenol, 3,4-dichlorophenol, and 4-chlorophenol did not serve as substrates. PMID:2793827

  9. Selection and validation of enzymatic activities as functional markers in wood biotechnology and fungal ecology.

    PubMed

    Mathieu, Yann; Gelhaye, Eric; Dumarçay, Stéphane; Gérardin, Philippe; Harvengt, Luc; Buée, Marc

    2013-02-15

    The dead wood and forest soils are sources of diversity and under-explored fungal strains with biotechnological potential, which require to be studied. Numerous enzymatic tests have been proposed to investigate the functional potential of the soil microbial communities or to test the functional abilities of fungal strains. Nevertheless, the diversity of these functional markers and their relevance in environmental studies or biotechnological screening does still have not been demonstrated. In this work, we assessed ten different extracellular enzymatic activities involved in the wood decaying process including β-etherase that specifically cleaves the β-aryl ether linkages in the lignin polymer. For this purpose, a collection of 26 fungal strains, distributed within three ecological groups (white, brown and soft rot fungi), has been used. Among the ten potential functional markers, the combinatorial use of only six of them allowed separation between the group of white and soft rot fungi from the brown rot fungi. Moreover, our results suggest that extracellular β-etherase is a rare and dispensable activity among the wood decay fungi. Finally, we propose that this set of markers could be useful for the analysis of fungal communities in functional and environmental studies, and for the selection of strains with biotechnological interests. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. A high-throughput assay for enzymatic polyester hydrolysis activity by fluorimetric detection.

    PubMed

    Wei, Ren; Oeser, Thorsten; Billig, Susan; Zimmermann, Wolfgang

    2012-12-01

    A fluorimetric assay for the fast determination of the activity of polyester-hydrolyzing enzymes in a large number of samples has been developed. Terephthalic acid (TPA) is a main product of the enzymatic hydrolysis of polyethylene terephthalate (PET), a synthetic polyester. Terephthalate has been quantified following its conversion to the fluorescent 2-hydroxyterephthalate by an iron autoxidation-mediated generation of free hydroxyl radicals. The assay proved to be robust at different buffer concentrations, reaction times, pH values, and in the presence of proteins. A validation of the assay was performed by analyzing TPA formation from PET films and nanoparticles catalyzed by a polyester hydrolase from Thermobifida fusca KW3 in a 96-well microplate format. The results showed a close correlation (R(2) = 0.99) with those obtained by a considerably more tedious and time-consuming HPLC method, suggesting the aptness of the fluorimetric assay for a high-throughput screening for polyester hydrolases. The method described in this paper will facilitate the detection and development of biocatalysts for the modification and degradation of synthetic polymers. The fluorimetric assay can be used to quantify the amount of TPA obtained as the final degradation product of the enzymatic hydrolysis of PET. In a microplate format, this assay can be applied for the high-throughput screening of polyester hydrolases. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Substitutions in PBP2b from β-Lactam-resistant Streptococcus pneumoniae Have Different Effects on Enzymatic Activity and Drug Reactivity*

    PubMed Central

    Calvez, Philippe; Breukink, Eefjan; Roper, David I.; Dib, Mélanie; Contreras-Martel, Carlos; Zapun, André

    2017-01-01

    Pneumococcus resists β-lactams by expressing variants of its target enzymes, the penicillin-binding proteins (PBPs), with many amino acid substitutions. Up to 10% of the sequence can be modified. These altered PBPs have a much reduced reactivity with the drugs but retain their physiological activity of cross-linking the peptidoglycan, the major constituent of the bacterial cell wall. However, because β-lactams are chemical and structural mimics of the natural substrate, resistance mediated by altered PBPs raises the following paradox: how PBPs that react poorly with the drugs maintain a sufficient level of activity with the physiological substrate. This question is addressed for the first time in this study, which compares the peptidoglycan cross-linking activity of PBP2b from susceptible and resistant strains with their inhibition by different β-lactams. Unexpectedly, the enzymatic activity of the variants did not correlate with their antibiotic reactivity. This finding indicates that some of the numerous amino acid substitutions were selected to restore a viable level of enzymatic activity by a compensatory molecular mechanism. PMID:28062575

  12. 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. © 2013. Published by Elsevier B.V. All rights reserved.

  13. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Enzymatic activity of anthropogenic proto-organic soils in soilless farming

    NASA Astrophysics Data System (ADS)

    Bireescu, Geanina; Dazzi, Carmelo; Laudicina, Vito Armando; Lo Papa, Giuseppe

    2017-04-01

    In soilless agriculture and horticulture coir is the more used substratum to grow plants because it is widely available and more environmentally friendly than sphagnum or peat. In Italy, soilless agriculture concerns an area of about 1,000 hectares, particularly concentrated in Sicily. The southern coastal belt of this region is the area interested by the most significant experiences in the application of techniques of soilless cultivation that, recently, has been used also for growing table grapes. Starting from the above consideration we suppose that the features of the coconut fiber underlay an evident transformation and that even after few years of table grape cultivation, such organic material undergone to a transformation that allows for the formation of a proto-organic soil (a proto-Histosol, we supposed). If this is true, we believe that, in this case, to speak about soilless cultivation is for sure misleading for the common people, as we should define this cultivation "on anthropogenic soils" instead. To fit the aims of this survey we used a big greenhouse devoted to soilless cultivation of table grape in a farm in the Southern Sicily We have considered the enzymatic activity that characterized the coconut fiber after 3 cycles of cultivation of table grapes. We used as a control the coconut fiber that the farmer used to prepare pots for soilless cultivation and coconut fiber of: 6 pots at the end of the first productive cycle 6 pots at the end of the second cycle and 3 pots at the end of the third cycle. On these organic samples we investigated three enzymes, belonging to oxydoreductase (catalase and dehydrogenase) and hydrolase (urease) classes. Statistical analysis of the investigated enzymes was developed using IBM Statistic SPSS v20 by ANOVA, Tukey test HSD for p ≤ 0.01 and Multivariate Statistical Analysis. Results have shown significant differences in enzymes content and quality among coir tests. The use of the coco fiber, as nutritive substratum

  15. Differential Expression and Enzymatic Activity of DPPIV/CD26 Affects Migration Ability of Cervical Carcinoma Cells

    PubMed Central

    Beckenkamp, Aline; Willig, Júlia Biz; Santana, Danielle Bertodo; Nascimento, Jéssica; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Bruno, Alessandra Nejar; Pilger, Diogo André; Wink, Márcia Rosângela; Buffon, Andréia

    2015-01-01

    Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane glycoprotein that inactivates or degrades some bioactive peptides and chemokines. For this reason, it regulates cell proliferation, migration and adhesion, showing its role in cancer processes. This enzyme is found mainly anchored onto the cell membrane, although it also has a soluble form, an enzymatically active isoform. In the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and non-tumorigenic HaCaT cells. The effect of the DPPIV/CD26 inhibitor (sitagliptin phosphate) on cell migration and adhesion was also evaluated. Cervical cancer cells and keratinocytes exhibited DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form. DPPIV/CD26 expression was observed in HaCaT, SiHa and C33A, while in HeLa cells it was almost undetectable. We observed higher migratory capacity of HeLa, when compared to SiHa. But in the presence of sitagliptin SiHa showed an increase in migration, indicating that, at least in part, cell migration is regulated by DPPIV/CD26 activity. Furthermore, in the presence of sitagliptin phosphate, SiHa and HeLa cells exhibited a significant reduction in adhesion. However this mechanism seems to be mediated independent of DPPIV/CD26. This study demonstrates, for the first time, the activity and expression of DPPIV/CD26 in cervical cancer cells and the effect of sitagliptin phosphate on cell migration and adhesion. PMID:26222679

  16. Differential Expression and Enzymatic Activity of DPPIV/CD26 Affects Migration Ability of Cervical Carcinoma Cells.

    PubMed

    Beckenkamp, Aline; Willig, Júlia Biz; Santana, Danielle Bertodo; Nascimento, Jéssica; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Bruno, Alessandra Nejar; Pilger, Diogo André; Wink, Márcia Rosângela; Buffon, Andréia

    2015-01-01

    Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane glycoprotein that inactivates or degrades some bioactive peptides and chemokines. For this reason, it regulates cell proliferation, migration and adhesion, showing its role in cancer processes. This enzyme is found mainly anchored onto the cell membrane, although it also has a soluble form, an enzymatically active isoform. In the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and non-tumorigenic HaCaT cells. The effect of the DPPIV/CD26 inhibitor (sitagliptin phosphate) on cell migration and adhesion was also evaluated. Cervical cancer cells and keratinocytes exhibited DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form. DPPIV/CD26 expression was observed in HaCaT, SiHa and C33A, while in HeLa cells it was almost undetectable. We observed higher migratory capacity of HeLa, when compared to SiHa. But in the presence of sitagliptin SiHa showed an increase in migration, indicating that, at least in part, cell migration is regulated by DPPIV/CD26 activity. Furthermore, in the presence of sitagliptin phosphate, SiHa and HeLa cells exhibited a significant reduction in adhesion. However this mechanism seems to be mediated independent of DPPIV/CD26. This study demonstrates, for the first time, the activity and expression of DPPIV/CD26 in cervical cancer cells and the effect of sitagliptin phosphate on cell migration and adhesion.

  17. Identification of three novel loci of ALDH2 Gene for Serum Folate levels in a Male Chinese Population by Genome-Wide Association Study.

    PubMed

    Deng, Caiwang; Tang, Shaomei; Huang, Xiaoliang; Gao, Jiamin; Tian, Jiarong; Zhou, Xianguo; Xie, Yuanliang; Liao, Ming; Mo, Zengnan; Wang, Qiuyan

    2018-06-25

    Serum folate is important in clinical researches and DNA synthesis and methylation. Some loci and genes that are associated with folate levels had been detected by genome-wide association studies (GWAS), such as rs1801133 in MTHFR and rs1979277 in SHMT1. Nevertheless, only a small part of variants has been clearly identified for serum folate. Hence, we conducted a GWAS to discover new inherited susceptibility and gene-environment interactions on serum folate concentration. In a healthy Chinese population of 1999 men, genotyping was performed using Illumina HumanOmni1-Quad BeadChip. Serum folate levels were measured by enzyme-linked immunosorbent assay (ELISA), pathway enrichment analysis and statistical analysis were performed by Database for Annotation, Visualization and Integrated Discovery (DAVID) and Statistic Package for Social Science (SPSS). We validated that rs1801133 in MTHFR was significantly involved in serum folate (P = 4.21 × 10 -19 ). Surprisingly, we discovered three novel loci rs3782886, rs671, and rs4646776 of ALDH2 gene were suggestively significantly associated with folate serum folate levels in the male population studied (P = 2.17 × 10 -7 , P = 3.60 × 10 -7 , P = 3.99 × 10 -7 , respectively) after adjusting for population stratification, BMI and age. Men with the AA genotype had significantly higher serum folate levels compared with men with the GG/AG genotype. But we found ALDH2 gene mutation no relation to part of environmental factors on serum folate levels. In a male Chinese population, genome-wide association study discovered that three novel SNPs rs3782886, rs671 and rs4646776 of ALDH2 gene were suggestively significantly associated with serum folate levels. Copyright © 2018. Published by Elsevier B.V.

  18. Enzymatic Processes in Marine Biotechnology.

    PubMed

    Trincone, Antonio

    2017-03-25

    In previous review articles the attention of the biocatalytically oriented scientific community towards the marine environment as a source of biocatalysts focused on the habitat-related properties of marine enzymes. Updates have already appeared in the literature, including marine examples of oxidoreductases, hydrolases, transferases, isomerases, ligases, and lyases ready for food and pharmaceutical applications. Here a new approach for searching the literature and presenting a more refined analysis is adopted with respect to previous surveys, centering the attention on the enzymatic process rather than on a single novel activity. Fields of applications are easily individuated: (i) the biorefinery value-chain, where the provision of biomass is one of the most important aspects, with aquaculture as the prominent sector; (ii) the food industry, where the interest in the marine domain is similarly developed to deal with the enzymatic procedures adopted in food manipulation; (iii) the selective and easy extraction/modification of structurally complex marine molecules, where enzymatic treatments are a recognized tool to improve efficiency and selectivity; and (iv) marine biomarkers and derived applications (bioremediation) in pollution monitoring are also included in that these studies could be of high significance for the appreciation of marine bioprocesses.

  19. Enzymatic Processes in Marine Biotechnology

    PubMed Central

    Trincone, Antonio

    2017-01-01

    In previous review articles the attention of the biocatalytically oriented scientific community towards the marine environment as a source of biocatalysts focused on the habitat-related properties of marine enzymes. Updates have already appeared in the literature, including marine examples of oxidoreductases, hydrolases, transferases, isomerases, ligases, and lyases ready for food and pharmaceutical applications. Here a new approach for searching the literature and presenting a more refined analysis is adopted with respect to previous surveys, centering the attention on the enzymatic process rather than on a single novel activity. Fields of applications are easily individuated: (i) the biorefinery value-chain, where the provision of biomass is one of the most important aspects, with aquaculture as the prominent sector; (ii) the food industry, where the interest in the marine domain is similarly developed to deal with the enzymatic procedures adopted in food manipulation; (iii) the selective and easy extraction/modification of structurally complex marine molecules, where enzymatic treatments are a recognized tool to improve efficiency and selectivity; and (iv) marine biomarkers and derived applications (bioremediation) in pollution monitoring are also included in that these studies could be of high significance for the appreciation of marine bioprocesses. PMID:28346336

  20. Micromechanical Modeling Study of Mechanical Inhibition of Enzymatic Degradation of Collagen Tissues

    PubMed Central

    Tonge, Theresa K.; Ruberti, Jeffrey W.; Nguyen, Thao D.

    2015-01-01

    This study investigates how the collagen fiber structure influences the enzymatic degradation of collagen tissues. We developed a micromechanical model of a fibrous collagen tissue undergoing enzymatic degradation based on two central hypotheses. The collagen fibers are crimped in the undeformed configuration. Enzymatic degradation is an energy activated process and the activation energy is increased by the axial strain energy density of the fiber. We determined the intrinsic degradation rate and characteristic energy for mechanical inhibition from fibril-level degradation experiments and applied the parameters to predict the effect of the crimped fiber structure and fiber properties on the degradation of bovine cornea and pericardium tissues under controlled tension. We then applied the model to examine the effect of the tissue stress state on the rate of tissue degradation and the anisotropic fiber structures that developed from enzymatic degradation. PMID:26682825

  1. Biofunctional Properties of Enzymatic Squid Meat Hydrolysate

    PubMed Central

    Choi, Joon Hyuk; Kim, Kyung-Tae; Kim, Sang Moo

    2015-01-01

    Squid is one of the most important commercial fishes in the world and is mainly utilized or consumed as sliced raw fish or as processed products. The biofunctional activities of enzymatic squid meat hydrolysate were determined to develop value-added products. Enzymatic squid hydrolysate manufactured by Alcalase effectively quenched 1,1-diphenyl-2-picrylhydrazyl radical, hydroxyl radical, and hydrogen peroxide radical with IC50 values of 311, 3,410, and 111.5 μg/mL, respectively. Angiotensin I-converting enzyme inhibitory activity of squid hydrolysate was strong with an IC50 value of 145.1 μg/mL, while tyrosinase inhibitory activity with an IC50 value of 4.72 mg/mL was moderately low. Overall, squid meat hydrolysate can be used in food or cosmetic industries as a bioactive ingredient and possibly be used in the manufacture of seasoning, bread, noodle, or cosmetics. PMID:25866752

  2. Enhanced enzymatic stability and antitumor activity of daunorubicin-GnRH-III bioconjugates modified in position 4.

    PubMed

    Manea, Marilena; Leurs, Ulrike; Orbán, Erika; Baranyai, Zsuzsa; Öhlschläger, Peter; Marquardt, Andreas; Schulcz, Ákos; Tejeda, Miguel; Kapuvári, Bence; Tóvári, József; Mezo, Gábor

    2011-07-20

    Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), (4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the (4)Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which (4)Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.

  3. Improved antimelanogenesis and antioxidant effects of polysaccharide from Cuscuta chinensis Lam seeds after enzymatic hydrolysis.

    PubMed

    Liu, Zi-Jun; Wang, Ya-Lan; Li, Qi-Ling; Yang, Liu

    2018-01-01

    Cuscuta chinensis polysaccharide (CPS) was extracted using hot water and enzymatically hydrolyzed C. chinensis polysaccharide (ECPS) was produced by the mannase enzymatic hydrolysis process. The purpose of this research was to investigate the antimelanogenic activity of ECPS and CPS in B16F10 melanoma cells. The in vitro antioxidant activity was assessed by their ferric iron reducing power and DPPH free radical scavenging activities. The molecular mass distribution of polysaccharides was determined using SEC-MALLS-RI. CPS was successfully enzymatically degraded using mannase and the weighted average molecular weights of CPS and ECPS were 434.6 kDa and 211.7 kDa. The results of biological activity assays suggested that the enzymatically hydrolyzed polysaccharide had superior antimelanogenic activity and antioxidant effect than the original polysaccharide. ECPS exhibited antimelanogenic activity by down-regulating the expression of tyrosinase, MITF, and TRP-1 without cytotoxic effects in B16F10 melanoma cells. In conclusion, ECPS have the potential to become a skin whitening product.

  4. Enzymatic activity profile of a Brazilian culture collection of Candida albicans isolated from diabetics and non-diabetics with oral candidiasis.

    PubMed

    Sanitá, Paula Volpato; Zago, Chaiene Evelin; Pavarina, Ana Cláudia; Jorge, Janaina Habib; Machado, Ana Lúcia; Vergani, Carlos Eduardo

    2014-06-01

    The secretion of hydrolytic enzymes is a fundamental virulence factor of Candida albicans to develop disease. The objective of this study was to characterise the virulence of 148 clinical isolates of C. albicans from oral candidiasis by assessing the expression of phospholipase (PL) and secreted aspartyl proteinase (SAP). Isolates were obtained from healthy subjects (HS) and diabetics (DOC) and non-diabetics with oral candidiasis (NDOC). An aliquot (5 μl) of each cell suspension was inoculated on PL and SAP agar plates and incubated. Enzymes secretion was detected by the formation of an opaque halo around the colonies and enzymatic activity (PZ) was determined by the ratio between colony diameter and colony diameter plus the halo zone. Statistical comparisons were made by a one-way anova followed by Tukey's post hoc test (α = 0.05). The clinical sources of C. albicans had significant effect (P < 0.001) on the PZ values of both enzymes. For PL, clinical isolates from NDOC and DOC had highest enzymatic activity than those from HS (P < 0.05), with no significant differences between them (P = 0.506). For SAP, C. albicans from NDOC showed the lower enzymatic activity (P < 0.001). There were no significant differences between isolates from HS and DOC (P = 0.7051). C. albicans isolates from NDOC and DOC patients showed an increased production of PL. © 2013 Blackwell Verlag GmbH.

  5. Enzymatic mechanisms of biological magnetic sensitivity.

    PubMed

    Letuta, Ulyana G; Berdinskiy, Vitaly L; Udagawa, Chikako; Tanimoto, Yoshifumi

    2017-10-01

    Primary biological magnetoreceptors in living organisms is one of the main research problems in magnetobiology. Intracellular enzymatic reactions accompanied by electron transfer have been shown to be receptors of magnetic fields, and spin-dependent ion-radical processes can be a universal mechanism of biological magnetosensitivity. Magnetic interactions in intermediate ion-radical pairs, such as Zeeman and hyperfine (HFI) interactions, in accordance with proposed strict quantum mechanical theory, can determine magnetic-field dependencies of reactions that produce biologically important molecules needed for cell growth. Hyperfine interactions of electrons with nuclear magnetic moments of magnetic isotopes can explain the most important part of biomagnetic sensitivities in a weak magnetic field comparable to the Earth's magnetic field. The theoretical results mean that magnetic-field dependencies of enzymatic reaction rates in a weak magnetic field that can be independent of HFI constant a, if H < a, and are determined by the rate constant of chemical transformations in the enzyme active site. Both Zeeman and HFI interactions predict strong magnetic-field dependence in weak magnetic fields and magnetic-field independence of enzymatic reaction rate constants in strong magnetic fields. The theoretical results can explain the magnetic sensitivity of E. coli cell and demonstrate that intracellular enzymatic reactions are primary magnetoreceptors in living organisms. Bioelectromagnetics. 38:511-521, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  6. Hyposialylation of neprilysin possibly affects its expression and enzymatic activity in hereditary inclusion-body myopathy muscle.

    PubMed

    Broccolini, Aldobrando; Gidaro, Teresa; De Cristofaro, Raimondo; Morosetti, Roberta; Gliubizzi, Carla; Ricci, Enzo; Tonali, Pietro A; Mirabella, Massimiliano

    2008-05-01

    Autosomal recessive hereditary inclusion-body myopathy (h-IBM) is caused by mutations of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, a rate-limiting enzyme in the sialic acid metabolic pathway. Previous studies have demonstrated an abnormal sialylation of glycoproteins in h-IBM. h-IBM muscle shows the abnormal accumulation of proteins including amyloid-beta (Abeta). Neprilysin (NEP), a metallopeptidase that cleaves Abeta, is characterized by the presence of several N-glycosylation sites, and changes in these sugar moieties affect its stability and enzymatic activity. In the present study, we found that NEP is hyposialylated and its expression and enzymatic activity reduced in all h-IBM muscles analyzed. In vitro, the experimental removal of sialic acid by Vibrio Cholerae neuraminidase in cultured myotubes resulted in reduced expression of NEP. This was most likely because of a post-translational modification consisting in an abnormal sialylation of the protein that leads to its reduced stability. Moreover, treatment with Vibrio Cholerae neuraminidase was associated with an increased immunoreactivity for Abeta mainly in the form of distinct cytoplasmic foci within myotubes. We hypothesize that, in h-IBM muscle, hyposialylated NEP has a role in hampering the cellular Abeta clearing system, thus contributing to its abnormal accumulation within vulnerable fibers and possibly promoting muscle degeneration.

  7. 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.

  8. Exome-wide association study identifies genetic polymorphisms of C12orf51, MYL2, and ALDH2 associated with blood lead levels in the general Korean population.

    PubMed

    Eom, Sang-Yong; Hwang, Myung Sil; Lim, Ji-Ae; Choi, Byung-Sun; Kwon, Ho-Jang; Park, Jung-Duck; Kim, Yong-Dae; Kim, Heon

    2017-02-17

    Lead (Pb) is a ubiquitous toxic metal present in the environment that poses adverse health effects to humans. Inter-individual variation in blood Pb levels is affected by various factors, including genetic makeup. However, limited data are available on the association between genetic variation and blood Pb levels. The purpose of this study was to identify the genetic markers associated with blood Pb levels in the Korean population. The study subjects consisted of 1,483 healthy adults with no history of occupational exposure to Pb. We measured blood Pb levels and calculated probable daily intake of Pb according to dietary data collected using 24-hour recall. We conducted exome-wide association screening using Illumina Human Exome-12v1.2 platform (n = 500) and a replication analysis using VeraCode Goldengate assay (n = 1,483). Among the 244,770 single nucleotide polymorphisms (SNPs) tested, 12 SNPs associated with blood Pb level were identified, with suggestive significance level (P < 1 × 10 -4 ). In the Goldengate assay for replication, three SNPs (C12orf51 rs11066280, MYL2 rs12229654, and ALDH2 rs671) were associated with statistically suggestively significant differences in blood Pb levels. When stratified by drinking status, a potential association of C12orf51 rs11066280, MYL2 rs12229654, and ALDH2 rs671 with blood Pb level was observed only in drinkers. A marginally significant gene-environment interaction between ALDH2 rs671 and alcohol consumption was observed in relation to blood Pb levels. The effects of the three suggestively significant SNPs on blood Pb levels was dependent on daily calcium intake amounts. This exome-wide association study indicated that C12orf51 rs11066280, MYL2 rs12229654, and ALDH2 rs671 polymorphisms are linked to blood Pb levels in the Korean population. Our results suggest that these three SNPs are involved in the determination of Pb levels in Koreans via the regulation of alcohol drinking behavior, and that their

  9. Asparatic acid 221 is critical in the calcium-induced modulation of the enzymatic activity of human aminopeptidase A.

    PubMed

    Goto, Yoshikuni; Hattori, Akira; Mizutani, Shigehiko; Tsujimoto, Masafumi

    2007-12-21

    Aminopeptidase A (APA) plays an important role in the regulation of blood pressure by mediating angiotensin II degradation in the renin-angiotensin system. The Ca2+-induced modulation of enzymatic activity is the most characteristic feature of APA among the M1 family of aminopeptidases. In this study, we used site-directed mutagenesis for any residues responsible for the Ca2+ modulation of human APA. Alignment of sequences of the M1 family members led to the identification of Asp-221 as a significant residue of APA among the family members. Replacement of Asp-221 with Asn or Gln resulted in a loss of Ca2+ responsiveness toward synthetic substrates. These enzymes were also unresponsive to Ca2+ when peptide hormones, such as angiotensin II, cholecystokinin-8, neurokinin B, and kallidin, were employed as substrates. These results suggest that the negative charge of Asp-221 is essential for Ca2+ modulation of the enzymatic activity of APA and causes preferential cleavage of acidic amino acid at the N-terminal end of substrate peptides.

  10. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.

    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, andmore » 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.« less

  11. 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.

  12. A Comparison of Protein Kinases Inhibitor Screening Methods Using Both Enzymatic Activity and Binding Affinity Determination

    PubMed Central

    Rudolf, Amalie Frederikke; Skovgaard, Tine; Knapp, Stefan; Jensen, Lars Juhl; Berthelsen, Jens

    2014-01-01

    Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening conditions. Furthermore a marked improvement in the correlation was found by using kinase constructs containing the catalytic domain in presence of additional domains or subunits. PMID:24915177

  13. Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks

    DOE PAGES

    Zhao, Suwen; Sakai, Ayano; Zhang, Xinshuai; ...

    2014-06-30

    Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins inmore » 12 families in the PRS that represent ~85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes.« less

  14. Proinflammatory Actions of Visfatin/Nicotinamide Phosphoribosyltransferase (Nampt) Involve Regulation of Insulin Signaling Pathway and Nampt Enzymatic Activity*

    PubMed Central

    Jacques, Claire; Holzenberger, Martin; Mladenovic, Zvezdana; Salvat, Colette; Pecchi, Emilie; Berenbaum, Francis; Gosset, Marjolaine

    2012-01-01

    Visfatin (also termed pre-B-cell colony-enhancing factor (PBEF) or nicotinamide phosphoribosyltransferase (Nampt)) is a pleiotropic mediator acting on many inflammatory processes including osteoarthritis. Visfatin exhibits both an intracellular enzymatic activity (nicotinamide phosphoribosyltransferase, Nampt) leading to NAD synthesis and a cytokine function via the binding to its hypothetical receptor. We recently reported the role of visfatin in prostaglandin E2 (PGE2) synthesis in chondrocytes. Here, our aim was to characterize the signaling pathways involved in this response in exploring both the insulin receptor (IR) signaling pathway and Nampt activity. IR was expressed in human and murine chondrocytes, and visfatin triggered Akt phosphorylation in murine chondrocytes. Blocking IR expression with siRNA or activity using the hydroxy-2-naphthalenyl methyl phosphonic acid tris acetoxymethyl ester (HNMPA-(AM)3) inhibitor diminished visfatin-induced PGE2 release in chondrocytes. Moreover, visfatin-induced IGF-1R−/− chondrocytes released higher concentration of PGE2 than IGF-1R+/+ cells, a finding confirmed with an antibody that blocked IGF-1R. Using RT-PCR, we found that visfatin did not regulate IR expression and that an increased insulin release was also unlikely to be involved because insulin was unable to increase PGE2 release. Inhibition of Nampt activity using the APO866 inhibitor gradually decreased PGE2 release, whereas the addition of exogenous nicotinamide increased it. We conclude that the proinflammatory actions of visfatin in chondrocytes involve regulation of IR signaling pathways, possibly through the control of Nampt enzymatic activity. PMID:22399297

  15. 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.

  16. 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

  17. 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.

  18. [Enzymatic characteristics of peroxidase from Chrysanthemum morifolium cv. Bo-ju].

    PubMed

    Zhu, Yu-Yun; Lyu, Xin-Lin; Li, Xiang-Wei; Zhang, Dong; Dong, Li-Hua; Zhu, Jing-Jing; Wang, Zhi-Min; Zhang, Jin-Zhen

    2018-04-01

    The enzymatic browning is one of the main reasons for affecting the quality of medicinal flowers. In the process of chrysanthemum harvesting and processing, improper treatment will lead to the browning and severely impact the appearance and quality of chrysanthemum. Peroxidase enzyme is one of the oxidoreductases that cause enzymatic browning of fresh chrysanthemum. The enzymatic characteristics of peroxidase (POD) in chrysanthemum were studied in this paper. In this experiment, the effects of different reaction substrates and their concentrations, PH value of buffer and reaction temperatures on the activity of POD enzyme were investigated. The results showed that the optimal substrate of POD was guaiacol, and the optimal concentration of POD was 50 mmol·L⁻¹. The optimal pH value and reaction temperature were 4.4 and 30-35 °C, respectively. Michaelis-Menten equation was obtained to express the kinetics of enzyme-catalyzed reaction of POD, Km=0.193 mol·L⁻¹, Vmax=0.329 D·min⁻¹. In addition, the results of POD enzyme thermal stability test showed that the POD enzyme activity was inhibited when being treated at 80 °C for 4 min or at 100 °C for 2 min. The above results were of practical significance to reveal the enzymatic browning mechanism, control the enzymatic browning and improve the quality of chrysanthemum, and can also provide the basis for the harvesting and processing of medicinal materials containing polyphenols. Copyright© by the Chinese Pharmaceutical Association.

  19. Ultraviolet Radiation: Cellular Antioxidant Response and the Role of Ocular Aldehyde Dehydrogenase Enzymes

    PubMed Central

    Marchitti, Satori A.; Chen, Ying; Thompson, David C.; Vasiliou, Vasilis

    2011-01-01

    Solar ultraviolet radiation (UVR) exposes the human eye to near constant oxidative stress. Evidence suggests that UVR is the most important environmental insult leading to the development of a variety of ophthalmoheliosis disorders. UVR-induced reactive oxygen species are highly reactive with DNA, proteins and cellular membranes, resulting in cellular and tissue damage. Antioxidant defense systems present in ocular tissues function to combat reactive oxygen species and protect the eye from oxidative damage. Important enzymatic antioxidants are the superoxide dismutases, catalase, glutathione peroxidases, glutathione reductase and members of the aldehyde dehydrogenase (ALDH) superfamily. Glutathione, ascorbic and uric acids, α-tocopherol, NADPH and ferritin serve as small molecule, nonenzymatic antioxidants. Ocular tissues have high levels of these antioxidants which are essential for the maintenance of redox homeostasis in the eye and protection against oxidative damage. ALDH1A1 and ALDH3A1, present abundantly in the cornea and lens, have been shown to have unique roles in the defense against UVR and the downstream effects of oxidative stress. This review presents the properties and functions of ocular antioxidants that play critical roles in the cellular response to UVR exposure, including a focused discussion of the unique roles that the ALDH1A1 and ALDH3A1 enzymes have as multi-functional ocular antioxidants. PMID:21670692

  20. Swimming training attenuates oxidative damage and increases enzymatic but not non-enzymatic antioxidant defenses in the rat brain.

    PubMed

    Nonato, L F; Rocha-Vieira, E; Tossige-Gomes, R; Soares, A A; Soares, B A; Freitas, D A; Oliveira, M X; Mendonça, V A; Lacerda, A C; Massensini, A R; Leite, H R

    2016-09-29

    Although it is well known that physical training ameliorates brain oxidative function after injuries by enhancing the levels of neurotrophic factors and oxidative status, there is little evidence addressing the influence of exercise training itself on brain oxidative damage and data is conflicting. This study investigated the effect of well-established swimming training protocol on lipid peroxidation and components of antioxidant system in the rat brain. Male Wistar rats were randomized into trained (5 days/week, 8 weeks, 30 min; n=8) and non-trained (n=7) groups. Forty-eight hours after the last session of exercise, animals were euthanized and the brain was collected for oxidative stress analysis. Swimming training decreased thiobarbituric acid reactive substances (TBARS) levels (P<0.05) and increased the activity of the antioxidant enzyme superoxide dismutase (SOD) (P<0.05) with no effect on brain non-enzymatic total antioxidant capacity, estimated by FRAP (ferric-reducing antioxidant power) assay (P>0.05). Moreover, the swimming training promoted metabolic adaptations, such as increased maximal workload capacity (P<0.05) and maintenance of body weight. In this context, the reduced TBARS content and increased SOD antioxidant activity induced by 8 weeks of swimming training are key factors in promoting brain resistance. In conclusion, swimming training attenuated oxidative damage and increased enzymatic antioxidant but not non-enzymatic status in the rat brain.

  1. Enzymatic hydrolysis of potato pulp.

    PubMed

    Lesiecki, Mariusz; Białas, Wojciech; Lewandowicz, Grażyna

    2012-01-01

    Potato pulp constitutes a complicated system of four types of polysaccharides: cellulose, hemicellulose, pectin and starch. Its composition makes it a potential and attractive raw material for the production of the second generation bioethanol. The aim of this research project was to assess the usefulness of commercial enzymatic preparations for the hydrolysis of potato pulp and to evaluate the effectiveness of hydrolysates obtained in this way as raw materials for ethanol fermentation. Sterilised potato pulp was subjected to hydrolysis with commercial enzymatic preparations. The effectiveness of the preparations declared as active towards only one fraction of potato pulp (separate amylase, pectinase and cellulase activity) and mixtures of these preparations was analysed. The monomers content in hydrolysates was determined using HPLC method. The application of amylolytic enzymes for potato pulp hydrolysis resulted in the release of only 18% of raw material with glucose as the dominant (77%) constituent of the formed product. In addition, 16% galactose was also determined in it. The hydrolysis of the cellulose fraction yielded up to 35% raw material and the main constituents of the obtained hydrolysate were glucose (46%) and arabinose (40%). Simultaneous application of amylolytic, cellulolytic and pectinolytic enzymes turned out to be the most effective way of carrying out the process as its efficiency in this case reached 90%. The obtained hydrolysate contained 63% glucose, 25% arabinose and 12% other simple substances. The application of commercial enzymatic preparations made it possible to perform potato pulp hydrolysis with 90% effectiveness. This was achieved by the application of a complex of amylolytic, cellulolytic and pectinolytic enzymes and the hydrolysate obtained in this way contained, primarily, glucose making it a viable substrate for ethanol fermentation.

  2. Allergenicity, trypsin inhibitor activity and nutritive quality of enzymatically modified soy proteins.

    PubMed

    De La Barca, Ana María Calderón; Wall, Abraham; López-Díaz, José Alberto

    2005-05-01

    Two ultrafiltered soy flour protein fractions were evaluated; the first was obtained by hydrolysis (0.5-3 kDa, F(0.5-3)), and the second was an enzymatically methionine-enriched fraction (1-10 kDa, F(1-10)E). Amino acid profiles, protein quality, allergenicity (against soy-sensitive infant sera) and trypsin inhibitor activity were determined. Fraction F(1-10)E fulfilled amino acid requirements for infants, whereas the F(0.5-3) fraction was methionine deficient. Both fractions were similar in net protein utilization, and F(1-10)E digestibility was comparable with casein and higher (P?activity with respect to soy flour was 8.1%, 3.3% and 1% for hydrolysate, F(1-10)E and F(0.5-3), respectively. Both fractions presented high nutritive quality and reduced or null allergenicity. The trypsin inhibitor activity decreased along processing and could be a useful indicator for production of hypoallergenic proteins.

  3. Role of low density lipoprotein in the activation of plasma lysolecithin acyltransferase activity. Effect of chemical and enzymatic modifications of the lipoprotein on enzyme activity.

    PubMed

    Subbaiah, P V; Chen, C H; Bagdade, J D; Albers, J J

    1985-01-01

    The effect of various chemical and enzymatic modifications of low density lipoprotein (LDL) on its ability to activate the isolated human plasma lysolecithin acyltransferase (LAT) was studied. Removal of all lipids from LDL resulted in the complete loss of LAT activation. Removal of only neutral lipids by extraction with heptane retained up to 50% of the original activity, which was not increased further by reconstitution of the LDL with the extracted lipids. Hydrolysis of the diacylphosphoglycerides of the LDL with phospholipases resulted in complete loss of LAT activation which was partially restored by the addition of egg lecithin. Hydrolysis of more than 4% of LDL protein by trypsin led to a linear decrease in activity with complete loss of activity occurring when about 25% of the LDL protein is hydrolyzed. Modification of the arginine groups of LDL reversibly inhibited the activation of LAT. Modification of lysine residues of LDL by acetylation, acetoacetylation or succinylation also abolished its ability to activate lysolecithin acylation.

  4. STAT3 signaling pathway is necessary for cell survival and tumorsphere forming capacity in ALDH{sup +}/CD133{sup +} stem cell-like human colon cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lin, Li, E-mail: lin.796@osu.edu; Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030; Fuchs, James

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer The phosphorylated or activated form of STAT3 was expressed in colon cancer stem-like cells. Black-Right-Pointing-Pointer STAT3 inhibitor, FLLL32 inhibits P-STAT3 and STAT3 target genes in colon cancer stem-like cells. Black-Right-Pointing-Pointer Inhibition of STAT3 resulted in decreased cell viability and reduced numbers of tumorspheres. Black-Right-Pointing-Pointer STAT3 is required for survival and tumorsphere forming capacity in colon cancer stem-like cells. Black-Right-Pointing-Pointer Targeting STAT3 in cancer stem-like cells may offer a novel treatment approach for colon cancer. -- Abstract: Persistent activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequently detected in colon cancer. Increasing evidence suggests the existencemore » of a small population of colon cancer stem or cancer-initiating cells may be responsible for tumor initiation, metastasis, and resistance to chemotherapy and radiation. Whether STAT3 plays a role in colon cancer-initiating cells and the effect of STAT3 inhibition is still unknown. Flow cytometry was used to isolate colon cancer stem-like cells from three independent human colon cancer cell lines characterized by both aldehyde dehydrogenase (ALDH)-positive and CD133-positive subpopulation (ALDH{sup +}/CD133{sup +}). The effects of STAT3 inhibition in colon cancer stem-like cells were examined. The phosphorylated or activated form of STAT3 was expressed in colon cancer stem-like cells and was reduced by a STAT3-selective small molecular inhibitor, FLLL32. FLLL32 also inhibited the expression of potential STAT3 downstream target genes in colon cancer stem-like cells including survivin, Bcl-XL, as well as Notch-1, -3, and -4, which may be involved in stem cell function. Furthermore, FLLL32 inhibited cell viability and tumorsphere formation as well as induced cleaved caspase-3 in colon cancer stem-like cells. FLLL32 is more potent than curcumin as evidenced

  5. Downregulation of Rubisco Activity by Non-enzymatic Acetylation of RbcL.

    PubMed

    Gao, Xiang; Hong, Hui; Li, Wei-Chao; Yang, Lili; Huang, Jirong; Xiao, You-Li; Chen, Xiao-Ya; Chen, Gen-Yun

    2016-07-06

    Atmospheric carbon dioxide (CO2) is assimilated by the most abundant but sluggish enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Here we show that acetylation of lysine residues of the Rubisco large subunit (RbcL), including Lys201 and Lys334 in the active sites, may be an important mechanism in the regulation of Rubisco activities. It is well known that Lys201 reacts with CO2 for carbamylation, a prerequisite for both carboxylase and oxygenase activities of Rubisco, and Lys334 contacts with ribulose-1,5-bisphosphate (RuBP). The acetylation level of RbcL in plants is lower during the day and higher at night, inversely correlating with the Rubisco carboxylation activity. A search of the chloroplast proteome database did not reveal a canonical acetyltransferase; instead, we found that a plant-derived metabolite, 7-acetoxy-4-methylcoumarin (AMC), can non-enzymatically acetylate both native Rubisco and synthesized RbcL peptides spanning Lys334 or Lys201. Furthermore, lysine residues were modified by synthesized 4-methylumbelliferone esters with different electro- and stereo-substitutes, resulting in varied Rubisco activities. 1-Chloroethyl 4-methylcoumarin-7-yl carbonate (ClMC) could transfer the chloroethyl carbamate group to lysine residues of RbcL and completely inactivate Rubisco, whereas bis(4-methylcoumarin-7-yl) carbonate (BMC) improved Rubisco activity through increasing the level of Lys201 carbamylation. Our findings indicate that RbcL acetylation negatively regulates Rubisco activity, and metabolic derivatives can be designed to dissect and improve CO2 fixation efficiency of plants through lysine modification. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  6. Cloning of a cDNA encoding rat aldehyde dehydrogenase with high activity for retinal oxidation.

    PubMed

    Bhat, P V; Labrecque, J; Boutin, J M; Lacroix, A; Yoshida, A

    1995-12-12

    Retinoic acid (RA), an important regulator of cell differentiation, is biosynthesized from retinol via retinal by a two-step oxidation process. We previously reported the purification and partial amino acid (aa) sequence of a rat kidney aldehyde dehydrogenase (ALDH) isozyme that catalyzed the oxidation of 9-cis and all-trans retinal to corresponding RA with high efficiency [Labrecque et al. Biochem. J. 305 (1995) 681-684]. A rat kidney cDNA library was screened using a 291-bp PCR product generated from total kidney RNA using a pair of oligodeoxyribonucleotide primers matched with the aa sequence. The full-length rat kidney ALDH cDNA contains a 2315-bp (501 aa) open reading frame (ORF). The aa sequence of rat kidney ALDH is 89, 96 and 87% identical to that of the rat cytosolic ALDH, the mouse cytosolic ALDH and human cytosolic ALDH, respectively. Northern blot and RT-PCR-mediated analysis demonstrated that rat kidney ALDH is strongly expressed in kidney, lung, testis, intestine, stomach and trachea, but weakly in the liver.

  7. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Beneficial effect of mixture of additives amendment on enzymatic activities, organic matter degradation and humification during biosolids co-composting.

    PubMed

    Awasthi, Mukesh Kumar; Wang, Quan; Chen, Hongyu; Awasthi, Sanjeev Kumar; Wang, Meijing; Ren, Xiuna; Zhao, Junchao; Zhang, Zengqiang

    2018-01-01

    The objective of this study was to identify the effect of mixture of additives to improve the enzymatic activities, organic matter humification and diminished the bioavailability of heavy metals (HMs) during biosolids co-composting. In this study, zeolite (Z) (10%, 15% and 30%) with 1%lime (L) (dry weight basis of biosolids) was blended into the mixture of biosolids and wheat straw, respectively. The without any amendment and 1%lime applied treatments were run for comparison (Control). The Z+L addition resulted rapid organic matter degradation and humification with maximum enzymatic activities. In addition, higher dosage of Z+1%L amendment reduced the bioavailability of HMs (Cu and Zn) and improved the end product quality as compared to control and 1%L applied treatments. However, the 30%Z+1%L applied treatment showed maximum humification and low bioavailability of HMs but considering the economic feasibility and compost quality results, the treatment with 10%Z+1%L is recommended for biosolids co-composting. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. A Multiplex Enzymatic Machinery for Cellular Protein S-nitrosylation.

    PubMed

    Seth, Divya; Hess, Douglas T; Hausladen, Alfred; Wang, Liwen; Wang, Ya-Juan; Stamler, Jonathan S

    2018-02-01

    S-nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), modifies all main classes of proteins and provides a fundamental redox-based cellular signaling mechanism. However, in contrast to other post-translational protein modifications, S-nitrosylation is generally considered to be non-enzymatic, involving multiple chemical routes. We report here that endogenous protein S-nitrosylation in the model organism E. coli depends principally upon the enzymatic activity of the hybrid cluster protein Hcp, employing NO produced by nitrate reductase. Anaerobiosis on nitrate induces both Hcp and nitrate reductase, thereby resulting in the S-nitrosylation-dependent assembly of a large interactome including enzymes that generate NO (NO synthase), synthesize SNO-proteins (SNO synthase), and propagate SNO-based signaling (trans-nitrosylases) to regulate cell motility and metabolism. Thus, protein S-nitrosylation by NO in E. coli is essentially enzymatic, and the potential generality of the multiplex enzymatic mechanism that we describe may support a re-conceptualization of NO-based cellular signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Enzymatic processing of pigmented and non pigmented rice bran on changes in oryzanol, polyphenols and antioxidant activity.

    PubMed

    Prabhu, Ashish A; Jayadeep, A

    2015-10-01

    Bran from different rice varieties is a treasure of nutrients and nutraceuticals, and its use is limited due to the poor sensory and functional properties. Application of enzymes can alter the functional and phytochemical properties. So the effect of endo-xylanase, cellulase and their combination on microstructural, nutraceutical and antioxidant properties of pigmented (Jyothi) and non-pigmented (IR64) rice bran were investigated. Scanning electron micrograph revealed micro structural changes in fibre structures on processing. All the enzymatic processing methods resulted in an increase in the content of oryzanol, soluble, bound and total polyphenols, flavonoid and tannin. It also showed an increase in the bioactivity with respect to free radical scavenging activity and total antioxidant activity. However, extent of the increase in bio-actives varied with the type of bran and enzyme application method. Endo-xylanase showed higher percentage difference compared to controls of Jyothi and IR64 bran extracts respectively in the content of the bound (10 & 19 %) and total (20 & 14 %) polyphenols. Combination of both the enzymes resulted in higher percentage increase of bioactive components and properties. It resulted in greater percentage difference compared to controls of Jyothi and IR64 extracts respectively in the content of soluble (58 & 17 %) and total (21 & 14 %) polyphenols, flavonoids (12 & 38 %), γ-oryzanol (10 & 12 %), free radical scavenging activity (64 & 30 %) and total antioxidant activity (82 & 136 %). It may be concluded that enzymatic bio-processing of bran with cellulose and hemicellulose degrading enzymes can improve its nutraceutical properties, and it may be used for development of functional foods.

  11. 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

  12. Improved measurement of extracellular enzymatic activities in subsurface sediments using competitive desorption treatment

    NASA Astrophysics Data System (ADS)

    Hoarfrost, Adrienne; Snider, Rachel; Arnosti, Carol

    2017-02-01

    Extracellular enzymatic activities initiate microbially-driven heterotrophic carbon cycling in subsurface sediments. While measurement of hydrolytic activities in sediments is fundamental to our understanding of carbon cycling, these measurements are often technically difficult due to sorption of organic substrates to the sediment matrix. Most methods that measure hydrolysis of organic substrates in sediments rely on recovery of a fluorophore or fluorescently-labeled target substrate from a sediment incubation. The tendency for substrates to sorb to sediments results in lower recovery of an added substrate, and can result in data that are unusable or difficult to interpret. We developed a treatment using competitive desorption of a fluorescently-labeled, high molecular weight organic substrate that improves recovery of the labeled substrate from sediment subsamples. Competitive desorption treatment improved recovery of the fluorescent substrate by a median of 66%, expanded the range of sediments for which activity measurements could be made, and was effective in sediments from a broad range of geochemical contexts. More reliable measurements of hydrolytic activities in sediments will yield usable and more easily interpretable data from a wider range of sedimentary environments, enabling better understanding of microbially-catalyzed carbon cycling in subsurface environments.

  13. Microbial production of metabolites and associated enzymatic reactions under high pressure.

    PubMed

    Dong, Yongsheng; Jiang, Hua

    2016-11-01

    High environmental pressure exerts an external stress on the survival of microorganisms that are commonly found under normal pressure. In response, many growth traits alter, including cell morphology and physiology, cellular structure, metabolism, physical and chemical properties, the reproductive process, and defense mechanisms. The high-pressure technology (HP) has been industrially utilized in pressurized sterilization, synthesis of stress-induced products, and microbial/enzymatic transformation of chemicals. This article reviews current research on pressure-induced production of metabolites in normal-pressure microbes and their enzymatic reactions. Factors that affect the production of such metabolites are summarized, as well as the effect of pressure on the performance of microbial fermentation and the yield of flavoring compounds, different categories of induced enzymatic reactions and their characteristics in the supercritical carbon dioxide fluid, effects on enzyme activity, and the selection of desirable bacterial strains. Technological challenges are discussed, and future research directions are proposed. Information presented here will benefit the research, development, and application of the HP technology to improve microbial fermentation and enzymatic production of biologically active substances, thereby help to meet their increasing demand from the ever-expanding market.

  14. Enzymatic browning and antioxidant activities in harvested litchi fruit as influenced by apple polyphenols.

    PubMed

    Zhang, Zhengke; Huber, Donald J; Qu, Hongxia; Yun, Ze; Wang, Hui; Huang, Zihui; Huang, Hua; Jiang, Yueming

    2015-03-15

    'Guiwei' litchi fruit were treated with 5 ga.i. L(-1) apple polyphenols (APP) and then stored at 25°C to investigate the effects on pericarp browning. APP treatment effectively reduced pericarp browning and retarded the loss of red colour. APP-treated fruit exhibited higher levels of anthocyanins and cyanidin-3-rutinoside, which correlated with suppressed anthocyanase activity. APP treatment also maintained membrane integrity and reduced oxidative damage, as indicated by a lower relative leakage rate, malondialdehyde content, and reactive oxygen species (ROS) generation. The data suggest that decompartmentalisation of peroxidase and polyphenoloxidase and respective browning substrates was reduced. In addition, APP treatment enhanced the activities of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), as well as non-enzymatic antioxidant capacity (DPPH radical-scavenging activity and reducing power), which might be beneficial in scavenging ROS. We propose that APP treatment is a promising safe strategy for controlling postharvest browning of litchi fruit. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Quinolone resistance-associated amino acid substitutions affect enzymatic activity of Mycobacterium leprae DNA gyrase.

    PubMed

    Yamaguchi, Tomoyuki; Yokoyama, Kazumasa; Nakajima, Chie; Suzuki, Yasuhiko

    2017-07-01

    Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.

  16. Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase.

    PubMed

    Coetzer, C; Corsini, D; Love, S; Pavek, J; Tumer, N

    2001-02-01

    Polyphenol oxidase (PPO) activity of Russet Burbank potato was inhibited by sense and antisense PPO RNAs expressed from a tomato PPO cDNA under the control of the 35S promoter from the cauliflower mosaic virus. Transgenic Russet Burbank potato plants from 37 different lines were grown in the field. PPO activity and the level of enzymatic browning were measured in the tubers harvested from the field. Of the tubers from 28 transgenic lines that were sampled, tubers from 5 lines exhibited reduced browning. The level of PPO activity correlated with the reduction in enzymatic browning in these lines. These results indicate that expression of tomato PPO RNA in sense or antisense orientation inhibits PPO activity and enzymatic browning in the major commercial potato cultivar. Expression of tomato PPO RNA in sense orientation led to the greatest decrease in PPO activity and enzymatic browning, possibly due to cosuppression. These results suggest that expression of closely related heterologous genes can be used to prevent enzymatic browning in a wide variety of food crops without the application of various food additives.

  17. Epidermal growth factor/heat shock protein 27 pathway regulates vasculogenic mimicry activity of breast cancer stem/progenitor cells.

    PubMed

    Lee, Che-Hsin; Wu, Yu-Ting; Hsieh, Hung-Chun; Yu, Yun; Yu, Alice L; Chang, Wen-Wei

    2014-09-01

    Tumor vascularization, which is mainly contributed by angiogenesis and vascularization, is necessary for tumor maintenance and progression. Vasculogenic mimicry (VM), vascular-like channels which are lack of the involvement of endothelial cells, has been observed in aggressive cancers and also involves in tumor vascularization. Breast cancer stem/progenitor cells (BCSCs) have been identified as a subpopulation of breast cancer cells with markers of CD24(-)CD44(+), high aldehyde dehydrogenase activity (ALDH(+)) or could be enriched by mammosphere cultivation. These cells have been proven to be associated with tumor vascularization. Here we investigated the molecular mechanisms in VM activity of BCSCs. By periodic acid-Schiff or hematoxylin-eosin stain, we found that there were VM structures in two xenografted human breast cancer tissues established from CD24(-)CD44(+) or ALDH(+) cells. Only ALDH(+) or mammosphere-forming BCSCs could form tube structures on matrigel-coated surface as similar as microvascular endothelial cells. Inhibition of the phosphorylation of epidermal growth factor receptor (EGFR) by gefitinib or knockdown of EGFR by lentiviral shRNA abolished the in vitro VM activity of BCSCs. By quercetin treatment, a plant flavonoid compound which is known to suppress heat shock proteins, or siRNA-mediated gene silencing, both Hsp27 expression and VM capability of BCSCs were suppressed. Forced expression of phosphor-mimic form of Hsp27 in ALDH(+) BCSCs could overcome the inhibitory effect of gefitinib. In conclusion, our data demonstrate that VM activity of BCSCs is mediated by EGF/Hsp27 signaling and targeting this pathway may benefit to breast cancer therapy. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  18. 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.

  19. White grape pomace extracts, obtained by a sequential enzymatic plus ethanol-based extraction, exert antioxidant, anti-tyrosinase and anti-inflammatory activities.

    PubMed

    Ferri, Maura; Rondini, Greta; Calabretta, Maria Maddalena; Michelini, Elisa; Vallini, Veronica; Fava, Fabio; Roda, Aldo; Minnucci, Giordano; Tassoni, Annalisa

    2017-10-25

    The present work aimed at optimizing a two-step enzymatic plus solvent-based process for the recovery of bioactive compounds from white grape (Vitis vinifera L., mix of Trebbiano and Verdicchio cultivars) pomace, the winemaking primary by-product. Phenolic compounds solubilised by water enzyme-assisted and ethanol-based extractions of wet (WP) and dried (DP) pomace were characterised for composition and tested for antioxidant, anti-tyrosinase and anti-inflammatory bioactivities. Ethanol treatment led to higher phenol yields than water extraction, while DP samples showed the highest capacity of releasing polyphenols, most probably as a positive consequence of the pomace drying process. Different compositions and bioactivities were observed between water and ethanol extracts and among different treatments and for the first time the anti-tyrosinase activity of V. vinifera pomace extracts, was here reported. Enzymatic treatments did not significantly improve the total amount of solubilised compounds; Celluclast in DP led to the recovery of extracts enriched in specific compounds, when compared to control. The best extracts (enzymatic plus ethanol treatment total levels) were obtained from DP showing significantly higher amounts of polyphenols, flavonoids, flavanols and tannins and exerted higher antioxidant and anti-tyrosinase activities than WP total extracts. Conversely, anti-inflammatory capacity was only detected in water (with and without enzyme) extracts, with WP samples showing on average a higher activity than DP. The present findings demonstrate that white grape pomace constitute a sustainable source for the extraction of phytochemicals that might be exploited as functional ingredients in the food, nutraceutical, pharmaceutical or cosmetic industries. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Ultrasound assisted enzymatic depolymerization of aqueous guar gum solution.

    PubMed

    Prajapat, Amrutlal L; Subhedar, Preeti B; Gogate, Parag R

    2016-03-01

    The present work investigates the effectiveness of application of low intensity ultrasonic irradiation for the intensification of enzymatic depolymerization of aqueous guar gum solution. The extent of depolymerization of guar gum has been analyzed in terms of intrinsic viscosity reduction. The effect of ultrasonic irradiation on the kinetic and thermodynamic parameters related to the enzyme activity as well as the intrinsic viscosity reduction of guar gum using enzymatic approach has been evaluated. The kinetic rate constant has been found to increase with an increase in the temperature and cellulase loading. It has been observed that application of ultrasound not only enhances the extent of depolymerization but also reduces the time of depolymerization as compared to conventional enzymatic degradation technique. In the presence of cellulase enzyme, the maximum extent of depolymerization of guar gum has been observed at 60 W of ultrasonic rated power and ultrasonic treatment time of 30 min. The effect of ultrasound on the kinetic and thermodynamic parameters as well as the molecular structure of cellulase enzyme was evaluated with the help of the chemical reaction kinetics model and fluorescence spectroscopy. Application of ultrasound resulted in a reduction in the thermodynamic parameters of activation energy (Ea), enthalpy (ΔH), entropy (ΔS) and free energy (ΔG) by 47%, 50%, 65% and 1.97%, respectively. The changes in the chemical structure of guar gum treated using ultrasound assisted enzymatic approach in comparison to the native guar gum were also characterized by FTIR. The results revealed that enzymatic depolymerization of guar gum resulted in a polysaccharide with low degree of polymerization, viscosity and consistency index without any change in the core chemical structure which could make it useful for incorporation in food products. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas

    PubMed Central

    Rovira, Meritxell; Scott, Sherri-Gae; Liss, Andrew S.; Jensen, Jan; Thayer, Sarah P.; Leach, Steven D.

    2009-01-01

    The question of whether dedicated progenitor cells exist in adult vertebrate pancreas remains controversial. Centroacinar cells and terminal duct (CA/TD) cells lie at the junction between peripheral acinar cells and the adjacent ductal epithelium, and are frequently included among cell types proposed as candidate pancreatic progenitors. However these cells have not previously been isolated in a manner that allows formal assessment of their progenitor capacities. We have found that a subset of adult CA/TD cells are characterized by high levels of ALDH1 enzymatic activity, related to high-level expression of both Aldh1a1 and Aldh1a7. This allows their isolation by FACS using a fluorogenic ALDH1 substrate. FACS-isolated CA/TD cells are relatively depleted of transcripts associated with differentiated pancreatic cell types. In contrast, they are markedly enriched for transcripts encoding Sca1, Sdf1, c-Met, Nestin, and Sox9, markers previously associated with progenitor populations in embryonic pancreas and other tissues. FACS-sorted CA/TD cells are uniquely able to form self-renewing “pancreatospheres” in suspension culture, even when plated at clonal density. These spheres display a capacity for spontaneous endocrine and exocrine differentiation, as well as glucose-responsive insulin secretion. In addition, when injected into cultured embryonic dorsal pancreatic buds, these adult cells display a unique capacity to contribute to both the embryonic endocrine and exocrine lineages. Finally, these cells demonstrate dramatic expansion in the setting of chronic epithelial injury. These findings suggest that CA/TD cells are indeed capable of progenitor function and may contribute to the maintenance of tissue homeostasis in adult mouse pancreas. PMID:20018761

  2. Enzymatic activity and partial purification of solanapyrone synthase: first enzyme catalyzing Diels-Alder reaction.

    PubMed

    Katayama, K; Kobayashi, T; Oikawa, H; Honma, M; Ichihara, A

    1998-05-19

    In cell-free extracts of Alternaria solani, an enzymatic activity converting prosolanapyrone II to solanapyrones A and D via oxidation and subsequent Diels-Alder reaction has been found. Chromatography with DEAE-Sepharose provided two active fractions, pools 1 and 2. The former fraction converted prosolanapyrone II to solanapyrones A and D in a ratio of 2.2:1 with optical purities of 99% and 45% ee, respectively. The latter fraction did so in a ratio of 7.6:1 with 99% and nearly 0% ee, respectively. The enzyme partially purified from pool 2 native molecular weight of 40-62 kD and a pl of 4.25. The high reactivity of prosolanapyrone III in aqueous solution and the chromatographic behavior of the enzyme in pool 2 suggest that a single enzyme catalyzes both the oxidation and Diels-Alder reaction.

  3. Production of xylooligosaccharide from wheat bran by microwave assisted enzymatic hydrolysis.

    PubMed

    Wang, Tseng-Hsing; Lu, Shin

    2013-06-01

    The effective production of xylooligosaccharides (XOS) from wheat bran was investigated. Wheat bran contains rich hemicellulose which can be hydrolyzed by enzyme; the XOS were obtained by microwave assisted enzymatic hydrolysis. To improve the productivity of XOS, repeated microwave assisted enzymatic hydrolysis and activated carbon adsorption method was chosen to eliminate macromolecules in the XOS. On the basis of experimental data, an industrial XOS production process consisting of pretreatment, repeated microwave assisted enzymatic treatment and purification was designed. Using the designed process, 3.2g dry of purified XOS was produced from 50 g dry wheat bran powder. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Enzymatic generation of galactose-rich oligosaccharides/oligomers from potato rhamnogalacturonan I pectic polysaccharides.

    PubMed

    Khodaei, Nastaran; Karboune, Salwa

    2016-04-15

    Potato pulp by-product rich in galactan-rich rhamnogalacturonan I (RG I) was investigated as a new source of oligosaccharides with potential prebiotic properties. The efficiency of selected monocomponent enzymes and multi-enzymatic preparations to generate oligosaccharides/oligomers from potato RG I was evaluated. These overall results of yield were dependent on the activity profile of the multi-enzymatic preparations. Highest oligo-RG I yield of 93.9% was achieved using multi-enzymatic preparation (Depol 670L) with higher hydrolytic activity toward side chains of RG I as compared to its backbone. Main oligo-RG I products were oligosaccharides with DP of 2-12 (79.8-100%), while the oligomers with DP of 13-70 comprised smaller proportion (0.0-20.2%). Galactose (58.9-91.2%, w/w) was the main monosaccharide of oligo-RG I, while arabinose represented 0.0-12.1%. An understanding of the relationship between the activity profile of multi-enzymatic preparations and the yield/DP of oligo-RG I was achieved. This is expected to provide the capability to generate galacto- and galacto(arabino) oligosaccharides and their corresponding oligomers from an abundant by-product. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Novel enzymatic method for assaying Lp-PLA2 in serum.

    PubMed

    Yamaura, Saki; Sakasegawa, Shin-Ichi; Koguma, Emisa; Ueda, Shigeru; Kayamori, Yuzo; Sugimori, Daisuke; Karasawa, Ken

    2018-06-01

    Measurement of lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) can be used as an adjunct to traditional cardiovascular risk factors for identifying individuals at higher risk of cardiovascular events. This can be performed by quantification of the protein concentration using an ELISA platform or by measuring Lp-PLA 2 activity using platelet-activating factor (PAF) analog as substrate. Here, an enzymatic Lp-PLA 2 activity assay method using 1-O-Hexadecyl-2-acetyl-rac-glycero-3-phosphocholine (rac C 16 PAF) was developed. The newly revealed substrate specificity of lysoplasmalogen-specific phospholipase D (lysophospholipase D (LysoPLD)) was exploited. Lp-PLA 2 hydrolyzes 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C 16 PAF) to 1-O-Hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPAF). LysoPLD acted on LysoPAF, and the hydrolytically released choline was detected by choline oxidase. Regression analysis of Lp-PLA 2 activity measured by the enzymatic Lp-PLA 2 activity assay vs. two chemical Lp-PLA 2 activity assays, i.e. LpPLA 2 FS and PLAC® test, and ELISA, gave the following correlation coefficients: 0.990, 0.893 and 0.785, respectively (n = 30). Advantages of this enzymatic Lp-PLA 2 activity assay compared with chemical Lp-PLA 2 methods include the following; (i) only requires two reagents enabling a simple two-point linear calibration method with one calibrator (ii) no need for inhibitors of esterase-like activity in serum. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Impact of the redox-cycling herbicide diquat on transcript expression and antioxidant enzymatic activities of the freshwater snail Lymnaea stagnalis.

    PubMed

    Bouétard, Anthony; Besnard, Anne-Laure; Vassaux, Danièle; Lagadic, Laurent; Coutellec, Marie-Agnès

    2013-01-15

    The presence of pesticides in the environment results in potential unwanted effects on non-target species. Freshwater organisms inhabiting water bodies adjacent to agricultural areas, such as ditches, ponds and marshes, are good models to test such effects as various pesticides may reach these habitats through several ways, including aerial drift, run-off, and drainage. Diquat is a non-selective herbicide used for crop protection or for weed control in such water bodies. In this study, we investigated the effects of diquat on a widely spread aquatic invertebrate, the holarctic freshwater snail Lymnaea stagnalis. Due to the known redox-cycling properties of diquat, we studied transcript expression and enzymatic activities relative to oxidative and general stress in the haemolymph and gonado-digestive complex (GDC). As diquat is not persistent, snails were exposed for short times (5, 24, and 48 h) to ecologically relevant concentrations (22.2, 44.4, and 222.2 μg l(-1)) of diquat dibromide. RT-qPCR was used to quantify the transcription of genes encoding catalase (cat), a cytosolic superoxide dismutase (Cu/Zn-sod), a selenium-dependent glutathione peroxidase (gpx), a glutathione reductase (gred), the retinoid X receptor (rxr), two heat shock proteins (hsp40 and hsp70), cortactin (cor) and the two ribosomal genes r18S and r28s. Enzymatic activities of SOD, Gpx, Gred and glutathione S-transferase (GST) were investigated in the GDC using spectrophoto/fluorometric methods. Opposite trends were obtained in the haemolymph depending on the herbicide concentration. At the lowest concentration, effects were mainly observed after 24 h of exposure, with over-transcription of cor, hsp40, rxr, and sod, whereas higher concentrations down-regulated the expression of most of the studied transcripts, especially after 48 h of exposure. In the GDC, earlier responses were observed and the fold-change magnitude was generally much higher: transcription of all target genes increased

  7. Kinetic and biophysical investigation of the inhibitory effect of caffeine on human salivary aldehyde dehydrogenase: Implications in oral health and chemotherapy

    NASA Astrophysics Data System (ADS)

    Laskar, Amaj Ahmed; Alam, Md Fazle; Ahmad, Mohammad; Younus, Hina

    2018-04-01

    Human salivary aldehyde dehydrogenase (hsALDH) is primarily a class 3 ALDH (ALDH3A1), and is an important antioxidant enzyme present in the saliva which maintains healthy oral cavity. It detoxifies toxic aldehydes into non-toxic carboxylic acids in the oral cavity. Reduced level of hsALDH activity is a risk factor for oral cancer development. It is involved in the resistance of certain chemotherapeutic drugs. Coffee has been reported to affect the activity of salivary ALDH. In this study, the effect of caffeine on the activity (dehydrogenase and esterase) of hsALDH was investigated. The binding of caffeine to hsALDH was studied using different biophysical methods and molecular docking analysis. Caffeine was found to inhibit both crude and purified hsALDH. The Km increased and the Vmax decreased showing a mixed type of inhibition. Caffeine decreased the nucleophilicity of the catalytic cysteine residue. It binds to the active site of ALDH3A1 by forming a complex through non-covalent interactions with some highly conserved amino acid residues. It partially alters the secondary structure of the enzyme. Therefore, it is very likely that caffeine binds and inhibits the activity of hsALDH by decreasing substrate binding affinity and the catalytic efficiency of the enzyme. The study indicates that oral intake of caffeine may have a harmful effect on the oral health and may increase the risk of carcinogenesis through the inhibition of this important enzyme. Further, the inactivation of oxazaphosphorine based chemotherapeutic drugs by ALDH3A1 may be prevented by using caffeine as an adjuvant during medication which is expected to increase the sensitivity of these drugs through its inhibitory effect on the enzyme.

  8. [Enzymatic degradation of organophosphorus insecticide chlorpyrifos by fungus WZ-I].

    PubMed

    Xie, Hui; Zhu, Lu-sheng; Wang, Jun; Wang, Xiu-guo; Liu, Wei; Qian, Bo; Wang, Qian

    2005-11-01

    Degradation characteristics of chlorpyrifos insecticides was determined by the crude enzyme extracted from the isolated strain WZ-I ( Fusarium LK. ex Fx). The best separating condition and the degrading characteristic of chlorpyrifos were studied. Rate of degradation for chlorpyrifos by its intracellular enzyme, extracellular enzyme and cell fragment was 60.8%, 11.3% and 48%, respectively. The degrading enzyme was extracted after this fungus was incubated for 8 generations in the condition of noninducement, and its enzymic activity lost less, the results show that this enzyme is an intracellular and connatural enzyme. The solubility protein of the crude enzyme was determined with Albumin (bovine serum) as standard protein and the solubility protein of the crude enzyme was 3.36 mg x mL(-1). The pH optimum for crude enzyme was 6.8 for enzymatic degradation of chlorpyrifos, and it had comparatively high activity in the range of pH 6.0 - 9.0. The optimum temperature for enzymatic activity was at 40 degrees C, it still had comparatively high activity in the range of temperature 20-50 degrees C, the activity of enzyme rapidly reduced at 55 degrees C, its activity was 41% of the maximal activity. The crude enzyme showed Km value for chlorpyrifos of 1.049 26 mmol x L(-1), and the maximal enzymatic degradation rate was 0.253 5 micromol x (mg x min)(-1). Additional experimental evidence suggests that the enzyme had the stability of endure for temperature and pH, the crude enzyme of fungus WZ-I could effectively degrade chlorpyrifos.

  9. Ectomycorrhizal Fungal Communities and Enzymatic Activities Vary across an Ecotone between a Forest and Field.

    PubMed

    Rúa, Megan A; Moore, Becky; Hergott, Nicole; Van, Lily; Jackson, Colin R; Hoeksema, Jason D

    2015-08-28

    Extracellular enzymes degrade macromolecules into soluble substrates and are important for nutrient cycling in soils, where microorganisms, such as ectomycorrhizal (ECM) fungi, produce these enzymes to obtain nutrients. Ecotones between forests and fields represent intriguing arenas for examining the effect of the environment on ECM community structure and enzyme activity because tree maturity, ECM composition, and environmental variables may all be changing simultaneously. We studied the composition and enzymatic activity of ECM associated with loblolly pine (Pinus taeda) across an ecotone between a forest where P. taeda is established and an old field where P. taeda saplings had been growing for <5 years. ECM community and environmental characteristics influenced enzyme activity in the field, indicating that controls on enzyme activity may be intricately linked to the ECM community, but this was not true in the forest. Members of the Russulaceae were associated with increased phenol oxidase activity and decreased peroxidase activity in the field. Members of the Atheliaceae were particularly susceptible to changes in their abiotic environment, but this did not mediate differences in enzyme activity. These results emphasize the complex nature of factors that dictate the distribution of ECM and activity of their enzymes across a habitat boundary.

  10. A novel ALDH5A1 mutation is associated with succinic semialdehyde dehydrogenase deficiency and severe intellectual disability in an Iranian family.

    PubMed

    Püttmann, Lucia; Stehr, Henning; Garshasbi, Masoud; Hu, Hao; Kahrizi, Kimia; Lipkowitz, Bettina; Jamali, Payman; Tzschach, Andreas; Najmabadi, Hossein; Ropers, Hans-Hilger; Musante, Luciana; Kuss, Andreas W

    2013-08-01

    Succinic semialdehyde dehydrogenase (SSADH) deficiency is a disorder of the catabolism of the neurotransmitter gamma-aminobutyric acid (GABA) with a very variable clinical phenotype ranging from mild intellectual disability to severe neurological defects. We report here on a large Iranian family with four affected patients presenting with severe intellectual disability, developmental delay and generalized tonic-clonic seizures. Molecular genetic analysis revealed a missense mutation c.901A>G (p.K301E, RefSeq number NM_001080) in ALDH5A1 co-segregating with the disease in the family. The missense mutation affects an amino acid residue that is highly conserved across the animal kingdom. Protein modeling showed that p.K301E most likely leads to a loss of NAD(+) binding and a predicted decrease in the free energy by 6.67 kcal/mol furthermore suggests a severe destabilization of the protein. In line with these in silico observations, no SSADH enzyme activity could be detected in patient lymphoblasts. Copyright © 2013 Wiley Periodicals, Inc.

  11. Enzymatic activities in different strains isolated from healthy and brittle leaf disease affected date palm leaves: study of amylase production conditions.

    PubMed

    Mouna, Jrad; Imen, Fendri; Choba Ines, Ben; Nourredine, Drira; Adel, Kadri; Néji, Gharsallah

    2015-02-01

    The present study aimed to investigate and compare the enzymatic production of endophytic bacteria isolated from healthy and brittle leaf disease affected date palm leaves (pectinase, cellulase, lipase, and amylase). The findings revealed that the enzymatic products from the bacterial isolates of healthy date palm leaves were primarily 33% amylolytic enzyme, 33 % cellulase, 25 % pectinase, and 25 % lipase. The isolates from brittle leaf disease date palm leaves, on the other hand, were noted to produce 16 % amylolytic enzyme, 20 % cellulose, 50 % pectinase, and 50 % lipase. The effects of temperature and pH on amylase, pectinase, and cellulose activities were investigated. The Bacillus subtilis JN934392 strain isolated from healthy date palm leaves produced higher levels of amylase activity at pH 7. A Box Behnken Design (BBD) was employed to optimize amylase extraction. Maximal activity was observed at pH and temperature ranges of pH 6-6.5 and 37-39 °C, respectively. Under those conditions, amylase activity was noted to be attained 9.37 U/ml. The results showed that the enzyme was able to maintain more than 50 % of its activity over a temperature range of 50-80 °C, with an optimum at 70 °C. This bacterial amylase showed high activity compared to other bacteria, which provides support for its promising candidacy for future industrial application.

  12. Use of enzymatic tools for biomonitoring inorganic pollution in aquatic sediments: a case study (Bor, Serbia)

    PubMed Central

    2013-01-01

    Background Sediment bacterial communities are key players in biogeochemical cycling of elements in the aquatic environment. Copper mining, smelting, and processing operations located in Bor area (Serbia) are major environmental hot spots in the lower Danube Basin and Western Balkans. In the present study, we evaluate the influence of trace element (TE) concentration in sediments and physico-chemical properties of water on sediment microbial communities in water streams adjacent to the Copper Smelter Complex Bor (RTB Bor, Serbia). The degree to which metabolic activities of bacterial biota inhabiting differently polluted sites is inhibited by inorganic pollution were compared using selected enzymatic bioindicators. Results Cu, Zn, Pb, and As concentrations systematically exceeded the target values for metal loadings in aquatic sediments. Water electrical conductivity (WEC) followed the same pattern of spatial variation, irrespective of season. Interestingly, the most intense enzymatic activity occurred at the reference site although this site showed the greatest TE levels in aquatic sediments. Catalase activity (CA), potential dehydrogenase activity (PDA), actual dehydrogenase activity (ADA), urease activity (UA), and phosphatase activity (PA) in aquatic sediments displayed heterogeneous patterns of spatio-temporal variation. Inorganic pollution greatly affected CA, ADA, and PDA, but much less so UA and PA. Canonical correlation analysis showed that pH and WEC were the strongest determinants of enzymatic activity in bacterial biota, with the latter variable being reversely correlated with the enzymatic indicator of sediment quality (EISQ). The median values of EISQ increased with distance from the major sources of pollution. In addition, it was found that sites with different degrees of inorganic pollution can be appropriately classified by applying cluster analysis to EISQ, TE levels in sediments, and physico-chemical properties of water. Conclusions Because EISQ

  13. Modulation of the pharmacological effects of enzymatically-active PLA2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

    PubMed Central

    Oliveira, Simone CB; Fonseca, Fabiana V; Antunes, Edson; Camargo, Enilton A; Morganti, Rafael P; Aparício, Ricardo; Toyama, Daniela O; Beriam, Luís OS; Nunes, Eudismar V; Cavada, Benildo S; Nagano, Celso S; Sampaio, Alexandre H; Nascimento, Kyria S; Toyama, Marcos H

    2008-01-01

    Background An interaction between lectins from marine algae and PLA2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA2 and its complex. Results This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24–26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion The

  14. 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

  15. Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

    PubMed Central

    2011-01-01

    Background Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Results Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. Conclusions This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug

  16. Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs).

    PubMed

    Yu, Shann S; Scherer, Randy L; Ortega, Ryan A; Bell, Charleson S; O'Neil, Conlin P; Hubbell, Jeffrey A; Giorgio, Todd D

    2011-02-27

    Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative

  17. Integration of Inhibition Kinetics and Molecular Dynamics Simulations: A Urea-Mediated Folding Study on Acetaldehyde Dehydrogenase 1.

    PubMed

    Xu, Yingying; Lee, Jinhyuk; Lü, Zhi-Rong; Mu, Hang; Zhang, Qian; Park, Yong-Doo

    2016-07-01

    Understanding the mechanism of acetaldehyde dehydrogenase 1 (ALDH1) folding is important because this enzyme is directly involved in several types of cancers and other diseases. We investigated the urea-mediated unfolding of ALDH1 by integrating kinetic inhibition studies with computational molecular dynamics (MD) simulations. Conformational changes in the enzyme structure were also analyzed using intrinsic and 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence measurements. Kinetic studies revealed that the direct binding of urea to ALDH1 induces inactivation of ALDH1 in a manner of mixed-type inhibition. Tertiary structural changes associated with regional hydrophobic exposure of the active site were observed. The urea binding regions on ALDH1 were predicted by docking simulations and were partly shared with active site residues of ALDH1 and with interface residues of the oligomerization domain for tetramer formation. The docking results suggest that urea prevents formation of the ALDH1 normal shape for the tetramer state as well as entrance of the substrate into the active site. Our study provides insight into the structural changes that accompany urea-mediated unfolding of ALDH1 and the catalytic role associated with conformational changes.

  18. Cardiac-specific overexpression of aldehyde dehydrogenase 2 exacerbates cardiac remodeling in response to pressure overload.

    PubMed

    Dassanayaka, Sujith; Zheng, Yuting; Gibb, Andrew A; Cummins, Timothy D; McNally, Lindsey A; Brittian, Kenneth R; Jagatheesan, Ganapathy; Audam, Timothy N; Long, Bethany W; Brainard, Robert E; Jones, Steven P; Hill, Bradford G

    2018-06-01

    Pathological cardiac remodeling during heart failure is associated with higher levels of lipid peroxidation products and lower abundance of several aldehyde detoxification enzymes, including aldehyde dehydrogenase 2 (ALDH2). An emerging idea that could explain these findings concerns the role of electrophilic species in redox signaling, which may be important for adaptive responses to stress or injury. The purpose of this study was to determine whether genetically increasing ALDH2 activity affects pressure overload-induced cardiac dysfunction. Mice subjected to transverse aortic constriction (TAC) for 12 weeks developed myocardial hypertrophy and cardiac dysfunction, which were associated with diminished ALDH2 expression and activity. Cardiac-specific expression of the human ALDH2 gene in mice augmented myocardial ALDH2 activity but did not improve cardiac function in response to pressure overload. After 12 weeks of TAC, ALDH2 transgenic mice had larger hearts than their wild-type littermates and lower capillary density. These findings show that overexpression of ALDH2 augments the hypertrophic response to pressure overload and imply that downregulation of ALDH2 may be an adaptive response to certain forms of cardiac pathology. Copyright © 2018. Published by Elsevier B.V.

  19. Ultrasonic-assisted enzymatic extraction of phenolics from broccoli (Brassica oleracea L. var. italica) inflorescences and evaluation of antioxidant activity in vitro.

    PubMed

    Wu, Hao; Zhu, Junxiang; Yang, Long; Wang, Ran; Wang, Chengrong

    2015-06-01

    An efficient ultrasonic-assisted enzymatic extraction technique was applied to extracting phenolics from broccoli inflorescences without organic solvents. The synergistic model of enzymolysis and ultrasonication simultaneously was selected, and the enzyme combination was optimized by orthogonal test: cellulase 7.5 mg/g FW (fresh weight), pectinase 10 mg/g FW, and papain 1.0 mg/g FW. The operating parameters in ultrasonic-assisted enzymatic extraction were optimized with response surface methodology using Box-Behnken design. The optimal extraction conditions were as follows: ultrasonic power, 440 W; liquid to material ratio, 7.0:1 mL/g; pH value of 6.0 at 54.5 ℃ for 10 min. Under these conditions, the extraction yield of phenolics achieved 1.816 ± 0.0187 mg gallic acid equivalents/gram FW. The free radical scavenging activity of ultrasonic-assisted enzymatic extraction extracts was determined by 1,1-diphenyl-2-picrylhydrazyl·assay with EC50 values of 0.25, and total antioxidant activity was determined by ferric reducing antioxidant power assay with ferric reducing antioxidant power value of 0.998 mmol FeSO4/g compared with the referential ascorbic acid of 1.184 mmol FeSO4/g. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  20. Enzymatic and non-enzymatic comparison of two different industrial tomato (Solanum lycopersicum) varieties against drought stress.

    PubMed

    Çelik, Özge; Ayan, Alp; Atak, Çimen

    2017-12-01

    The aim of this study is to compare the tolerance mechanisms of two industrial tomato varieties (X5671R and 5MX12956) under drought stress. 14 days-old tomato seedlings were subjected to 7 days-long drought stress by withholding irrigation. The effects of stress were determined by enzymatic and non-enzymatic parameters. The physiological damages were evaluated via lipid peroxidation ratio, total protein content, relative water content, chlorophyll content and proline accumulation. Enzymatic responses were determined by biochemical analysis and electrophoresis of SOD, APX, POX and CAT enzymes. Relative water contents of X5671R and 5MX12956 varieties at 7th day of drought were decreased to 8.4 and 12.2%, respectively. Applied drought decreased all photosynthetic pigments of X5671R and 5MX12956 varieties during the treatment period significantly comparing to the Day 0 as the control. Total protein content, lipid peroxidation and proline accumulation presented increased values in both varieties in accordance with the increasing stress intensity. According to lipid peroxidation analysis, 5MX12956 tomato variety was found more drought sensitive than X5671R variety. Antioxidative enzyme activities showed increases in both varieties as a response to drought stress, although CAT and APX activities presented decrease on the 7th day of applied stress. 7 days long drought stress differentially altered POX, APX and SOD isozyme patterns. Same POX bands were observed in both varieties with different band intensities. However, main isozyme pattern differences were obtained for SOD and APX. APX1, Fe-SOD and Cu/Zn-SOD2 isozyme bands should be evaluated to define their main role in the tolerance mechanism of both tomato varieties.

  1. The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity*

    PubMed Central

    Peters, Diane E.; Szabo, Roman; Friis, Stine; Shylo, Natalia A.; Uzzun Sales, Katiuchia; Holmbeck, Kenn; Bugge, Thomas H.

    2014-01-01

    The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. Prostasin null (Prss8−/−) mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast, mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders prostasin catalytically inactive (Prss8Cat−/Cat− mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8−/− and Prss8Cat−/Cat− mice born within the same litter. Furthermore, Prss8Cat−/Cat− mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this unique membrane-anchored serine protease. PMID:24706745

  2. Mechanistic investigation in ultrasound induced enhancement of enzymatic hydrolysis of invasive biomass species.

    PubMed

    Borah, Arup Jyoti; Agarwal, Mayank; Poudyal, Manisha; Goyal, Arun; Moholkar, Vijayanand S

    2016-08-01

    This study has assessed four invasive weeds, viz. Saccharum spontaneum (SS), Mikania micrantha (MM), Lantana camara (LC) and Eichhornia crassipes (EC) for enzymatic hydrolysis prior to bioalcohol fermentation. Enzymatic hydrolysis of pretreated biomasses of weeds has been conducted with mechanical agitation and sonication under constant (non-optimum) conditions. Profiles of total reducible sugar release have been fitted to HCH-1 model of enzymatic hydrolysis using Genetic Algorithm. Trends in parameters of this model reveal physical mechanism of ultrasound-induced enhancement of enzymatic hydrolysis. Sonication accelerates hydrolysis kinetics by ∼10-fold. This effect is contributed by several causes, attributed to intense micro-convection generated during sonication: (1) increase in reaction velocity, (2) increase in enzyme-substrate affinity, (3) reduction in product inhibition, and (4) enhancement of enzyme activity due to conformational changes in its secondary structure. Enhancement effect of sonication is revealed to be independent of conditions of enzymatic hydrolysis - whether optimum or non-optimum. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Salinomycin possesses anti-tumor activity and inhibits breast cancer stem-like cells via an apoptosis-independent pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    An, Hyunsook; Kim, Ji Young; Lee, Nahyun

    Cancer stem cells (CSCs) play important roles in the formation, growth and recurrence of tumors, particularly following therapeutic intervention. Salinomycin has received recent attention for its ability to target breast cancer stem cells (BCSCs), but the mechanisms of action involved are not fully understood. In the present study, we sought to investigate the mechanisms responsible for salinomycin's selective targeting of BCSCs and its anti-tumor activity. Salinomycin suppressed cell viability, concomitant with the downregulation of cyclin D1 and increased p27{sup kip1} nuclear accumulation. Mammosphere formation assays revealed that salinomycin suppresses self-renewal of ALDH1-positive BCSCs and downregulates the transcription factors Nanog, Oct4more » and Sox2. TUNEL analysis of MDA-MB-231-derived xenografts revealed that salinomycin administration elicited a significant reduction in tumor growth with a marked downregulation of ALDH1 and CD44 levels, but seemingly without the induction of apoptosis. Our findings shed further light on the mechanisms responsible for salinomycin's effects on BCSCs. - Highlights: • Salinomycin suppresses mammosphere formation. • Salinomycin reduces ALDH1 activity and downregulates Nanog, Oct4 and Sox2. • Salinomycin targets BCSCs via an apoptosis-independent pathway.« less

  4. 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.

  5. cPLA2α Gene Activation by IL-1β is Dependent on an Upstream Kinase pathway, Enzymatic Activation and Downstream 15-lipoxygenase Activity: A Positive Feedback Loop

    PubMed Central

    Walters, Jewell N.; Bickford, Justin S.; Beachy, Dawn E.; Newsom, Kimberly J.; Herlihy, John-David H.; Peck, Molly V.; Qiu, Xiaolei; Nick, Harry S.

    2011-01-01

    Cytosolic phospholipase A2α (cPLA2α) is the most widely studied member of the Group IV PLA2 family. The enzyme is Ca2+-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA2α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences. cPLA2α activity is regulated by pro-inflammatory stimuli through pathways involving increased intracellular Ca2+ levels, phosphorylation coupled to increased enzymatic activity and de novo gene transcription. This study addresses the signal transduction pathways for protein phosphorylation and gene induction following IL-1β stimulation in human fetal lung fibroblasts. Our results utilizing both inhibitors and kinase-deficient cells demonstrate that cPLA2α is phosphorylated within 10 min of IL-1β treatment, an event requiring p38 MAPK as well as the upstream kinase, MKK3/MKK6. Inhibition of p38 MAPK also blocks the phosphorylation of a downstream, nuclear kinase, MSK-1. Our results further demonstrate that the activities of both cPLA2α and a downstream lipoxygenase (15-LOX2) are required for IL-1β-dependent induction of cPLA2α mRNA expression. Overall, these data support an MKK3/MKK6→p38 MAPK→MSK-1→cPLA2α→15-LOX2-dependent, positive feedback loop where a protein’s enzymatic activity is required to regulate its own gene induction by a pro-inflammatory stimulus. PMID:21771656

  6. Optimized dosing schedule based on circadian dynamics of mouse breast cancer stem cells improves the anti-tumor effects of aldehyde dehydrogenase.

    PubMed

    Matsunaga, Naoya; Ogino, Takashi; Hara, Yukinori; Tanaka, Takahiro; Koyanagi, Satoru; Ohdo, Shigehiro

    2018-05-07

    Although malignant phenotypes of triple-negative breast cancer (TNBC) are subject to circadian alterations, the role of cancer stem cells (CSC) in defining this circadian change remains unclear. CSC are often characterized by high aldehyde dehydrogenase (ALDH) activity, which is associated with the malignancy of cancer cells and used for identification and isolation of CSC. Here we show that the popultation of ALDH-positive cells in a mouse 4T1 breast tumor model exhibits pronounced circadian alterations. Alterations in the number of ALDH-positive cells was generated by time-dependent increases and decreases in the expression of Aldh3a1. Importantly, circadian clock genes were rhythmically expressed in ALDH-negative cells, but not in ALDH-positive cells. Circadian expression of Aldh3a1 in ALDH-positive cells was dependent on the time-dependent release of Wingless-type mmtv integration site family 10a (WNT10a) from ALDH-negative cells. Furthermore, anti-tumor and anti-metastatic effects of ALDH inhibitor N,N-diethylaminobenzaldehyde were enhanced by administration at the time of day when ALDH activity was increased in 4T1 tumor cells. Our findings reveal a new role for the circadian clock within the tumor microenvironment in regulating the circadian dynamics of CSC. These results should enable the development of novel therapeutic strategies for treatment of TNBC with ALDH inhibitors. Copyright ©2018, American Association for Cancer Research.

  7. Evaluation of enzymatic and non-enzymatic antioxidants in seminal plasma of men with genitourinary infections, varicocele and idiopathic infertility.

    PubMed

    Micheli, L; Cerretani, D; Collodel, G; Menchiari, A; Moltoni, L; Fiaschi, A I; Moretti, E

    2016-05-01

    This study was aimed to assess the antioxidant enzymatic and non-enzymatic compounds in semen of infertile men. Seventy-four infertile patients were grouped according to their clinical diagnosis: genitourinary infection, varicocele, idiopathic infertility. Semen samples of fertile men represent the control. Semen characteristics were evaluated by light and transmission electron microscopy (TEM). TEM data was quantified with a mathematical formula, which provides numerical scores. Spectrophotometric and HPLC methods were used to measure the amount of reduced (GSH), oxidised glutathione (GSSG), ascorbic acid (AA) and malondialdehyde (MDA, marker of lipid peroxidation) and the activity of glutathione reductase, catalase (CAT), glutathione peroxidase. Infertile groups showed significantly decreased values of sperm parameters vs. In infection and varicocele groups, the seminal MDA levels were significantly increased when compared to controls (p < 0.001), indicating an alteration of oxidative status and a peroxidative damage. In infection and varicocele groups, AA levels were reduced (p < 0.05) vs. control; in the varicocele group, the GSH levels were also decreased (p < 0.05). Significantly higher CAT activity was observed in infection and varicocele groups vs. fertile men (p < 0.001 and p < 0.05 respectively). The GSH/GSSG ratio was significantly decreased in varicocele and idiopathic infertility groups vs. control (p < 0.01). The study of the alteration of a single parameter of oxidative stress or of the antioxidant system may not have a relevant clinical value to estimate male fertilising potential and the background of infertility causes, since complex and multifactorial mechanisms are involved in different pathologies. In our study, each pathology is characterised by a definite pattern of markers such as MDA and enzymatic and non-enzymatic antioxidant compounds. In the different pathologies related to infertility, the identification of the complex of

  8. Impacts of dissolved organic matter on aqueous behavior of nano/micron-titanium nitride and their induced enzymatic/non-enzymatic antioxidant activities in Scenedesmus obliquus.

    PubMed

    Zhang, Xin; Wang, Zhuang; Wang, Se; Fang, Hao; Zhang, Fan; Wang, De-Gao

    2017-01-02

    Freshwater dispersion stability and ecotoxicological effects of titanium nitride (TiN) with particle size of 20 nm, 50 nm, and 2-10 μm in the presence of dissolved organic matter (DOM) at various concentrations were studied. The TiN particles that had a more negative zeta potential and smaller hydrodynamic size showed more stable dispersion in an aqueous medium when DOM was present than when DOM was absent. Biochemical assays indicated that relative to the control, the TiN particles in the presence of DOM alleviated to some extent the antioxidative stress enzyme activity in Scenedesmus obliquus. In addition, it was found that the TiN with a primary size of 50 nm at a high concentration presented a significant impact on non-enzymatic antioxidant defense in algal cells.

  9. THE ENZYMATIC RESPONSE OF ASTROCYTES TO VARIOUS IONS IN VITRO

    PubMed Central

    Friede, Reinhard L.

    1964-01-01

    The effect of environmental ion concentration on the enzyme activity of astrocytes was investigated in tissue cultures of rat cerebral cortex. It was found that the oxidative enzymatic activity (succinic dehydrogenase, DPN-diaphorase, and several other enzymes) of astrocytes depended on the concentration of NaCl in the environment. This response was not specific for NaCl, but was also elicited by MgCl2 and LiCl; the response was less consistent, and often questionable for KCl. However, only NaCl could elicit enzymatic changes in astrocytes at concentrations known to be present in a living organism. Astrocytes were the only cells which responded this way; it appeared that the foot-plates were particularly involved in the response since increase of enzyme activity occurred earlier in the foot-plates than in the perikarya. It was concluded that astrocytes are metabolically involved in the maintenance of the ionic and osmotic environment of the central nervous system, particularly in regard to the active transport of sodium. PMID:14105217

  10. Allergenic Properties of Enzymatically Hydrolyzed Peanut Flour Extracts

    USDA-ARS?s Scientific Manuscript database

    Peanut flour is a high protein, low oil, powdered material prepared from roasted 21 peanut seed. In addition to being a well-established food ingredient, peanut flour is also the 22 active ingredient in peanut oral immunotherapy trials. Enzymatic hydrolysis was evaluated as a 23 processing strategy ...

  11. Ectomycorrhizal Fungal Communities and Enzymatic Activities Vary across an Ecotone between a Forest and Field

    PubMed Central

    Rúa, Megan A.; Moore, Becky; Hergott, Nicole; Van, Lily; Jackson, Colin R.; Hoeksema, Jason D.

    2015-01-01

    Extracellular enzymes degrade macromolecules into soluble substrates and are important for nutrient cycling in soils, where microorganisms, such as ectomycorrhizal (ECM) fungi, produce these enzymes to obtain nutrients. Ecotones between forests and fields represent intriguing arenas for examining the effect of the environment on ECM community structure and enzyme activity because tree maturity, ECM composition, and environmental variables may all be changing simultaneously. We studied the composition and enzymatic activity of ECM associated with loblolly pine (Pinus taeda) across an ecotone between a forest where P. taeda is established and an old field where P. taeda saplings had been growing for <5 years. ECM community and environmental characteristics influenced enzyme activity in the field, indicating that controls on enzyme activity may be intricately linked to the ECM community, but this was not true in the forest. Members of the Russulaceae were associated with increased phenol oxidase activity and decreased peroxidase activity in the field. Members of the Atheliaceae were particularly susceptible to changes in their abiotic environment, but this did not mediate differences in enzyme activity. These results emphasize the complex nature of factors that dictate the distribution of ECM and activity of their enzymes across a habitat boundary. PMID:29376908

  12. Cloning and characterization of microbial activated Aedes aegypti MEK4 (AaMEK4): influences of noncatalytic domains on enzymatic activity.

    PubMed

    Wu, R C-C; Cho, W-L

    2014-10-01

    Protein kinases are known to be involved in a number of signal transduction cascades. Both the stress-activated Jun N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) p38 pathways have been shown to correlate with the insect immune response to microbial infection. MAP kinase kinase 4 (MEK4) is an upstream kinase of JNK and p38 kinase. The cDNA of AaMEK4 was cloned and characterized. AaMEK4 was activated by microbial lysates of Gram-positive, Gram-negative bacteria and yeast. The conserved lysine (K112 ) and the putative phosphorylation sites (S238 and T242 ) were shown to be important for kinase activity by site-directed mutagenesis. A common MAPK docking site (MAPK_dsA) was found and in addition, a new nearby docking site, MAPK_dsB, was identified in the N-terminal noncatalytic domain of AaMEK4. MAPK_dsB was shown to be a unique element in the MEK4 family. In this study, both MAPK_dsA and _dsB were demonstrated to be important to AaMEK4 enzymatic activity for the downstream protein kinase, Aap38. © 2014 The Royal Entomological Society.

  13. Antagonists' impact on enzymatic response in wilt infected cotton plants

    USDA-ARS?s Scientific Manuscript database

    A number of PR-proteins possess enzymatic activity. As such, these proteins maybe indicators of defensive response of plants. Thus, we have conducted a comparative analysis of beta-1,3-glucanase, peroxidase and xylanase activity in cotton plants to determine how these enzymes are affected by the pat...

  14. Continuous enzymatic hydrolysis of lignocellulosic biomass with simultaneous detoxification and enzyme recovery.

    PubMed

    Gurram, Raghu N; Menkhaus, Todd J

    2014-07-01

    Recovering hydrolysis enzymes and/or alternative enzyme addition strategies are two potential mechanisms for reducing the cost during the biochemical conversion of lignocellulosic materials into renewable biofuels and biochemicals. Here, we show that enzymatic hydrolysis of acid-pretreated pine wood with continuous and/or fed-batch enzyme addition improved sugar conversion efficiencies by over sixfold. In addition, specific activity of the hydrolysis enzymes (cellulases, hemicellulases, etc.) increased as a result of continuously washing the residual solids with removal of glucose (avoiding the end product inhibition) and other enzymatic inhibitory compounds (e.g., furfural, hydroxymethyl furfural, organic acids, and phenolics). As part of the continuous hydrolysis, anion exchange resin was tested for its dual application of simultaneous enzyme recovery and removal of potential enzymatic and fermentation inhibitors. Amberlite IRA-96 showed favorable adsorption profiles of inhibitors, especially furfural, hydroxymethyl furfural, and acetic acid with low affinity toward sugars. Affinity of hydrolysis enzymes to adsorb onto the resin allowed for up to 92 % of the enzymatic activity to be recovered using a relatively low-molar NaCl wash solution. Integration of an ion exchange column with enzyme recovery into the proposed fed-batch hydrolysis process can improve the overall biorefinery efficiency and can greatly reduce the production costs of lignocellulosic biorenewable products.

  15. Biological evaluation and 3D-QSAR studies of curcumin analogues as aldehyde dehydrogenase 1 inhibitors.

    PubMed

    Wang, Hui; Du, Zhiyun; Zhang, Changyuan; Tang, Zhikai; He, Yan; Zhang, Qiuyan; Zhao, Jun; Zheng, Xi

    2014-05-16

    Aldehyde dehydrogenase 1 (ALDH1) is reported as a biomarker for identifying some cancer stem cells, and down-regulation or inhibition of the enzyme can be effective in anti-drug resistance and a potent therapeutic for some tumours. In this paper, the inhibitory activity, mechanism mode, molecular docking and 3D-QSAR (three-dimensional quantitative structure activity relationship) of curcumin analogues (CAs) against ALDH1 were studied. Results demonstrated that curcumin and CAs possessed potent inhibitory activity against ALDH1, and the CAs compound with ortho di-hydroxyl groups showed the most potent inhibitory activity. This study indicates that CAs may represent a new class of ALDH1 inhibitor.

  16. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Enzymatic reactions in confined environments

    NASA Astrophysics Data System (ADS)

    Küchler, Andreas; Yoshimoto, Makoto; Luginbühl, Sandra; Mavelli, Fabio; Walde, Peter

    2016-05-01

    Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.

  18. Murine hepatic aldehyde dehydrogenase 1a1 is a major contributor to oxidation of aldehydes formed by lipid peroxidation

    PubMed Central

    Makia, Ngome L.; Bojang, Pasano; Falkner, K. Cameron; Conklin, Daniel J.; Prough, Russell A.

    2015-01-01

    Reactive lipid aldehydes are implicated in the pathogenesis of various oxidative stress-mediated diseases, including non-alcoholic steatohepatitis, atherosclerosis, Alzheimer’s and cataract. In the present study, we sought to define which hepatic Aldh isoform plays a major role in detoxification of lipid-derived aldehydes, such as acrolein and HNE by enzyme kinetic and gene expression studies. The catalytic efficiencies for metabolism of acrolein by Aldh1a1 was comparable to that of Aldh3a1 (Vmax/Km = 23). However, Aldh1a1 exhibits far higher affinity for acrolein (Km = 23.2 μM) compared to Aldh3a1 (Km = 464 μM). Aldh1a1 displays a 3-fold higher catalytic efficiency for HNE than Aldh3a1 (218 vs 69 ml/min/mg). The endogenous Aldh1a1 gene was highly expressed in mouse liver and a liver-derived cell line (Hepa-1c1c7) compared to Aldh2, Aldh1b1 and Aldh3a1. Aldh1a1 mRNA levels was 34-fold and 73-fold higher than Aldh2 in mouse liver and Hepa-1c1c7 cells respectively. Aldh3a1 gene was absent in mouse liver, but moderately expressed in Hepa-1c1c7 cells compared to Aldh1a1. We demonstrated that knockdown of Aldh1a1 expression by siRNA caused Hepa-1c1c7 cells to be more sensitive to acrolein-induced cell death and resulted in increased accumulation of acrolein-protein adducts and caspase 3 activation. These results indicate that Aldh1a1 plays a major role in cellular defense against oxidative damage induced by reactive lipid aldehydes in mouse liver. We also noted that hepatic Aldh1a1 mRNA levels were significantly increased (≈ 3 fold) in acrolein-fed mice compared to control. In addition, hepatic cytosolic ALDH activity was induced by acrolein when 1 mM NAD+ was used as cofactor, suggesting an Aldh1a1-protective mechanism against acrolein toxicity in mice liver. Thus, mechanisms to induce Aldh1a1 gene expression may provide a useful rationale for therapeutic protection against oxidative stress-induced pathologies. PMID:21256123

  19. Enzymatic activity of a subtilisin homolog, Tk-SP, from Thermococcus kodakarensis in detergents and its ability to degrade the abnormal prion protein

    PubMed Central

    2013-01-01

    Background Tk-SP is a member of subtilisin-like serine proteases from a hyperthermophilic archaeon Thermococcus kodakarensis. It has been known that the hyper-stable protease, Tk-SP, could exhibit enzymatic activity even at high temperature and in the presence of chemical denaturants. In this work, the enzymatic activity of Tk-SP was measured in the presence of detergents and EDTA. In addition, we focused to demonstrate that Tk-SP could degrade the abnormal prion protein (PrPSc), a protease-resistant isoform of normal prion protein (PrPC). Results Tk-SP was observed to maintain its proteolytic activity with nonionic surfactants and EDTA at 80°C. We optimized the condition in which Tk-SP functions efficiently, and demonstrated that the enzyme is highly stable in the presence of 0.05% (w/v) nonionic surfactants and 0.01% (w/v) EDTA, retaining up to 80% of its activity. Additionally, we also found that Tk-SP can degrade PrPSc to a level undetectable by western-blot analysis. Conclusions Our results indicate that Tk-SP has a great potential for technological applications, such as thermo-stable detergent additives. In addition, it is also suggested that Tk-SP-containing detergents can be developed to decrease the secondary infection risks of transmissible spongiform encephalopathies (TSE). PMID:23448268

  20. Bio-conversion of apple pomace into ethanol and acetic acid: Enzymatic hydrolysis and fermentation.

    PubMed

    Parmar, Indu; Rupasinghe, H P Vasantha

    2013-02-01

    Enzymatic hydrolysis of cellulose present in apple pomace was investigated using process variables such as enzyme activity of commercial cellulase, pectinase and β-glucosidase, temperature, pH, time, pre-treatments and end product separation. The interaction of enzyme activity, temperature, pH and time had a significant effect (P<0.05) on release of glucose. Optimal conditions of enzymatic saccharification were: enzyme activity of cellulase, 43units; pectinase, 183units; β-glucosidase, 41units/g dry matter (DM); temperature, 40°C; pH 4.0 and time, 24h. The sugars were fermented using Saccharomyces cerevisae yielding 19.0g ethanol/100g DM. Further bio-conversion using Acetobacter aceti resulted in the production of acetic acid at a concentration of 61.4g/100g DM. The present study demonstrates an improved process of enzymatic hydrolysis of apple pomace to yield sugars and concomitant bioconversion to produce ethanol and acetic acid. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function.

    PubMed

    Zaidman-Rémy, Anna; Poidevin, Mickael; Hervé, Mireille; Welchman, David P; Paredes, Juan C; Fahlander, Carina; Steiner, Hakan; Mengin-Lecreulx, Dominique; Lemaitre, Bruno

    2011-02-18

    Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs) and, in some cases, to efficiently degrade it (catalytic PGRPs). In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.

  2. 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. Copyright © 2012 John Wiley & Sons, Ltd.

  3. 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

  4. Comparative study of enzymatic activities of new KatG mutants from low- and high-level isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.

    PubMed

    Brossier, Florence; Boudinet, Marlène; Jarlier, Vincent; Petrella, Stéphanie; Sougakoff, Wladimir

    2016-09-01

    Resistance to isoniazid (INH-R) in Mycobacterium tuberculosis is mainly due to mutations at position 315 (S315T) of the catalase-peroxidase KatG. We identified 16 mutations (including 13 biochemically uncharacterized mutations) in KatG from INH-R clinical isolates of M. tuberculosis showing mutations other than S315T. The KatG enzymatic activities (catalase, peroxidase, free radical production and isonicotinoyl-NAD formation) of wild-type KatG and the 16 mutants were determined and correlated to their spatial location in a KatG model structure. Of all mutations studied, H270R, which conferred a high level of INH-R and results in the disruption of a coordination bond with the heme, caused complete loss of all enzymatic KatG activities. The mutants generally associated with a very high level of INH-R were all characterized by a drastic reduction in catalase activity and a marked decrease in INH activation activities. One mutant, A162E, displayed a behavior similar to S315T, i.e. a moderate decrease in catalase activity and a drastic decrease in the formation of the radical form of INH. Finally, the mutants associated with a low level of INH-R showed a moderate reduction in the four catalytic activities, likely stemming from an overall alteration of the folding and/or stability of the KatG protein. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Isoprene Production on Enzymatic Hydrolysate of Peanut Hull Using Different Pretreatment Methods.

    PubMed

    Wang, Sumeng; Li, Ruichao; Yi, Xiaohua; Fang, Tigao; Yang, Jianming; Bae, Hyeun-Jong

    2016-01-01

    The present study is about the use of peanut hull for isoprene production. In this study, two pretreatment methods, hydrogen peroxide-acetic acid (HPAC) and popping, were employed prior to enzymatic hydrolysis, which could destroy the lignocellulosic structure and accordingly improve the efficiency of enzymatic hydrolysis. It is proven that the isoprene production on enzymatic hydrolysate with HPAC pretreatment is about 1.9-fold higher than that of popping pretreatment. Moreover, through High Performance Liquid Chromatography (HPLC) analysis, the amount and category of inhibitors such as formic acid, acetic acid, and HMF were assayed and were varied in different enzymatic hydrolysates, which may be the reason leading to a decrease in isoprene production during fermentation. To further increase the isoprene yield, the enzymatic hydrolysate of HPAC was detoxified by activated carbon. As a result, using the detoxified enzymatic hydrolysate as the carbon source, the engineered strain YJM21 could accumulate 297.5 mg/L isoprene, which accounted for about 90% of isoprene production by YJM21 fermented on pure glucose (338.6 mg/L). This work is thought to be the first attempt on isoprene production by E. coli using peanut hull as the feedstock. More importantly, it also shows the prospect of peanut hull to be considered as an alternative feedstock for bio-based chemicals or biofuels production due to its easy access and high polysaccharide content.

  6. Macrophage Migration Inhibitory Factor Enzymatic Activity, Lung Inflammation, and Cystic Fibrosis

    PubMed Central

    Adamali, Huzaifa; Armstrong, Michelle E.; McLaughlin, Anne Marie; Cooke, Gordon; McKone, Edward; Costello, Christine M.; Gallagher, Charles G.; Leng, Lin; Baugh, John A.; Fingerle-Rowson, Günter; Bucala, Richard J.; McLoughlin, Paul

    2012-01-01

    Rationale: Macrophage migration inhibitory factor (MIF) is a proinflammatory mediator with unique tautomerase enzymatic activity; the precise function has not been clearly defined. We previously demonstrated that individual patients with cystic fibrosis (CF) who are genetically predisposed to be high MIF producers develop accelerated end-organ injury. Objectives: To characterize the effects of the MIF-CATT polymorphism in patients with CF ex vivo. To investigate the role of MIF’s tautomerase activity in a murine model of Pseudomonas aeruginosa infection. Methods: MIF and tumor necrosis factor (TNF)-α protein levels were assessed in plasma or peripheral blood mononuclear cell (PBMC) supernatants by ELISA. A murine pulmonary model of chronic Pseudomonas infection was used in MIF wild-type mice (mif+/+) and in tautomerase-null, MIF gene knockin mice (mif P1G/P1G). Measurements and Main Results: MIF protein was measured in plasma and PBMCs from 5- and 6-CATT patients with CF; LPS-induced TNF-α production from PBMCs was also assessed. The effect of a specific inhibitor of MIF-tautomerase activity, ISO-1, was investigated in PBMCs. In the murine infection model, total weight loss, differential cell counts, bacterial load, and intraacinar airspace/tissue volume were measured. MIF and TNF-α levels were increased in 6-CATT compared with 5-CATT patients with CF. LPS-induced TNF-α production from PBMCs was attenuated in the presence of ISO-1. In a murine model of Pseudomonas infection, significantly less pulmonary inflammation and bacterial load was observed in mifP1G/P1G compared with mif+/+ mice. Conclusions: MIF-tautomerase activity may provide a novel therapeutic target in patients with chronic inflammatory diseases such as CF, particularly those patients who are genetically predisposed to produce increased levels of this cytokine. PMID:22592805

  7. Substitution-inert trinuclear platinum complexes efficiently condense/aggregate nucleic acids and inhibit enzymatic activity.

    PubMed

    Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor

    2014-11-17

    The trinuclear platinum complexes (TriplatinNC-A [{Pt(NH3 )3 }2 -μ-{trans-Pt(NH3 )2 (NH2 (CH2 )6 NH2 )2 }](6+) , and TriplatinNC [{trans-Pt(NH3 )2 (NH2 (CH2 )6 NH3 (+) )}2 -μ-{trans-Pt(NH3 )2 (NH2 (CH2 )6 NH2 )2 }](8+) ) are biologically active agents that bind to DNA through noncovalent (hydrogen bonding, electrostatic) interactions. Herein, we show that TriplatinNC condenses DNA with a much higher potency than conventional DNA condensing agents. Both complexes induce aggregation of small transfer RNA molecules, and TriplatinNC in particular completely inhibits DNA transcription at lower concentrations than naturally occurring spermine. Topoisomerase I-mediated relaxation of supercoiled DNA was inhibited by TriplatinNC-A and TriplatinNC at concentrations which were 60 times and 250 times lower than that of spermine. The mechanisms for the biological activity of TriplatinNC-A and TriplatinNC may be associated with their ability to condense/aggregate nucleic acids with consequent inhibitory effects on crucial enzymatic activities. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Determination of the Activation Energy of the Enzymatic Biodegradation Process in Microfabricated Polyhydroxyalkanoate Thin Films Using In-Situ, Real Time Atomic Force Microscopy

    NASA Astrophysics Data System (ADS)

    Morse, Clinton; Latuga, Brian M.; Delfaus, Stephen; Devore, Thomas C.; Augustine, Brian H.; Hughes, W. Christopher; Warne, Paul G.

    2003-11-01

    Using the liquid cell capability of the atomic force microscope (AFM), we report the determination of the activation energy of the biodegradation process of the enzymatic biodegradation of poly 3-hydroxybutyrate / poly 3-hydroxyvalerate [P(3HB-HV)] thin films. We have prepared P(3HB-3HV) copolymer microstructures by the selective dewetting of soft lithographically patterned gold substrates with features sizes down to 10 mm. These have been then used as an internal height standard to measure the volume of material as a function of biodegradation time. Biodegradation is measured in-situ and real time using contact mode AFM in an enzymatic solution produced from Streptomyces sp. bacteria. The temperature dependent biodegradation has been measured over a temperature range from 23oC to 40oC. We will discuss the calculation of the activation energy of this process as well as a physical model to describe three distinct regions in the biodegradation process that have been observed.

  9. Recent Research Trends on the Enzymatic Synthesis of Structured Lipids.

    PubMed

    Kim, Byung Hee; Akoh, Casimir C

    2015-08-01

    Structured lipids (SLs) are lipids that have been chemically or enzymatically modified from their natural biosynthetic form. Because SLs are made to possess desired nutritional, physicochemical, or textural properties for various applications in the food industry, many research activities have been aimed at their commercialization. The production of SLs by enzymatic procedures has a great potential in the future market because of the specificity of lipases and phospholipases used as the biocatalysts. The aim of this review is to provide concise information on the recent research trends on the enzymatic synthesis of SLs of commercial interest, such as medium- and long-chain triacylglycerols, human milk fat substitutes, cocoa butter equivalents, trans-free or low-trans plastic fats (such as margarines and shortenings), low-calorie fats/oils, health-beneficial fatty acid-rich fats/oils, mono- or diacylglycerols, and structurally modified phospholipids. This limited review covers 108 research articles published between 2010 and 2014 which were searched in Web of Science. © 2015 Institute of Food Technologists®

  10. A singular enzymatic megacomplex from Bacillus subtilis.

    PubMed

    Straight, Paul D; Fischbach, Michael A; Walsh, Christopher T; Rudner, David Z; Kolter, Roberto

    2007-01-02

    Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

  11. Ink-native electrophoresis: an alternative to blue-native electrophoresis more suitable for in-gel detection of enzymatic activity.

    PubMed

    Kaneko, Keisuke; Sueyoshi, Noriyuki; Kameshita, Isamu; Ishida, Atsuhiko

    2013-09-15

    Blue-native electrophoresis (BNE) is a useful technique for analyzing protein complexes, but the Coomassie brilliant blue (CBB) dye used in BNE often hampers in-gel detection of enzymatic activity. Here we report an improved method, termed ink-native electrophoresis (INE), in which Pelikan 4001 fountain pen ink is used as a charge-shifting agent instead of CBB. INE is more suitable than BNE for in-gel detection of protein kinase activity after polyacrylamide gel electrophoresis (PAGE), and its performance in protein complex separation is comparable to that of conventional BNE. INE may provide a powerful tool to isolate and analyze various protein complexes. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Iron(II) supramolecular helicates condense plasmid DNA and inhibit vital DNA-related enzymatic activities.

    PubMed

    Malina, Jaroslav; Hannon, Michael J; Brabec, Viktor

    2015-07-27

    The dinuclear iron(II) supramolecular helicates [Fe2 L3 ]Cl4 (L=C25 H20 N4 ) bind to DNA through noncovalent (i.e., hydrogen-bonding, electrostatic) interactions and exhibit antimicrobial and anticancer effects. In this study, we show that the helicates condense plasmid DNA with a much higher potency than conventional DNA-condensing agents. Notably, molecules of DNA in the presence of the M enantiomer of [Fe2 L3 ]Cl4 do not form intermolecular aggregates typically formed by other condensing agents, such as spermidine or spermine. The helicates inhibit the activity of several DNA-processing enzymes, such as RNA polymerase, DNA topoisomerase I, deoxyribonuclease I, and site-specific restriction endonucleases. However, the results also indicate that the DNA condensation induced by the helicates does not play a crucial role in these inhibition reactions. The mechanisms for the inhibitory effects of [Fe2 L3 ]Cl4 helicates on DNA-related enzymatic activities have been proposed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Cold enzymatic bleaching of fluid whey.

    PubMed

    Campbell, R E; Drake, M A

    2013-01-01

    Chemical bleaching of fluid whey and retentate with hydrogen peroxide (HP) alone requires high concentrations (100-500 mg of HP/kg) and recent studies have demonstrated that off-flavors are generated during chemical bleaching that carry through to spray-dried whey proteins. Bleaching of fluid whey and retentate with enzymes such as naturally present lactoperoxidase or an exogenous commercial peroxidase (EP) at cold temperatures (4°C) may be a viable alternative to traditional chemical bleaching of whey. The objective of this study was to determine the optimum level of HP for enzymatic bleaching (both lactoperoxidase and EP) at 4°C and to compare bleaching efficacy and sensory characteristics to HP chemical bleaching at 4°C. Selected treatments were subsequently applied for whey protein concentrate with 80% protein (WPC80) manufacture. Fluid Cheddar whey and retentate (80% protein) were manufactured in triplicate from pasteurized whole milk. The optimum concentration of HP (0 to 250 mg/kg) to activate enzymatic bleaching at 4°C was determined by quantifying the loss of norbixin. In subsequent experiments, bleaching efficacy, descriptive sensory analysis, and volatile compounds were monitored at selected time points. A control with no bleaching was also evaluated. Enzymatic bleaching of fluid whey and retentate at 4°C resulted in faster bleaching and higher bleaching efficacy (color loss) than bleaching with HP alone at 250 mg/kg. Due to concentrated levels of naturally present lactoperoxidase, retentate bleached to completion (>80% norbixin destruction in 30 min) faster than fluid whey at 4°C (>80% norbixin destruction in 12h). In fluid whey, the addition of EP decreased bleaching time. Spray-dried WPC80 from bleached wheys, regardless of bleaching treatment, were characterized by a lack of sweet aromatic and buttery flavors, and the presence of cardboard flavor concurrent with higher relative abundance of 1-octen-3-ol and 1-octen-3-one. Among enzymatically

  14. Associations of common variants at ALDH2 gene and the risk of stroke in patients with coronary artery diseases undergoing percutaneous coronary intervention.

    PubMed

    You, Ling; Li, Chenze; Zhao, Jinzhao; Wang, Dao Wen; Cui, Wei

    2018-05-01

    Limited data are available about the role of common variants at the aldehyde dehydrogenase 2 gene (ALDH2) on the clinical outcome in Chinese patients with coronary heart disease (CHD) undergoing percutaneous coronary intervention (PCI). In the present study, a total of 1089 patients were consecutively enrolled from January 2012 and July 2013. Six common variants at ALDH2 gene, including rs2339840, rs4648328, rs4767939, rs11066028, rs16941669, and rs671, were selected to test the associations of those polymorphisms with the cardiovascular outcome in patients with CHD after PCI. The clinical endpoints included cardiovascular death, nonfatal myocardial infarction, and nonfatal stroke. The composite of clinical endpoints was defined as the primary endpoint, and every endpoint alone was considered as the secondary endpoints. The median follow-up time was 38.27 months. Our results showed that the common variant rs2339840 was independently associated with a lower risk of stroke in patients with CHD after PCI (codominant model, HR = 0.32, 95% CI, 0.11-0.91, P = .074 for heterozygotes; HR = 0.25, 95% CI, 0.06-1.14, P = .033 for homozygotes; dominant model, HR = 0.32, 95% CI, 0.14-0.74, P = .007). However, no significant associations were found between other 5 single nucleotide polymorphisms (SNPs) and the clinical endpoints. For the first time, the common variant rs2339840 was reported to be a protective factor against stroke in CHD patients with PCI.

  15. Isoprene Production on Enzymatic Hydrolysate of Peanut Hull Using Different Pretreatment Methods

    PubMed Central

    Wang, Sumeng; Li, Ruichao; Yi, Xiaohua; Fang, Tigao

    2016-01-01

    The present study is about the use of peanut hull for isoprene production. In this study, two pretreatment methods, hydrogen peroxide-acetic acid (HPAC) and popping, were employed prior to enzymatic hydrolysis, which could destroy the lignocellulosic structure and accordingly improve the efficiency of enzymatic hydrolysis. It is proven that the isoprene production on enzymatic hydrolysate with HPAC pretreatment is about 1.9-fold higher than that of popping pretreatment. Moreover, through High Performance Liquid Chromatography (HPLC) analysis, the amount and category of inhibitors such as formic acid, acetic acid, and HMF were assayed and were varied in different enzymatic hydrolysates, which may be the reason leading to a decrease in isoprene production during fermentation. To further increase the isoprene yield, the enzymatic hydrolysate of HPAC was detoxified by activated carbon. As a result, using the detoxified enzymatic hydrolysate as the carbon source, the engineered strain YJM21 could accumulate 297.5 mg/L isoprene, which accounted for about 90% of isoprene production by YJM21 fermented on pure glucose (338.6 mg/L). This work is thought to be the first attempt on isoprene production by E. coli using peanut hull as the feedstock. More importantly, it also shows the prospect of peanut hull to be considered as an alternative feedstock for bio-based chemicals or biofuels production due to its easy access and high polysaccharide content. PMID:27847814

  16. Bacterial communities and enzymatic activities in the vegetation-activated sludge process (V-ASP) and related advantages by comparison with conventional constructed wetland.

    PubMed

    Yuan, Jiajia; Dong, Wenyi; Sun, Feiyun; Zhao, Ke; Du, Changhang; Shao, Yunxian

    2016-11-01

    A new-developed vegetation-activated sludge process (V-ASP) was implemented for decentralized domestic wastewater treatment, and studied in lab-scale and full-scale. The main purpose of this work was the investigation of biomass activities and microbial communities in V-ASP by comparison with conventional constructed wetland (CW), to unveil the causations of its consistently higher pollutants removal efficiencies. Compared with CWs, V-ASP has greater vegetation nitrogen and phosphorus uptake rates, higher biomass and enzymatic activities, and more bacteria community diversity. The microbial community structure was comprehensively analyzed by using high-throughput sequencing. It was observed that Proteobacteria was dominated in both CWs and V-ASPs, while their subdivisions distribution was rather different. V-ASPs contained a higher nitrite-oxidizing bacteria (Nitrospira) abundances that resulted in a consistently better nitrogen removal efficiency. Hence, a long-term experiment of full-scale V-ASP displayed stably excellent capability in resistance of influent loading shocks and seasonal temperature effect. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Autoantibody against aldehyde dehydrogenase 2 could be a biomarker to monitor progression of Graves' orbitopathy.

    PubMed

    Cheng, Kai-Chun; Wu, Yu-Jen; Cheng, Kai-Hung; Cheng, Kai-Yuan; Chen, Kuo-Jen; Wu, Wen-Chuan; Lee, Po-Yen; Chang, Cheng-Hsien

    2018-06-01

    This study surveyed the novel autoantigens expressed in the orbital fat tissue of patients with Graves' orbitopathy (GO) and explored the possibility of the autoantibodies against novel autoantigens as biomarkers for GO. We used immuno-proteomic methods to survey novel autoantigens expressed in the orbit fat tissue of GO patients and confirmed by enzyme-linked immunosorbent assay (ELISA). One protein spot (aldehyde dehydrogenase 2 (ALDH2)) revealed high reactivity with the GO serum than did the healthy control serum and was further verified by ELISA. We found that the plasma anti-ALDH2 antibody level was increased in GO patients compared to healthy control donors. In addition, anti-ALDH2 antibody level was correlated with GO activity classified by clinical activity score(r = 0.588, p < 0.001, using Pearson's correlation). These increased levels of anti-ALDH2 antibody in GO serum suggested that ALDH2 could attribute target autoantigen in GO, and anti-ALDH2 autoantibody might serve as a biomarker for GO and help to predict disease activity.

  18. Aldehyde dehydrogenase expression in Metaphire posthuma as a bioindicator to monitor heavy metal pollution in soil.

    PubMed

    Panday, Raju; Bhatt, Padam Shekhar; Bhattarai, Tribikram; Shakya, Kumudini; Sreerama, Lakshmaiah

    2016-11-21

    Soil contamination and associated pollution plays a detrimental role in soil flora and fauna. Soil is processed and remodeled by subterranean earthworms, accordingly are referred to as soil chemical engineers. These worms, besides processing carbon and nitrogen, serve as minors for processing metals. In heavy metal contaminated soils, they accumulate heavy metals, which in turn cause altered gene expression, including aldehyde dehydrogenase (ALDH) enzymes. This study explores the possibility of ALDH expression in earthworms as a novel biomarker for the heavy metal contamination of soil. Earthworms cultured in contaminated soils accumulated significantly higher levels of Pb and Cd. Similarly, significantly higher levels of ALDH enzyme activities were observed in earthworms cultured in soils contaminated with Pb and Cd. The ALDH activity was found to be highest in worms cultured in 5 ppm heavy metal contaminated soils. Although, ALDH activities decreased as the heavy metal concentration in soil increased, they were significantly higher when compared to control worms cultured in uncontaminated soils. The accumulation of heavy metal in earthworms measured after 28 days decreased as the heavy metal concentration in soil increased. Levels of ALDH expression correlated with total Pb and Cd concentration in the earthworm tissue. This study showed that the ALDH activity in earthworms could potentially be used as a biomarker to show heavy metal pollution in soil.

  19. [Enzymatic analysis of the quality of foodstuffs].

    PubMed

    Kolesnov, A Iu

    1997-01-01

    Enzymatic analysis is an independent and separate branch of enzymology and analytical chemistry. It has become one of the most important methodologies used in food analysis. Enzymatic analysis allows the quick, reliable determination of many food ingredients. Often these contents cannot be determined by conventional methods, or if methods are available, they are determined only with limited accuracy. Today, methods of enzymatic analysis are being increasingly used in the investigation of foodstuffs. Enzymatic measurement techniques are used in industry, scientific and food inspection laboratories for quality analysis. This article describes the requirements of an optimal analytical method: specificity, sample preparation, assay performance, precision, sensitivity, time requirement, analysis cost, safety of reagents.

  20. 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

  1. Enzymatic regeneration of adenosine triphosphate cofactor

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1974-01-01

    Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

  2. D-Amino acid oxidase bio-functionalized platforms: Toward an enhanced enzymatic bio-activity

    NASA Astrophysics Data System (ADS)

    Herrera, Elisa; Valdez Taubas, Javier; Giacomelli, Carla E.

    2015-11-01

    The purpose of this work is to study the adsorption process and surface bio-activity of His-tagged D-amino acid oxidase (DAAO) from Rhodotorula gracilis (His6-RgDAAO) as the first step for the development of an electrochemical bio-functionalized platform. With such a purpose this work comprises: (a) the His6-RgDAAO bio-activity in solution determined by amperometry, (b) the adsorption mechanism of His6-RgDAAO on bare gold and carboxylated modified substrates in the absence (substrate/COO-) and presence of Ni(II) (substrate/COO- + Ni(II)) determined by reflectometry, and (c) the bio-activity of the His6-RgDAAO bio-functionalized platforms determined by amperometry. Comparing the adsorption behavior and bio-activity of His6-RgDAAO on these different solid substrates allows understanding the contribution of the diverse interactions responsible for the platform performance. His6-RgDAAO enzymatic performance in solution is highly improved when compared to the previously used pig kidney (pk) DAAO. His6-RgDAAO exhibits an amperometrically detectable bio-activity at concentrations as low as those expected on a bio-functional platform; hence, it is a viable bio-recognition element of D-amino acids to be coupled to electrochemical platforms. Moreover, His6-RgDAAO bio-functionalized platforms exhibit a higher surface activity than pkDAAO physically adsorbed on gold. The platform built on Ni(II) modified substrates present enhanced bio-activity because the surface complexes histidine-Ni(II) provide with site-oriented, native-like enzymes. The adsorption mechanism responsible of the excellent performance of the bio-functionalized platform takes place in two steps involving electrostatic and bio-affinity interactions whose prevalence depends on the degree of surface coverage.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25903123

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.

    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 sitemore » 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.« less

  5. 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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Clinical manifestations and enzymatic activities of mitochondrial respiratory chain complexes in Pearson marrow-pancreas syndrome with 3-methylglutaconic aciduria: a case report and literature review.

    PubMed

    Sato, Takeshi; Muroya, Koji; Hanakawa, Junko; Iwano, Reiko; Asakura, Yumi; Tanaka, Yukichi; Murayama, Kei; Ohtake, Akira; Hasegawa, Tomonobu; Adachi, Masanori

    2015-12-01

    Pearson marrow-pancreas syndrome (PS) is a rare mitochondrial disorder. Impaired mitochondrial respiratory chain complexes (MRCC) differ among individuals and organs, which accounts for variable clinical pictures. A subset of PS patients develop 3-methylglutaconic aciduria (3-MGA-uria), but the characteristic symptoms and impaired MRCC remain unknown. Our patient, a girl, developed pancytopenia, hyperlactatemia, steatorrhea, insulin-dependent diabetes mellitus, liver dysfunction, Fanconi syndrome, and 3-MGA-uria. She died from cerebral hemorrhage at 3 years of age. We identified a novel 5.4-kbp deletion of mitochondrial DNA. The enzymatic activities of MRCC I and IV were markedly reduced in the liver and muscle and mildly reduced in skin fibroblasts and the heart. To date, urine organic acid analysis has been performed on 29 PS patients, including our case. Eight patients had 3-MGA-uria, while only one patient did not. The remaining 20 patients were not reported to have 3-MGA-uria. In this paper, we included these 20 patients as PS patients without 3-MGA-uria. PS patients with and without 3-MGA-uria have similar manifestations. Only a few studies have examined the enzymatic activities of MRCC. No clinical characteristics distinguish between PS patients with and without 3-MGA-uria. The correlation between 3-MGA-uria and the enzymatic activities of MRCC remains to be elucidated. • The clinical characteristics of patients with Pearson marrow-pancreas syndrome and 3-methylglutaconic aciduria remain unknown. • No clinical characteristics distinguish between Pearson marrow-pancreas syndrome patients with and without 3-methylglutaconic aciduria.

  7. How Metal Substitution Affects the Enzymatic Activity of Catechol-O-Methyltransferase

    PubMed Central

    Sparta, Manuel; Alexandrova, Anastassia N.

    2012-01-01

    Catechol-O-methyltransferase (COMT) degrades catecholamines, such as dopamine and epinephrine, by methylating them in the presence of a divalent metal cation (usually Mg(II)), and S-adenosyl-L-methionine. The enzymatic activity of COMT is known to be vitally dependent on the nature of the bound metal: replacement of Mg(II) with Ca(II) leads to a complete deactivation of COMT; Fe(II) is slightly less than potent Mg(II), and Fe(III) is again an inhibitor. Considering the fairly modest role that the metal plays in the catalyzed reaction, this dependence is puzzling, and to date remains an enigma. Using a quantum mechanical / molecular mechanical dynamics method for extensive sampling of protein structure, and first principle quantum mechanical calculations for the subsequent mechanistic study, we explicate the effect of metal substitution on the rate determining step in the catalytic cycle of COMT, the methyl transfer. In full accord with experimental data, Mg(II) bound to COMT is the most potent of the studied cations and it is closely followed by Fe(II), whereas Fe(III) is unable to promote catalysis. In the case of Ca(II), a repacking of the protein binding site is observed, leading to a significant increase in the activation barrier and higher energy of reaction. Importantly, the origin of the effect of metal substitution is different for different metals: for Fe(III) it is the electronic effect, whereas in the case of Ca(II) it is instead the effect of suboptimal protein structure. PMID:23056605

  8. Ultrasonic accelerates asparagine-glucose non-enzymatic browning reaction without acrylamide formation.

    PubMed

    Gao, Zhiqiang; Zheng, Junfeng; Chen, Lian

    2017-01-01

    Ultrasonic accelerated the asparagine-glucose non-enzymatic browning reaction with significant decrease of glucose and asparagine concentrations, and marked increase of intermediate products (UV-absorbance value at 294nm, Abs 294 ), melanoidins (UV-absorbance value at 420nm, Abs 420 ) and in vitro antioxidant activity (DPPH free radical scavenging activity). As the ultrasonic intensity was 17.83W/cm 2 , the asparagine-glucose solution's Abs 294 , Abs 420 and antioxidant activity increased from 0 to 1.26, 0.88 and 21.56%, respectively, and the glucose and asparagine concentrations of the asparagine-glucose solution reduced 58.97 and 12.57%, respectively. The high performance liquid chromatography (HPLC)-Diode Array Detector (DAD) analyses showed that no acrylamide was detected after 50-min ultrasonic reaction. This study suggested that ultrasonic at higher intensity was a potential method to accelerate the non-enzymatic browning reaction in the asparagine-glucose solution without acrylamide production. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Maltodextrin-powered enzymatic fuel cell through a non-natural enzymatic pathway

    NASA Astrophysics Data System (ADS)

    Zhu, Zhiguang; Wang, Yiran; Minteer, Shelley D.; Percival Zhang, Y.-H.

    Enzymatic fuel cells (EFCs) use a variety of fuels to generate electricity through oxidoreductase enzymes, such as oxidases or dehydrogenases, as catalysts on electrodes. We have developed a novel synthetic enzymatic pathway containing two free enzymes (maltodextrin phosphorylase and phosphoglucomutase) and one immobilized glucose-6-phosphate dehydrogenase that can utilize an oligomeric substrate maltodextrin for producing electrons mediated via a diaphorase and vitamin K 3 electron shuttle system. Three different enzyme immobilization approaches were compared based on electrostatic force entrapment, chemical cross-linking, and cross-linking with the aid of carbon nanotubes. At 10 mM glucose-6-phosphate (G6P) as a substrate concentration, the maximum power density of 0.06 mW cm -2 and retaining 42% of power output after 11 days were obtained through the method of chemical cross-linking with carbon nanotubes, approximately 6-fold and 3.5-fold better than those of the electrostatic force-based method, respectively. When changed to maltodextrin (degree of polymerization = 19) as the substrate, the EFC achieved a maximum power density of 0.085 mW cm -2. With the advantages of stable, low cost, high energy density, non-inhibitor to enzymes, and environmental friendly, maltodextrin is suggested to be an ideal fuel to power enzymatic fuel cells.

  10. 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). © 2015 FEBS.

  11. 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.

  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. [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.

  14. Optimization of Process Parameters and Kinetic Model of Enzymatic Extraction of Polyphenols from Lonicerae Flos

    PubMed Central

    Kong, Fansheng; Yu, Shujuan; Bi, Yongguang; Huang, Xiaojun; Huang, Mengqian

    2016-01-01

    Objective: To optimize and verify the cellulase extraction of polyphenols from honeysuckle and provide a reference for enzymatic extracting polyphenols from honeysuckle. Materials and Methods: The uniform design was used According to Fick's first law and kinetic model, fitting analysis of the dynamic process of enzymatic extracting polyphenols was conducted. Results: The optimum enzymatic extraction parameters for polyphenols from honeysuckle are found to be 80% (v/v) of alcohol, 35:1 (mL/g) of liquid-solid ratio, 80°C of extraction temperature, 8.5 of pH, 6.0 mg of enzyme levels, and 130 min of extraction time. Under the optimal conditions, the extraction rate of polyphenols was 3.03%. The kinetic experiments indicated kinetic equation had a good linear relationship with t even under the conditions of different levels of enzyme and temperature, which means fitting curve tallies well with the experimental values. Conclusion: The results of quantification showed that the results provide a reference for enzymatic extracting polyphenols from honeysuckle. SUMMARY Lonicerae flos (Lonicera japonica Thunb.) is a material of traditional Chinese medicine and healthy drinks, of which active compounds mainly is polyphenols. At present, plant polyphenols are the hotspots centents of food, cosmetic and medicine, because it has strong bioactivity. Several traditional methods are available for the extraction of plant polyphenols including impregnation, solvent extraction, ultrasonic extraction, hot-water extraction, alkaline dilute alcohol or alkaline water extraction, microwave extraction and Supercritical CO2 extraction. But now, an increasing number of research on using cellulase to extract active ingredients from plants. Enzymatic method is widely used for enzyme have excellent properties of high reaction efficiency and specificity, moderate reaction conditions, shorter extraction time and easier to control, less damage to the active ingredient. At present, the enzymatic

  15. Inhibition of enzymatic browning of chlorogenic acid by sulfur-containing compounds.

    PubMed

    Kuijpers, Tomas F M; Narváez-Cuenca, Carlos-Eduardo; Vincken, Jean-Paul; Verloop, Annewieke J W; van Berkel, Willem J H; Gruppen, Harry

    2012-04-04

    The antibrowning activity of sodium hydrogen sulfite (NaHSO(3)) was compared to that of other sulfur-containing compounds. Inhibition of enzymatic browning was investigated using a model browning system consisting of mushroom tyrosinase and chlorogenic acid (5-CQA). Development of brown color (spectral analysis), oxygen consumption, and reaction product formation (RP-UHPLC-PDA-MS) were monitored in time. It was found that the compounds showing antibrowning activity either prevented browning by forming colorless addition products with o-quinones of 5-CQA (NaHSO(3), cysteine, and glutathione) or inhibiting the enzymatic activity of tyrosinase (NaHSO(3) and dithiothreitol). NaHSO(3) was different from the other sulfur-containing compounds investigated, because it showed a dual inhibitory effect on browning. Initial browning was prevented by trapping the o-quinones formed in colorless addition products (sulfochlorogenic acid), while at the same time, tyrosinase activity was inhibited in a time-dependent way, as shown by pre-incubation experiments of tyrosinase with NaHSO(3). Furthermore, it was demonstrated that sulfochlorogenic and cysteinylchlorogenic acids were not inhibitors of mushroom tyrosinase.

  16. Histone Deacetylase Inhibitors Stimulate Dedifferentiation of Human Breast Cancer Cells through WNT/β-catenin Signaling

    PubMed Central

    Debeb, Bisrat G; Lacerda, Lara; Xu, Wei; Larson, Richard; Solley, Travis; Atkinson, Rachel; Sulman, Erik P.; Ueno, Naoto T; Krishnamurthy, Savitri; Reuben, James M; Buchholz, Thomas A; Woodward, Wendy A

    2015-01-01

    Recent studies have shown that differentiated cancer cells can de-differentiate into cancer stem cells (CSCs) although to date no studies have reported whether this transition is influenced by systemic anti-cancer agents. Valproic acid (VA) is a histone deacetylase (HDAC) inhibitor that promotes self renewal and expansion of hematopietic stem cells and facilitates the generation of induced pluripotent stem cells from somatic cells and is currently being investigated in breast cancer clinical trials. We hypothesized that HDAC inhibitors reprogram differentiated cancer cells towards the more resistant stem cell-like state. Two highly aggressive breast cancer cell lines, SUM159 and MDA-231, were FACS-sorted based on ALDH activity and subsequently ALDH-negative and ALDH-positive cells were treated with one of two known HDAC inhibitors, VA or SAHA (suberoylanilide hydroxamic acid). In addition, primary tumor cells from patients with metastatic breast cancer were evaluated for ALDH activity following treatment with HDAC inhibitors. We demonstrate that single cell sorted ALDH- negative cells spontaneously generated ALDH-positive cells in vitro. Treatment of ALDH-negative cells with HDAC inhibitors promoted the expansion of ALDH-positive cells and increased mammosphere forming efficiency. Most importantly, it significantly increased the tumor-initiating capacity of ALDH- negative cells in limiting dilution outgrowth assays. Moreover, while HDAC inhibitors upregulated β-catenin expression and significantly increased WNT reporter activity, a TCF4 dominant negative construct abolished HDAC-inhibitor induced expansion of CSCs. These results demonstrate that HDAC inhibitors promote the expansion of breast CSCs through dedifferentiation and have important clinical implications for the use of HDAC inhibitors in the treatment of cancer. PMID:22961641

  17. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Morgan, Cynthia A.; Hurley, Thomas D.

    Aldehyde dehydrogenases (ALDH) catalyze the irreversible oxidation of aldehydes to their corresponding carboxylic acid. Alterations in ALDH1A1 activity are associated with such diverse diseases as cancer, Parkinson’s disease, obesity, and cataracts. Inhibitors of ALDH1A1 could aid in illuminating the role of this enzyme in disease processes. However, there are no commercially available selective inhibitors for ALDH1A1. Here we characterize two distinct chemical classes of inhibitors that are selective for human ALDH1A1 compared to eight other ALDH isoenzymes. The prototypical members of each structural class, CM026 and CM037, exhibit sub-micromolar inhibition constants, but have different mechanisms of inhibition. The crystal structuresmore » of these compounds bound to ALDH1A1 demonstrate that they bind within the aldehyde binding pocket of ALDH1A1 and exploit the presence of a unique Glycine residue to achieve their selectivity. Lastly, these two novel and selective ALDH1A1 inhibitors may serve as chemical tools to better understand the contributions of ALDH1A1 to normal biology and to disease states.« less

  18. Characterization of Two Distinct Structural Classes of Selective Aldehyde Dehydrogenase 1A1 Inhibitors

    DOE PAGES

    Morgan, Cynthia A.; Hurley, Thomas D.

    2015-01-29

    Aldehyde dehydrogenases (ALDH) catalyze the irreversible oxidation of aldehydes to their corresponding carboxylic acid. Alterations in ALDH1A1 activity are associated with such diverse diseases as cancer, Parkinson’s disease, obesity, and cataracts. Inhibitors of ALDH1A1 could aid in illuminating the role of this enzyme in disease processes. However, there are no commercially available selective inhibitors for ALDH1A1. Here we characterize two distinct chemical classes of inhibitors that are selective for human ALDH1A1 compared to eight other ALDH isoenzymes. The prototypical members of each structural class, CM026 and CM037, exhibit sub-micromolar inhibition constants, but have different mechanisms of inhibition. The crystal structuresmore » of these compounds bound to ALDH1A1 demonstrate that they bind within the aldehyde binding pocket of ALDH1A1 and exploit the presence of a unique Glycine residue to achieve their selectivity. Lastly, these two novel and selective ALDH1A1 inhibitors may serve as chemical tools to better understand the contributions of ALDH1A1 to normal biology and to disease states.« less

  19. Enzymatic treatment to improve the quality of black tea extracts.

    PubMed

    Chandini, S K; Rao, L Jaganmohan; Gowthaman, M K; Haware, D J; Subramanian, R

    2011-08-01

    Enzymatic extraction was investigated to improve the quality of black tea extracts with pretreatment of pectinase and tannase independently, successively and simultaneously. Pectinase improved the extractable-solids-yield (ESY) up to 11.5%, without much of an improvement in polyphenols recovery, while tannase pre-treatment showed a significant improvement in polyphenols recovery (14.3%) along with an 11.1% improvement in ESY. Among the four treatments, tannase-alone treatment showed the maximum improvement in tea quality, with higher polyphenols-in-extracted solids. Treatments involving tannase resulted in the significant release of gallic acid, due to its hydrolytic activity, leading to greater solubility besides favourably improving TF/TR ratio. The results suggested that employing a single enzyme, tannase, for the pre-treatment of black tea is desirable. Enzymatic extraction may be preferred over enzymatic clarification as it not only displayed reduction in tea cream and turbidity but also improved the recovery of polyphenols and ESY in the extract, as well as maintaining a good balance of tea quality. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin.

    PubMed

    Opelt, Marissa; Eroglu, Emrah; Waldeck-Weiermair, Markus; Russwurm, Michael; Koesling, Doris; Malli, Roland; Graier, Wolfgang F; Fassett, John T; Schrammel, Astrid; Mayer, Bernd

    2016-11-11

    Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 μm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production.

    PubMed

    Zarei, Adel; Trobacher, Christopher P; Shelp, Barry J

    2016-10-11

    Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress. Terminal catabolism of putrescine in Arabidopsis thaliana involves amine oxidase and the production of 4-aminobutanal, which is a substrate for NAD + -dependent aminoaldehyde dehydrogenase (AMADH). Here, two AMADH homologs were chosen (AtALDH10A8 and AtALDH10A9) as candidates for encoding 4-aminobutanal dehydrogenase activity for GABA synthesis. The two genes were cloned and soluble recombinant proteins were produced in Escherichia coli. The pH optima for activity and catalytic efficiency of recombinant AtALDH10A8 with 3-aminopropanal as substrate was 10.5 and 8.5, respectively, whereas the optima for AtALDH10A9 were approximately 9.5. Maximal activity and catalytic efficiency were obtained with NAD + and 3-aminopropanal, followed by 4-aminobutanal; negligible activity was obtained with betaine aldehyde. NAD + reduction was accompanied by the production of GABA and β-alanine, respectively, with 4-aminobutanal and 3-aminopropanal as substrates. Transient co-expression systems using Arabidopsis cell suspension protoplasts or onion epidermal cells and several organelle markers revealed that AtALDH10A9 was peroxisomal, but AtALDH10A8 was cytosolic, although the N-terminal 140 amino acid sequence of AtALDH10A8 localized to the plastid. Root growth of single loss-of-function mutants was more sensitive to salinity than wild-type plants, and this was accompanied by reduced GABA accumulation.

  2. What’s New in Enzymatic Halogenations

    PubMed Central

    Fujimori, Danica Galoniæ; Walsh, Christopher T.

    2007-01-01

    Summary The halogenation of thousands of natural products occurs during biosynthesis and often confers important functional properties. While haloperoxidases had been the default paradigm for enzymatic incorporation of halogens, via X+ equivalents into organic scaffolds, a combination of microbial genome sequencing, enzymatic studies and structural biology have provided deep new insights into enzymatic transfer of halide equivalents in three oxidation states. These are: (1) the halide ions (X−) abundant in nature, (2) halogen atoms (X•), and (3) the X+ equivalents. The mechanism of halogen incorporation is tailored to the electronic demands of specific substrates and involves enzymes with distinct redox coenzyme requirements. PMID:17881282

  3. Modification of chemical properties, Cu fractionation and enzymatic activities in an acid vineyard soil amended with winery wastes: A field study.

    PubMed

    Rodríguez-Salgado, Isabel; Pérez-Rodríguez, Paula; Gómez-Armesto, Antía; Díaz-Raviña, Montserrat; Nóvoa-Muñoz, Juan Carlos; Arias-Estévez, Manuel; Fernández-Calviño, David

    2017-11-01

    The effects of adding two winery wastes, perlite waste (PW) and bentonite waste (BW), to an acid vineyard soil were assessed using some chemical and biological soil properties in a field study that lasted 18 months. The addition of PW (up to 81 Mg ha -1 ) had neither significant nor permanent effects on soil characteristics such as the pH, organic matter content or nutrient concentrations, the amounts of copper or zinc, or the electrical conductivity. Moreover, no persistent negative effects were found on the enzymatic activities after PW application. In contrast, soil that was amended with up to 71 Mg BW ha -1 showed increases in its soil pH values, exchangeable potassium and water soluble potassium and phosphorus contents. In addition, it caused significant increases in the electrical conductivity and water-soluble Cu. In addition, the phosphomonoesterase enzymatic activity decreased significantly (up to 28%) in response to the amendment with 71 Mg BW ha -1 . These results showed that adding BW and PW to the soil may be a good agronomic practice for recycling these types of wastes. However, in the case of PW, its use as a soil amendment must be performed with caution to control its possible harmful effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Extraction and quantitation of coumarin from cinnamon and its effect on enzymatic browning in fresh apple juice: a bioinformatics approach to illuminate its antibrowning activity.

    PubMed

    Thada, Rajarajeshwari; Chockalingam, Shivashri; Dhandapani, Ramesh Kumar; Panchamoorthy, Rajasekar

    2013-06-05

    Enzymatic browning by polyphenoloxidase (PPO) affects food quality and taste in fruits and vegetables. Thus, the study was designed to reduce browning in apple juice by coumarin. The ethanolic extract of cinnamon was prepared and its coumarin content was quantitated by HPLC, using authentic coumarin (AC) as standard. The effect of cinnamon extract (CE) and AC on enzymatic browning, its time dependent effects, and the specific activity of PPO and peroxidase (POD) were studied in apple juice. The docking of coumarin with PPO and POD was also performed to elucidate its antibrowning mechanism. The CE (73%) and AC (82%) showed better reduction in browning, maintained its antibrowning effect at all time points, and significantly (p < 0.05) reduced the specific activity of PPO and POD when compared with controls. Coumarin showed strong interaction with binding pockets of PPO and POD, suggesting its potential use as inhibitor to enzyme mediated browning in apple juice.

  5. Algal extracellular release in river-floodplain dissolved organic matter: response of extracellular enzymatic activity during a post-flood period

    PubMed Central

    Sieczko, Anna; Maschek, Maria; Peduzzi, Peter

    2015-01-01

    River-floodplain systems are susceptible to rapid hydrological events. Changing hydrological connectivity of the floodplain generates a broad range of conditions, from lentic to lotic. This creates a mixture of allochthonously and autochthonously derived dissolved organic matter (DOM). Autochthonous DOM, including photosynthetic extracellular release (PER), is an important source supporting bacterial secondary production (BSP). Nonetheless, no details are available regarding microbial extracellular enzymatic activity (EEA) as a response to PER under variable hydrological settings in river-floodplain systems. To investigate the relationship between bacterial and phytoplankton components, we therefore used EEA as a tool to track the microbial response to non-chromophoric, but reactive and ecologically important DOM. The study was conducted in three floodplain subsystems with distinct hydrological regimes (Danube Floodplain National Park, Austria). The focus was on the post-flood period. Enhanced %PER (up to 48% of primary production) in a hydrologically isolated subsystem was strongly correlated with β-glucosidase, which was related to BSP. This shows that—in disconnected floodplain backwaters with high terrestrial input—BSP can also be driven by autochthonous carbon sources (PER). In a semi-isolated section, in the presence of fresh labile material from primary producers, enhanced activity of phenol oxidase was observed. In frequently flooded river-floodplain systems, BSP was mainly driven by enzymatic degradation of particulate primary production. Our research demonstrates that EEA measurements are an excellent tool to describe the coupling between bacteria and phytoplankton, which cannot be deciphered when focusing solely on chromophoric DOM. PMID:25741326

  6. Dual role of the carboxyl-terminal region of pig liver L-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity.

    PubMed

    Hirai, Kumiko; Kuroyanagi, Hidehito; Tatebayashi, Yoshitaka; Hayashi, Yoshitaka; Hirabayashi-Takahashi, Kanako; Saito, Kuniaki; Haga, Seiich; Uemura, Tomihiko; Izumi, Susumu

    2010-12-01

    l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMOΔC50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (ΔC20, ΔC30 and ΔC50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.

  7. Similar potential ATP-P production and enzymatic activities in the microplankton community off Concepción (Chile) under oxic and suboxic conditions

    NASA Astrophysics Data System (ADS)

    González, Rodrigo R.; Gutiérrez, Marcelo H.; Quiñones, Renato A.

    2007-11-01

    The effects of the oxygen minimum zone on the metabolism of the heterotrophic microplankton community (0.22-100 μm) in the Humboldt Current System, as well as the factors controlling its biomass production, remain unknown. Here we compare the effect of four sources of dissolved organic carbon (glucose, oxaloacetate, glycine, leucine) on microbial biomass production (such as ATP-P) and the potential enzymatic activities involved in catabolic pathways under oxic and suboxic conditions. Our results show significant differences ( p < 0.05) in the ATP-P production when induced by the different substrates that are used as dissolved organic carbon herein. The induction of ATP-P production is enhanced from glucose < oxaloacetate < glycine < leucine. Nevertheless, for individual substrates, no significant differences were found between incubation under oxic and suboxic conditions except in the case of leucine. For this amino acid, the induction of ATP-P synthesis was higher under suboxic than oxic conditions. The data sets of all the substrates used showed greater potential ATP-P production under suboxic than oxic conditions. The results of the potential enzymatic activities suggest that malate dehydrogenase has the highest signal of NADH oxidization activity in the microbial assemblage. Furthermore, for all experiments, the malate dehydrogenase activity data set had a significant relationship with ATP-P production. These findings suggest that the microbial community inhabiting the oxygen minimum zone has the same or greater potential growth than the community inhabiting more oxygenated strata of the water column and that malate dehydrogenase is the activity that best represents the metabolic potential of the community.

  8. Mechanism of lignin inhibition of enzymatic biomass deconstruction

    DOE PAGES

    Vermaas, Josh V.; Petridis, Loukas; Qi, Xianghong; ...

    2015-12-01

    The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process. By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose bindingmore » of TrCel7A (Y466, Y492, and Y493). In conclusion, lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.« less

  9. Changes on lipid peroxidation,enzymatic activities and gene expression in planarian (Dugesia japonica) following exposure to perfluorooctanoic acid.

    PubMed

    Yuan, Zuoqing; Miao, Zili; Gong, Xiaoning; Zhao, Baoying; Zhang, Yuanyuan; Ma, Hongdou; Zhang, Jianyong; Zhao, Bosheng

    2017-11-01

    We investigated perfluorooctanoic acid (PFOA)-induced stress response in planarians. We administered different concentrations of PFOA to planarians for up to 10 d. PFOA exposure resulted in significant concentration-dependent elevations in lipid peroxidation, glutathione S-transferase and caspase-3 protease activities, and a significant decline in glutathione peroxidase activities compared with control groups. Exposure to PFOA significantly up-regulated the heat shock proteins hsp70 and hsp90, and p53, and down-regulated hsp40 compared with controls. PFOA exposure also increased HSP70 protein levels, as demonstrated by western blot analysis. These alterations indicated that PFOA exposure induced a stress response and affected the regulation of oxidative stress, enzymatic activities and gene expression. These results suggest that these sensitive parameters, together with other biomarkers, could be used for evaluating toxicity, for ecological risk assessment of PFOA in freshwaters. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Arabidopsis and tobacco plants ectopically expressing the soybean antiquitin-like ALDH7 gene display enhanced tolerance to drought, salinity, and oxidative stress.

    PubMed

    Rodrigues, Simone M; Andrade, Maxuel O; Gomes, Ana Paula Soares; Damatta, Fabio M; Baracat-Pereira, Maria C; Fontes, Elizabeth P B

    2006-01-01

    Despite extensive studies in eukaryotic aldehyde dehydrogenases, functional information about the ALDH7 antiquitin-like proteins is lacking. A soybean antiquitin homologue gene, designated GmTP55, has been isolated which encodes a dehydrogenase motif-containing 55 kDa protein induced by dehydration and salt stress. GmTP55 is closely related to the stress-induced plant antiquitin-like proteins that belong to the ALDH7 family. Transgenic tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana) plants constitutively expressing GmTP55 have been obtained in order to examine the physiological role of this enzyme under a variety of stress conditions. Ectopic expression of GmTP55 in both Arabidopsis and tobacco conferred tolerance to salinity during germination and to water deficit during plant growth. Under salt stress, the germination efficiency of both transgenic tobacco and Arabidopsis seeds was significantly higher than that of their control counterparts. Likewise, under progressive drought, the transgenic tobacco lines apparently kept the shoot turgidity to a normal level, which contrasted with the leaf wilt phenotype of control plants. The transgenic plants also exhibited an enhanced tolerance to H(2)O(2)- and paraquat-induced oxidative stress. Both GmTP55-expressing Arabidopsis and tobacco seeds germinated efficiently in medium supplemented with H(2)O(2), whereas the germination of control seeds was drastically impaired. Similarly, transgenic tobacco leaf discs treated with paraquat displayed a significant reduction in the necrotic lesions as compared with control leaves. These transgenic lines also exhibited a lower concentration of lipid peroxidation-derived reactive aldehydes under oxidative stress. These results suggest that antiquitin may be involved in adaptive responses mediated by a physiologically relevant detoxification pathway in plants.

  11. A multiple mediator analysis approach to quantify the effects of the ADH1B and ALDH2 genes on hepatocellular carcinoma risk.

    PubMed

    Shih, Stephannie; Huang, Yen-Tsung; Yang, Hwai-I

    2018-06-01

    Previous work suggested a genetic component affecting the risk of hepatocellular carcinoma (HCC) and mediation analyses have elucidated potential indirect pathways of these genetic effects. Specifically, the effects of alcohol dehydrogenase (ADH1B) and aldehyde dehydrogenase (ALDH2) genes on HCC risk vary based on alcohol consumption habits. However, alcohol consumption may not be the only mediator in the identified pathway: factors related to alcohol consumption may contribute to the same indirect pathway. Thus, we developed a multimediator model to quantify the genetic effects on HCC risk through sequential dichotomous mediators under the counterfactual framework. Our method provided a closed form formula for the mediation effects through different indirect paths, which requires no assumption for the rarity of outcome. In simulation studies of a finite sample, we presented the utility of the method with the variance of the effects estimated using the delta method and bootstrapping. We applied our method to data from participants in Taiwan (580 cases and 3,207 controls) and quantified the mediation effects of single nucleotide polymorphisms (SNPs) in the ADH1B and ALDH2 genes on HCC through alcohol consumption (yes/no) and high alanine transaminase (ALT) levels (greater than or equal to 45 U/L or below 45 U/L). Assuming a dominant risk model, we identified that the SNPs' effects through alcohol consumption is more significant than through ALT levels on HCC risk. This new method provides insight to the magnitude of various casual mechanisms as a closed form solution and can be readily applied in other genomic studies. © 2018 WILEY PERIODICALS, INC.

  12. Active Sites of Reduced Epidermal Fluorescence1 (REF1) Isoforms Contain Amino Acid Substitutions That Are Different between Monocots and Dicots

    PubMed Central

    Missihoun, Tagnon D.; Kotchoni, Simeon O.; Bartels, Dorothea

    2016-01-01

    Plant aldehyde dehydrogenases (ALDHs) play important roles in cell wall biosynthesis, growth, development, and tolerance to biotic and abiotic stresses. The Reduced Epidermal Fluorescence1 is encoded by the subfamily 2C of ALDHs and was shown to oxidise coniferaldehyde and sinapaldehyde to ferulic acid and sinapic acid in the phenylpropanoid pathway, respectively. This knowledge has been gained from works in the dicotyledon model species Arabidopsis thaliana then used to functionally annotate ALDH2C isoforms in other species, based on the orthology principle. However, the extent to which the ALDH isoforms differ between monocotyledons and dicotyledons has rarely been accessed side-by-side. In this study, we used a phylogenetic approach to address this question. We have analysed the ALDH genes in Brachypodium distachyon, alongside those of other sequenced monocotyledon and dicotyledon species to examine traits supporting either a convergent or divergent evolution of the ALDH2C/REF1-type proteins. We found that B. distachyon, like other grasses, contains more ALDH2C/REF1 isoforms than A. thaliana and other dicotyledon species. Some amino acid residues in ALDH2C/REF1 isoforms were found as being conserved in dicotyledons but substituted by non-equivalent residues in monocotyledons. One example of those substitutions concerns a conserved phenylalanine and a conserved tyrosine in monocotyledons and dicotyledons, respectively. Protein structure modelling suggests that the presence of tyrosine would widen the substrate-binding pocket in the dicotyledons, and thereby influence substrate specificity. We discussed the importance of these findings as new hints to investigate why ferulic acid contents and cell wall digestibility differ between the dicotyledon and monocotyledon species. PMID:27798665

  13. Active Sites of Reduced Epidermal Fluorescence1 (REF1) Isoforms Contain Amino Acid Substitutions That Are Different between Monocots and Dicots.

    PubMed

    Missihoun, Tagnon D; Kotchoni, Simeon O; Bartels, Dorothea

    2016-01-01

    Plant aldehyde dehydrogenases (ALDHs) play important roles in cell wall biosynthesis, growth, development, and tolerance to biotic and abiotic stresses. The Reduced Epidermal Fluorescence1 is encoded by the subfamily 2C of ALDHs and was shown to oxidise coniferaldehyde and sinapaldehyde to ferulic acid and sinapic acid in the phenylpropanoid pathway, respectively. This knowledge has been gained from works in the dicotyledon model species Arabidopsis thaliana then used to functionally annotate ALDH2C isoforms in other species, based on the orthology principle. However, the extent to which the ALDH isoforms differ between monocotyledons and dicotyledons has rarely been accessed side-by-side. In this study, we used a phylogenetic approach to address this question. We have analysed the ALDH genes in Brachypodium distachyon, alongside those of other sequenced monocotyledon and dicotyledon species to examine traits supporting either a convergent or divergent evolution of the ALDH2C/REF1-type proteins. We found that B. distachyon, like other grasses, contains more ALDH2C/REF1 isoforms than A. thaliana and other dicotyledon species. Some amino acid residues in ALDH2C/REF1 isoforms were found as being conserved in dicotyledons but substituted by non-equivalent residues in monocotyledons. One example of those substitutions concerns a conserved phenylalanine and a conserved tyrosine in monocotyledons and dicotyledons, respectively. Protein structure modelling suggests that the presence of tyrosine would widen the substrate-binding pocket in the dicotyledons, and thereby influence substrate specificity. We discussed the importance of these findings as new hints to investigate why ferulic acid contents and cell wall digestibility differ between the dicotyledon and monocotyledon species.

  14. Histamine H4-Receptors Inhibit Mast Cell Renin Release in Ischemia/Reperfusion via Protein Kinase Cε-Dependent Aldehyde Dehydrogenase Type-2 Activation

    PubMed Central

    Aldi, Silvia; Takano, Ken-ichi; Tomita, Kengo; Koda, Kenichiro; Chan, Noel Y.-K.; Marino, Alice; Salazar-Rodriguez, Mariselis; Thurmond, Robin L.

    2014-01-01

    Renin released by ischemia/reperfusion (I/R) from cardiac mast cells (MCs) activates a local renin-angiotensin system (RAS) causing arrhythmic dysfunction. Ischemic preconditioning (IPC) inhibits MC renin release and consequent activation of this local RAS. We postulated that MC histamine H4-receptors (H4Rs), being Gαi/o-coupled, might activate a protein kinase C isotype–ε (PKCε)–aldehyde dehydrogenase type-2 (ALDH2) cascade, ultimately eliminating MC-degranulating and renin-releasing effects of aldehydes formed in I/R and associated arrhythmias. We tested this hypothesis in ex vivo hearts, human mastocytoma cells, and bone marrow–derived MCs from wild-type and H4R knockout mice. We found that activation of MC H4Rs mimics the cardioprotective anti-RAS effects of IPC and that protection depends on the sequential activation of PKCε and ALDH2 in MCs, reducing aldehyde-induced MC degranulation and renin release and alleviating reperfusion arrhythmias. These cardioprotective effects are mimicked by selective H4R agonists and disappear when H4Rs are pharmacologically blocked or genetically deleted. Our results uncover a novel cardioprotective pathway in I/R, whereby activation of H4Rs on the MC membrane, possibly by MC-derived histamine, leads sequentially to PKCε and ALDH2 activation, reduction of toxic aldehyde-induced MC renin release, prevention of RAS activation, reduction of norepinephrine release, and ultimately to alleviation of reperfusion arrhythmias. This newly discovered protective pathway suggests that MC H4Rs may represent a new pharmacologic and therapeutic target for the direct alleviation of RAS-induced cardiac dysfunctions, including ischemic heart disease and congestive heart failure. PMID:24696042

  15. Enzymatic reaction paths as determined by transition path sampling

    NASA Astrophysics Data System (ADS)

    Masterson, Jean Emily

    Enzymes are biological catalysts capable of enhancing the rates of chemical reactions by many orders of magnitude as compared to solution chemistry. Since the catalytic power of enzymes routinely exceeds that of the best artificial catalysts available, there is much interest in understanding the complete nature of chemical barrier crossing in enzymatic reactions. Two specific questions pertaining to the source of enzymatic rate enhancements are investigated in this work. The first is the issue of how fast protein motions of an enzyme contribute to chemical barrier crossing. Our group has previously identified sub-picosecond protein motions, termed promoting vibrations (PVs), that dynamically modulate chemical transformation in several enzymes. In the case of human heart lactate dehydrogenase (hhLDH), prior studies have shown that a specific axis of residues undergoes a compressional fluctuation towards the active site, decreasing a hydride and a proton donor--acceptor distance on a sub-picosecond timescale to promote particle transfer. To more thoroughly understand the contribution of this dynamic motion to the enzymatic reaction coordinate of hhLDH, we conducted transition path sampling (TPS) using four versions of the enzymatic system: a wild type enzyme with natural isotopic abundance; a heavy enzyme where all the carbons, nitrogens, and non-exchangeable hydrogens were replaced with heavy isotopes; and two versions of the enzyme with mutations in the axis of PV residues. We generated four separate ensembles of reaction paths and analyzed each in terms of the reaction mechanism, time of barrier crossing, dynamics of the PV, and residues involved in the enzymatic reaction coordinate. We found that heavy isotopic substitution of hhLDH altered the sub-picosecond dynamics of the PV, changed the favored reaction mechanism, dramatically increased the time of barrier crossing, but did not have an effect on the specific residues involved in the PV. In the mutant systems

  16. Substitution-inert trinuclear platinum complexes efficiently condense/aggregate nucleic acids and inhibit enzymatic activity**

    PubMed Central

    Malina, Jaroslav; Farrell, Nicholas P.; Brabec, Viktor

    2015-01-01

    The trinuclear platinum complexes ([{Pt(NH3)3}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}]6+, TriplatinNC‐A; [{trans-Pt(NH3)2(NH2(CH2)6NH3+)}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}]8+, TriplatinNC) belong to a class of biologically active agents that bind to DNA via nonbonding noncovalent (hydrogen bonding, electrostatic) interactions. Charge delocalization (6+ to 8+) in these linear trinuclear platinum complexes results in a high cellular uptake and promising cytotoxic activity in several carcinoma cell lines. We show in the present work with the aid of the methods of biophysical chemistry that in particular TriplatinNC condenses DNA with unprecedented potency which is much higher than that of conventional DNA condensing agents. In addition, in contrast to other DNA condensing agents, both platinum complexes induce aggregation of small transfer RNA molecules. We also demonstrate for the first time that TriplatinNC-A and TriplatinNC in particular completely inhibit DNA transcriptional activity at markedly lower concentration than naturally occurring spermine. Notably, the topoisomerase I-mediated relaxation of supercoiled DNA was inhibited by TriplatinNC-A and TriplatinNC at ~60-fold and ~250-fold lower concentration than that of spermine, respectively. We suggest that the general mechanisms of biological activity of TriplatinNC-A and TriplatinNC may be associated with their unique ability to condense/aggregate nucleic acids with consequent inhibitory effect on crucial enzymatic activities. PMID:25256921

  17. Influence of crop rotation, intermediate crops, and organic fertilizers on the soil enzymatic activity and humus content in organic farming systems

    NASA Astrophysics Data System (ADS)

    Marcinkeviciene, A.; Boguzas, V.; Balnyte, S.; Pupaliene, R.; Velicka, R.

    2013-02-01

    The influence of crop rotation systems with different portions of nitrogen-fixing crops, intermediate crops, and organic fertilizers on the enzymatic activity and humus content of soils in organic farming was studied. The highest activity of the urease and invertase enzymes was determined in the soil under the crop rotation with 43% nitrogen-fixing crops and with perennial grasses applied twice per rotation. The application of manure and the growing of intermediate crops for green fertilizers did not provide any significant increase in the content of humus. The activity of urease slightly correlated with the humus content ( r = 0.30 at the significance level of 0.05 and r = 0.39 at the significance level of 0.01).

  18. Enzymatic approaches to rare sugar production.

    PubMed

    Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    Rare sugars have recently attracted much attention because of their potential applications in the food, nutraceutical, and pharmaceutical industries. A systematic strategy for enzymatic production of rare sugars, named Izumoring, was developed >10years ago. The strategy consists of aldose-ketose isomerization, ketose C-3 epimerization, and monosaccharide oxidation-reduction. Recent development of the Izumoring strategy is reviewed herein, especially the genetic approaches to the improvement of rare sugar-producing enzymes and the applications of target-oriented bioconversion. In addition, novel non-Izumoring enzymatic approaches are also summarized, including enzymatic condensation, phosphorylation-dephosphorylation cascade reaction, aldose epimerization, ulosonic acid decarboxylation, and biosynthesis of rare disaccharides. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Altered enzymatic activity and allele frequency of OMI/HTRA2 in Alzheimer's disease

    PubMed Central

    Westerlund, Marie; Behbahani, Homira; Gellhaar, Sandra; Forsell, Charlotte; Belin, Andrea Carmine; Anvret, Anna; Zettergren, Anna; Nissbrandt, Hans; Lind, Charlotta; Sydow, Olof; Graff, Caroline; Olson, Lars; Ankarcrona, Maria; Galter, Dagmar

    2011-01-01

    The serine-protease OMI/HTRA2, required for several cellular processes, including mitochondrial function, autophagy, chaperone activity, and apoptosis, has been implicated in the pathogenesis of both Alzheimer's disease (AD) and Parkinson's disease (PD). Western blot quantification of OMI/HTRA2 in frontal cortex of patients with AD (n=10) and control subjects (n=10) in two separate materials indicated reduced processed (active, 35 kDa) OMI/HTRA2 levels, whereas unprocessed (50 kDa) enzyme levels were not significantly different between the groups. Interestingly, the specific protease activity of OMI/HTRA2 was found to be significantly increased in patients with AD (n=10) compared to matched control subjects (n=10) in frontal cortex in two separate materials. Comparison of OMI/HTRA2 mRNA levels in frontal cortex and hippocampus, two brain areas particularly affected by AD, indicated similar levels in patients with AD (n=10) and matched control subjects (n=10). In addition, we analyzed the occurrence of the OMI/HTRA2 variants A141S and G399S in Swedish case-control materials for AD and PD and found a weak association of A141S with AD, but not with PD. In conclusion, our genetic, histological, and biochemical findings give further support to an involvement of OMI/HTRA2 in the pathology of AD; however, further studies are needed to clarify the role of this gene in neurodegeneration.—Westerlund, M., Behbahani, H., Gellhaar, S., Forsell, C., Carmine Belin, A., Anvret, A., Zettergren, A., Nissbrandt, H., Lind, C., Sydow, O., Graff, C., Olson, L., Ankarcrona, M., Galter, D. Altered enzymatic activity and allele frequency of OMI/HTRA2 in Alzheimer's disease. PMID:21163861

  20. Kinetic properties of the human liver cytosolic aldehyde dehydrogenase for retinal isomers.

    PubMed

    Bhat, P V; Samaha, H

    1999-01-15

    Retinoic acid exerts pleiotropic effects by acting through two families of nuclear receptors, RAR and RXR. All-trans and 9-cis retinoic acid bind RARs, whereas 9-cis retinoic acid binds and activates only the RXRs. To understand the role of human liver cytosolic aldehyde dehydrogenase (ALDH1) in retinoic acid synthesis, we examined the ability of ALDH 1 to catalyze the oxidation of the naturally occurring retinal isomers. ALDH1 catalyzed the oxidation of all-trans, 9-cis, and 13-cis retinal with equal efficiency. However, the affinity to all-trans retinal (Km = 2.2 microM) was twofold higher than to 9-cis (Km = 5.5 microM) and 13-cis (Km = 4.6 microM) retinal. All-trans retinol was a potent inhibitor of ALDH1 activity, and inhibited all-trans retinal oxidation uncompetitively. Comparison of the kinetic properties of ALDH1 for retinal isomers with those of previously reported rat kidney retinal dehydrogenase showed distinct differences, suggesting that ALDH1 may play a different role in retinal metabolism in liver.

  1. Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis.

    PubMed

    Stiti, Naim; Missihoun, Tagnon D; Kotchoni, Simeon O; Kirch, Hans-Hubert; Bartels, Dorothea

    2011-01-01

    Aldehyde dehydrogenases (ALDHs) are a family of enzymes which catalyze the oxidation of reactive aldehydes to their corresponding carboxylic acids. Here we summarize molecular genetic and biochemical analyses of selected ArabidopsisALDH genes. Aldehyde molecules are very reactive and are involved in many metabolic processes but when they accumulate in excess they become toxic. Thus activity of aldehyde dehydrogenases is important in regulating the homeostasis of aldehydes. Overexpression of some ALDH genes demonstrated an improved abiotic stress tolerance. Despite the fact that several reports are available describing a role for specific ALDHs, their precise physiological roles are often still unclear. Therefore a number of genetic and biochemical tools have been generated to address the function with an emphasis on stress-related ALDHs. ALDHs exert their functions in different cellular compartments and often in a developmental and tissue specific manner. To investigate substrate specificity, catalytic efficiencies have been determined using a range of substrates varying in carbon chain length and degree of carbon oxidation. Mutational approaches identified amino acid residues critical for coenzyme usage and enzyme activities.

  2. Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis

    PubMed Central

    Stiti, Naim; Missihoun, Tagnon D.; Kotchoni, Simeon O.; Kirch, Hans-Hubert; Bartels, Dorothea

    2011-01-01

    Aldehyde dehydrogenases (ALDHs) are a family of enzymes which catalyze the oxidation of reactive aldehydes to their corresponding carboxylic acids. Here we summarize molecular genetic and biochemical analyses of selected Arabidopsis ALDH genes. Aldehyde molecules are very reactive and are involved in many metabolic processes but when they accumulate in excess they become toxic. Thus activity of aldehyde dehydrogenases is important in regulating the homeostasis of aldehydes. Overexpression of some ALDH genes demonstrated an improved abiotic stress tolerance. Despite the fact that several reports are available describing a role for specific ALDHs, their precise physiological roles are often still unclear. Therefore a number of genetic and biochemical tools have been generated to address the function with an emphasis on stress-related ALDHs. ALDHs exert their functions in different cellular compartments and often in a developmental and tissue specific manner. To investigate substrate specificity, catalytic efficiencies have been determined using a range of substrates varying in carbon chain length and degree of carbon oxidation. Mutational approaches identified amino acid residues critical for coenzyme usage and enzyme activities. PMID:22639603

  3. Glycated cholecystokinin-8 has an enhanced satiating activity and is protected against enzymatic degradation.

    PubMed

    O'Harte, F P; Mooney, M H; Kelly, C M; Flatt, P R

    1998-10-01

    Monoglycated cholecystokinin octapeptide (CCK-8) (glucitol-Asp1 adduct) modified at the NH2-terminus was prepared under hyperglycemic conditions, purified by high-performance liquid chromatography, and characterized by mass spectrometry (Mr 1228.4 Da) and peptide sequencing. CCK-8 (100 nmol/kg, i.p.) significantly (P < 0.001) reduced voluntary food intake of fasted mice for up to 30 min after its administration, compared with saline-administered controls. Glycated CCK-8 reduced food intake at 30-120 min (P < 0.01 to P < 0.001) and significantly reduced feeding compared with CCK-8 from 60 to 120 min (P < 0.01). In vitro plasma degradation studies indicated that glycated CCK-8 was resistant to the normal rapid enzymatic conversion to CCK fragments. This study demonstrated that CCK-8 is a potent short-term inhibitor of food intake, and that structural modification of this peptide by amino-terminal glycation leads to enhanced satiating activity, partially due to increased resistance to serum aminopeptidase degradation.

  4. Topical formulations with superoxide dismutase: influence of formulation composition on physical stability and enzymatic activity.

    PubMed

    Di Mambro, Valéria M; Borin, Maria F; Fonseca, Maria J V

    2003-04-24

    Three different topical formulations were supplemented with superoxide dismutase (SOD) and evaluated concerning physical and chemical stabilities in order to determine the most stable formulation that would maintain SOD activity. Physical stability was evaluated by storing the formulation at room temperature, and at 37 and 45 degrees C for 28 days. Samples were collected at 7-day intervals for assessment of rheological behavior. Chemical stability was evaluated by the measurement of enzymatic activity in formulations stored at room temperature and at 45 degrees C for 75 days. The formulations showed a pseudoplastic behavior, with a flow index of less than 1. There was no significant difference in the initial values of flow index, hysteresis loop or minimum apparent viscosity. The simple emulsion and the one stabilized with hydroxyethylcellulose showed decreased viscosity by the 21st day and with higher temperature, but no significant changes concerning the presence of SOD. Although there were no significant changes concerning storage time or temperature, the formulation stabilized with hydroxyethylcellulose showed a marked loss of SOD activity. The addition of SOD to the formulations studied did not affect their physical stability. Simple emulsions or emulsions stabilized with carboxypolymethylene seem to be better bases for enzyme addition than emulsion stabilized with hydroxyethylcellulose.

  5. Enzymatic hydrolysis of biomimetic bacterial cellulose-hemicellulose composites.

    PubMed

    Penttilä, Paavo A; Imai, Tomoya; Hemming, Jarl; Willför, Stefan; Sugiyama, Junji

    2018-06-15

    The production of biofuels and other chemicals from lignocellulosic biomass is limited by the inefficiency of enzymatic hydrolysis. Here a biomimetic composite material consisting of bacterial cellulose and wood-based hemicelluloses was used to study the effects of hemicelluloses on the enzymatic hydrolysis with a commercial cellulase mixture. Bacterial cellulose synthesized in the presence of hemicelluloses, especially xylan, was found to be more susceptible to enzymatic hydrolysis than hemicellulose-free bacterial cellulose. The reason for the easier hydrolysis could be related to the nanoscale structure of the substrate, particularly the packing of cellulose microfibrils into ribbons or bundles. In addition, small-angle X-ray scattering was used to show that the average nanoscale morphology of bacterial cellulose remained unchanged during the enzymatic hydrolysis. The reported easier enzymatic hydrolysis of bacterial cellulose produced in the presence of wood-based xylan offers new insights to overcome biomass recalcitrance through genetic engineering. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Enzymatic Decontamination of Environmental Organophosphorus Compounds

    DTIC Science & Technology

    2006-12-04

    ABSTRACT (Maximum 200 words) The abstract is below since many authors do not follow the 200 word limit 14. SUBJECT TERMS organophosphorus compounds ...5404 Enzymatic decontamination of environmental organophosphorus compounds REPORT DOCUMENTATION PAGE 18. SECURITY CLASSIFICATION ON THIS PAGE...239-18 298-102 15. NUMBER OF PAGES 20. LIMITATION OF ABSTRACT UL - 4-Dec-2006 Enzymatic decontamination of environmental organophosphorus compounds

  7. Enzymatic production of biodiesel from microalgal oil using ethyl acetate as an acyl acceptor.

    PubMed

    Alavijeh, Razieh Shafiee; Tabandeh, Fatemeh; Tavakoli, Omid; Karkhane, Aliasghar; Shariati, Parvin

    2015-01-01

    Microalgae have become an important source of biomass for biodiesel production. In enzymatic transesterification reaction, the enzyme activity is decreased in presence of alcohols. The use of different acyl acceptors such as methyl/ethyl acetate is suggested as an alternative and effective way to overcome this problem. In this study, ethyl acetate was used for the first time in the enzymatic production of biodiesel by using microalga, Chlorella vulgaris, as a triglyceride source. Enzymatic conversion of such fatty acids to biodiesel was catalyzed by Novozym 435 as an efficient immobilized lipase which is extensively used in biodiesel production. The best conversion yield of 66.71% was obtained at the ethyl acetate to oil molar ratio of 13:1 and Novozym 435 concentration of 40%, based on the amount of oil, and a time period of 72 h at 40℃. The results showed that ethyl acetate have no adverse effect on lipase activity and the biodiesel amount was not decreased even after seven transesterification cycles, so ethyl acetate has a great potential to be substituted for short-chain alcohols in transesterification reaction.

  8. Enzymatic extraction of star gooseberry (Phyllanthus acidus) juice with high antioxidant level

    NASA Astrophysics Data System (ADS)

    Loan, Do Thi Thanh; Tra, Tran Thi Thu; Nguyet, Ton Nu Minh; Man, Le Van Viet

    2017-09-01

    Ascorbic acid and phenolic compounds are main antioxidants in star gooseberry (Phyllanthus acidus) fruit. In this study, Pectinex Ultra SP-L preparation with pectinase activity was used in the extraction of star gooseberry juice. The effects of pectinase concentration and biocatalytic time on the content of ascorbic acid, phenolic compounds and antioxidant activity of the fruit juice were firstly investigated. Response surface methodology was then used to optimize the conditions of enzymatic extraction for maximizing the antioxidant activity of the star gooseberry juice. The optimal pectinase concentration and biocatalytic time were 19 polygalacturonase units per 100g pulp dry weight and 67 min, respectively under which the maximal antioxidant activity achieved 5595±6 µmol Trolox equivalent per 100g juice dry weight. On the basis of kinetic model of second-order extraction, the extraction rate constant of ascorbic acid and phenolic compounds in the enzymatic extraction increased approximately 21% and 157%, respectively in comparison with that in the conventional extraction. Application of pectinase preparation to the fruit juice extraction was therefore potential for improvement in antioxidant level of the product.

  9. Enhanced enzymatic hydrolysis of kenaf core using irradiation and dilute acid

    NASA Astrophysics Data System (ADS)

    Lee, Byoung-Min; Jeun, Joon-Pyo; Kang, Phil-Hyun

    2017-01-01

    This study was performed to determine the effect of electron beam dose and enzymatic hydrolysis time for production of sugar such as glucose and xylose. After kenaf core was exposed to an irradiation dose that ranged from 0 to 500 kGy, the irradiated kenaf core was treated with a 3% (v/v) sulfuric acid solution using an autoclave for 5 h at 120 °C. The pretreated kenaf core was subsequently subjected to enzymatic hydrolysis at 50 °C in a shaking water bath at 150 rpm for 12, 24, 48, and 72 h. The determined enzyme activity rates were 70 FPU (Celluclast 1.5 L) and 40 CBU (Novozyme-188). The crystallinity index decreased from 50.6% in a non-pretreated kenaf core to 27.7% in kenaf core that was subjected to the two-stage pretreatment at dose of 500 kGy. The sugar yield of the two-stage pretreated kenaf core increased with an increase in irradiation dose. The sugar yield after 72 h of enzymatic hydrolysis was 73.6% at its highest with an irradiation dose of 500 kGy. The enhancement of enzymatic hydrolysis by two-stage pretreatment was more effective than non- and single pretreatment (36.9%, 40.6% and 44.0% in non-pretreatment, electron beam and dilute acid, respectively).

  10. Oxygen isotope ratios of PO4: An inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life

    PubMed Central

    Blake, Ruth E.; Alt, Jeffrey C.; Martini, Anna M.

    2001-01-01

    The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (δ18Op) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO4–H2O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that δ18OP values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of δ18Op as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, δ18Op may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars. PMID:11226207

  11. Oxygen isotope ratios of PO4: an inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life.

    PubMed

    Blake, R E; Alt, J C; Martini, A M

    2001-02-27

    The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (delta(18)O(p)) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO(4)-H(2)O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that delta(18)O(P) values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of delta(18)O(p) as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, delta(18)O(p) may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars.

  12. 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.

  13. Characterization of enzymatic micromachining for construction of variable cross-section microchannel topologies

    PubMed Central

    Ruggles, Molly E.; Jayaraman, Arul; Ugaz, Victor M.

    2016-01-01

    The ability to harness enzymatic activity as an etchant to precisely machine biodegradable substrates introduces new possibilities for microfabrication. This flow-based etching is straightforward to implement, enabling patterning of microchannels with topologies that incorporate variable depth along the cross-sectional dimension. Additionally, unlike conventional small-molecule formulations, the macromolecular nature of enzymatic etchants enables features to be precisely positioned. Here, we introduce a kinetic model to characterize the enzymatic machining process and its localization by co-injection of a macromolecular inhibitor species. Our model captures the interaction between enzyme, inhibitor, and substrate under laminar flow, enabling rational prediction of etched microchannel profiles so that cross-sectional topologies incorporating complex lateral variations in depth can be constructed. We also apply this approach to achieve simultaneous widening of an entire network of microchannels produced in the biodegradable polymeric substrate poly(lactic acid), laying a foundation to construct systems incorporating a broad range of internal cross-sectional dimensions by manipulating the process conditions. PMID:27190566

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

    PubMed Central

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

    2016-01-01

    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. PMID:26582203

  15. JMJD6 and U2AF65 co-regulate alternative splicing in both JMJD6 enzymatic activity dependent and independent manner

    PubMed Central

    Yi, Jia; Shen, Hai-Feng; Qiu, Jin-Song; Huang, Ming-Feng; Zhang, Wen-Juan; Ding, Jian-Cheng; Zhu, Xiao-Yan; Zhou, Yu

    2017-01-01

    Abstract JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes. PMID:27899633

  16. 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

  17. TMPRSS4 induces cancer stem cell-like properties in lung cancer cells and correlates with ALDH expression in NSCLC patients.

    PubMed

    de Aberasturi, Arrate L; Redrado, Miriam; Villalba, Maria; Larzabal, Leyre; Pajares, Maria J; Garcia, Javier; Evans, Stephanie R; Garcia-Ros, David; Bodegas, Maria Elena; Lopez, Lissett; Montuenga, Luis; Calvo, Alfonso

    2016-01-28

    Metastasis involves a series of changes in cancer cells that promote their escape from the primary tumor and colonization to a new organ. This process is related to the transition from an epithelial to a mesenchymal phenotype (EMT). Recently, some authors have shown that migratory cells with an EMT phenotype share properties of cancer stem cells (CSCs), which allow them to form a new tumor mass. The type II transmembrane serine protease TMPRSS4 is highly expressed in some solid tumors, promotes metastasis and confers EMT features to cancer cells. We hypothesized that TMPRSS4 could also provide CSC properties. Overexpression of TMPRSS4 reduces E-cadherin and induces N-cadherin and vimentin in A549 lung cancer cells, supporting an EMT phenotype. These changes are accompanied by enhanced migration, invasion and tumorigenicity in vivo. TMPRSS4 expression was highly increased in a panel of lung cancer cells cultured as tumorspheres (a typical assay to enrich for CSCs). H358 and H441 cells with knocked-down TMPRSS4 levels were significantly less able to form primary and secondary tumorspheres than control cells. Moreover, they showed a lower proportion of ALDH+ cells (examined by FACS analysis) and lower expression of some CSC markers than controls. A549 cells overexpressing TMPRSS4 conferred the opposite phenotype and were also more sensitive to the CSC-targeted drug salinomycin than control cells, but were more resistant to regular chemotherapeutic drugs (cisplatin, gemcitabine and 5-fluorouracil). Analysis of 70 NSCLC samples from patients revealed a very significant correlation between TMPRSS4 expression and CSC markers ALDH (p = 0.0018) and OCT4 (p = 0.0004), suggesting that TMPRSS4 is associated with a CSC phenotype in patients' tumors. These results show that TMPRSS4, in addition to inducing EMT, can also promote CSC features in lung cancer; therefore, CSC-targeting drugs could be an appropriate treatment for TMPRSS4+ tumors. Copyright © 2015 Elsevier

  18. 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.

  19. Bioelectrocatalytic NAD+/NADH inter-conversion: transformation of an enzymatic fuel cell into an enzymatic redox flow battery.

    PubMed

    Quah, Timothy; Milton, Ross D; Abdellaoui, Sofiene; Minteer, Shelley D

    2017-07-25

    Diaphorase and a benzylpropylviologen redox polymer were combined to create a bioelectrode that can both oxidize NADH and reduce NAD + . We demonstrate how bioelectrocatalytic NAD + /NADH inter-conversion can transform a glucose/O 2 enzymatic fuel cell (EFC) with an open circuit potential (OCP) of 1.1 V into an enzymatic redox flow battery (ERFB), which can be rapidly recharged by operation as an EFC.

  20. Extracellular enzymatic activity of two hydrolases in wastewater treatment for biological nutrient removal.

    PubMed

    Berrio-Restrepo, Jorge Mario; Saldarriaga, Julio César; Correa, Mauricio Andrés; Aguirre, Néstor Jaime

    2017-10-01

    Due to the complex nature of the wastewater (both domestic and non-domestic) composition, biological processes are widely used to remove nutrients, such as carbon (C), nitrogen (N), and phosphorous (P), which cause instability and hence contribute to the damage of water bodies. Systems with different configurations have been developed (including anaerobic, anoxic, and aerobic conditions) for the joint removal of carbon, nitrogen, and phosphorus. The goal of this research is to evaluate the extracellular activity of β-glucosidase and phosphatase enzymes in a University of Cape Town (UCT) system fed with two synthetic wastewaters of different molecular complexity. Both types of waters have medium strength characteristics similar to those of domestic wastewater with a mean C/N/P ratio of 100:13:1. The operation parameters were hydraulic retention time (HRT) of 10 h, solid retention time (SRT) of 12 days, mean concentration of the influent in terms of chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN), and total phosphorus (TP) of 600, 80, and 6 mg/L, respectively. According to the results obtained, statistically significant differences have been found in the extracellular enzyme activities with the evaluated wastewaters and in the units comprising the treatment system in some of the cases. An analysis of principal components showed that the extracellular enzymatic activity has been correlated to nutrient concentration in wastewater, biomass concentration in the system, and metabolic conditions of treatment phases. Additionally, this research has allowed determining an inverse relationship between wastewater biodegradability and the extracellular enzyme activity of β-glucosidase and phosphatase. These results highlight the importance of including the analysis of biomass biochemical characteristics as control methods in wastewater treatment systems for the nutrient removal.

  1. PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

    PubMed

    Kostova, Maya B; Brennen, William Nathaniel; Lopez, David; Anthony, Lizamma; Wang, Hao; Platz, Elizabeth; Denmeade, Samuel R

    2018-08-01

    Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA <0.1 ng/mL. PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate

  2. Single-Molecule Probing the Energy Landscape of Enzymatic Reaction and Non-Covalent Interactions

    NASA Astrophysics Data System (ADS)

    Lu, H. Peter; Hu, Dehong; Chen, Yu; Vorpagel, Erich R.

    2002-03-01

    We have applied single-molecule spectroscopy under physiological conditions to study the mechanisms and dynamics of T4 lysozyme enzymatic reactions, characterizing mode-specific protein conformational dynamics. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time. The overall reaction rates were found to vary widely from molecule-to-molecule, and the initial non-specific binding of the enzyme to the substrate was seen to dominate this inhomogeneity. The reaction steps subsequent to the initial binding were found to have homogeneous rates. Molecular dynamics simulation has been applied to elucidate the mechanism and intermediate states of the single-molecule enzymatic reaction. Combining the analysis of single-molecule experimental trajectories, MD simulation trajectories, and statistical modeling, we have revealed the nature of multiple intermediate states involved in the active enzyme-substrate complex formation and the associated conformational change mechanism and dynamics.

  3. Rational design of functional and tunable oscillating enzymatic networks

    NASA Astrophysics Data System (ADS)

    Semenov, Sergey N.; Wong, Albert S. Y.; van der Made, R. Martijn; Postma, Sjoerd G. J.; Groen, Joost; van Roekel, Hendrik W. H.; de Greef, Tom F. A.; Huck, Wilhelm T. S.

    2015-02-01

    Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks.

  4. Methods for improving enzymatic trans-glycosylation for synthesis of human milk oligosaccharide biomimetics.

    PubMed

    Zeuner, Birgitte; Jers, Carsten; Mikkelsen, Jørn Dalgaard; Meyer, Anne S

    2014-10-08

    Recently, significant progress has been made within enzymatic synthesis of biomimetic, functional glycans, including, for example, human milk oligosaccharides. These compounds are mainly composed of N-acetylglucosamine, fucose, sialic acid, galactose, and glucose, and their controlled enzymatic synthesis is a novel field of research in advanced food ingredient chemistry, involving the use of rare enzymes, which have until now mainly been studied for their biochemical significance, not for targeted biosynthesis applications. For the enzymatic synthesis of biofunctional glycans reaction parameter optimization to promote "reverse" catalysis with glycosidases is currently preferred over the use of glycosyl transferases. Numerous methods exist for minimizing the undesirable glycosidase-catalyzed hydrolysis and for improving the trans-glycosylation yields. This review provides an overview of the approaches and data available concerning optimization of enzymatic trans-glycosylation for novel synthesis of complex bioactive carbohydrates using sialidases, α-l-fucosidases, and β-galactosidases as examples. The use of an adequately high acceptor/donor ratio, reaction time control, continuous product removal, enzyme recycling, and/or the use of cosolvents may significantly improve trans-glycosylation and biocatalytic productivity of the enzymatic reactions. Protein engineering is also a promising technique for obtaining high trans-glycosylation yields, and proof-of-concept for reversing sialidase activity to trans-sialidase action has been established. However, the protein engineering route currently requires significant research efforts in each case because the structure-function relationship of the enzymes is presently poorly understood.

  5. Complex inhibition of autophagy by mitochondrial aldehyde dehydrogenase shortens lifespan and exacerbates cardiac aging.

    PubMed

    Zhang, Yingmei; Wang, Cong; Zhou, Jingmin; Sun, Aijun; Hueckstaedt, Lindsay K; Ge, Junbo; Ren, Jun

    2017-08-01

    Autophagy, a conservative degradation process for long-lived and damaged proteins, participates in a cascade of biological processes including aging. A number of autophagy regulators have been identified. Here we demonstrated that mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme with the most common single point mutation in humans, governs cardiac aging through regulation of autophagy. Myocardial mechanical and autophagy properties were examined in young (4months) and old (26-28months) wild-type (WT) and global ALDH2 transgenic mice. ALDH2 overexpression shortened lifespan by 7.7% without affecting aging-associated changes in plasma metabolic profiles. Myocardial function was compromised with aging associated with cardiac hypertrophy, the effects were accentuated by ALDH2. Aging overtly suppressed autophagy and compromised autophagy flux, the effects were exacerbated by ALDH2. Aging dampened phosphorylation of JNK, Bcl-2, IKKβ, AMPK and TSC2 while promoting phosphorylation of mTOR, the effects of which were exaggerated by ALDH2. Co-immunoprecipitation revealed increased dissociation between Bcl-2 and Beclin-1 (result of decreased Bcl-2 phosphorylation) in aging, the effect of which was exacerbated with ALDH2. Chronic treatment of the autophagy inducer rapamycin alleviated aging-induced cardiac dysfunction in both WT and ALDH2 mice. Moreover, activation of JNK and inhibition of either Bcl-2 or IKKβ overtly attenuated ALDH2 activation-induced accentuation of cardiomyocyte aging. Examination of the otherwise elderly individuals revealed a positive correlation between cardiac function/geometry and ALDH2 gene mutation. Taken together, our data revealed that ALDH2 enzyme may suppress myocardial autophagy possibly through a complex JNK-Bcl-2 and IKKβ-AMPK-dependent mechanism en route to accentuation of myocardial remodeling and contractile dysfunction in aging. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure

  6. A comparison of the distribution of enzymatically and non-enzymatically produced lead phosphate in insect flight muscle.

    PubMed

    Tice, L W

    1969-01-01

    Lead phosphate precipitates were produced in indirect flight muscles of Phormia regina by sequential incubation in solutions containing lead and inorganic phosphate and their distribution was compared with those produced by ATP hydrolysis in the presence of lead. Enzymatically produced precipitates were associated almost exclusively with thick filaments. Non-enzymatically produced precipitates were associated with thick filaments but were also found associated with thin filaments in significant numbers.

  7. Enzymatic DNA molecules

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F. (Inventor); Breaker, Ronald R. (Inventor)

    1998-01-01

    The present invention discloses deoxyribonucleic acid enzymes--catalytic or enzymatic DNA molecules--capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

  8. Nature and position of functional group on thiopurine substrates influence activity of xanthine oxidase--enzymatic reaction pathways of 6-mercaptopurine and 2-mercaptopurine are different.

    PubMed

    Tamta, Hemlata; Kalra, Sukirti; Thilagavathi, Ramasamy; Chakraborti, Asit K; Mukhopadhyay, Anup K

    2007-02-01

    Xanthine oxidase-catalyzed hydroxylation reactions of the anticancer drug 6-mercaptopurine (6-MP) and its analog 2-mercaptopurine (2-MP) as well as 6-thioxanthine (6-TX) and 2-thioxanthine (2-TX) have been studied using UV-spectroscopy, high pressure liquid chromatography, photodiode array, and liquid chromatography-based mass spectral analysis. It is shown that 6-MP and 2-MP are oxidatively hydroxylated through different pathways. Enzymatic hydroxylation of 6-MP forms 6-thiouric acid in two steps involving 6-TX as the intermediate, whereas 2-MP is converted to 8-hydroxy-2-mercaptopurine as the expected end product in one step. Surprisingly, in contrast to the other thiopurines, enzymatic hydroxylation of 2-MP showed a unique hyperchromic effect at 264 nm as the reaction proceeded. However, when 2-TX is used as the substrate, it is hydroxylated to 2-thiouric acid. The enzymatic hydroxylation of 2-MP is considerably faster than that of 6-MP, while 6-TX and 2-TX show similar rates under identical reaction conditions. The reason why 2-MP is a better substrate than 6-MP and how the chemical nature and position of the functional groups present on the thiopurine substrates influence xanthine oxidase activity are discussed.

  9. Structure, enzymatic transformation, anticancer activity of fucoidan and sulphated fucooligosaccharides from Sargassum horneri.

    PubMed

    Silchenko, Artem S; Rasin, Anton B; Kusaykin, Mikhail I; Kalinovsky, Anatoly I; Miansong, Zhang; Changheng, Liu; Malyarenko, Olesya; Zueva, Anastasiya O; Zvyagintseva, Tatyana N; Ermakova, Svetlana P

    2017-11-01

    Structure and anticancer activity of fucoidan from Sargassum horneri and from products of its enzymatic transformation were investigated. A gene that encodes fucoidanase ffa1 in the marine bacteria F. algae was identified, cloned and the protein (FFA1) was produced in Escherichia coli. The mass of the gene product FFA1 is 111kDa. Sequence analysis has revealed that fucoidanase FFA1 belongs to the GH107 (CAZy) family. Recombinant fucoidanase FFA1 was used to produce fucooligosaccharides. Structure of 5 sulphated oligosaccharides with polymerization degree 4-10 was established by NMR-spectroscopy. The fucoidan extracted from S. horneri is almost pure fucan. The main chain of the fucoidan is established to consist mostly of the repeating →3-α-l-Fucp(2SO 3 - )-1→4-α-l-Fucp(2,3SO 3 - )-1→ fragment, with insertions of →3-α-l-Fucp(2,4SO 3 - )-1→ fragment. Unsulphated side chains with the α-l-Fucp-1→2-α-l-Fucp-1→ structure connect to the main one at the C4 of monosaccharide residue. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Fluorescence lifetime analysis and effect of magnesium ions on binding of NADH to human aldehyde dehydrogenase 1

    USDA-ARS?s Scientific Manuscript database

    Aldehyde dehydrogenase 1 (ALDH1) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence ALDH1 activity in part by increasing NADH binding affinity to the enzyme thus reducing activity. By using time-resolved fluorescence spectroscopy, we have resolved t...

  11. Classical dendritic cells mediate fibrosis directly via the retinoic acid pathway in severe eye allergy

    PubMed Central

    Ahadome, Sarah D.; Mathew, Rose; Reyes, Nancy J.; Mettu, Priyatham S.; Cousins, Scott W.; Calder, Virginia L.; Saban, Daniel R.

    2016-01-01

    Fibrosis is a shared end-stage pathway to lung, liver, and heart failure. In the ocular mucosa (conjunctiva), fibrosis leads to blindness in trachoma, pemphigoid, and allergy. The indirect fibrogenic role of DCs via T cell activation and inflammatory cell recruitment is well documented. However, here we demonstrate that DCs can directly induce fibrosis. In the mouse model of allergic eye disease (AED), classical CD11b+ DCs in the ocular mucosa showed increased activity of aldehyde dehydrogenase (ALDH), the enzyme required for retinoic acid synthesis. In vitro, CD11b+ DC–derived ALDH was associated with 9-cis-retinoic acid ligation to retinoid x receptor (RXR), which induced conjunctival fibroblast activation. In vivo, stimulating RXR led to rapid onset of ocular mucosal fibrosis, whereas inhibiting ALDH activity in DCs or selectively depleting DCs markedly reduced fibrosis. Collectively, these data reveal a profibrotic ALDH-dependent pathway by DCs and uncover a role for DC retinoid metabolism. PMID:27595139

  12. Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies

    PubMed Central

    Mirabelli, Peppino; Di Noto, Rosa; Lo Pardo, Catia; Morabito, Paolo; Abate, Giovanna; Gorrese, Marisa; Raia, Maddalena; Pascariello, Caterina; Scalia, Giulia; Gemei, Marica; Mariotti, Elisabetta; Del Vecchio, Luigi

    2008-01-01

    Background Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. Results In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). Conclusion Our study, comparing surface antigen expression of ALDH+/CD34+, ALDH

  13. Aldehyde dehydrogenase-2 genotypes and HLA haplotypes in Japanese patients with esophageal cancer.

    PubMed

    Watanabe, Seishiro; Sasahara, Katsuyuki; Kinekawa, Fumihiko; Uchida, Naohito; Masaki, Tsutomu; Kurokohchi, Kazutaka; Murota, Masayuki; Touge, Tetsuo; Kawauchi, Kazuyoshi; Oda, Syuji; Kuriyama, Shigeki

    2002-01-01

    The aim of this study was to examine how aldehyde dehydrogenase-2 (ALDH2) genotypes and human leukocyte antigen (HLA) haplotypes contribute to the risk for esophageal cancer. We examined ALDH2 genotypes and HLA haplotypes in 29 Japanese patients with esophageal cancer. The ratio of patients who experienced current or former intense vasodilatation upon consuming alcohol (flushing type) was much higher in individuals with the inactive form of ALDH2 encoded by the ALDH2(2)/2(2) or ALDH2(1)/2(2) genotype than in those with the active form of ALDH2 encoded by the ALDH2(1)/2(1) genotype. The ratio of inactive ALDH2 was significantly higher in patients with esophageal cancer than in control normal subjects, suggesting that alcoholics with inactive ALDH2 were susceptible to esophageal cancer. HLA haplotypes A24, A26, B54, B61 and DR9 were prevalent in patients with esophageal cancer (82.8, 24.1, 34.5, 37.9 and 44.8%, respectively). HLA haplotype of A24 and inactive ALDH2 were simultaneously found in 58.6% of patients with esophageal cancer. Furthermore, we found other primary malignancies in 6 of 29 (20.7%) patients with esophageal cancer, and 4 of these 6 patients had both the inactive form of ALDH2 and the HLA A24 haplotype. The present study showed the high prevalence of the inactive form of ALDH2 and HLA haplotypes A24, A26, B54, B61 and DR9 in Japanese patients with esophageal cancer. Therefore, the examination of genotypes of ALDH2 loci and HLA haplotypes may allow the early detection of esophageal cancer in the Japanese population.

  14. Volatile Compound, Physicochemical, and Antioxidant Properties of Beany Flavor-Removed Soy Protein Isolate Hydrolyzates Obtained from Combined High Temperature Pre-Treatment and Enzymatic Hydrolysis.

    PubMed

    Yoo, Sang-Hun; Chang, Yoon Hyuk

    2016-12-01

    The present study investigated the volatile compound, physicochemical, and antioxidant properties of beany flavor-removed soy protein isolate (SPI) hydrolyzates produced by combined high temperature pre-treatment and enzymatic hydrolysis. Without remarkable changes in amino acid composition, reductions of residual lipoxygenase activity and beany flavor-causing volatile compounds such as hexanol, hexanal, and pentanol in SPI were observed after combined heating and enzymatic treatments. The degree of hydrolysis, emulsion capacity and stability, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, and superoxide radical scavenging activity of SPI were significantly increased, but the magnitudes of apparent viscosity, consistency index, and dynamic moduli (G', G″) of SPI were significantly decreased after the combined heating and enzymatic treatments. Based on these results, it was suggested that the enzymatic hydrolysis in combination with high temperature pre-treatment may allow for the production of beany flavor-removed SPI hydrolyzates with superior emulsifying and antioxidant functionalities.

  15. Comparison of enzymatic activities in different Candida species isolated from women with vulvovaginitis.

    PubMed

    Fatahinia, M; Halvaeezadeh, M; Rezaei-Matehkolaei, A

    2017-06-01

    Comparing the activities of secreted enzymes in different fungal species can improve our understanding of their pathogenic role. Secretion of various enzymes by Candida species has been considered for determination of their virulence in different Candida infections including vulvovaginitis. The aim of this study was to determine and compare the activity of secreted enzymes in Candidia strains isolated from women suspected to vulvovaginal candidiasis (VVC) and referred to some health centers in Khuzestan, Southwestern Iran. The vaginal secretion samples were taken by swap from 250 suspected women with symptoms of vulvovaginal candidiasis and cultured on CHROMagar Candida medium. Identification of the isolated Candida from culture positive samples performed by the color of colonies and some standard mycological procedures. Activities of phospholipase, hemolysin-α, hemolysin-β, esterase and proteinase were measured in vitro by standard laboratory protocols. The enzymatic activity index (EAI) was calculated for each enzyme in accordance to relevant protocols. Totally in eighty cases (32%), a Candida strain was isolated which found to be as 52 (65%) Candida albicans; 12 (15%) C. glabrata; 10 (12.5%) C. dubliniensis; 4 (5%) C. krusei, C. tropicalis and C. parapsilosis species (each=1; 1.3%). Among C. albicans strains, 89.1% produced all studied enzymes, while 86% of C. glabrata strains failed to produce proteinase and phospholipase. The EAIs in decreasing order were as hemolysin-β=0.2895, hemolysin-α=0.5420, esterase=0.5753, proteinase=0.7413, and phospholipase=0.7446, respectively. Activity of phospholipase, esterase and proteinase secreted by C. albicans and C. dubliniensis were significantly more than those released by C. glabrata and C. krusei, while 86% of C. glabrata strains did not show esterase activity. On the other hand, the activity rates of hemolysin α and β among all studied isolates were almost similar. In the present study, the prevalence

  16. JMJD6 and U2AF65 co-regulate alternative splicing in both JMJD6 enzymatic activity dependent and independent manner.

    PubMed

    Yi, Jia; Shen, Hai-Feng; Qiu, Jin-Song; Huang, Ming-Feng; Zhang, Wen-Juan; Ding, Jian-Cheng; Zhu, Xiao-Yan; Zhou, Yu; Fu, Xiang-Dong; Liu, Wen

    2017-04-07

    JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Enzymatic modification of egg lecithin to improve properties.

    PubMed

    Asomaning, Justice; Curtis, Jonathan M

    2017-04-01

    This research studied the enzymatic modification of egg yolk phospholipids and its effect on physicochemical properties. Egg yolk lipids were extracted with food grade ethanol and egg phospholipids (ePL) produced by deoiling with acetone. Vegetable oils were used to interesterify ePL utilizing Lipozyme®: sn-1,3 specific lipase. The enzymatic interesterification resulted in a single phase liquid product, whereas simple blending of the ePL and vegetable oil resulted in a product with two phases. In addition solid fat content decreased by 50% at -10°C and 94% at 35°C when compared with egg yolk lipids extract. A decrease in melting temperature resulted from the interesterification process. Interesterification improved emulsion stability index when used as an emulsifier in oil-in-water emulsion and compared to the native and soy lecithin. Enzyme reusability test showed retention of 63% activity after 10 cycles. Overall, the properties of native egg phospholipids were significantly enhanced in a potentially useful manner through interesterification. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Enzymatic and chemical synthesis of new anticoagulant peptides.

    PubMed

    Origone, Anabella; Bersi, Grisel; Illanes, Andrés; Sturniolo, Héctor; Liggieri, Constanza; Guzmán, Fanny; Barberis, Sonia

    2018-06-08

    In this study we report the enzymatic synthesis of N-α-[Carbobenzyloxy]-Tyr-Gln-Gln (Z-YQQ), a new anticoagulant tripeptide. It was obtained using phytoproteases from the stems and petioles of Asclepias curassavica L. as catalyst in an aqueous-organic biphasic system formed by 50% (v/v) ethyl acetate and 0.1 M Tris - HCl buffer pH 8. The resulting peptide was compared with the analogous peptide Tyr-Gln-Gln (YQQ) produced by solid-phase chemical synthesis. The in vitro anticoagulant activity of the above mentioned peptides was determined using Wiener Lab Test (Wiener, Argentina). The toxicological activity of the peptides was also determined. The enzymatically synthesized Z-YQQ peptide acted on the extrinsic pathway of the coagulation cascade, delaying the conversion time of prothrombin to thrombin and fibrinogen to fibrin by 136% and 50%, respectively, with respect to the controls. The chemically synthesized YQQ peptide acted specifically on the intrinsic pathway of the coagulation cascade, affecting factors VIII, IX, XI and XII from such cascade, and increasing the coagulation time by 105% with respect to the control. The results suggest that two new anticoagulant peptides (Z-YQQ and YQQ) can be useful for safe pharmaceutical applications. Nevertheless, some aspects related to peptide production should be optimized. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

  19. Effect of alkali lignins with different molecular weights from alkali pretreated rice straw hydrolyzate on enzymatic hydrolysis.

    PubMed

    Li, Yun; Qi, Benkun; Luo, Jianquan; Wan, Yinhua

    2016-01-01

    This study investigated the effect of alkali lignins with different molecular weights on enzymatic hydrolysis of lignocellulose. Different alkali lignins fractions, which were obtained from cascade ultrafiltration, were added into the dilute acid pretreated (DAP) and alkali pretreated (AP) rice straws respectively during enzymatic hydrolysis. The results showed that the addition of alkali lignins enhanced the hydrolysis and the enhancement for hydrolysis increased with increasing molecular weights of alkali lignins, with maximum enhancement being 28.69% for DAP and 20.05% for AP, respectively. The enhancement was partly attributed to the improved cellulase activity, and filter paper activity increased by 18.03% when adding lignin with highest molecular weight. It was found that the enhancement of enzymatic hydrolysis was correlated with the adsorption affinity of cellulase on alkali lignins, and the difference in surface charge and hydrophobicity of alkali lignins were responsible for the difference in affinity between cellulase and lignins. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Scale-up and integration of alkaline hydrogen peroxide pretreatment, enzymatic hydrolysis, and ethanolic fermentation.

    PubMed

    Banerjee, Goutami; Car, Suzana; Liu, Tongjun; Williams, Daniel L; Meza, Sarynna López; Walton, Jonathan D; Hodge, David B

    2012-04-01

    Alkaline hydrogen peroxide (AHP) has several attractive features as a pretreatment in the lignocellulosic biomass-to-ethanol pipeline. Here, the feasibility of scaling-up the AHP process and integrating it with enzymatic hydrolysis and fermentation was studied. Corn stover (1 kg) was subjected to AHP pretreatment, hydrolyzed enzymatically, and the resulting sugars fermented to ethanol. The AHP pretreatment was performed at 0.125 g H(2) O(2) /g biomass, 22°C, and atmospheric pressure for 48 h with periodic pH readjustment. The enzymatic hydrolysis was performed in the same reactor following pH neutralization of the biomass slurry and without washing. After 48 h, glucose and xylose yields were 75% and 71% of the theoretical maximum. Sterility was maintained during pretreatment and enzymatic hydrolysis without the use of antibiotics. During fermentation using a glucose- and xylose-utilizing strain of Saccharomyces cerevisiae, all of the Glc and 67% of the Xyl were consumed in 120 h. The final ethanol titer was 13.7 g/L. Treatment of the enzymatic hydrolysate with activated carbon prior to fermentation had little effect on Glc fermentation but markedly improved utilization of Xyl, presumably due to the removal of soluble aromatic inhibitors. The results indicate that AHP is readily scalable and can be integrated with enzyme hydrolysis and fermentation. Compared to other leading pretreatments for lignocellulosic biomass, AHP has potential advantages with regard to capital costs, process simplicity, feedstock handling, and compatibility with enzymatic deconstruction and fermentation. Biotechnol. Bioeng. 2012; 109:922-931. © 2011 Wiley Periodicals, Inc. Copyright © 2011 Wiley Periodicals, Inc.

  1. Enzymatic Inverse Opal Hydrogel Particles for Biocatalyst.

    PubMed

    Wang, Huan; Gu, Hongcheng; Chen, Zhuoyue; Shang, Luoran; Zhao, Ze; Gu, Zhongze; Zhao, Yuanjin

    2017-04-19

    Enzymatic carriers have a demonstrated value for chemical reactions and industrial applications. Here, we present a novel kind of inverse opal hydrogel particles as the enzymatic carriers. The particles were negatively replicated from spherical colloidal crystal templates by using magnetic nanoparticles tagged acrylamide hydrogel. Thus, they were endowed with the features of monodispersity, small volume, complete penetrating structure, and controllable motion, which are all beneficial for improving the efficiency of biocatalysis. In addition, due to the ordered porous nanostructure, the inverse opal hydrogel particles were imparted with unique photonic band gaps (PBGs) and vivid structural colors for encoding varieties of immobilized enzymes and for constructing a multienzymes biocatalysis system. These features of the inverse opal hydrogel particles indicate that they are ideal enzymatic carriers for biocatalysis.

  2. Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi.

    PubMed

    Dias, Albino A; Freitas, Gil S; Marques, Guilhermina S M; Sampaio, Ana; Fraga, Irene S; Rodrigues, Miguel A M; Evtuguin, Dmitry V; Bezerra, Rui M F

    2010-08-01

    Wheat straw was submitted to a pre-treatment by the basidiomycetous fungi Euc-1 and Irpex lacteus, aiming to improve the accessibility of cellulose towards enzymatic hydrolysis via previous selective bio-delignification. This allowed the increase of substrate saccharification nearly four and three times while applying the basidiomycetes Euc-1 and I. lacteus, respectively. The cellulose/lignin ratio increased from 2.7 in the untreated wheat straw to 5.9 and 4.6 after the bio-treatment by the basidiomycetes Euc-1 and I. lacteus, respectively, thus evidencing the highly selective lignin biodegradation. The enzymatic profile of both fungi upon bio-treatment of wheat straw have been assessed including laccase, manganese-dependent peroxidase, lignin peroxidase, carboxymethylcellulase, xylanase, avicelase and feruloyl esterase activities. The difference in efficiency and selectivity of delignification within the two fungi treatments was interpreted in terms of specific lignolytic enzyme profiles and moderate xylanase and cellulolytic activities. (c) 2010 Elsevier Ltd. All rights reserved.

  3. Printing enzymatic reactions.

    PubMed

    Tian, Junfei; Shen, Wei

    2011-02-07

    We used relief and planographic printing methods to print the catalytic effect of an enzyme, but not the enzyme molecules, onto paper. Printing enzymatic reactions have applications in bioactive papers, low-cost diagnostics, anti-counterfeiting devices and advanced packaging materials. These methods can create novel printing effects on commodity surfaces for advanced applications.

  4. Genome-Scale Architecture of Small Molecule Regulatory Networks and the Fundamental Trade-Off between Regulation and Enzymatic Activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Reznik, Ed; Christodoulou, Dimitris; Goldford, Joshua E.

    Metabolic flux is in part regulated by endogenous small molecules that modulate the catalytic activity of an enzyme, e.g., allosteric inhibition. In contrast to transcriptional regulation of enzymes, technical limitations have hindered the production of a genome-scale atlas of small molecule-enzyme regulatory interactions. Here, we develop a framework leveraging the vast, but fragmented, biochemical literature to reconstruct and analyze the small molecule regulatory network (SMRN) of the model organism Escherichia coli, including the primary metabolite regulators and enzyme targets. Using metabolic control analysis, we prove a fundamental trade-off between regulation and enzymatic activity, and we combine it with metabolomic measurementsmore » and the SMRN to make inferences on the sensitivity of enzymes to their regulators. By generalizing the analysis to other organisms, we identify highly conserved regulatory interactions across evolutionarily divergent species, further emphasizing a critical role for small molecule interactions in the maintenance of metabolic homeostasis.« less

  5. Genome-Scale Architecture of Small Molecule Regulatory Networks and the Fundamental Trade-Off between Regulation and Enzymatic Activity

    DOE PAGES

    Reznik, Ed; Christodoulou, Dimitris; Goldford, Joshua E.; ...

    2017-09-12

    Metabolic flux is in part regulated by endogenous small molecules that modulate the catalytic activity of an enzyme, e.g., allosteric inhibition. In contrast to transcriptional regulation of enzymes, technical limitations have hindered the production of a genome-scale atlas of small molecule-enzyme regulatory interactions. Here, we develop a framework leveraging the vast, but fragmented, biochemical literature to reconstruct and analyze the small molecule regulatory network (SMRN) of the model organism Escherichia coli, including the primary metabolite regulators and enzyme targets. Using metabolic control analysis, we prove a fundamental trade-off between regulation and enzymatic activity, and we combine it with metabolomic measurementsmore » and the SMRN to make inferences on the sensitivity of enzymes to their regulators. By generalizing the analysis to other organisms, we identify highly conserved regulatory interactions across evolutionarily divergent species, further emphasizing a critical role for small molecule interactions in the maintenance of metabolic homeostasis.« less

  6. Imbalanced nutrient recycling in a warmer ocean driven by differential response of extracellular enzymatic activities.

    PubMed

    Ayo, Begoña; Abad, Naiara; Artolozaga, Itxaso; Azua, Iñigo; Baña, Zuriñe; Unanue, Marian; Gasol, Josep M; Duarte, Carlos M; Iriberri, Juan

    2017-10-01

    Ocean oligotrophication concurrent with warming weakens the capacity of marine primary producers to support marine food webs and act as a CO 2 sink, and is believed to result from reduced nutrient inputs associated to the stabilization of the thermocline. However, nutrient supply in the oligotrophic ocean is largely dependent on the recycling of organic matter. This involves hydrolytic processes catalyzed by extracellular enzymes released by bacteria, which temperature dependence has not yet been evaluated. Here, we report a global assessment of the temperature-sensitivity, as represented by the activation energies (E a ), of extracellular β-glucosidase (βG), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) enzymatic activities, which enable the uptake by bacteria of substrates rich in carbon, nitrogen, and phosphorus, respectively. These E a were calculated from two different approaches, temperature experimental manipulations and a space-for-time substitution approach, which generated congruent results. The three activities showed contrasting E a in the subtropical and tropical ocean, with βG increasing the fastest with warming, followed by LAP, while AP showed the smallest increase. The estimated activation energies predict that the hydrolysis products under projected warming scenarios will have higher C:N, C:P and N:P molar ratios than those currently generated, and suggest that the warming of oceanic surface waters leads to a decline in the nutrient supply to the microbial heterotrophic community relative to that of carbon, particularly so for phosphorus, slowing down nutrient recycling and contributing to further ocean oligotrophication. © 2017 John Wiley & Sons Ltd.

  7. Volatile Compound, Physicochemical, and Antioxidant Properties of Beany Flavor-Removed Soy Protein Isolate Hydrolyzates Obtained from Combined High Temperature Pre-Treatment and Enzymatic Hydrolysis

    PubMed Central

    Yoo, Sang-Hun; Chang, Yoon Hyuk

    2016-01-01

    The present study investigated the volatile compound, physicochemical, and antioxidant properties of beany flavor-removed soy protein isolate (SPI) hydrolyzates produced by combined high temperature pre-treatment and enzymatic hydrolysis. Without remarkable changes in amino acid composition, reductions of residual lipoxygenase activity and beany flavor-causing volatile compounds such as hexanol, hexanal, and pentanol in SPI were observed after combined heating and enzymatic treatments. The degree of hydrolysis, emulsion capacity and stability, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, and superoxide radical scavenging activity of SPI were significantly increased, but the magnitudes of apparent viscosity, consistency index, and dynamic moduli (G′, G″) of SPI were significantly decreased after the combined heating and enzymatic treatments. Based on these results, it was suggested that the enzymatic hydrolysis in combination with high temperature pre-treatment may allow for the production of beany flavor-removed SPI hydrolyzates with superior emulsifying and antioxidant functionalities. PMID:28078256

  8. Enzymatic Conversion of CO2 to Bicarbonate in Functionalized Mesoporous Silica

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yu, Yuehua; Chen, Baowei; Qi, Wen N.

    2012-05-01

    We report here that carbonic anhydrase (CA), the fastest enzyme that can covert carbon dioxide to bicarbonate, can be spontaneously entrapped in functionalized mesoporous silica (FMS) with super-high loading density (up to 0.5 mg of protein/mg of FMS) due to the dominant electrostatic interaction. The binding of CA to HOOC-FMS can result in the protein’s conformational change comparing to the enzyme free in solution, but can be overcome with increased protein loading density. The higher the protein loading density, the less conformational change, hence the higher enzymatic activity and the higher enzyme immobilization efficiency. The electrostatically bound CA can bemore » released by changing pH. The released enzyme still displayed the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. This work opens up a new approach converting carbon dioxide to biocarbonate in a biomimetic nanoconfiguration that can be integrated with the other part of biosynthesis process for the assimilation of carbon dioxide.« less

  9. Co-composting of organic fraction of municipal solid waste mixed with different bulking waste: characterization of physicochemical parameters and microbial enzymatic dynamic.

    PubMed

    Awasthi, Mukesh Kumar; Pandey, Akhilesh Kumar; Bundela, Pushpendra Singh; Khan, Jamaluddin

    2015-04-01

    The effect of various bulking waste such as wood shaving, agricultural and yard trimming waste combined with organic fraction of municipal solid waste (OFMSW) composting was investigated through assessing their influence on microbial enzymatic activities and quality of finished compost. All three piles of OFMSW with different bulking waste were inoculated with microbial consortium. The results revealed that OFMSW combined with wood shaving and microbial consortium (Phanerochaete chrysosporium, Trichoderma viride and Pseudomonas aeruginosa) were helpful tool to facilitate the enzymatic activity and shortened composting period within 4 weeks. Maximum enzymatic activity were observed in pile 1 and 3 during the first 3 weeks, while in pile 2 relatively very low. But phosphatase activity was relatively higher in all piles until the end of the process. Maturity parameters of compost quality also favored the pile 1 as the best formulation for OFMSW composting. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Physicochemical structural changes of cellulosic substrates during enzymatic saccharification

    DOE PAGES

    Meng, Xianzhi; Yoo, Chang Geun; Li, Mi; ...

    2016-12-30

    Enzymatic hydrolysis represents one of the major steps and barriers in the commercialization process of converting cellulosic substrates into biofuels and other value added products. It is usually achieved by a synergistic action of enzyme mixture typically consisting of multiple enzymes such as glucanase, cellobiohydrolase and β-glucosidase with different mode of actions. Due to the innate biomass recalcitrance, enzymatic hydrolysis normally starts with an initial fast rate of hydrolysis followed by a rapid decrease of rate toward the end of hydrolysis. With majority of literature studies focusing on the effect of key substrate characteristics on the initial rate or finalmore » yield of enzymatic hydrolysis, information about physicochemical structural changes of cellulosic substrates during enzymatic hydrolysis is still quite limited. Consequently, what slows down the reaction rate toward the end of hydrolysis is not well understood. Lastly, this review highlights recent advances in understanding the structural changes of cellulosic substrates during the hydrolysis process, to better understand the fundamental mechanisms of enzymatic hydrolysis.« less

  11. Physicochemical structural changes of cellulosic substrates during enzymatic saccharification

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Meng, Xianzhi; Yoo, Chang Geun; Li, Mi

    Enzymatic hydrolysis represents one of the major steps and barriers in the commercialization process of converting cellulosic substrates into biofuels and other value added products. It is usually achieved by a synergistic action of enzyme mixture typically consisting of multiple enzymes such as glucanase, cellobiohydrolase and β-glucosidase with different mode of actions. Due to the innate biomass recalcitrance, enzymatic hydrolysis normally starts with an initial fast rate of hydrolysis followed by a rapid decrease of rate toward the end of hydrolysis. With majority of literature studies focusing on the effect of key substrate characteristics on the initial rate or finalmore » yield of enzymatic hydrolysis, information about physicochemical structural changes of cellulosic substrates during enzymatic hydrolysis is still quite limited. Consequently, what slows down the reaction rate toward the end of hydrolysis is not well understood. Lastly, this review highlights recent advances in understanding the structural changes of cellulosic substrates during the hydrolysis process, to better understand the fundamental mechanisms of enzymatic hydrolysis.« less

  12. Enzymatic induction of supramolecular order and bioactivity

    NASA Astrophysics Data System (ADS)

    Yang, Chengbiao; Ren, Xinrui; Ding, Dan; Wang, Ling; Yang, Zhimou

    2016-05-01

    We showed in this study that enzymatic triggering is a totally different pathway for the preparation of self-assembling nanomaterials to the heating-cooling process. Because the molecules were under lower energy levels and the molecular conformation was more ordered during the enzymatic triggeration under mild conditions, nanomaterials with higher supramolecular order could be obtained through biocatalytic control. In this study, nanoparticles were obtained by an enzymatic reaction and nanofibers were observed through the heating-cooling process. We observed a distinct trough at 318 nm from the CD spectrum of a particle sample but not a fiber sample, suggesting the long range arrangement of molecules and helicity in the nanoparticles. The nanoparticles with higher supramolecular order possessed much better potency as a protein vaccine adjuvant because it accelerated the DC maturation and elicited stronger T-cells cytokine production than the nanofibers. Our study demonstrated that biocatalytic triggering is a useful method for preparing supramolecular nanomaterials with higher supramolecular order and probably better bioactivity.We showed in this study that enzymatic triggering is a totally different pathway for the preparation of self-assembling nanomaterials to the heating-cooling process. Because the molecules were under lower energy levels and the molecular conformation was more ordered during the enzymatic triggeration under mild conditions, nanomaterials with higher supramolecular order could be obtained through biocatalytic control. In this study, nanoparticles were obtained by an enzymatic reaction and nanofibers were observed through the heating-cooling process. We observed a distinct trough at 318 nm from the CD spectrum of a particle sample but not a fiber sample, suggesting the long range arrangement of molecules and helicity in the nanoparticles. The nanoparticles with higher supramolecular order possessed much better potency as a protein vaccine

  13. Enzymatic Synthesis of Psilocybin.

    PubMed

    Fricke, Janis; Blei, Felix; Hoffmeister, Dirk

    2017-09-25

    Psilocybin is the psychotropic tryptamine-derived natural product of Psilocybe carpophores, the so-called "magic mushrooms". Although its structure has been known for 60 years, the enzymatic basis of its biosynthesis has remained obscure. We characterized four psilocybin biosynthesis enzymes, namely i) PsiD, which represents a new class of fungal l-tryptophan decarboxylases, ii) PsiK, which catalyzes the phosphotransfer step, iii) the methyltransferase PsiM, catalyzing iterative N-methyl transfer as the terminal biosynthetic step, and iv) PsiH, a monooxygenase. In a combined PsiD/PsiK/PsiM reaction, psilocybin was synthesized enzymatically in a step-economic route from 4-hydroxy-l-tryptophan. Given the renewed pharmaceutical interest in psilocybin, our results may lay the foundation for its biotechnological production. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. In Situ Enzymatically Generated Photoswitchable Oxidase Mimetics and Their Application for Colorimetric Detection of Glucose Oxidase.

    PubMed

    Cao, Gen-Xia; Wu, Xiu-Ming; Dong, Yu-Ming; Li, Zai-Jun; Wang, Guang-Li

    2016-07-09

    In this study, a simple and amplified colorimetric assay is developed for the detection of the enzymatic activity of glucose oxidase (GOx) based on in situ formation of a photoswitchable oxidase mimetic of PO₄(3-)-capped CdS quantum dots (QDs). GOx catalyzes the oxidation of 1-thio-β-d-glucose to give 1-thio-β-d-gluconic acid which spontaneously hydrolyzes to β-d-gluconic acid and H₂S; the generated H₂S instantly reacts with Cd(2+) in the presence of Na₃PO₄ to give PO₄(3-)-stabilized CdS QDs in situ. Under visible-light (λ ≥ 400 nm) stimulation, the PO₄(3-)-capped CdS QDs are a new style of oxidase mimic derived by producing some active species, such as h⁺, (•)OH, O₂(•-) and a little H₂O₂, which can oxidize the typical substrate (3,3,5,5-tetramethylbenzydine (TMB)) with a color change. Based on the GOx-triggered growth of the oxidase mimetics of PO₄(3-)-capped CdS QDs in situ, we developed a simple and amplified colorimetric assay to probe the enzymatic activity of GOx. The proposed method allowed the detection of the enzymatic activity of GOx over the range from 25 μg/L to 50 mg/L with a low detection limit of 6.6 μg/L. We believe the PO₄(3-)-capped CdS QDs generated in situ with photo-stimulated enzyme-mimicking activity may find wide potential applications in biosensors.

  15. Enzymatic Enantioselective Decarboxylative Protonation of Heteroaryl Malonates

    PubMed Central

    Lewin, Ross; Goodall, Mark; Thompson, Mark L; Leigh, James; Breuer, Michael; Baldenius, Kai; Micklefield, Jason

    2015-01-01

    The enzyme aryl/alkenyl malonate decarboxylase (AMDase) catalyses the enantioselective decarboxylative protonation (EDP) of a range of disubstituted malonic acids to give homochiral carboxylic acids that are valuable synthetic intermediates. AMDase exhibits a number of advantages over the non-enzymatic EDP methods developed to date including higher enantioselectivity and more environmentally benign reaction conditions. In this report, AMDase and engineered variants have been used to produce a range of enantioenriched heteroaromatic α-hydroxycarboxylic acids, including pharmaceutical precursors, from readily accessible α-hydroxymalonates. The enzymatic method described here represents an improvement upon existing synthetic chemistry methods that have been used to produce similar compounds. The relationship between the structural features of these new substrates and the kinetics associated with their enzymatic decarboxylation is explored, which offers further insight into the mechanism of AMDase. PMID:25766433

  16. Relationship between the enzymatic browning and phenylalanine ammonia-lyase activity of cut lettuce, and the prevention of browning by inhibitors of polyphenol biosynthesis.

    PubMed

    Hisaminato, H; Murata, M; Homma, S

    2001-05-01

    Cut lettuce stored at 4 degrees C gradually turned brown on the cut section after several days of storage. Three factors for enzymatic browning, the polyphenol content, polyphenol oxidase activity, and phenylalanine ammonia-lyase (PAL) activity, were examined during the cold storage of cut lettuce. A relationship between the browning and PAL activity was apparent. We tried to prevent this browning by using the two enzyme inhibitors, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the phenylpropanoid pathway, and glyphosate, an inhibitor of the shikimate pathway. AIP and glyphosate significantly inhibited the browning of cut lettuce. The polyphenol content and PAL activity were both reduced by the treatment with AIP. These results show that regulating the biosynthesis of polyphenols is essential to prevent the browning of cut lettuce.

  17. How Mg2+ ions lower the SN2@P barrier in enzymatic triphosphate hydrolysis.

    PubMed

    van Bochove, Marc A; Roos, Goedele; Fonseca Guerra, Célia; Hamlin, Trevor A; Bickelhaupt, F Matthias

    2018-04-03

    Our quantum chemical activation strain analyses demonstrate how Mg2+ lowers the barrier of the enzymatic triphosphate hydrolysis through two distinct mechanisms: (a) weakening of the leaving-group bond, thereby decreasing activation strain; and (b) transition state (TS) stabilization through enhanced electrophilicity of the triphosphate PPP substrate, thereby strengthening the interaction with the nucleophile.

  18. Performance of glucose/O2 enzymatic fuel cell based on supporting electrodes over-coated by polymer-nanogold particle composite with entrapped enzymes

    NASA Astrophysics Data System (ADS)

    Huo, W. S.; Zeng, H.; Yang, Y.; Zhang, Y. H.

    2017-03-01

    Enzymatic electrodes over-coated by thin film of nano-composite made up of polymer and functionalized nano-gold particle was prepared. Glucose/O2 membrane-free enzymatic fuel cell based on nano-composite based electrodes with incorporated glucose oxidase and laccase was assembled. This enzymatic fuel cell exhibited high energy out-put density even when applied in human serum. Catalytic cycle involved in enzymatic fuel cell was limited by oxidation of glucose occurred on bioanode resulting from impact of sophisticated interaction between active site in glucose oxidase and nano-gold particle on configuration of redox center of enzyme molecule which crippled catalytic efficiency of redox protein.

  19. Enzymatic modification of schizophyllan

    USDA-ARS?s Scientific Manuscript database

    An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of ß-endoglucanase with specif...

  20. Polymersome nanoreactors for enzymatic ring-opening polymerization.

    PubMed

    Nallani, Madhavan; de Hoog, Hans-Peter M; Cornelissen, Jeroen J L M; Palmans, Anja R A; van Hest, Jan C M; Nolte, Roeland J M

    2007-12-01

    Polystyrene-polyisocyanopeptide (PS-PIAT) polymersomes containing CALB in two different locations, one in the aqueous inner compartment and one in the bilayer, were investigated for enzymatic ring-opening polymerization of lactones in water. It is shown that the monomers 8-octanolactone and dodecalactone yield oligomers with this polymersome system. It is also observed that the polymerization activity is dependent on the position of the enzyme in the polymersome. SEM investigations show that the polymersome structures were destabilized during the polymerization. Further investigations show that the vesicular morphology of the polymersomes was destabilized only in the case of polymer product formation.

  1. Enzymatic biomarkers can portray nanoCuO-induced oxidative and neuronal stress in freshwater shredders.

    PubMed

    Pradhan, Arunava; Silva, Carla O; Silva, Carlos; Pascoal, Cláudia; Cássio, Fernanda

    2016-11-01

    Commercial applications of nanometal oxides have increased concern about their release into natural waters and consequent risks to aquatic biota and the processes they drive. In forest streams, the invertebrate shredder Allogamus ligonifer plays a key role in detritus food webs by transferring carbon and energy from plant litter to higher trophic levels. We assessed the response profiles of oxidative and neuronal stress enzymatic biomarkers in A. ligonifer after 96h exposure to nanoCuO at concentration ranges enzymatic responses to Cu 2+ exposure at similar effective concentrations were compared. The highest activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) were observed at concentrations enzymatic activities decreased at effective concentrations between LC 10 and LC 30 . GR activity remained higher than in control at all concentrations. The activity of glutathione S-transferase (GST) increased whereas that of catalase (CAT) decreased at concentrations between LC 10 and LC 30 . The response patterns suggested that antioxidant enzymes could prevent oxidative stress at low concentrations (activities were concentration-dependent, and led to oxidative stress and even to animal death. The activity of acetylcholinesterase (AChE) was strongly inhibited even at concentrations

  2. Unraveling the effects of laccase treatment on enzymatic hydrolysis of steam-exploded wheat straw.

    PubMed

    Oliva-Taravilla, Alfredo; Moreno, Antonio D; Demuez, Marie; Ibarra, David; Tomás-Pejó, Elia; González-Fernández, Cristina; Ballesteros, Mercedes

    2015-01-01

    Laccase enzymes are promising detoxifying agents during lignocellulosic bioethanol production from wheat straw. However, they affect the enzymatic hydrolysis of this material by lowering the glucose recovery yields. This work aimed at explaining the negative effects of laccase on enzymatic hydrolysis. Relative glucose recovery in presence of laccase (10IU/g substrate) with model cellulosic substrate (Sigmacell) at 10% (w/v) was almost 10% points lower (P<0.01) than in the absence of laccase. This fact could be due to an increase in the competition of cellulose binding sites between the enzymes and a slight inhibition of β-glucosidase activity. However, enzymatic hydrolysis and infrared spectra of laccase-treated and untreated wheat straw filtered pretreated residue (WS-FPR), revealed that a grafting process of phenoxy radicals onto the lignin fiber could be the cause of diminished accessibility of cellulases to cellulose in pretreated wheat straw. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Fractionation of enzymatic hydrolysis lignin by sequential extraction for enhancing antioxidant performance.

    PubMed

    An, Liangliang; Wang, Guanhua; Jia, Hongyu; Liu, Cuiyun; Sui, Wenjie; Si, Chuanling

    2017-06-01

    The heterogeneity of lignin chemical structure and molecular weight results in the lignin inhomogeneous properties which also covers the antioxidant performance. In order to evaluate the effects of lignin heterogeneity on its antioxidant activity, four lignin fractions from enzymatic hydrolysis lignin were classified by sequential organic solvent extraction and further evaluated by DPPH (1,1-Diphenyl-2-Picrylhydrazyl) free radical scavenging capacity and reducing power analysis. The characterization including FTIR, 1 H NMR and GPC showed that the fractionation process could effectively separate lignin fractions with distinctly different molecular weight and weaken the heterogeneity of unfractionated lignin. The antioxidant performance comparison of lignin fractions indicated that the dichloromethane fraction (F1) with lowest molecular weight (4585g/mol) and highest total phenolics content (246.13mg GAE/g) exhibited the highest antioxidant activity whose value was close to commercial antioxidant BHT (butylated hydroxytoluene). Moreover, the relationship between the antioxidant activity and the structure of lignin was further discussed to elucidate the mechanism of antioxidant activity improvement of lignin fractionation. Consequently, this study suggested that the sequential extraction was an effective way to obtain relatively homogeneous enzymatic hydrolysis lignin fractions which showed the potential for the value-added antioxidant application. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Ceramic microsystem incorporating a microreactor with immobilized biocatalyst for enzymatic spectrophotometric assays.

    PubMed

    Baeza, Mireia; López, Carmen; Alonso, Julián; López-Santín, Josep; Alvaro, Gregorio

    2010-02-01

    Low-temperature cofired ceramics (LTCC) technology is a versatile fabrication technique used to construct microflow systems. It permits the integration of several unitary operations (pretreatment, separation, (bio)chemical reaction, and detection stage) of an analytical process in a modular or monolithic way. Moreover, because of its compatibility with biological material, LTCC is adequate for analytical applications based on enzymatic reactions. Here we present the design, construction, and evaluation of a LTCC microfluidic system that integrates a microreactor (internal volume, 24.28 microL) with an immobilized beta-galactosidase from Escherichia coli (0.479 activity units) and an optical flow cell to measure the product of the enzymatic reaction. The enzyme was immobilized on a glyoxal-agarose support, maintaining its activity along the time of the study. As a proof of concept, the LTCC-beta-galactosidase system was tested by measuring the conversion of ortho-nitrophenyl beta-D-galactopyranoside, the substrate usually employed for activity determinations. Once packed in a monolithically integrated microcolumn, the miniaturized flow system was characterized, the operational conditions optimized (flow rate and injection volume), and its performance successfully evaluated by determining the beta-galactosidase substrate concentration at the millimolar level.

  5. Kinetic study of the activation of banana juice enzymatic browning by the addition of maltosyl-beta-cyclodextrin.

    PubMed

    López-Nicolás, José M; Pérez-López, Antonio J; Carbonell-Barrachina, Angel; García-Carmona, Francisco

    2007-11-14

    In recent years, the use of cyclodextrins (CDs) as antibrowning agents in fruit juices has received growning attention. However, there has been no detailed study of the behavior of these molecules as substances, which can lead to the darkening of foods. In this paper, when the color of fresh banana juice was evaluated in the presence of different CDs, the evolution of several color parameters was the opposite of that observed in other fruit juices. Moreover, a kinetic model based on the complexation by CDs of the natural browning inhibitors present in banana is developed for the first time to clarify the enzymatic browning activation of banana juice. Finally, the apparent complexation constant between the natural polyphenoloxidase inhibitors present in banana juice and maltosyl-beta-CD was calculated (Kci = 27.026 +/- 0.212 mM (-1)).

  6. Developmental lead exposure induces opposite effects on ethanol intake and locomotion in response to central vs. systemic cyanamide administration.

    PubMed

    Mattalloni, Mara Soledad; Deza-Ponzio, Romina; Albrecht, Paula Alejandra; Cancela, Liliana Marina; Virgolini, Miriam Beatriz

    2017-02-01

    Lead (Pb) is a developmental neurotoxicant that elicits differential responses to drugs of abuse. Particularly, ethanol consumption has been demonstrated to be increased as a consequence of environmental Pb exposure, with catalase (CAT) and brain acetaldehyde (ACD, the first metabolite of ethanol) playing a role. The present study sought to interfere with ethanol metabolism by inhibiting ALDH2 (mitochondrial aldehyde dehydrogenase) activity in both liver and brain from control and Pb-exposed rats as a strategy to accumulate ACD, a substance that plays a major role in the drug's reinforcing and/or aversive effects. To evaluate the impact on a 2-h chronic voluntary ethanol intake test, developmentally Pb-exposed and control rats were administered with cyanamide (CY, an ALDH inhibitor) either systemically or intracerebroventricularly (i.c.v.) on the last 4 sessions of the experiment. Furthermore, on the last session and after locomotor activity was assessed, all animals were sacrificed to obtain brain and liver samples for ALDH2 and CAT activity determination. Systemic CY administration reduced the elevated ethanol intake already reported in the Pb-exposed animals (but not in the controls) accompanied by liver (but not brain) ALDH2 inactivation. On the other hand, a 0.3 mg i.c.v. CY administration enhanced both ethanol intake and locomotor activity accompanied by brain ALDH2 inactivation in control animals, while an increase in ethanol consumption was also observed in the Pb-exposed group, although in the absence of brain ALDH2 blockade. No changes were observed in CAT activity as a consequence of CY administration. These results support the participation of liver and brain ACD in ethanol intake and locomotor activity, responses that are modulated by developmental Pb exposure. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Simultaneous sonochemical-enzymatic coating of medical textiles with antibacterial ZnO nanoparticles.

    PubMed

    Petkova, Petya; Francesko, Antonio; Perelshtein, Ilana; Gedanken, Aharon; Tzanov, Tzanko

    2016-03-01

    The antimicrobial finishing is a must for production of medical textiles, aiming at reducing the bioburden in clinical wards and consequently decreasing the risk of hospital-acquired infections. This work reports for the first time on a simultaneous sonochemical/enzymatic process for durable antibacterial coating of cotton with zinc oxide nanoparticles (ZnO NPs). The novel technology goes beyond the "stepwise" concept we proposed recently for enzymatic pre-activation of the fabrics and subsequent sonochemical nano-coating, and is designed to produce "ready-to-use" antibacterial medical textiles in a single step. A multilayer coating of uniformly dispersed NPs was obtained in the process. The enzymatic treatment provides better adhesion of the ZnO NPs and, as a consequence, enhanced coating stability during exploitation. The NPs-coated cotton fabrics inhibited the growth of the medically relevant Staphylococcus aureus and Escherichia coli respectively by 67% and 100%. The antibacterial efficiency of these textile materials resisted the intensive laundry regimes used in hospitals, though only 33% of the initially deposited NPs remained firmly fixed onto the fabrics after multiple washings. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Electrochemical enzymatic biosensors using carbon nanofiber nanoelectrode arrays

    NASA Astrophysics Data System (ADS)

    Li, Jun; Li, Yi-fen; Swisher, Luxi Z.; Syed, Lateef U.; Prior, Allan M.; Nguyen, Thu A.; Hua, Duy H.

    2012-10-01

    The reduction of electrode size down to nanometers could dramatically enhance detection sensitivity and temporal resolution. Nanoelectrode arrays (NEAs) are of particular interest for ultrasensitive biosensors. Here we report the study of two types of biosensors for measuring enzyme activities using NEAs fabricated with vertically aligned carbon nanofibers (VACNFs). VACNFs of ~100 nm in average diameter and 3-5 μm in length were grown on conductive substrates as uniform vertical arrays which were then encapsulated in SiO2 matrix leaving only the tips exposed. We demonstrate that such VACNF NEAs can be used in profiling enzyme activities through monitoring the change in electrochemical signals induced by enzymatic reactions to the peptides attached to the VACNF tip. The cleavage of the tetrapeptide with a ferrocene tag by a cancerrelated protease (legumain) was monitored with AC voltammetry. Real-time electrochemical impedance spectroscopy (REIS) was used for fast label-free detection of two reversible processes, i.e. phosphorylation by c-Src tyrosine kinase and dephosphorylation by protein tyrosine phosphatase 1B (PTP1B). The REIS data of phosphorylation were slow and unreliable, but those of dephosphorylation showed large and fast exponential decay due to much higher activity of phosphatase PTP1B. The kinetic data were analyzed with a heterogeneous Michaelis-Menten model to derive the "specificity constant" kcat/Km, which is 8.2x103 M-1s-1 for legumain and (2.1 ± 0.1) x 107 M-1s-1 for phosphatase (PTP1B), well consistent with literature. It is promising to develop VACNF NEA based electrochemical enzymatic biosensors as portable multiplex electronic techniques for rapid cancer diagnosis and treatment monitoring.

  9. Evaluation of the enzymatic activity and stability of commercial bromelain incorporated in topical formulations.

    PubMed

    Lourenço, C B; Ataide, J A; Cefali, L C; Novaes, L C D L; Moriel, P; Silveira, E; Tambourgi, E B; Mazzola, P G

    2016-10-01

    Bromelain is a mixture of proteolytic enzymes found in various tissues of the pineapple plant (Ananas comosus) and other species of Bromeliaceae. Owing to its proteolytic activity, bromelain has been used in the food, medical, pharmaceutical and cosmetic industries, for its cell renewal, anti-ageing, whitening and anti-cellulite properties. This study evaluated the stability of bromelain (commercial powder) incorporated in topical formulations. Bromelain was incorporated at three concentrations, 0.5%, 1.0% and 2.0%, in oil-in-water emulsion and gel, and stored for six months at varying stress conditions. Stability was accessed by measuring the changes in the protein content, enzymatic activity, viscosity, rheology, pH and colour of the selected formulations. The colour of all the samples changed after 180 days of incubation, indicating the concentration-dependence and temperature-sensitive nature of these formulations. No relationship was observed between the changes in the pH, temperature and luminosity exposure in all the samples. Gels proved to be the least preferred base for incorporation of bromelain for use as a topical formulation, owing to its inability to maintain the integrity of bromelain, thereby affecting the formulation characteristics. The emulsion-based formulations at all the concentrations of bromelain were more stable than the gel-based formulation over 180 days of evaluation, at a temperature of 5°C, protected from light. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  10. Sulfate radicals enable a non-enzymatic Krebs cycle precursor

    PubMed Central

    Keller, Markus A.; Kampjut, Domen; Harrison, Stuart A.; Ralser, Markus

    2017-01-01

    The evolutionary origins of the tricarboxylic acid cycle (TCA), or Krebs cycle, are so far unclear. Despite a few years ago, the existence of a simple non-enzymatic Krebs-cycle catalyst has been dismissed ‘as an appeal to magic’, citrate and other intermediates have meanwhile been discovered on a carbonaceous meteorite and do interconvert non-enzymatically. To identify the non-enzymatic Krebs cycle catalyst, we used combinatorial, quantitative high-throughput metabolomics to systematically screen iron and sulfate reaction milieus that orient on Archean sediment constituents. TCA cycle intermediates are found stable in water and in the presence of most iron and sulfate species, including simple iron-sulfate minerals. However, we report that TCA intermediates undergo 24 interconversion reactions in the presence of sulfate radicals that form from peroxydisulfate. The non-enzymatic reactions critically cover a topology as present in the Krebs cycle, the glyoxylate shunt and the succinic semialdehyde pathways. Assembled in a chemical network, the reactions achieve more than ninety percent carbon recovery. Our results show that a non-enzymatic precursor for the Krebs cycle is biologically sensible, efficient, and forms spontaneously in the presence of sulfate radicals. PMID:28584880

  11. In situ enzymatic activity of transglutaminase isoforms on brain tissue sections of rodents: A new approach to monitor differences in post-translational protein modifications during neurodegeneration.

    PubMed

    Schulze-Krebs, Anja; Canneva, Fabio; Schnepf, Rebecca; Dobner, Julia; Dieterich, Walburga; von Hörsten, Stephan

    2016-01-15

    Mammalian transglutaminases (TGs) catalyze the irreversible post-translational modifications of proteins, the most prominent of which is the calcium-dependent formation of covalent acyl transfers between the γ-carboxamide group of glutamine and the ε-amino-group of lysine (GGEL-linkage). In the central nervous system, at least four TG isoforms are present and some of them are differentially expressed under pathological conditions in human patients. However, the precise TG-isoform-dependent enzymatic activities in the brain as well as their anatomical distribution are unknown. Specificity of the used biotinylated peptides was analyzed using an in vitro assay. Isoform-specific TG activity was evaluated in in vitro and in situ studies, using brain extracts and native brain tissue obtained from rodents. Our method allowed us to reveal in vitro and in situ TG-isoform-dependent enzymatic activity in brain extracts and tissue of rats and mice, with a specific focus on TG6. In situ activity of this isoform varied between BACHD mice in comparison to their wt controls. TG isozyme-specific activity can be detected by isoform-specific biotinylated peptides in brain tissue sections of rodents to reveal differences in the anatomical and/or subcellular distribution of TG activity. Our findings yield the basis for a broader application of this method for the screening of pathological expression and activity of TGs in a variety of animal models of human diseases, as in the case of neurodegenerative conditions such as Huntington׳s, Parkinson׳s and Alzheimer׳s, where protein modification is involved as a key mechanism of disease progression. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Analysis of non-enzymatically glycated peptides: neutral-loss-triggered MS3 versus multi-stage activation tandem mass spectrometry

    PubMed Central

    Zhang, Qibin; Petyuk, Vladislav A.; Schepmoes, Athena A.; Orton, Daniel J.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Metz, Thomas O.

    2009-01-01

    Non-enzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. While electron transfer dissociation (ETD) has been shown to outperform collision-induced dissociation (CID) in sequencing glycated peptides by tandem mass spectrometry, ETD instrumentation is not yet widely available and often suffers from significantly lower sensitivity than CID. In this study, we evaluated different advanced CID techniques (i.e., neutral-loss-triggered MS3 and multi-stage activation) during liquid chromatography/multi-stage mass spectrometric (LC/MSn) analyses of Amadori-modified peptides enriched from human serum glycated in vitro. During neutral-loss-triggered MS3 experiments, MS3 scans triggered by neutral losses of 3 H2O or 3 H2O + HCHO produced similar results in terms of glycated peptide identifications. However, neutral losses of 3 H2O resulted in significantly more glycated peptide identifications during multi-stage activation experiments. Overall, the multi-stage activation approach produced more glycated peptide identifications, while the neutral-loss-triggered MS3 approach resulted in much higher specificity. Both techniques are viable alternatives to ETD for identifying glycated peptides. PMID:18763275

  13. Perturbation theory in the catalytic rate constant of the Henri-Michaelis-Menten enzymatic reaction.

    PubMed

    Bakalis, Evangelos; Kosmas, Marios; Papamichael, Emmanouel M

    2012-11-01

    The Henry-Michaelis-Menten (HMM) mechanism of enzymatic reaction is studied by means of perturbation theory in the reaction rate constant k (2) of product formation. We present analytical solutions that provide the concentrations of the enzyme (E), the substrate (S), as well as those of the enzyme-substrate complex (C), and the product (P) as functions of time. For k (2) small compared to k (-1), we properly describe the entire enzymatic activity from the beginning of the reaction up to longer times without imposing extra conditions on the initial concentrations E ( o ) and S ( o ), which can be comparable or much different.

  14. Homogeneous, Heterogeneous, and Enzymatic Catalysis.

    ERIC Educational Resources Information Center

    Oyama, S. Ted; Somorjai, Gabor A.

    1988-01-01

    Discusses three areas of catalysis: homegeneous, heterogeneous, and enzymatic. Explains fundamentals and economic impact of catalysis. Lists and discusses common industrial catalysts. Provides a list of 107 references. (MVL)

  15. 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

  16. Development of enzymatically-active bacterial cellulose membranes through stable immobilization of an engineered β-galactosidase.

    PubMed

    Estevinho, Berta N; Samaniego, Nuria; Talens-Perales, David; Fabra, Maria José; López-Rubio, Amparo; Polaina, Julio; Marín-Navarro, Julia

    2018-08-01

    Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a β-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus. TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme. However, enzyme performance was similar in all three cases for the hydrolysis of 5% lactose to a high extent. Reuse of the immobilized enzyme was limited by the stability of the β-galactosidase module, whereas the CBM2 module provided stable attachment of the hybrid enzyme to the BC support, after long incubation periods (3 h) at 75 °C. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

    PubMed

    Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

    2016-02-01

    A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  18. Full inhibition of enzymatic browning in the presence of thiol-functionalised silica nanomaterial.

    PubMed

    Muñoz-Pina, Sara; Ros-Lis, José V; Argüelles, Ángel; Coll, Carmen; Martínez-Máñez, Ramón; Andrés, Ana

    2018-02-15

    Darkening processed fruits and vegetables is caused mainly by enzymatic browning through polyphenol oxidase (PPO) action. Accordingly, we explored the potential of four silica-based materials (MCM-41 nanometric size, MCM-41 micrometric size, UVM-7 and aerosil), non-functionalised and functionalised with thiol groups, to inhibit PPO activity in the model system and apple juice. All materials showed relevant performance when immobilising and inhibiting PPO in model systems, and support topology is a main factor for enzyme immobilisation and inhibition. Thiol-containing silica UVM7-SH showed the greatest inactivation, and similar browning values to those obtained by acidification. The enzyme's kinetic parameters in the presence of UVM-7-SH suggested non-competitive inhibition, which indicated that the material interacted with the enzyme, but beyond the active centre. In real systems, UVM-7-SH completely inhibited enzymatic browning in apple juice (cv. Granny Smith and cv. Golden Delicious) up to 9days after 5min of contact. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. [The effect of enzymatic treatment using proteases on properties of persistent sodium current in CA1 pyramidal neurons of rat hippocampus].

    PubMed

    Lun'ko, O O; Isaiev, D S; Maxymiuk, O P; Kryshtal', O O; Isaieva, O V

    2014-01-01

    We investigated the effect of proteases, widely used for neuron isolation in electrophysiological studies, on the amplitude and kinetic characteristics of persistent sodium current (I(NaP)) in hippocampal CA1 pyramidal neurons. Properties of I(NaP) were studied on neurons isolated by mechanical treatment (control group) and by mechanical and enzymatic treatment using pronase E (from Streptomyces griseus) or protease type XXIII (from Aspergillus oryzae). We show that in neurons isolated with pronase E kinetic of activation and density of I(NaP) was unaltered. Enzymatic treatment with protease type XXIII did not alter I(NaP) activation but result in significant decrease in I(NaP) density. Our data indicates that enzymatic treatment using pronase E for neuron isolation is preferable for investigation of I(NaP).

  20. Green-synthesized CdS nano-pesticides: Toxicity on young instars of malaria vectors and impact on enzymatic activities of the non-target mud crab Scylla serrata.

    PubMed

    Sujitha, Vasu; Murugan, Kadarkarai; Dinesh, Devakumar; Pandiyan, Amuthvalli; Aruliah, Rajasekar; Hwang, Jiang-Shiou; Kalimuthu, Kandasamy; Panneerselvam, Chellasamy; Higuchi, Akon; Aziz, Al Thabiani; Kumar, Suresh; Alarfaj, Abdullah A; Vaseeharan, Baskaralingam; Canale, Angelo; Benelli, Giovanni

    2017-07-01

    Currently, nano-formulated mosquito larvicides have been widely proposed to control young instars of malaria vector populations. However, the fate of nanoparticles in the aquatic environment is scarcely known, with special reference to the impact of nanoparticles on enzymatic activity of non-target aquatic invertebrates. In this study, we synthesized CdS nanoparticles using a green protocol relying on the cheap extract of Valoniopsis pachynema algae. CdS nanoparticles showed high toxicity on young instars of the malaria vectors Anopheles stephensi and A. sundaicus. The antimalarial activity of the nano-synthesized product against chloroquine-resistant (CQ-r) Plasmodium falciparum parasites was investigated. From a non-target perspective, we focused on the impact of this novel nano-pesticide on antioxidant enzymes acetylcholinesterase (AChE) and glutathione S-transferase (GST) activities of the mud crab Scylla serrata. The characterization of nanomaterials was carried out by UV-vis and FTIR spectroscopy, as well as SEM and XRD analyses. In mosquitocidal assays, LC 50 of V. pachynema-synthesized CdS nanoparticles on A. stephensi ranged from 16.856 (larva I), to 30.301μg/ml (pupa), while for An. sundaicus they ranged from 13.584 to 22.496μg/ml. The antiplasmodial activity of V. pachynema extract and CdS nanoparticles was evaluated against CQ-r and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. IC 50 of V. pachynema extract was 58.1μg/ml (CQ-s) and 71.46μg/ml (CQ-r), while nano-CdS IC 50 was 76.14μg/ml (CQ-s) and 89.21μg/ml (CQ-r). In enzymatic assays, S. serrata crabs were exposed to sub-lethal concentrations, i.e. 4, 6 and 8μg/ml of CdS nanoparticles, assessing changes in GST and AChE activity after 16days. We observed significantly higher activity of GST, if compared to the control, during the whole experiment period. In addition, a single treatment with CdS nanoparticles led to a significant decrease in AChE activity over time. The toxicity of Cd