Nickel compounds induce histone ubiquitination by inhibiting histone deubiquitinating enzyme activity

International Nuclear Information System (INIS)

Ke Qingdong; Ellen, Thomas P.; Costa, Max

2008-01-01

Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis

A high-confidence interaction map identifies SIRT1 as a mediator of acetylation of USP22 and the SAGA coactivator complex.

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Armour, Sean M; Bennett, Eric J; Braun, Craig R; Zhang, Xiao-Yong; McMahon, Steven B; Gygi, Steven P; Harper, J Wade; Sinclair, David A

2013-04-01

Although many functions and targets have been attributed to the histone and protein deacetylase SIRT1, a comprehensive analysis of SIRT1 binding proteins yielding a high-confidence interaction map has not been established. Using a comparative statistical analysis of binding partners, we have assembled a high-confidence SIRT1 interactome. Employing this method, we identified the deubiquitinating enzyme ubiquitin-specific protease 22 (USP22), a component of the deubiquitinating module (DUBm) of the SAGA transcriptional coactivating complex, as a SIRT1-interacting partner. We found that this interaction is highly specific, requires the ZnF-UBP domain of USP22, and is disrupted by the inactivating H363Y mutation within SIRT1. Moreover, we show that USP22 is acetylated on multiple lysine residues and that alteration of a single lysine (K129) within the ZnF-UBP domain is sufficient to alter interaction of the DUBm with the core SAGA complex. Furthermore, USP22-mediated recruitment of SIRT1 activity promotes the deacetylation of individual SAGA complex components. Our results indicate an important role of SIRT1-mediated deacetylation in regulating the formation of DUBm subcomplexes within the larger SAGA complex.

Autophagy-Related Deubiquitinating Enzymes Involved in Health and Disease

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Fouzi El Magraoui

2015-10-01

Full Text Available Autophagy is an evolutionarily-conserved process that delivers diverse cytoplasmic components to the lysosomal compartment for either recycling or degradation. This involves the removal of protein aggregates, the turnover of organelles, as well as the elimination of intracellular pathogens. In this situation, when only specific cargoes should be targeted to the lysosome, the potential targets can be selectively marked by the attachment of ubiquitin in order to be recognized by autophagy-receptors. Ubiquitination plays a central role in this process, because it regulates early signaling events during the induction of autophagy and is also used as a degradation-tag on the potential autophagic cargo protein. Here, we review how the ubiquitin-dependent steps of autophagy are balanced or counteracted by deubiquitination events. Moreover, we highlight the functional role of the corresponding deubiquitinating enzymes and discuss how they might be involved in the occurrence of cancer, neurodegenerative diseases or infection with pathogenic bacteria.

Inhibitors of Deubiquitinating Enzymes Block HIV-1 Replication and Augment the Presentation of Gag-Derived MHC-I Epitopes.

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Setz, Christian; Friedrich, Melanie; Rauch, Pia; Fraedrich, Kirsten; Matthaei, Alina; Traxdorf, Maximilian; Schubert, Ulrich

2017-08-12

In recent years it has been well established that two major constituent parts of the ubiquitin proteasome system (UPS)-the proteasome holoenzymes and a number of ubiquitin ligases-play a crucial role, not only in virus replication but also in the regulation of the immunogenicity of human immunodeficiency virus type 1 (HIV-1). However, the role in HIV-1 replication of the third major component, the deubiquitinating enzymes (DUBs), has remained largely unknown. In this study, we show that the DUB-inhibitors (DIs) P22077 and PR-619, specific for the DUBs USP7 and USP47, impair Gag processing and thereby reduce the infectivity of released virions without affecting viral protease activity. Furthermore, the replication capacity of X4- and R5-tropic HIV-1 NL4-3 in human lymphatic tissue is decreased upon treatment with these inhibitors without affecting cell viability. Most strikingly, combinatory treatment with DIs and proteasome inhibitors synergistically blocks virus replication at concentrations where mono-treatment was ineffective, indicating that DIs can boost the therapeutic effect of proteasome inhibitors. In addition, P22077 and PR-619 increase the polyubiquitination of Gag and thus its entry into the UPS and the major histocompatibility complex (MHC)-I pathway. In summary, our data point towards a model in which specific inhibitors of DUBs not only interfere with virus spread but also increase the immune recognition of HIV-1 expressing cells.

USP22 acts as an oncogene by regulating the stability of cyclooxygenase-2 in non-small cell lung cancer

Energy Technology Data Exchange (ETDEWEB)

Xiao, Haibo [Department of Cardiothoracic Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200092 (China); Tian, Yue [Institute of Orthopaedics, Chinese PLA General Hospital, Beijing 100853 (China); Yang, Yang; Hu, Fengqing; Xie, Xiao; Mei, Ju [Department of Cardiothoracic Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200092 (China); Ding, Fangbao, E-mail: drnail@sina.com [Department of Cardiothoracic Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200092 (China)

2015-05-08

The histone ubiquitin hydrolase ubiquitin-specific protease 22 (USP22) is an epigenetic modifier and an oncogene that is upregulated in many types of cancer. In non-small cell lung cancer (NSCLC), aberrant expression of USP22 is a predictor of poor survival, as is high expression of cyclooxygenase-2 (COX-2). Despite its oncogenic role, few substrates of USP22 have been identified and its mechanism of action in cancer remains unclear. Here, we identified COX-2 as a direct substrate of USP22 and showed that its levels are modulated by USP22 mediated deubiquitination. Silencing of USP22 downregulated COX-2, decreased its half-life, and inhibited lung carcinoma cell proliferation by directly interacting with and modulating the stability and activity of COX-2 through the regulation of its ubiquitination status. The findings of the present study suggest a potential mechanism underlying the oncogenic role of USP22 mediated by the modulation of the stability and activity of COX-2. - Highlights: • USP22 interacts with COX-2. • USP22 deubiquitinates and stabilizes COX-2. • USP22 is required for COX-2-mediated upregulation of prostaglandin E2.

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites.

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Hunt, Paul; Afonso, Ana; Creasey, Alison; Culleton, Richard; Sidhu, Amar Bir Singh; Logan, John; Valderramos, Stephanie G; McNae, Iain; Cheesman, Sandra; do Rosario, Virgilio; Carter, Richard; Fidock, David A; Cravo, Pedro

2007-07-01

Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed.

Fine-tuning the ubiquitin code at DNA double-strand breaks: deubiquitinating enzymes at work

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Elisabetta eCitterio

2015-09-01

Full Text Available Ubiquitination is a reversible protein modification broadly implicated in cellular functions. Signaling processes mediated by ubiquitin are crucial for the cellular response to DNA double-strand breaks (DSBs, one of the most dangerous types of DNA lesions. In particular, the DSB response critically relies on active ubiquitination by the RNF8 and RNF168 ubiquitin ligases at the chromatin, which is essential for proper DSB signaling and repair. How this pathway is fine-tuned and what the functional consequences are of its deregulation for genome integrity and tissue homeostasis are subject of intense investigation. One important regulatory mechanism is by reversal of substrate ubiquitination through the activity of specific deubiquitinating enzymes (DUBs, as supported by the implication of a growing number of DUBs in DNA damage response (DDR processes. Here, we discuss the current knowledge of how ubiquitin-mediated signaling at DSBs is controlled by deubiquitinating enzymes, with main focus on DUBs targeting histone H2A and on their recent implication in stem cell biology and cancer.

Quantitative analysis by next generation sequencing of hematopoietic stem and progenitor cells (LSK and of splenic B cells transcriptomes from wild-type and Usp3-knockout mice

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Cesare Lancini

2016-03-01

Full Text Available The data described here provide genome-wide expression profiles of murine primitive hematopoietic stem and progenitor cells (LSK and of B cell populations, obtained by high throughput sequencing. Cells are derived from wild-type mice and from mice deficient for the ubiquitin-specific protease 3 (USP3; Usp3Δ/Δ. Modification of histone proteins by ubiquitin plays a crucial role in the cellular response to DNA damage (DDR (Jackson and Durocher, 2013 [1]. USP3 is a histone H2A deubiquitinating enzyme (DUB that regulates ubiquitin-dependent DDR in response to DNA double-strand breaks (Nicassio et al., 2007; Doil et al., 2008 [2,3]. Deletion of USP3 in mice increases the incidence of spontaneous tumors and affects hematopoiesis [4]. In particular, Usp3-knockout mice show progressive loss of B and T cells and decreased functional potential of hematopoietic stem cells (HSCs during aging. USP3-deficient cells, including HSCs, display enhanced histone ubiquitination, accumulate spontaneous DNA damage and are hypersensitive to ionizing radiation (Lancini et al., 2014 [4]. To address whether USP3 loss leads to deregulation of specific molecular pathways relevant to HSC homeostasis and/or B cell development, we have employed the RNA-sequencing technology and investigated transcriptional differences between wild-type and Usp3Δ/Δ LSK, naïve B cells or in vitro activated B cells. The data relate to the research article “Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells” (Lancini et al., 2014 [4]. The RNA-sequencing and analysis data sets have been deposited in NCBI׳s Gene Expression Omnibus (Edgar et al., 2002 [5] and are accessible through GEO Series accession number GSE58495 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58495. With this article, we present validation of the RNA-seq data set through quantitative real-time PCR and comparative analysis. Keywords: B

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites§

Science.gov (United States)

Hunt, Paul; Afonso, Ana; Creasey, Alison; Culleton, Richard; Sidhu, Amar Bir Singh; Logan, John; Valderramos, Stephanie G; McNae, Iain; Cheesman, Sandra; do Rosario, Virgilio; Carter, Richard; Fidock, David A; Cravo, Pedro

2007-01-01

Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed. PMID:17581118

Generation and Validation of Intracellular Ubiquitin Variant Inhibitors for USP7 and USP10

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Zhang, Wei; Sartori, Maria A.; Makhnevych, Taras; Federowicz, Kelly E.; Dong, Xiaohui; Liu, Li; Nim, Satra; Dong, Aiping; Yang, Jingsong; Li, Yanjun; Haddad, Dania; Ernst, Andreas; Heerding, Dirk; Tong, Yufeng; Moffat, Jason; Sidhu, Sachdev S.

2017-11-01

Post-translational modification of the p53 signaling pathway plays an important role in cell cycle progression and stress-induced apoptosis. Indeed, a large body of work has shown that dysregulation of p53 and its E3 ligase MDM2 by the ubiquitin-proteasome system (UPS) promotes carcinogenesis and malignant transformation. Thus, drug discovery efforts have focused on the restoration of wild-type p53 activity or inactivation of oncogenic mutant p53 by targeted inhibition of UPS components, particularly key deubiquitinases (DUBs) of the ubiquitin-specific protease (USP) class. However, development of selective small-molecule USP inhibitors has been challenging, partly due to the highly conserved structural features of the catalytic sites across the class. To tackle this problem, we devised a protein engineering strategy for rational design of inhibitors for DUBs and other UPS proteins. We employed a phage-displayed ubiquitin variant (UbV) library to develop inhibitors targeting the DUBs USP7 and USP10, which are involved in regulating levels of p53 and MDM2. We were able to identify UbVs that bound USP7 or USP10 with high affinity and inhibited deubiquitination activity. We solved the crystal structure of UbV.7.2 and rationalized the molecular basis for enhanced affinity and specificity for USP7. Finally, cell death was increased significantly by UbV.7.2 expression in a colon cancer cell line that was treated with the chemotherapy drug cisplatin, demonstrating the therapeutic potential of inhibiting USP7 by this approach

OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo

DEFF Research Database (Denmark)

Iglesias-Gato, Diego; Chuan, Yin-Choy; Jiang, Ning

2015-01-01

BackgroundUbiquitination is a highly dynamic and reversible process with a central role in cell homeostasis. Deregulation of several deubiquitinating enzymes has been linked to tumor development but their specific role in prostate cancer progression remains unexplored.MethodsRNAi screening was us...

Nucleophosmin/B23 regulates ubiquitin dynamics in nucleoli by recruiting deubiquitylating enzyme USP36.

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Endo, Akinori; Kitamura, Naomi; Komada, Masayuki

2009-10-09

The nucleolus is a subnuclear compartment with multiple cellular functions, including ribosome biogenesis. USP36 is a deubiquitylating enzyme that localizes to nucleoli and plays an essential role in regulating the structure and function of the organelle. However, how the localization of USP36 is regulated remains unknown. Here, we identified a short stretch of basic amino acids (RGKEKKIKKFKREKRR) that resides in the C-terminal region of USP36 and serves as a nucleolar localization signal for the protein. We found that this motif interacts with a central acidic region of nucleophosmin/B23, a major nucleolar protein involved in various nucleolar functions. Knockdown of nucleophosmin/B23 resulted in a significant reduction in the amount of USP36 in nucleoli, without affecting the cellular USP36 level. This was associated with elevated ubiquitylation levels of fibrillarin, a USP36 substrate protein in nucleoli. We conclude that nucleophosmin/B23 recruits USP36 to nucleoli, thereby serving as a platform for the regulation of nucleolar protein functions through ubiquitylation/deubiquitylation.

Usp7 promotes medulloblastoma cell survival and metastasis by activating Shh pathway

International Nuclear Information System (INIS)

Zhan, Meixiao; Sun, Xiaohan; Liu, Jinxiao; Li, Yan; Li, Yong; He, Xu; Zhou, Zizhang; Lu, Ligong

2017-01-01

The ubiquitin-specific protease Usp7 plays roles in multiple cellular processes through deubiquitinating and stabilizing numerous substrates, including P53, Pten and Gli. Aberrant Usp7 activity has been implicated in many disorders and tumorigenesis, making it as a potential target for therapeutic intervention. Although it is clear that Usp7 is involved in many types of cancer, its role in regulating medulloblastoma (MB) is still unknown. In this study, we show that knockdown of Usp7 inhibits the proliferation and migration of MB cells, while Usp7 overexpression exerts an opposite effect. Furthermore, we establish Usp7 knockout MB cell line using the CRISPR/Cas9 system and further confirm that Usp7 knockout also blocks MB cell proliferation and metastasis. In addition, we reveal that knockdown of Usp7 compromises Shh pathway activity and decrease Gli protein levels, while P53 level and P53 target gene expression have no obvious changes. Finally, we find that Usp7 inhibitors apparently inhibit MB cell viability and migration. Taken together, our findings suggest that Usp7 is important for MB cell proliferation and metastasis by activating Shh pathway, and is a putative therapeutic target for MBs. - Highlights: • Loss of usp7 blocks the proliferation and metastasis of MB cells. • Usp7 regulates MB cell growth and migration through stimulating Shh pathway. • Usp7 inhibitors hamper MB cell proliferation and migration. • Usp7 inhibitors could attenuate Shh pathway activity.

USP7/HAUSP: A SUMO deubiquitinase at the heart of DNA replication.

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Smits, Veronique A J; Freire, Raimundo

2016-09-01

DNA replication is both highly conserved and controlled. Problematic DNA replication can lead to genomic instability and therefore carcinogenesis. Numerous mechanisms work together to achieve this tight control and increasing evidence suggests that post-translational modifications (phosphorylation, ubiquitination, SUMOylation) of DNA replication proteins play a pivotal role in this process. Here we discuss such modifications in the light of a recent article that describes a novel role for the deubiquitinase (DUB) USP7/HAUSP in the control of DNA replication. USP7 achieves this function by an unusual and novel mechanism, namely deubiquitination of SUMOylated proteins at the replication fork, making USP7 also a SUMO DUB (SDUB). This work extends previous observations of increased levels of SUMO and low levels of ubiquitin at the on-going replication fork. Here, we discuss this novel study, its contribution to the DNA replication and genomic stability field and what questions arise from this work. © 2016 WILEY Periodicals, Inc.

Deubiquitinase USP12 promotes LPS induced macrophage responses through inhibition of IκBα

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Nayak, Tapan Kumar Singh; Alamuru-Yellapragada, Neeraja P.; Parsa, Kishore V.L.

2017-01-01

Post translational modifications, ubiquitination and its reversal by deubiquitination play an important role in regulating innate immune system. USP12 is a poorly studied deubiquitinase reported to regulate T-cell receptor signalling however the functional role of USP12 in macrophages, the principal architects of inflammation, is unknown. Thus, in this study we probed the involvement of USP12 in macrophage mediated inflammatory responses using bacterial endotoxin, LPS, as the model system. Here, we observed that the expression of USP12 was altered in time dependent manner in LPS stimulated RAW 264.7 macrophages at both mRNA and protein levels as revealed by qPCR and western blot analysis, respectively. Further analysis showed that LPS reduced the levels of Sp1 which enhanced the transcriptional levels of USP12. We observed that siRNA mediated ablation of USP12 expression in mouse macrophages suppressed the induction of LPS-induced iNOS and IL-6 expression but failed to alter IFN-β synthesis, oxidative stress and phagocytic ability of macrophages. Mechanistic analysis suggest that USP12 may be required for the activation of NFκB pathway as knockdown of USP12 reduced the inhibitory phosphorylation of IκBα, a well characterized inhibitor of NFκB nuclear translocation. Further, USP12 was observed to be required for LPS elicited phosphorylation of ERK1/2 and p38. Collectively, our data suggest that USP12 may be a key mediator of LPS stimulated macrophage responses. - Highlights: • USP12 levels are significantly altered in LPS stimulated macrophages. • USP12 is required for LPS induced iNOS and IL6 expression. • USP12 is crucial for LPS induced phosphorylation of IκBα, ERK1/2, p38.

The deubiquitylating enzyme USP44 counteracts the DNA double-strand break response mediated by the RNF8 and RNF168 ubiquitin ligases

DEFF Research Database (Denmark)

Mosbech, Anna; Lukas, Claudia; Bekker-Jensen, Simon

2013-01-01

Protein recruitment to DNA double-strand breaks (DSBs) relies on ubiquitylation of the surrounding chromatin by the RING finger ubiquitin ligases RNF8 and RNF168. Flux through this pathway is opposed by several deubiquitylating enzymes (DUBs), including OTUB1 and USP3. By analyzing the effect...... of individually overexpressing the majority of human DUBs on RNF8/RNF168-mediated 53BP1 retention at DSB sites, we found that USP44 and USP29 powerfully inhibited this response at the level of RNF168 accrual. Both USP44 and USP29 promoted efficient deubiquitylation of histone H2A, but unlike USP44, USP29...... displayed non-specific reactivity towards ubiquitylated substrates. Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites. Individual depletion of these DUBs only mildly enhanced accumulation of ubiquitin conjugates and 53BP1 at DSBs, suggesting...

Quantitative analysis by next generation sequencing of hematopoietic stem and progenitor cells (LSK) and of splenic B cells transcriptomes from wild-type and Usp3-knockout mice.

Science.gov (United States)

Lancini, Cesare; Gargiulo, Gaetano; van den Berk, Paul C M; Citterio, Elisabetta

2016-03-01

The data described here provide genome-wide expression profiles of murine primitive hematopoietic stem and progenitor cells (LSK) and of B cell populations, obtained by high throughput sequencing. Cells are derived from wild-type mice and from mice deficient for the ubiquitin-specific protease 3 (USP3; Usp3Δ/Δ). Modification of histone proteins by ubiquitin plays a crucial role in the cellular response to DNA damage (DDR) (Jackson and Durocher, 2013) [1]. USP3 is a histone H2A deubiquitinating enzyme (DUB) that regulates ubiquitin-dependent DDR in response to DNA double-strand breaks (Nicassio et al., 2007; Doil et al., 2008) [2], [3]. Deletion of USP3 in mice increases the incidence of spontaneous tumors and affects hematopoiesis [4]. In particular, Usp3-knockout mice show progressive loss of B and T cells and decreased functional potential of hematopoietic stem cells (HSCs) during aging. USP3-deficient cells, including HSCs, display enhanced histone ubiquitination, accumulate spontaneous DNA damage and are hypersensitive to ionizing radiation (Lancini et al., 2014) [4]. To address whether USP3 loss leads to deregulation of specific molecular pathways relevant to HSC homeostasis and/or B cell development, we have employed the RNA-sequencing technology and investigated transcriptional differences between wild-type and Usp3Δ/Δ LSK, naïve B cells or in vitro activated B cells. The data relate to the research article "Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells" (Lancini et al., 2014) [4]. The RNA-sequencing and analysis data sets have been deposited in NCBI׳s Gene Expression Omnibus (Edgar et al., 2002) [5] and are accessible through GEO Series accession number GSE58495 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58495). With this article, we present validation of the RNA-seq data set through quantitative real-time PCR and comparative analysis.

OTUD5 regulates p53 stability by deubiquitinating p53.

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Judong Luo

Full Text Available The p53 tumour suppressor protein is a transcription factor that prevents oncogenic progression by activating the expression of apoptosis and cell-cycle arrest genes in stressed cells. The stability of p53 is tightly regulated by ubiquitin-dependent degradation, driven mainly by its negative regulators ubiquitin ligase MDM2.In this study, we have identified OTUD5 as a DUB that interacts with and deubiquitinates p53. OTUD5 forms a direct complex with p53 and controls level of ubiquitination. The function of OTUD5 is required to allow the rapid activation of p53-dependent transcription and a p53-dependent apoptosis in response to DNA damage stress.As a novel deubiquitinating enzyme for p53, OTUD5 is required for the stabilization and the activation of a p53 response.

USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

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Aman Wang

2017-05-01

Full Text Available Resistance to platinum-based chemotherapy is one of the most important reasons for treatment failure in advanced non-small cell lung cancer, but the underlying mechanism is extremely complex and unclear. The present study aimed to investigate the correlation of ubiquitin-specific peptidase 22 (USP22 with acquired resistance to cisplatin in lung adenocarcinoma. In this study, we found that overexpression of USP22 could lead to cisplatin resistance in A549 cells. USP22 and its downstream proteins γH2AX and Sirt1 levels are upregulated in the cisplatin- resistant A549/CDDP cell line. USP22 enhances DNA damage repair and induce cisplatin resistance by promoting the phosphorylation of histone H2AX via deubiquitinating histone H2A. In addition, USP22 decreases the acetylation of Ku70 by stabilizing Sirt1, thus inhibiting Bax-mediated apoptosis and inducing cisplatin resistance. The cisplatin sensitivity in cisplatin-resistant A549/CDDP cells was restored by USP22 inhibition in vivo and vitro. In summary, our findings reveal the dual mechanism of USP22 involvement in cisplatin resistance that USP22 can regulate γH2AX-mediated DNA damage repair and Ku70/Bax-mediated apoptosis. USP22 is a potential target in cisplatin-resistant lung adenocarcinoma and should be considered in future therapeutic practice.

Quantum Mechanics and Molecular Mechanics Study of the Catalytic Mechanism of Human AMSH-LP Domain Deubiquitinating Enzymes.

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Zhu, Wenyou; Liu, Yongjun; Ling, Baoping

2015-08-25

Deubiquitinating enzymes (DUBs) catalyze the cleavage of the isopeptide bond in polyubiquitin chains to control and regulate the deubiquitination process in all known eukaryotic cells. The human AMSH-LP DUB domain specifically cleaves the isopeptide bonds in the Lys63-linked polyubiquitin chains. In this article, the catalytic mechanism of AMSH-LP has been studied using a combined quantum mechanics and molecular mechanics method. Two possible hydrolysis processes (Path 1 and Path 2) have been considered. Our calculation results reveal that the activation of Zn(2+)-coordinated water molecule is the essential step for the hydrolysis of isopeptide bond. In Path 1, the generated hydroxyl first attacks the carbonyl group of Gly76, and then the amino group of Lys63 is protonated, which is calculated to be the rate limiting step with an energy barrier of 13.1 kcal/mol. The energy barrier of the rate limiting step and the structures of intermediate and product are in agreement with the experimental results. In Path 2, the protonation of amino group of Lys63 is prior to the nucleophilic attack of activated hydroxyl. The two proton transfer processes in Path 2 correspond to comparable overall barriers (33.4 and 36.1 kcal/mol), which are very high for an enzymatic reaction. Thus, Path 2 can be ruled out. During the reaction, Glu292 acts as a proton transfer mediator, and Ser357 mainly plays a role in stabilizing the negative charge of Gly76. Besides acting as a Lewis acid, Zn(2+) also influences the reaction by coordinating to the reaction substrates (W1 and Gly76).

53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis.

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Fong, Chii Shyang; Mazo, Gregory; Das, Tuhin; Goodman, Joshua; Kim, Minhee; O'Rourke, Brian P; Izquierdo, Denisse; Tsou, Meng-Fu Bryan

2016-07-02

Mitosis occurs efficiently, but when it is disturbed or delayed, p53-dependent cell death or senescence is often triggered after mitotic exit. To characterize this process, we conducted CRISPR-mediated loss-of-function screens using a cell-based assay in which mitosis is consistently disturbed by centrosome loss. We identified 53BP1 and USP28 as essential components acting upstream of p53, evoking p21-dependent cell cycle arrest in response not only to centrosome loss, but also to other distinct defects causing prolonged mitosis. Intriguingly, 53BP1 mediates p53 activation independently of its DNA repair activity, but requiring its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. Moreover, 53BP1 can transduce prolonged mitosis to cell cycle arrest independently of the spindle assembly checkpoint (SAC), suggesting that while SAC protects mitotic accuracy by slowing down mitosis, 53BP1 and USP28 function in parallel to select against disturbed or delayed mitosis, promoting mitotic efficiency.

Mechanism of Ubiquitination and Deubiquitination in the Fanconi Anemia Pathway.

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van Twest, Sylvie; Murphy, Vincent J; Hodson, Charlotte; Tan, Winnie; Swuec, Paolo; O'Rourke, Julienne J; Heierhorst, Jörg; Crismani, Wayne; Deans, Andrew J

2017-01-19

Monoubiquitination and deubiquitination of FANCD2:FANCI heterodimer is central to DNA repair in a pathway that is defective in the cancer predisposition syndrome Fanconi anemia (FA). The "FA core complex" contains the RING-E3 ligase FANCL and seven other essential proteins that are mutated in various FA subtypes. Here, we purified recombinant FA core complex to reveal the function of these other proteins. The complex contains two spatially separate FANCL molecules that are dimerized by FANCB and FAAP100. FANCC and FANCE act as substrate receptors and restrict monoubiquitination to the FANCD2:FANCI heterodimer in only a DNA-bound form. FANCA and FANCG are dispensable for maximal in vitro ubiquitination. Finally, we show that the reversal of this reaction by the USP1:UAF1 deubiquitinase only occurs when DNA is disengaged. Our work reveals the mechanistic basis for temporal and spatial control of FANCD2:FANCI monoubiquitination that is critical for chemotherapy responses and prevention of Fanconi anemia. Copyright © 2017 Elsevier Inc. All rights reserved.

Deubiquitylating enzyme USP9x regulates hippo pathway activity by controlling angiomotin protein turnover

DEFF Research Database (Denmark)

Nguyen, Thanh Hung; Andrejeva, Diana; Gupta, Rajat

2016-01-01

/TAZ activity. We demonstrate that USPx regulates ubiquitin-mediated turnover of the YAP inhibitor, Angiomotin. USP9x acts to deubiquitylate Angiomotin at lysine 496, resulting in stabilization of Angiomotin and lower YAP/TAZ activity. USP9x mRNA levels were reduced in several cancers. Clinically, USP9x m......RNA levels were reduced in several cancers with low USPx expression correlating with poor prognosis in renal clear cell carcinoma. Our data indicate that USP9x may be a useful biomarker for renal clear cell carcinoma....

Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)

DEFF Research Database (Denmark)

Hendriks, Ivo A; Schimmel, Joost; Eifler, Karolin

2015-01-01

of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO...

USP22 regulates oncogenic signaling pathways to drive lethal cancer progression.

Science.gov (United States)

Schrecengost, Randy S; Dean, Jeffry L; Goodwin, Jonathan F; Schiewer, Matthew J; Urban, Mark W; Stanek, Timothy J; Sussman, Robyn T; Hicks, Jessica L; Birbe, Ruth C; Draganova-Tacheva, Rossitza A; Visakorpi, Tapio; DeMarzo, Angelo M; McMahon, Steven B; Knudsen, Karen E

2014-01-01

Increasing evidence links deregulation of the ubiquitin-specific proteases 22 (USP22) deubitiquitylase to cancer development and progression in a select group of tumor types, but its specificity and underlying mechanisms of action are not well defined. Here we show that USP22 is a critical promoter of lethal tumor phenotypes that acts by modulating nuclear receptor and oncogenic signaling. In multiple xenograft models of human cancer, modeling of tumor-associated USP22 deregulation demonstrated that USP22 controls androgen receptor accumulation and signaling, and that it enhances expression of critical target genes coregulated by androgen receptor and MYC. USP22 not only reprogrammed androgen receptor function, but was sufficient to induce the transition to therapeutic resistance. Notably, in vivo depletion experiments revealed that USP22 is critical to maintain phenotypes associated with end-stage disease. This was a significant finding given clinical evidence that USP22 is highly deregulated in tumors, which have achieved therapeutic resistance. Taken together, our findings define USP22 as a critical effector of tumor progression, which drives lethal phenotypes, rationalizing this enzyme as an appealing therapeutic target to treat advanced disease.

Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

International Nuclear Information System (INIS)

Nakada, Shinichiro

2016-01-01

The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway

Identification of a functional nuclear localization signal within the human USP22 protein

International Nuclear Information System (INIS)

Xiong, Jianjun; Wang, Yaqin; Gong, Zhen; Liu, Jianyun; Li, Weidong

2014-01-01

Highlights: • USP22 was accumulated in nucleus. • We identified of a functional USP22 NLS. • The KRRK amino acid residues are indispensable in NLS. • The KRRK motif is conserved in USP22 homologues. - Abstract: Ubiquitin-specific processing enzyme 22 (USP22), a member of the deubiquitinase family, is over-expressed in most human cancers and has been implicated in tumorigenesis. Because it is an enzymatic subunit of the human SAGA transcriptional cofactor, USP22 deubiquitylates histone H2A and H2B in the nucleus, thus participating in gene regulation and cell-cycle progression. However, the mechanisms regulating its nuclear translocation have not yet been elucidated. It was here demonstrated that USP22 is imported into the nucleus through a mechanism mediated by nuclear localization signal (NLS). The bipartite NLS sequence KRELELLKHNPKRRKIT (aa152–168), was identified as the functional NLS for its nuclear localization. Furthermore, a short cluster of basic amino acid residues KRRK within this bipartite NLS plays the primary role in nuclear localization and is evolutionarily conserved in USP22 homologues. In the present study, a functional NLS and the minimal sequences required for the active targeting of USP22 to the nucleus were identified. These findings may provide a molecular basis for the mechanism underlying USP22 nuclear trafficking and function

Identification of a functional nuclear localization signal within the human USP22 protein

Energy Technology Data Exchange (ETDEWEB)

Xiong, Jianjun [Key Laboratory of Jiangxi Province for the Systems Bio-Medicine, Jiujiang, Jiangxi Province 332000 (China); College of Basic Medical Science, Jiujiang University, Jiujiang, Jiangxi Province 332000 (China); Wang, Yaqin [Reproductive Medical Center, Renmin Hospital of Wuhan University, Wuhan, Hubei Province 430060 (China); Gong, Zhen [Key Laboratory of Jiangxi Province for the Systems Bio-Medicine, Jiujiang, Jiangxi Province 332000 (China); Liu, Jianyun [College of Basic Medical Science, Jiujiang University, Jiujiang, Jiangxi Province 332000 (China); Li, Weidong, E-mail: lwd626518@163.com [College of Basic Medical Science, Jiujiang University, Jiujiang, Jiangxi Province 332000 (China)

2014-06-20

Highlights: • USP22 was accumulated in nucleus. • We identified of a functional USP22 NLS. • The KRRK amino acid residues are indispensable in NLS. • The KRRK motif is conserved in USP22 homologues. - Abstract: Ubiquitin-specific processing enzyme 22 (USP22), a member of the deubiquitinase family, is over-expressed in most human cancers and has been implicated in tumorigenesis. Because it is an enzymatic subunit of the human SAGA transcriptional cofactor, USP22 deubiquitylates histone H2A and H2B in the nucleus, thus participating in gene regulation and cell-cycle progression. However, the mechanisms regulating its nuclear translocation have not yet been elucidated. It was here demonstrated that USP22 is imported into the nucleus through a mechanism mediated by nuclear localization signal (NLS). The bipartite NLS sequence KRELELLKHNPKRRKIT (aa152–168), was identified as the functional NLS for its nuclear localization. Furthermore, a short cluster of basic amino acid residues KRRK within this bipartite NLS plays the primary role in nuclear localization and is evolutionarily conserved in USP22 homologues. In the present study, a functional NLS and the minimal sequences required for the active targeting of USP22 to the nucleus were identified. These findings may provide a molecular basis for the mechanism underlying USP22 nuclear trafficking and function.

Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks.

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Satoshi Nakajima

Full Text Available During the DNA damage response (DDR, ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5, a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.

A Pseudomonas aeruginosa toxin that hijacks the host ubiquitin proteolytic system.

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Jennifer M Bomberger

2011-03-01

Full Text Available Pseudomonas aeruginosa (P. aeruginosa is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD, pneumonia, cystic fibrosis (CF, and bronchiectasis. Cif (PA2934, a bacterial toxin secreted in outer membrane vesicles (OMV by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB, USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens.

Somatic USP8 Gene Mutations Are a Common Cause of Pediatric Cushing Disease.

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Faucz, Fabio R; Tirosh, Amit; Tatsi, Christina; Berthon, Annabel; Hernández-Ramírez, Laura C; Settas, Nikolaos; Angelousi, Anna; Correa, Ricardo; Papadakis, Georgios Z; Chittiboina, Prashant; Quezado, Martha; Pankratz, Nathan; Lane, John; Dimopoulos, Aggeliki; Mills, James L; Lodish, Maya; Stratakis, Constantine A

2017-08-01

Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have been recently identified as the most common genetic alteration in patients with Cushing disease (CD). However, the frequency of these mutations in the pediatric population has not been extensively assessed. We investigated the status of the USP8 gene at the somatic level in a cohort of pediatric patients with corticotroph adenomas. The USP8 gene was fully sequenced in both germline and tumor DNA samples from 42 pediatric patients with CD. Clinical, biochemical, and imaging data were compared between patients with and without somatic USP8 mutations. Five different USP8 mutations (three missense, one frameshift, and one in-frame deletion) were identified in 13 patients (31%), all of them located in exon 14 at the previously described mutational hotspot, affecting the 14-3-3 binding motif of the protein. Patients with somatic mutations were older at disease presentation [mean 5.1 ± 2.1 standard deviation (SD) vs 13.1 ± 3.6 years, P = 0.03]. Levels of urinary free cortisol, midnight serum cortisol, and adrenocorticotropic hormone, as well as tumor size and frequency of invasion of the cavernous sinus, were not significantly different between the two groups. However, patients harboring somatic USP8 mutations had a higher likelihood of recurrence compared with patients without mutations (46.2% vs 10.3%, P = 0.009). Somatic USP8 gene mutations are a common cause of pediatric CD. Patients harboring a somatic mutation had a higher likelihood of tumor recurrence, highlighting the potential importance of this molecular defect for the disease prognosis and the development of targeted therapeutic options. Copyright © 2017 Endocrine Society

An AAA Motor-Driven Mechanical Switch in Rpn11 Controls Deubiquitination at the 26S Proteasome.

Science.gov (United States)

Worden, Evan J; Dong, Ken C; Martin, Andreas

2017-09-07

Poly-ubiquitin chains direct protein substrates to the 26S proteasome, where they are removed by the deubiquitinase Rpn11 during ATP-dependent substrate degradation. Rapid deubiquitination is required for efficient degradation but must be restricted to committed substrates that are engaged with the ATPase motor to prevent premature ubiquitin chain removal and substrate escape. Here we reveal the ubiquitin-bound structure of Rpn11 from S. cerevisiae and the mechanisms for mechanochemical coupling of substrate degradation and deubiquitination. Ubiquitin binding induces a conformational switch of Rpn11's Insert-1 loop from an inactive closed state to an active β hairpin. This switch is rate-limiting for deubiquitination and strongly accelerated by mechanical substrate translocation into the AAA+ motor. Deubiquitination by Rpn11 and ubiquitin unfolding by the ATPases are in direct competition. The AAA+ motor-driven acceleration of Rpn11 is therefore important to ensure that poly-ubiquitin chains are removed only from committed substrates and fast enough to prevent their co-degradation. Copyright © 2017 Elsevier Inc. All rights reserved.

A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells

International Nuclear Information System (INIS)

Liu, Shuai; Lv, Jiaju; Han, Liping; Ichikawa, Tomonaga; Wang, Wenjuan; Li, Siying; Wang, Xing Li; Tang, Dongqi; Cui, Taixing

2012-01-01

Highlights: ► Cyld deficiency suppresses pro-inflammatory phenotypic switch of VSMCs. ► Cyld deficiency inhibits MAPK rather than NF-kB activity in inflamed VSMCs. ► CYLD is up-regulated in the coronary artery with neointimal hyperplasia. -- Abstract: CYLD, a deubiquitinating enzyme (DUB), is a critical regulator of diverse cellular processes, ranging from proliferation and differentiation to inflammatory responses, via regulating multiple key signaling cascades such as nuclear factor kappa B (NF-κB) pathway. CYLD has been shown to inhibit vascular lesion formation presumably through suppressing NF-κB activity in vascular cells. However, herein we report a novel role of CYLD in mediating pro-inflammatory responses in vascular smooth muscle cells (VSMCs) via a mechanism independent of NF-κB activity. Adenoviral knockdown of Cyld inhibited basal and the tumor necrosis factor alpha (TNFα)-induced mRNA expression of pro-inflammatory cytokines including monocyte chemotactic protein-1 (Mcp-1), intercellular adhesion molecule (Icam-1) and interleukin-6 (Il-6) in rat adult aortic SMCs (RASMCs). The CYLD deficiency led to increases in the basal NF-κB transcriptional activity in RASMCs; however, did not affect the TNFα-induced NF-κB activity. Intriguingly, the TNFα-induced IκB phosphorylation was enhanced in the CYLD deficient RASMCs. While knocking down of Cyld decreased slightly the basal expression levels of IκBα and IκBβ proteins, it did not alter the kinetics of TNFα-induced IκB protein degradation in RASMCs. These results indicate that CYLD suppresses the basal NF-κB activity and TNFα-induced IκB kinase activation without affecting TNFα-induced NF-κB activity in VSMCs. In addition, knocking down of Cyld suppressed TNFα-induced activation of mitogen activated protein kinases (MAPKs) including extracellular signal-activated kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 in RASMCs. TNFα-induced RASMC migration and monocyte adhesion to

USP : Key Considerations and Changes for Health Systems

Science.gov (United States)

Sahadeo, Priya; Weber, Robert J.

2015-01-01

On March 28, 2014, The United States Pharmacopeia and The National Formulary (USP-NF) published USP General Chapter Hazardous Drugs–Handling in Healthcare Settings, as open for public comment in the USP Pharmacopeial Forum (PF) 40(3). Pharmacy directors must be proactive in understanding the impact that USP will have on their processes for preparing sterile products. USP General Chapter pertains to the compounding of both hazardous and nonhazardous drugs. USP serves as a new standard to guide the handling of hazardous drugs in order to protect patients, health care personnel, and the environment. USP describes hazardous drug handling related to the receipt, storage, compounding, dispensing, administration, and disposal of both sterile and nonsterile products and preparations. Regardless of all of the requirements listed in USP , there is no substitute for disciplined, consistent work practices regarding proper sterile technique. This point should be emphasized with all compounding personnel. Even if one is compounding in the most compliant USP cleanroom, improper technique can negate all the benefits of the physical structures. Pharmacy leaders at every level will play a key role in assisting an organization to achieve timely compliance with USP standards. Until the standard becomes official, it is important for pharmacists to become familiarized with the latest draft to identify potential barriers to compliance and to strategize a plan to overcome barriers. Although complying with USP may seem to be a daunting task, it can be manageable if approached in a systematic organized way. PMID:27729683

Ubiquitin specific protease 21 is dispensable for normal development, hematopoiesis and lymphocyte differentiation.

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Jaspreet Pannu

Full Text Available USP21 is a ubiquitin specific protease that catalyzes protein deubiquitination, however the identification of its physiological substrates remains challenging. USP21 is known to deubiquitinate transcription factor GATA3 and death-domain kinase RIPK1 in vitro, however the in vivo settings where this regulation plays a biologically significant role remain unknown. In order to determine whether USP21 is an essential and non-redundant regulator of GATA3 or RIPK1 activity in vivo, we characterized Usp21-deficient mice, focusing on mouse viability and development, hematopoietic stem cell function, and lymphocyte differentiation. The Usp21-knockout mice were found to be viable and fertile, with no significant dysmorphology, in contrast to the GATA3 and RIPK1 knockout lines that exhibit embryonic or perinatal lethality. Loss of USP21 also had no effect on hematopoietic stem cell function, lymphocyte development, or the responses of antigen presenting cells to TLR and TNFR stimulation. GATA3 levels in hematopoietic stem cells or T lymphocytes remained unchanged. We observed that aged Usp21-knockout mice exhibited spontaneous T cell activation, however this was not linked to altered GATA3 levels in the affected cells. The contrast in the phenotype of the Usp21-knockout line with the previously characterized GATA3 and RIPK1 knockout mice strongly indicates that USP21 is redundant for the regulation of GATA3 and RIPK1 activity during mouse development, in hematopoietic stem cells, and in lymphocyte differentiation. The Usp21-deficient mouse line characterized in this study may serve as a useful tool for the future characterization of USP21 physiological functions.

A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells

Energy Technology Data Exchange (ETDEWEB)

Liu, Shuai [Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012 (China); Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States); Lv, Jiaju [Department of Urology, Shandong Provincial Hospital, Shandong University, Jinan 250021 (China); Han, Liping; Ichikawa, Tomonaga; Wang, Wenjuan; Li, Siying [Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States); Wang, Xing Li [Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012 (China); Tang, Dongqi, E-mail: tangdq@pathology.ufl.edu [Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610-0275 (United States); Cui, Taixing, E-mail: taixing.cui@uscmed.sc.edu [Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States)

2012-03-30

Highlights: Black-Right-Pointing-Pointer Cyld deficiency suppresses pro-inflammatory phenotypic switch of VSMCs. Black-Right-Pointing-Pointer Cyld deficiency inhibits MAPK rather than NF-kB activity in inflamed VSMCs. Black-Right-Pointing-Pointer CYLD is up-regulated in the coronary artery with neointimal hyperplasia. -- Abstract: CYLD, a deubiquitinating enzyme (DUB), is a critical regulator of diverse cellular processes, ranging from proliferation and differentiation to inflammatory responses, via regulating multiple key signaling cascades such as nuclear factor kappa B (NF-{kappa}B) pathway. CYLD has been shown to inhibit vascular lesion formation presumably through suppressing NF-{kappa}B activity in vascular cells. However, herein we report a novel role of CYLD in mediating pro-inflammatory responses in vascular smooth muscle cells (VSMCs) via a mechanism independent of NF-{kappa}B activity. Adenoviral knockdown of Cyld inhibited basal and the tumor necrosis factor alpha (TNF{alpha})-induced mRNA expression of pro-inflammatory cytokines including monocyte chemotactic protein-1 (Mcp-1), intercellular adhesion molecule (Icam-1) and interleukin-6 (Il-6) in rat adult aortic SMCs (RASMCs). The CYLD deficiency led to increases in the basal NF-{kappa}B transcriptional activity in RASMCs; however, did not affect the TNF{alpha}-induced NF-{kappa}B activity. Intriguingly, the TNF{alpha}-induced I{kappa}B phosphorylation was enhanced in the CYLD deficient RASMCs. While knocking down of Cyld decreased slightly the basal expression levels of I{kappa}B{alpha} and I{kappa}B{beta} proteins, it did not alter the kinetics of TNF{alpha}-induced I{kappa}B protein degradation in RASMCs. These results indicate that CYLD suppresses the basal NF-{kappa}B activity and TNF{alpha}-induced I{kappa}B kinase activation without affecting TNF{alpha}-induced NF-{kappa}B activity in VSMCs. In addition, knocking down of Cyld suppressed TNF{alpha}-induced activation of mitogen activated protein

Deletion of creB in Aspergillus oryzae increases secreted hydrolytic enzyme activity.

Science.gov (United States)

Hunter, A J; Morris, T A; Jin, B; Saint, C P; Kelly, J M

2013-09-01

Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes.

Interaction of the deubiquitinating enzyme Ubp2 and the e3 ligase Rsp5 is required for transporter/receptor sorting in the multivesicular body pathway.

Directory of Open Access Journals (Sweden)

Mandy H Y Lam

Full Text Available Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.

The Translesion Polymerase Pol η Is Required for Efficient Epstein-Barr Virus Infectivity and Is Regulated by the Viral Deubiquitinating Enzyme BPLF1.

Science.gov (United States)

Dyson, Ossie F; Pagano, Joseph S; Whitehurst, Christopher B

2017-10-01

lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes and is well studied, but there are few studies of endogenous repair of viral DNA. This work evaluates how EBV's BPLF1 protein and its conserved deubiquitinating activity regulate the cellular DNA repair enzyme polymerase eta and recruit it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle, contributing to efficient infectious virion production. Copyright © 2017 American Society for Microbiology.

Screen for ISG15-crossreactive deubiquitinases.

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André Catic

2007-07-01

Full Text Available The family of ubiquitin-like molecules (UbLs comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs might exist, capable of recognizing both ubiquitin and ISG15.We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1, USP13 (IsoT3, and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43. USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome.By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice.

A Fat-Facets-Dscam1-JNK Pathway Enhances Axonal Growth in Development and after Injury

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Marta Koch

2018-02-01

Full Text Available Injury to the adult central nervous systems (CNS can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. Here, we conducted a gain-of-function genetic screen in Drosophila to identify strong inducers of axonal growth after injury. We focus on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1, the de-ubiquitinating enzyme Fat Facets (Faf/Usp9x and the Jun N-Terminal Kinase (JNK pathway transcription factor Kayak (Kay/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3′-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism.

Mice null for the deubiquitinase USP18 spontaneously develop leiomyosarcomas

International Nuclear Information System (INIS)

Chinyengetere, Fadzai; Sekula, David J.; Lu, Yun; Giustini, Andrew J.; Sanglikar, Aarti; Kawakami, Masanori; Ma, Tian; Burkett, Sandra S.; Eisenberg, Burton L.; Wells, Wendy A.; Hoopes, Paul J.; Demicco, Elizabeth G.; Lazar, Alexander J; Torres, Keila E.; Memoli, Vincent; Freemantle, Sarah J.; Dmitrovsky, Ethan

2015-01-01

USP18 (ubiquitin-specific protease 18) removes ubiquitin-like modifier interferon stimulated gene 15 (ISG15) from conjugated proteins. USP18 null mice in a FVB/N background develop tumors as early as 2 months of age. These tumors are leiomyosarcomas and thus represent a new murine model for this disease. Heterozygous USP18 +/− FVB/N mice were bred to generate wild-type, heterozygous and homozygous cohorts. Tumors were characterized immunohistochemically and two cell lines were derived from independent tumors. Cell lines were karyotyped and their responses to restoration of USP18 activity assessed. Drug testing and tumorigenic assays were also performed. USP18 immunohistochemical staining in a large series of human leiomyosacomas was examined. USP18 −/− FVB/N mice spontaneously develop tumors predominantly on the back of the neck with most tumors evident between 6–12 months (80 % penetrance). Immunohistochemical characterization of the tumors confirmed they were leiomyosarcomas, which originate from smooth muscle. Restoration of USP18 activity in sarcoma-derived cell lines did not reduce anchorage dependent or independent growth or xenograft tumor formation demonstrating that these cells no longer require USP18 suppression for tumorigenesis. Karyotyping revealed that both tumor-derived cell lines were aneuploid with extra copies of chromosomes 3 and 15. Chromosome 15 contains the Myc locus and MYC is also amplified in human leiomyosarcomas. MYC protein levels were elevated in both murine leiomyosarcoma cell lines. Stabilized P53 protein was detected in a subset of these murine tumors, another feature of human leiomyosarcomas. Immunohistochemical analyses of USP18 in human leiomyosarcomas revealed a range of staining intensities with the highest USP18 expression in normal vascular smooth muscle. USP18 tissue array analysis of primary leiomyosarcomas from 89 patients with a clinical database revealed cases with reduced USP18 levels had a significantly

青杄PwUSP2基因的克隆和表达分析%Cloning and Expression Analysis of PwUSP2 from Picea wilsonii

Institute of Scientific and Technical Information of China (English)

周燕妮; 李艳芳; 张通; 张凌云

2015-01-01

Universal stress proteins (USPs) involve in multiple abiotic responses including carbon starvation, O2 deprivation, drought and high salinity;however, it still remains elusive in plants. In this study, the full length cDNA of PwUSP2 was obtained using RACE-PCR method. Bioinformatics analysis showed that the full length cDNA of PwUSP2 is 987 bp, including the open reading frame (ORF) 723 bp. The PwUSP2 contains 240 ami-no acids with a theoretical molecular weight of 26.84 kDa and theoretical isoelectric points (pI) of 4.61. It is also a hydrophilic protein with serine and threonine binding sites and has non-transmembrane domain. Mean-while, it has the typical UspA domain of USP family but not the characteristic of ATP-binding site G-2X-G-9X-G[S/T]. RT-qPCR analysis showed that PwUSP2 was expressed in different tissues, while highly expressed in fruit. Furthermore, the expression of PwUSP2 signiifcantly changed under abiotic stresses such as abscisic acid (ABA) and methyl jasmonate (MeJA), indicating that PwUSP2 might be involved in response to multiple stresses in plants.%广泛逆境胁迫蛋白(USPs)参与碳缺乏、缺氧、干旱和高盐等多种非生物胁迫,但在植物中的研究尚不深入。本文通过RACE-PCR的方法获得青杄PwUSP2基因的cDNA全长,共987 bp,其中编码区723 bp,共编码240个氨基酸。利用生物信息学工具对其理化性质、二级结构和三级结构进行分析,结果显示,该蛋白理论分子质量为26.84 kDa,理论等电点为4.61,有丝氨酸和苏氨酸结合位点,为非跨膜的亲水蛋白。PwUSP2具有USP家族典型的UspA结构域,但无典型的ATP结合位点G-2X-G-9X-G[S/T]。RT-qPCR分析表明, PwUSP2在青杄花粉、果实、种子、成熟叶、幼叶、成茎中均有表达,在果实中表达量较高。同时, PwUSP2在脱落酸(ABA)、茉莉酸甲酯(MeJA)等非生物胁迫下表达量有明显变化,推测PwUSP2可能参与青杄对逆境胁迫的响应。

Regulation of behavioral circadian rhythms and clock protein PER1 by the deubiquitinating enzyme USP2

DEFF Research Database (Denmark)

Yang, Yaoming; Duguay, David; Bédard, Nathalie

2012-01-01

Endogenous 24-hour rhythms are generated by circadian clocks located in most tissues. The molecular clock mechanism is based on feedback loops involving clock genes and their protein products. Post-translational modifications, including ubiquitination, are important for regulating the clock...

WIDE AND THICK GRAIN 1, which encodes an otubain-like protease with deubiquitination activity, influences grain size and shape in rice.

Science.gov (United States)

Huang, Ke; Wang, Dekai; Duan, Penggen; Zhang, Baolan; Xu, Ran; Li, Na; Li, Yunhai

2017-09-01

Grain size and shape are two crucial traits that influence grain yield and grain appearance in rice. Although several factors that affect grain size have been described in rice, the molecular mechanisms underlying the determination of grain size and shape are still elusive. In this study we report that WIDE AND THICK GRAIN 1 (WTG1) functions as an important factor determining grain size and shape in rice. The wtg1-1 mutant exhibits wide, thick, short and heavy grains and also shows an increased number of grains per panicle. WTG1 determines grain size and shape mainly by influencing cell expansion. WTG1 encodes an otubain-like protease, which shares similarity with human OTUB1. Biochemical analyses indicate that WTG1 is a functional deubiquitinating enzyme, and the mutant protein (wtg1-1) loses this deubiquitinating activity. WTG1 is expressed in developing grains and panicles, and the GFP-WTG1 fusion protein is present in the nucleus and cytoplasm. Overexpression of WTG1 results in narrow, thin, long grains due to narrow and long cells, further supporting the role of WTG1 in determining grain size and shape. Thus, our findings identify the otubain-like protease WTG1 to be an important factor that determines grain size and shape, suggesting that WTG1 has the potential to improve grain size and shape in rice. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

ρ0 Cells Feature De-Ubiquitination of SLC Transporters and Increased Levels and Fluxes of Amino Acids

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André Bordinassi Medina

2017-04-01

Full Text Available Solute carrier (SLC transporters are a diverse group of membrane transporter proteins that regulate the cellular flux and distribution of endogenous and xenobiotic compounds. Post-translational modifications (PTMs, such as ubiquitination, have recently emerged as one of the major regulatory mechanisms in protein function and localization. Previously, we showed that SLC amino acid transporters were on average 6-fold de-ubiquitinated and increased amino acid levels were detected in ρ0 cells (lacking mitochondrial DNA, mtDNA compared to parental cells. Here, we elucidated the altered functionality of SLC transporters and their dynamic ubiquitination status by measuring the uptake of several isotopically labeled amino acids in both human osteosarcoma 143B.TK- and ρ0 cells. Our pulse chase analysis indicated that de-ubiquitinated amino acid transporters in ρ0 cells were accompanied by an increased transport rate, which leads to higher levels of amino acids in the cell. Finding SLC transport enhancers is an aim of the pharmaceutical industry in order to compensate for loss of function mutations in these genes. Thus, the ubiquitination status of SLC transporters could be an indicator for their functionality, but evidence for a direct connection between de-ubiquitination and transporter activity has to be further elucidated.

25 anos da revista Psicologia USP

OpenAIRE

Ramozzi-Chiarottino, Zelia

2016-01-01

Resumo Este pequeno artigo narra a fundação da revista Psicologia USP em 1990 e sua origem, inspirada no Jornal Brasileiro de Psicologia. Faz também, singela homenagem ao seu idealizador Professor Norberto de Abreu e Silva Neto. Abstract This short article is on the establishement of the Psicologia USP journal and its origins, which was inspired by the journal Jornal Brasileiro de Psicologia. It also renders a tribute to Doctor Norberto de Abreu e Silva Neto, the creator of the journal. ...

USP7 is a SUMO deubiquitinase essential for DNA replication

DEFF Research Database (Denmark)

Lecona, Emilio; Rodriguez-Acebes, Sara; Specks, Julia

2016-01-01

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication. We have previously shown that chromatin around replisomes is rich in SUMO and poor in Ub, whereas mature chromatin exhibits an opposite pattern. How this SUMO-rich, Ub-poor environment...... is maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads...... to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development of USP7...

Sérgio Buarque de Holanda na USP

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Rodrigo Ruiz Sanches

2011-04-01

Full Text Available O objetivo deste artigo é descrever e analisar a trajetória de Sérgio Buarque de Holanda na Universidade de São Paulo. Na USP, pôde dedicar-se inteiramente ao que mais gostava: pesquisar. Lá, formou um grupo de pesquisadores e mudou os rumos do ensino do curso de História que, à época, formava somente professores para o Ensino Fundamental e Médio. Idealizou e fundou, também, o Instituto de Estudos Brasileiros, órgão multidisciplinar responsável por importantes pesquisas que orbitam o tema Brasil. No IEB formou um amplo arquivo e uma importante biblioteca. Os 13 anos na USP se encerram num processo conturbado em 1969, quando pede sua aposentadoria em solidariedade aos outros professores aposentados compulsoriamente pelo AI-5.The main purpose of the article is to describe and to analyse Sérgio Buarque de Holanda's trajectory in Universidade de São Paulo (USP. At USP, he could dedicate himself entirely in what he liked to do: to research. There, Sérgio Buarque de Holanda formed a researchers' group and he changed the teaching routes in History course, which formed only Elementary and High School teachers. He idealized and founded the "Instituto de Estudos Brasileiros" (Brazilian Studies Intitute, a multi-disciplinary institution responsible for important researches included the theme "Brasil". In this Institute (IEB, he formed a ample archive and a important library. In 1969, the 13-years-job at USP finished itself in a careworm process, when he asked for his retirement in sympathy with other retired professors compulsorily by AI-5.

USP7 is a SUMO deubiquitinase essential for DNA replication

Science.gov (United States)

Lecona, Emilio; Rodriguez-Acebes, Sara; Specks, Julia; Lopez-Contreras, Andres J; Ruppen, Isabel; Murga, Matilde; Muñoz, Javier; Mendez, Juan; Fernandez-Capetillo, Oscar

2016-01-01

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates various aspects of DNA replication. We previously showed that the chromatin around replisomes is rich in SUMO and depleted in Ub, whereas an opposite pattern is observed in mature chromatin. How this SUMO-rich/Ub-low environment is maintained at sites of DNA replication is not known. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Chemical inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced to chromatin away from replisomes. Our findings provide a model to explain the differential accumulation of SUMO and Ub at replication forks, and identify an essential role of USP7 in DNA replication that should be taken into account for the use of USP7 inhibitors as anticancer agents. PMID:26950370

Behavioral Characteristics of Ubiquitin-Specific Peptidase 46-Deficient Mice

Science.gov (United States)

Imai, Saki; Kano, Makoto; Nonoyama, Keiko; Ebihara, Shizufumi

2013-01-01

We have previously identified Usp46, which encodes for ubiquitin-specific peptidase 46, as a quantitative trait gene affecting the immobility time of mice in the tail suspension test (TST) and forced swimming test. The mutation that we identified was a 3-bp deletion coding for lysine (Lys 92), and mice with this mutation (MT mice), as well as Usp46 KO mice exhibited shorter TST immobility times. Behavioral pharmacology suggests that the gamma aminobutyric acid A (GABAA) receptor is involved in regulating TST immobility time. In order to understand how far Usp46 controls behavioral phenotypes, which could be related to mental disorders in humans, we subjected Usp46 MT and KO mice to multiple behavioral tests, including the open field test, ethanol preference test, ethanol-induced loss of righting reflex test, sucrose preference test, novelty-suppressed feeding test, marble burying test, and novel object recognition test. Although behavioral phenotypes of the Usp46 MT and KO mice were not always identical, deficiency of Usp46 significantly affected performance in all these tests. In the open field test, activity levels were lower in Usp46 KO mice than wild type (WT) or MT mice. Both MT and KO mice showed lower ethanol preference and shorter recovery times after ethanol administration. Compared to WT mice, Usp46 MT and KO mice exhibited decreased sucrose preference, took longer latency periods to bite pellets, and buried more marbles in the sucrose preference test, novelty-suppressed feeding test, and marble burying test, respectively. In the novel object recognition test, neither MT nor KO mice showed an increase in exploration of a new object 24 hours after training. These findings indicate that Usp46 regulates a wide range of behavioral phenotypes that might be related to human mental disorders and provides insight into the function of USP46 deubiquitinating enzyme in the neural system. PMID:23472206

Behavioral characteristics of ubiquitin-specific peptidase 46-deficient mice.

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Saki Imai

Full Text Available We have previously identified Usp46, which encodes for ubiquitin-specific peptidase 46, as a quantitative trait gene affecting the immobility time of mice in the tail suspension test (TST and forced swimming test. The mutation that we identified was a 3-bp deletion coding for lysine (Lys 92, and mice with this mutation (MT mice, as well as Usp46 KO mice exhibited shorter TST immobility times. Behavioral pharmacology suggests that the gamma aminobutyric acid A (GABAA receptor is involved in regulating TST immobility time. In order to understand how far Usp46 controls behavioral phenotypes, which could be related to mental disorders in humans, we subjected Usp46 MT and KO mice to multiple behavioral tests, including the open field test, ethanol preference test, ethanol-induced loss of righting reflex test, sucrose preference test, novelty-suppressed feeding test, marble burying test, and novel object recognition test. Although behavioral phenotypes of the Usp46 MT and KO mice were not always identical, deficiency of Usp46 significantly affected performance in all these tests. In the open field test, activity levels were lower in Usp46 KO mice than wild type (WT or MT mice. Both MT and KO mice showed lower ethanol preference and shorter recovery times after ethanol administration. Compared to WT mice, Usp46 MT and KO mice exhibited decreased sucrose preference, took longer latency periods to bite pellets, and buried more marbles in the sucrose preference test, novelty-suppressed feeding test, and marble burying test, respectively. In the novel object recognition test, neither MT nor KO mice showed an increase in exploration of a new object 24 hours after training. These findings indicate that Usp46 regulates a wide range of behavioral phenotypes that might be related to human mental disorders and provides insight into the function of USP46 deubiquitinating enzyme in the neural system.

Heparin interferes with the radioenzymatic and homogeneous enzyme immunoassays for aminoglycosides

International Nuclear Information System (INIS)

Krogstad, D.J.; Granich, G.G.; Murray, P.R.; Pfaller, M.A.; Valdes, R.

1981-01-01

Heparin interferes with measurement of aminoglycosides in serum by biological, radioenzymatic, and homogeneous enzyme immunoassay techniques, but not with radioimmunoassay. At concentrations greater than or equal to 10 5 and greater than or equal to 3 X 10 6 USP units/L, respectively, it interferes with the radioenzymatic assay by inhibiting the gentamicin 3-acetyltransferase and kanamycin 6'-acetyltransferase enzymes used in the assay. It interferes with the homogeneous enzyme immunoassays for gentamicin and tobramycin (at concentrations greater than or equal to 10 5 and greater than or equal to10 4 USP units/L, respectively), but not with the commercially available homogeneous enzyme immunoassays for other drugs. Heparin interference with the homogeneous enzyme immunoassay for aminoglycosides requires both the heparin polyanion and glucose-6-phosphate dehydrogenase bound to a cationic aminoglycoside. This interference can be reproduced with dextran sulfate (but not dextran), and does not occur with free enzyme (glucose-6-phosphate dehydrogenase) alone. Heparin interference with these two assays and at concentrations that may be present in intravenous infusions or in seriously underfilled blood-collection tubes is described

The deubiquitinase USP9X regulates FBW7 stability and suppresses colorectal cancer

Science.gov (United States)

Khan, Omar M.; Carvalho, Joana; Spencer-Dene, Bradley; Mitter, Richard; Frith, David; Snijders, Ambrosius P.; Wood, Stephen A.

2018-01-01

The tumor suppressor FBW7 targets oncoproteins such as c-MYC for ubiquitylation and is mutated in several human cancers. We noted that in a substantial percentage of colon cancers, FBW7 protein is undetectable despite the presence of FBW7 mRNA. To understand the molecular mechanism of FBW7 regulation in these cancers, we employed proteomics and identified the deubiquitinase (DUB) USP9X as an FBW7 interactor. USP9X antagonized FBW7 ubiquitylation, and Usp9x deletion caused Fbw7 destabilization. Mice lacking Usp9x in the gut showed reduced secretory cell differentiation and increased progenitor proliferation, phenocopying Fbw7 loss. In addition, Usp9x inactivation impaired intestinal regeneration and increased tumor burden in colitis-associated intestinal cancer. c-Myc heterozygosity abrogated increased progenitor proliferation and tumor burden in Usp9x-deficient mice, suggesting that Usp9x suppresses tumor formation by regulating Fbw7 protein stability and thereby reducing c-Myc. Thus, we identify a tumor suppressor mechanism in the mammalian intestine that arises from the posttranslational regulation of FBW7 by USP9X independent of somatic FBW7 mutations. PMID:29346117

77 FR 18707 - USPS Package Intercept-New Product Offerings

Science.gov (United States)

2012-03-28

... commercial customers to register and request USPS Package Intercept service through the Business Customer... Intelligent Mail[supreg] package barcode will be included on the redirected new Priority Mail pieces. The USPS... requests are active for 10 business days from the date of the request. Interception of eligible mailpieces...

USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance.

Science.gov (United States)

Lee, Jin-Ku; Chang, Nakho; Yoon, Yeup; Yang, Heekyoung; Cho, Heejin; Kim, Eunhee; Shin, Yongjae; Kang, Wonyoung; Oh, Young Taek; Mun, Gyeong In; Joo, Kyeung Min; Nam, Do-Hyun; Lee, Jeongwu

2016-01-01

Clinical benefits from standard therapies against glioblastoma (GBM) are limited in part due to intrinsic radio- and chemoresistance of GBM and inefficient targeting of GBM stem-like cells (GSCs). Novel therapeutic approaches that overcome treatment resistance and diminish stem-like properties of GBM are needed. We determined the expression levels of ubiquitination-specific proteases (USPs) by transcriptome analysis and found that USP1 is highly expressed in GBM. Using the patient GBM-derived primary tumor cells, we inhibited USP1 by shRNA-mediated knockdown or its specific inhibitor pimozide and evaluated the effects on stem cell marker expression, proliferation, and clonogenic growth of tumor cells. USP1 was highly expressed in gliomas relative to normal brain tissues and more preferentially in GSC enrichment marker (CD133 or CD15) positive cells. USP1 positively regulated the protein stability of the ID1 and CHEK1, critical regulators of DNA damage response and stem cell maintenance. Targeting USP1 by RNA interference or treatment with a chemical USP1 inhibitor attenuated clonogenic growth and survival of GSCs and enhanced radiosensitivity of GBM cells. Finally, USP1 inhibition alone or in combination with radiation significantly prolonged the survival of tumor-bearing mice. USP1-mediated protein stabilization promotes GSC maintenance and treatment resistance, thereby providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Ubiquitination-Linked Phosphorylation of the FANCI S/TQ Cluster Contributes to Activation of the Fanconi Anemia I/D2 Complex

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Ronald S. Cheung

2017-06-01

Full Text Available Repair of interstrand crosslinks by the Fanconi anemia (FA pathway requires both monoubiquitination and de-ubiquitination of the FANCI/FANCD2 (FANCI/D2 complex. In the standing model, the phosphorylation of six sites in the FANCI S/TQ cluster domain occurs upstream of, and promotes, FANCI/D2 monoubiquitination. We generated phospho-specific antibodies against three different S/TQ cluster sites (serines 556, 559, and 565 on human FANCI and found that, in contrast to the standing model, distinct FANCI sites were phosphorylated either predominantly upstream (ubiquitination independent; serine 556 or downstream (ubiquitination-linked; serines 559 and 565 of FANCI/D2 monoubiquitination. Ubiquitination-linked FANCI phosphorylation inhibited FANCD2 de-ubiquitination and bypassed the need to de-ubiquitinate FANCD2 to achieve effective interstrand crosslink repair. USP1 depletion suppressed ubiquitination-linked FANCI phosphorylation despite increasing FANCI/D2 monoubiquitination, providing an explanation of why FANCD2 de-ubiquitination is important for function of the FA pathway. Our work results in a refined model of how FANCI phosphorylation activates the FANCI/D2 complex.

An enzymic method for the determination of [1-14C] lactose

International Nuclear Information System (INIS)

Davies, E.; Bourke, E.; Costello, J.

1975-01-01

A simple, rapid and specific method for the determination of [1- 14 C] lactose in biological fluids is described. It is based on the enzymic removal of the 1- 14 C atom of lactose as [ 14 C] carbon dioxide, using commercially available enzymes. The assay involves only one critical addition and the entire reaction can be carried out in a scintillation vial. (author)

Symposium 19: The contributions of the Department of Biochemistry/USP towards Biochemistry teaching

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Bayardo Baptista Torres

2014-08-01

Full Text Available K-Education(Portuguese Chair: V. Trindade Bayardo Torres; Clovis Wannmacher; Denise MacedoThe contributions of the Department of Biochemistry/USP towards Biochemistry teaching.O ensino de Bioquímica nos últimos 20 anosBayardo B. TorresDepartamento de Bioquímica, Instituto de Química, USP. São Paulo, Brazil.Among the contributions of the Department of Biochemistry/USP one must recall:1. Winter school for graduate studentsThis course, now at the ninth edition, is intended for students in the final stage of their Masters or PhD in Biochemistry or related areas from any institution of higher education.Modern and important techniques are offered as possible support to help the student’s projects.2. Summer courses for undergraduate studentsThe Department offers every year, since 1999, complementary courses for undergraduate students to extend their knowledge in biochemical subjects not ordinarily treated in introductory courses. Some examples:Plant Molecular Biology, Biochemistry and Diseases, Biochemistry of Mind, Biochemistry of Ageing, Cancer Biochemistry, Nutrition and Sports, Biochemistry of Beauty, Biochemistry of the Envenomation Response, etc.3. Summer courses for high school teachers. Some examples:Biochemistry of Nutrition, DNA – Techniques and Applications, Biochemistry in the kitchen.4. Software developmentMany software for biochemistry teaching/learning were developed and are freely available at the Biblioteca Digital de Ciências [http://www.bdc.ib.unicamp.br/bdc/index.php]. Some examples:Oxygen consumption by mitochondria, Muscle contraction, Electron transport chain and oxidative phosphorylation, Free radicals, Enzyme kinetics, cAMP signalization, Interactive study of protein structure, Leptin, Insulin and Obesity.5. A Biochemistry textbook. 

dos museus da USP (1895 - 2000

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Teresa Cristina Toledo de Paula

2005-01-01

Full Text Available The text presents all the original published contents on textiles and conservation as issues of interest and research by the present museums of São Paulo University (USP in the period of 1895 to 2000.

Seniority-conserving forces and USp(2j+1) partial dynamical symmetry

International Nuclear Information System (INIS)

Rosensteel, G.; Rowe, D.J.

2003-01-01

A quasispin tensor decomposition of the two-nucleon interaction determines the most general seniority-conserving rotationally invariant two-body interaction in a j shell. Such interactions define solvable and partially solvable shell model Hamiltonians for which the unitary symplectic algebra USp(2j+1) provides a complete set of quantum numbers for a subset of states. The matrix elements of seniority-conserving interactions are deduced from the matrix elements of USp(2j+1) operators. A new and powerful numerical technique is presented for computing irreps of the USp(2j+1) algebra. Applications are reported for the low-energy spectra of N=50 and N=126 isotones. The effects of including seniority nonconserving interactions are investigated

Symposium 19: The contributions of the Department of Biochemistry/USP towards Biochemistry teaching

OpenAIRE

Baptista Torres, Bayardo; Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo (USP)

2014-01-01

K-Education(Portuguese) Chair: V. Trindade Bayardo Torres; Clovis Wannmacher; Denise MacedoThe contributions of the Department of Biochemistry/USP towards Biochemistry teaching.O ensino de Bioquímica nos últimos 20 anosBayardo B. TorresDepartamento de Bioquímica, Instituto de Química, USP. São Paulo, Brazil.Among the contributions of the Department of Biochemistry/USP one must recall:1. Winter school for graduate studentsThis course, now at the ninth edition, is intended for students in the f...

USP10 Antagonizes c-Myc Transcriptional Activation through SIRT6 Stabilization to Suppress Tumor Formation

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Zhenghong Lin

2013-12-01

Full Text Available The reduced protein expression of SIRT6 tumor suppressor is involved in tumorigenesis. The molecular mechanisms underlying SIRT6 protein downregulation in human cancers remain unknown. Using a proteomic approach, we have identified the ubiquitin-specific peptidase USP10, another tumor suppressor, as one of the SIRT6-interacting proteins. USP10 suppresses SIRT6 ubiquitination to protect SIRT6 from proteasomal degradation. USP10 antagonizes the transcriptional activity of the c-Myc oncogene through SIRT6, as well as p53, to inhibit cell-cycle progression, cancer cell growth, and tumor formation. To support this conclusion, we detected significant reductions in both USP10 and SIRT6 protein expression in human colon cancers. Our study discovered crosstalk between two tumor-suppressive genes in regulating cell-cycle progression and proliferation and showed that dysregulated USP10 function promotes tumorigenesis through SIRT6 degradation.

In vitro adsorption of sodium pentobarbital by SuperChar, USP and Darco G-60 activated charcoals

International Nuclear Information System (INIS)

Curd-Sneed, C.D.; Parks, K.S.; Bordelon, J.G.; Stewart, J.J.

1987-01-01

This study was designed to examine the in vitro adsorption of sodium pentobarbital by three activated charcoals. Solutions of sodium pentobarbital (20 mM) were prepared in distilled water and in 70% sorbitol (w/v). Radiolabeled ( 14 C) sodium pentobarbital was added to each solution to serve as a concentration marker. Two ml of each drug solution was added to test tubes containing 40 mg of either Darco G-60, USP, or SuperChar activated charcoal. The drug-charcoal mixtures were incubated at 37 degrees C for O, 2.5, 5, 7.5 or 10 min. Equilibrium, indicated by a constant percentage of drug bound for two consecutive time periods, was established immediately for the aqueous mixtures and for Darco G-60 in sorbitol. The time to equilibrium was prolonged for USP (2.5 min) and SuperChar (5 min) in the presence of sorbitol. In the second series of experiments, solutions of sodium pentobarbital (1.25 to 160 mM) were prepared in either distilled water or sorbitol. Amount of drug bound by 10 to 320 mg of activated charcoal within a 10 min incubation period was determined. Scatchard analysis determined maximum binding capacity (Bmax) and dissociation constants (Kd) for each activated charcoal. In water, Bmax (mumoles/gm) was greatest for SuperChar (1141), followed by USP (580) and Darco G-60 (381), while the Kd's did not differ. Sorbitol did not change the Bmax or Kd of USP or Darco G-60, but the additive significantly decreased the Bmax (717) and increased the Kd for SuperChar (3.3 to 10.1 mM). The results suggest that relative binding capacity of activated charcoal is directly proportional to surface area, and that sorbitol significantly reduces sodium pentobarbital binding to SuperChar

Legibility of USP pictograms by clients of community pharmacies in Portugal.

Science.gov (United States)

Soares, Maria Augusta

2013-02-01

Effective and safe use of medicines depends on patients' good understanding of the pharmacotherapy. Pictograms are a communication tool, as complement of verbal/written information, to facilitate drug use and, avoiding errors, being useful for drug effectiveness and safety. To assess the ability of pharmacy's clients, in understanding USP pictograms, establishing the relationship with their socio demographic profiles. The study was developed in community pharmacies, in Lisbon region. Cross sectional study, carried out in pharmacies in Lisbon. A structured questionnaire was used in pharmacies clients of 18 years old and above. Legibility of fifteen USP pictograms was studied using ISO and ANSI criteria. Correlation between the legibility of pictograms and pharmacies clients' demographic profile (age, gender, scholarship, frequency of medicines use) was evaluated. SPSS data base version 18 was used for descriptive analysis. Legibility of fifteen United States Pharmacopeia (USP) pictograms and, its relationship with clients' demographic data (scholarship degree, age, frequency of medicines use and, gender) was studied. From 751 responders, ten pictograms were legible by ISO and seven by ANSI. More than 30 % of the responders weren't able to understand five of the pictograms. It was found statistically significant relationships between some the understanding of some pictograms and clients' scholarships degree, age and frequency of medicines use. It was found that not all the fifteen tested USP pictograms were correctly comprehended by Portuguese pharmacies' clients, having found correlations with scholarship degree, daily use of medicines and age group, for some pictograms. To ensure the effectiveness of USP pictograms it is advisable to test patients' comprehension, before their use in general practice.

USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level.

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Daniel Pouly

Full Text Available The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2, whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output effector. We report here that Usp2 is a clock output effector related to bodily Ca2+ homeostasis, a feature that is conserved across evolution. Drosophila with a whole-body knockdown of the orthologue of Usp2, CG14619 (dUsp2-kd, predominantly die during pupation but are rescued by dietary Ca2+ supplementation. Usp2-KO mice show hyperabsorption of dietary Ca2+ in small intestine, likely due to strong overexpression of the membrane scaffold protein NHERF4, a regulator of the Ca2+ channel TRPV6 mediating dietary Ca2+ uptake. In this tissue, USP2-45 is found in membrane fractions and negatively regulates NHERF4 protein abundance in a rhythmic manner at the protein level. In clock mutant animals (Cry1/Cry2-dKO, rhythmic USP2-45 expression is lost, as well as the one of NHERF4, confirming the inverse relationship between USP2-45 and NHERF4 protein levels. Finally, USP2-45 interacts in vitro with NHERF4 and endogenous Clathrin Heavy Chain. Taken together these data prompt us to define USP2-45 as the first clock output effector acting at the post-translational level at cell membranes and possibly regulating membrane permeability of Ca2+.

Synthesis of [21-14C]-fusarin C by enzymic demethylation and remethylation with [14C]-diazomethane

International Nuclear Information System (INIS)

Lu, S.-J.; Li, M.H.

1989-01-01

Fusarin C, a potent mutagen isolated from Fusarium moniliforme culture extracts, has been prepared radiolabeled in two steps by enzymic hydrolysis of the 21-methyl ester group, using phenobarbital induced microsomal preparations, followed by remethylation using [ 14 F]-diazomethane. Yields, based upon fusarin C, were essentially quantitative and approximately 10% of the [ 14 C]-methyl-nitrosourea, converted to diazomethane, reacted to yield [ 14 C]-fusarin C. (author)

Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response.

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Adi Ben Yehuda

Full Text Available Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington's disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality.

USP1 regulates AKT phosphorylation by modulating the stability of PHLPP1 in lung cancer cells.

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Zhiqiang, Zhang; Qinghui, Yang; Yongqiang, Zhang; Jian, Zhang; Xin, Zhao; Haiying, Ma; Yuepeng, Guo

2012-07-01

Hyperactivation of phosphatidylinositol 3-kinase/Akt signaling is commonly associated with human tumors including lung cancers. PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), which terminates Akt signaling by directly dephosphorylating and inactivating Akt, has been identified as a tumor suppressor. The protein level of PHLPP1 is regulated by E3 ligase beta-TRCP, however, the deubiquitinase for PHLPP1 is still not known. The mRNA levels of USP1 and PHLPP1 in lung cancer cells and tissues were determined by real-time PCR. The half-life of PHLPP1 was detected by CHX assay. The interaction between USP1 and PHLPP1 was examined by immunoprecipitation and GST pull-down assay. Both USP1 and PHLPP1 are low expressed in lung cancer cells and tissues and silencing of USP1 by RNA interference significantly decreased the half-life of PHLPP1, which in turn amplified Akt1 phosphorylation. Our data identified a novel USP1-PHLPP1-Akt signaling axis, and decreased USP1 level in lung cancer cells may play an important role in lung cancer progress.

High USP6NL levels in breast cancer sustain chronic AKT phosphorylation and GLUT1 stability fueling aerobic glycolysis.

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Avanzato, Daniele; Pupo, Emanuela; Ducano, Nadia; Isella, Claudio; Bertalot, Giovanni; Luise, Chiara; Pece, Salvatore; Bruna, Alejandra; Rueda, Oscar M; Caldas, Carlos; Di Fiore, Pier Paolo; Sapino, Anna; Lanzetti, Letizia

2018-04-24

USP6NL, also named RN-tre, is a GTPase activating protein (GAP) involved in control of endocytosis and signal transduction. Here we report that USP6NL is overexpressed in breast cancer (BC), mainly of the basal-like/integrative cluster 10 subtype. Increased USP6NL levels were accompanied by gene amplification and were associated with worse prognosis in the METABRIC dataset, retaining prognostic value in multivariable analysis. High levels of USP6NL in BC cells delayed endocytosis and degradation of the epidermal growth factor receptor (EGFR), causing chronic AKT activation. In turn, AKT stabilized the glucose transporter GLUT1 at the plasma membrane, increasing aerobic glycolysis. In agreement, elevated USP6NL sensitized BC cells to glucose deprivation, indicating that their glycolytic capacity relies on this protein. Depletion of USP6NL accelerated EGFR/AKT downregulation and GLUT1 degradation, impairing cell proliferation exclusively in BC cells that harbored increased levels of USP6NL. Overall, these findings argue that USP6NL overexpression generates a metabolic rewiring that is essential to foster the glycolytic demand of BC cells and promote their proliferation. Copyright ©2018, American Association for Cancer Research.

The C. elegans Ortholog of USP7 controls DAF-16 stability in Insulin/IGF-1-like signaling.

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Heimbucher, Thomas; Hunter, Tony

2015-01-01

FOXO family transcription factors are downstream effectors of Insulin/IGF-1 signaling (IIS) and are regulated by posttranslational modification and coregulators, including components of the ubiquitin-proteasome system (UPS). Cofactors promoting DAF-16/FOXO protein stability and function in IIS have not been described yet. In a recent study, we have identified the deubiquitylating enzyme MATH-33, the ortholog of mammalian USP7/HAUSP, as an essential DAF-16 coregulator. We found that MATH-33 actively stabilizes DAF-16 protein levels when IIS is downregulated. Here we discuss how DAF-16/FOXO transcription factors are regulated by the UPS, in particular by the interplay of E3-ubiquitin ligases and deubiquitylating enzymes, which is critical for balancing DAF-16/FOXO activity and degradation. Recent findings raise the intriguing possibility that regulated oscillations in DAF-16/FOXO steady state levels play an integral role in mechanisms controlling healthspan and lifespan extension.

The Promoter of AtUSP Is Co-regulated by Phytohormones and Abiotic Stresses in Arabidopsis thaliana.

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Bhuria, Monika; Goel, Parul; Kumar, Sanjay; Singh, Anil K

2016-01-01

Universal stress proteins (USPs) are known to be expressed in response to various abiotic stresses in a wide variety of organisms, such as bacteria, archaebacteria, protists, algae, fungi, plants, and animals. However, in plants, biological function of most of the USPs still remains obscure. In the present study, Arabidopsis USP gene ( AtUSP ) showed induction in response to abscisic acid (ABA) and various abiotic stresses viz . heat, dehydration, salt, osmotic, and cold stresses. Additionally, in silico analysis of AtUSP promoter identified several cis -elements responsive to phytohormones and abiotic stresses such as ABRE, ERE, DRE, and HSE, etc. To functionally validate the AtUSP promoter, the 1115 bp region of promoter was characterized under phytohormone and abiotic stress treatments. Deletion analysis of promoter was carried out by cloning the full length promoter (D0) and its three 5' deletion derivatives, D1 (964 bp), D2 (660 bp), and D3 (503 bp) upstream of the β-glucuronidase (GUS) reporter gene, which were then stably transformed in Arabidopsis plants. The AtUSP promoter (D0) showed minimal activity under non-stress conditions which was enhanced in response to phytohormone treatments (ABA and ACC) and abiotic stresses such as dehydration, heat, cold, salt, and osmotic stresses. The seedlings harboring D1 and D2 deletion fragments showed constitutive GUS expression even under control condition with increased activity almost under all the treatments. However, D3 seedlings exhibited complete loss of activity under control condition with induction under ACC treatment, dehydration, heat, oxidative, salt, and osmotic stresses. Thus, present study clearly showed that AtUSP promoter is highly inducible by phytohormones and multiple abiotic stresses and it can be exploited as stress inducible promoter to generate multi-stress tolerant crops with minimal effects on their other important traits.

Ubiquitin-specific protease 14 regulates cell proliferation and apoptosis in oral squamous cell carcinoma.

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Chen, Xiangyun; Wu, Jingjing; Chen, Yitian; Ye, Dongxia; Lei, Hu; Xu, Hanzhang; Yang, Li; Wu, Yingli; Gu, Wenli

2016-10-01

Ubiquitin-specific protease 14, a deubiquitinating enzyme, has been implicated in the tumorigenesis and progression of several cancers, but its role in oral squamous cell carcinoma remains to be elucidated. The aim of this study was to explore the expression pattern and roles of Ubiquitin-specific protease 14 in the occurrence and development of oral squamous cell carcinoma. Interestingly, Ubiquitin-specific protease 14 was overexpressed in oral cancer tissues and cell lines at both mRNA and protein levels. b-AP15, a specific inhibitor of Ubiquitin-specific protease 14, significantly inhibited the growth of cancer cells and increased cell apoptosis in a dose-dependent manner. Moreover, knockdown of Ubiquitin-specific protease 14 by shRNA significantly inhibited the proliferation and migration of cancer cells in vitro. Finally, using a xenograft mouse model of oral squamous cell carcinoma, knockdown of Ubiquitin-specific protease 14 markedly inhibited tumor growth and triggered the cancer cell apoptosis in vivo, supporting previous results. In conclusion, for the first time we have demonstrated the expression pattern of Ubiquitin-specific protease 14 in oral squamous cell carcinoma and verified a relationship with tumor growth and metastasis. These results may highlight new therapeutic strategies for tumor treatment, application of Ubiquitin-specific protease 14 selective inhibitor, such as b-AP15, or knockdown by shRNA. Collectively, Ubiquitin-specific protease 14 could be a potential therapeutic target for oral squamous cell carcinoma patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mechanisms of c-myc degradation by nickel compounds and hypoxia.

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Qin Li

2009-12-01

Full Text Available Nickel (Ni compounds have been found to cause cancer in humans and animal models and to transform cells in culture. At least part of this effect is mediated by stabilization of hypoxia inducible factor (HIF1a and activating its downstream signaling. Recent studies reported that hypoxia signaling might either antagonize or enhance c-myc activity depending on cell context. We investigated the effect of nickel on c-myc levels, and demonstrated that nickel, hypoxia, and other hypoxia mimetics degraded c-myc protein in a number of cancer cells (A549, MCF-7, MDA-453, and BT-474. The degradation of the c-Myc protein was mediated by the 26S proteosome. Interestingly, knockdown of both HIF-1alpha and HIF-2alpha attenuated c-Myc degradation induced by Nickel and hypoxia, suggesting the functional HIF-1alpha and HIF-2alpha was required for c-myc degradation. Further studies revealed two potential pathways mediated nickel and hypoxia induced c-myc degradation. Phosphorylation of c-myc at T58 was significantly increased in cells exposed to nickel or hypoxia, leading to increased ubiquitination through Fbw7 ubiquitin ligase. In addition, nickel and hypoxia exposure decreased USP28, a c-myc de-ubiquitinating enzyme, contributing to a higher steady state level of c-myc ubiquitination and promoting c-myc degradation. Furthermore, the reduction of USP28 protein by hypoxia signaling is due to both protein degradation and transcriptional repression. Nickel and hypoxia exposure significantly increased the levels of dimethylated H3 lysine 9 at the USP28 promoter and repressed its expression. Our study demonstrated that Nickel and hypoxia exposure increased c-myc T58 phosphorylation and decreased USP28 protein levels in cancer cells, which both lead to enhanced c-myc ubiquitination and proteasomal degradation.

Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue

OpenAIRE

Nanduri, Bindu; Shack, Leslie A.; Rai, Aswathy N.; Epperson, William B.; Baumgartner, Wes; Schmidt, Ty B.; Edelmann, Mariola J.

2016-01-01

To develop a reproducible tissue-lysis method that retains enzyme function for activity-based protein profiling, we compared four different tissue lysis methods of bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue and focused ultrasonication had also the fastest pr...

Zebrafish usp39 mutation leads to rb1 mRNA splicing defect and pituitary lineage expansion.

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Yesenia Ríos

2011-01-01

Full Text Available Loss of retinoblastoma (Rb tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39 mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130, and cdkn1a (p21 expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

[Translation and linguistic validation in classical Arabic of the urinary symptom profile (USP) questionnaire].

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Arabi, H; Bendeddouche, I; Khalfaoui, S; Louardi, N; Ameur, A; Lebreton, F; Amarenco, G

2013-04-01

The objective was to translate and linguistically validate in classical Arabic; the French version of the Urinary Symptom Profile (USP), the scale adapted to vesico-sphincter disorders. Prospective study of 30 patients suffering the vesico-sphincter disorders. The translation was obtained by the method: translation back-translation. Patients completed the final questionnaire on day 0 and day 15. The feasibility, acceptability, internal consistency using Cronbach's alpha and test-retest repeatability by the interclass correlation coefficient (ICC) with the confidence interval (CI) were studied. The sample consisted of 30 subjects including 20 men (66.6%) and 10 women (33.3%). The mean age was 48±18, 14 years ranging from 25 to 70 years. The questionnaire was feasible and acceptable. The Cronbach's alpha of the three dimensions, urinary stress incontinence, overactive bladder and voiding difficulties was respectively 0.9880, 0.9774 and 0.9683, respectively; the ICC was 0.9762 (95% CI: 0.9307-0.9919), 0.9558 (CI 95%: 0.8738-0.9849) and 0.9385 (95% CI: 0.8274-0.9789). The Arabic version of the classic USP had excellent internal consistency and excellent repeatability enable a full assessment of all urinary disorders and their severity. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Globule-size distribution in injectable 20% lipid emulsions: Compliance with USP requirements.

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Driscoll, David F

2007-10-01

The compliance of injectable 20% lipid emulsions with the globule-size limits in chapter 729 of the U.S. Pharmacopeia (USP) was examined. As established in chapter 729, dynamic light scattering was applied to determine mean droplet diameter (MDD), with an upper limit of 500 nm. Light obscuration was used to determine the size of fat globules found in the large-diameter tail, expressed as the volume-weighted percent fat exceeding 5 microm (PFAT(5)), with an upper limit of 0.05%. Compliance of seven different emulsions, six of which were stored in plastic bags, with USP limits was assessed. To avoid reaching coincidence limits during the application of method II from overly concentrated emulsion samples, a variable dilution scheme was used to optimize the globule-size measurements for each emulsion. One-way analysis of variance of globule-size distribution (GSD) data was conducted if any results of method I or II exceeded the respective upper limits. Most injectable lipid emulsions complied with limits established by USP chapter 729, with the exception of those of one manufacturer, which failed limits as proposed for to meet the PFAT(5) three of the emulsions tested. In contrast, all others studied (one packaged in glass and three packaged in plastic) met both criteria. Among seven injectable lipid emulsions tested for GSD, all met USP chapter 729 MDD requirements and three, all from the same manufacturer and packaged in plastic, did not meet PFAT(5) requirements.

USP22 knockdown enhanced chemosensitivity of hepatocellular carcinoma cells to 5-Fu by up-regulation of Smad4 and suppression of Akt.

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Zhang, Jing; Luo, Nan; Tian, Yu; Li, Jiazhi; Yang, Xiaozhou; Yin, Huimin; Xiao, Congshu; Sheng, Jie; Li, Yang; Tang, Bo; Li, Rongkuan

2017-04-11

USP22, a member of the deubiquitinases (DUBs) family, is known to be a key subunit of the human Spt-Ada-Gcn5 acetyltransferase (hSAGA) transcriptional cofactor complex. Within hSAGA, USP22 removes ubiquitin from histone proteins, thus regulating the transcription and expression of downstream genes. USP22 plays important roles in many cancers; however, its effect and the mechanism underlying HCC chemoresistance remain unclear. In the present study, we found that USP22 was highly expressed in chemoresistant HCC tissues and cells and was correlated with the prognosis of HCC patients who received chemotherapy. Silencing USP22 in chemoresistant HCC Bel/Fu cells dramatically inhibited proliferation, migration, invasion and epithelial-mesenchymal transition in vitro; suppressed tumorigenic and metastatic capacities in vivo; and inhibited drug resistance-related proteins (MDR1, LRP, MRP1). Mechanistically, we found that USP22 knockdown exerts its function through down-regulating PI3K and activating Smad4, which inhibited phosphorylation of Akt. Silencing Smad4 blocked USP22 knockdown-induced Akt inhibition in Bel/Fu cells. Our results, for the first time, provide evidence that USP22 plays a critical role in the development of chemoresistant HCC cells and that high USP22 expression serves as a molecular marker for the prognosis of HCC patients who undergo chemotherapy.

The downregulation of Mcl-1 via USP9X inhibition sensitizes solid tumors to Bcl-xl inhibition

International Nuclear Information System (INIS)

Peddaboina, Chander; Smythe, W Roy; Cao, Xiaobo; Jupiter, Daniel; Fletcher, Steven; Yap, Jeremy L; Rai, Arun; Tobin, Richard P; Jiang, Weihua; Rascoe, Philip; Rogers, M Karen Newell

2012-01-01

It has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. Mcl-1 is a critical survival protein in a variety of cell lineages and is critically regulated via ubiquitination. The Mcl-1, Bcl-xL and USP9X expression patterns in human lung and colon adenocarcinomas were evaluated via immunohistochemistry. Interaction between USP9X and Mcl-1 was demonstrated by immunoprecipitation-western blotting. The protein expression profiles of Mcl-1, Bcl-xL and USP9X in multiple cancer cell lines were determined by western blotting. Annexin-V staining and cleaved PARP western blotting were used to assay for apoptosis. The cellular toxicities after various treatments were measured via the XTT assay. In our current analysis of colon and lung cancer samples, we demonstrate that Mcl-1 and Bcl-xL are overexpressed and also co-exist in many tumors and that the expression levels of both genes correlate with the clinical staging. The downregulation of Mcl-1 or Bcl-xL via RNAi was found to increase the sensitivity of the tumor cells to chemotherapy. Furthermore, our analyses revealed that USP9X expression correlates with that of Mcl-1 in human cancer tissue samples. We additionally found that the USP9X inhibitor WP1130 promotes Mcl-1 degradation and increases tumor cell sensitivity to chemotherapies. Moreover, the combination of WP1130 and ABT-737, a well-documented Bcl-xL inhibitor, demonstrated a chemotherapeutic synergy and promoted apoptosis in different tumor cells. Mcl-1, Bcl-xL and USP9X overexpression are tumor survival mechanisms protective against chemotherapy. USP9X inhibition increases tumor cell sensitivity to various chemotherapeutic agents including Bcl-2/Bcl-xL inhibitors

T Cell Intrinsic USP15 Deficiency Promotes Excessive IFN-γ Production and an Immunosuppressive Tumor Microenvironment in MCA-Induced Fibrosarcoma

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Qiang Zou

2015-12-01

Full Text Available USP15 is a deubiquitinase that negatively regulates activation of naive CD4+ T cells and generation of IFN-γ-producing T helper 1 (Th1 cells. USP15 deficiency in mice promotes antitumor T cell responses in a transplantable cancer model; however, it has remained unclear how deregulated T cell activation impacts primary tumor development during the prolonged interplay between tumors and the immune system. Here, we find that the USP15-deficient mice are hypersensitive to methylcholantrene (MCA-induced fibrosarcomas. Excessive IFN-γ production in USP15-deficient mice promotes expression of the immunosuppressive molecule PD-L1 and the chemokine CXCL12, causing accumulation of T-bet+ regulatory T cells and CD11b+Gr-1+ myeloid-derived suppressor cells at tumor site. Mixed bone marrow adoptive transfer studies further reveals a T cell-intrinsic role for USP15 in regulating IFN-γ production and tumor development. These findings suggest that T cell intrinsic USP15 deficiency causes excessive production of IFN-γ, which promotes an immunosuppressive tumor microenvironment during MCA-induced primary tumorigenesis.

Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue.

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Nanduri, Bindu; Shack, Leslie A; Rai, Aswathy N; Epperson, William B; Baumgartner, Wes; Schmidt, Ty B; Edelmann, Mariola J

2016-12-15

To develop a reproducible tissue lysis method that retains enzyme function for activity-based protein profiling, we compared four different methods to obtain protein extracts from bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue, and focused ultrasonication also had the fastest processing time. We used focused-ultrasonicator for subsequent activity-based proteomic analysis of deubiquitinases to test the compatibility of this method in sample preparation for activity-based chemical proteomics. Copyright © 2016 Elsevier Inc. All rights reserved.

Anaerobic survival of Pseudomonas aeruginosa by pyruvate fermentation requires an Usp-type stress protein

DEFF Research Database (Denmark)

Schreiber, K; Boes, N; Escbach, M

2006-01-01

the induced synthesis of three enzymes involved in arginine fermentation, ArcA, ArcB, and ArcC, and the outer membrane protein OprL. Moreover, formation of two proteins of unknown function, PA3309 and PA4352, increased by factors of 72- and 22-fold, respectively. Both belong to the group of universal stress...... proteins (Usp). Long-term survival of a PA3309 knockout mutant by pyruvate fermentation was found drastically reduced. The oxygen-sensing regulator Anr controls expression of the PPA3309-lacZ reporter gene fusion after a shift to anaerobic conditions and further pyruvate fermentation. PA3309 expression...... was also found induced during the anaerobic and aerobic stationary phases. This aerobic stationary-phase induction is independent of the regulatory proteins Anr, RpoS, RelA, GacA, RhlR, and LasR, indicating a currently unknown mechanism of stationary-phase-dependent gene activation. PA3309 promoter...

In vitro release of ketoprofen suppositories using the USP basket and the flow-through cell dissolution methods.

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Medina, José Raúl; Padilla, Adrián Roberto; Hurtado, Marcela; Cortés, Alma Rosa; Domínguez-Ramírez, Adriana Miriam

2014-05-01

In order to study the release characteristics of ketoprofen suppositories under the hydrodynamic environment generated by USP Apparatus 1 and 4, the dissolution profiles of the Mexican reference product (100 mg) were determined. Phosphate buffer pH 8 and 1% sodium lauryl sulfate (SLS) aqueous solutions were proved as dissolution mediums. Baskets were rotated at 100 rpm with USP Apparatus 1 and different flow rates from 16-32 mL/min with USP Apparatus 4 were used. Drug samples were taken and quantified during 60 min by UV analysis at 260 nm. Mean dissolution time (MDT) and dissolution efficiency (DE) were calculated by model-independent methods. Data were also fitted to several kinetic models. Poor dissolution was found in both dissolution mediums when USP basket method was used ( 0.99). The results suggest the need to establish an adequate dissolution method to evaluate the release kinetics of ketoprofen from suppositories.

Comparative analyses of two thermophilic enzymes exhibiting both beta-1,4 mannosidic and beta-1,4 glucosidic cleavage activities from Caldanaerobius polysaccharolyticus.

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Han, Yejun; Dodd, Dylan; Hespen, Charles W; Ohene-Adjei, Samuel; Schroeder, Charles M; Mackie, Roderick I; Cann, Isaac K O

2010-08-01

The hydrolysis of polysaccharides containing mannan requires endo-1,4-beta-mannanase and 1,4-beta-mannosidase activities. In the current report, the biochemical properties of two endo-beta-1,4-mannanases (Man5A and Man5B) from Caldanaerobius polysaccharolyticus were studied. Man5A is composed of an N-terminal signal peptide (SP), a catalytic domain, two carbohydrate-binding modules (CBMs), and three surface layer homology (SLH) repeats, whereas Man5B lacks the SP, CBMs, and SLH repeats. To gain insights into how the two glycoside hydrolase family 5 (GH5) enzymes may aid the bacterium in energy acquisition and also the potential application of the two enzymes in the biofuel industry, two derivatives of Man5A (Man5A-TM1 [TM1 stands for truncational mutant 1], which lacks the SP and SLH repeats, and Man5A-TM2, which lacks the SP, CBMs, and SLH repeats) and the wild-type Man5B were biochemically analyzed. The Man5A derivatives displayed endo-1,4-beta-mannanase and endo-1,4-beta-glucanase activities and hydrolyzed oligosaccharides with a degree of polymerization (DP) of 4 or higher. Man5B exhibited endo-1,4-beta-mannanase activity and little endo-1,4-beta-glucanase activity; however, this enzyme also exhibited 1,4-beta-mannosidase and cellodextrinase activities. Man5A-TM1, compared to either Man5A-TM2 or Man5B, had higher catalytic activity with soluble and insoluble polysaccharides, indicating that the CBMs enhance catalysis of Man5A. Furthermore, Man5A-TM1 acted synergistically with Man5B in the hydrolysis of beta-mannan and carboxymethyl cellulose. The versatility of the two enzymes, therefore, makes them a resource for depolymerization of mannan-containing polysaccharides in the biofuel industry. Furthermore, on the basis of the biochemical and genomic data, a molecular mechanism for utilization of mannan-containing nutrients by C. polysaccharolyticus is proposed.

USP22 knockdown enhanced chemosensitivity of hepatocellular carcinoma cells to 5-Fu by up-regulation of Smad4 and suppression of Akt

OpenAIRE

Zhang, Jing; Luo, Nan; Tian, Yu; Li, Jiazhi; Yang, Xiaozhou; Yin, Huimin; Xiao, Congshu; Sheng, Jie; Li, Yang; Tang, Bo; Li, Rongkuan

2017-01-01

USP22, a member of the deubiquitinases (DUBs) family, is known to be a key subunit of the human Spt-Ada-Gcn5 acetyltransferase (hSAGA) transcriptional cofactor complex. Within hSAGA, USP22 removes ubiquitin from histone proteins, thus regulating the transcription and expression of downstream genes. USP22 plays important roles in many cancers; however, its effect and the mechanism underlying HCC chemoresistance remain unclear. In the present study, we found that USP22 was highly expressed in c...

Structure of the USP15 N-terminal domains: a β-hairpin mediates close association between the DUSP and UBL domains.

Science.gov (United States)

Harper, Stephen; Besong, Tabot M D; Emsley, Jonas; Scott, David J; Dreveny, Ingrid

2011-09-20

Ubiquitin specific protease 15 (USP15) functions in COP9 signalosome mediated regulation of protein degradation and cellular signaling through catalyzing the ubiquitin deconjugation reaction of a discrete number of substrates. It influences the stability of adenomatous polyposis coli, IκBα, caspase-3, and the human papillomavirus type 16 E6. USP15 forms a subfamily with USP4 and USP11 related through a shared presence of N-terminal "domain present in ubiquitin specific proteases" (DUSP) and "ubiquitin-like" (UBL) domains (DU subfamily). Here we report the 1.5 Å resolution crystal structure of the human USP15 N-terminal domains revealing a 80 Å elongated arrangement with the DU domains aligned in tandem. This architecture is generated through formation of a defined interface that is dominated by an intervening β-hairpin structure (DU finger) that engages in an intricate hydrogen-bonding network between the domains. The UBL domain is closely related to ubiquitin among β-grasp folds but is characterized by the presence of longer loop regions and different surface characteristics, indicating that this domain is unlikely to act as ubiquitin mimic. Comparison with the related murine USP4 DUSP-UBL crystal structure reveals that the main DU interdomain contacts are conserved. Analytical ultracentrifugation, small-angle X-ray scattering, and gel filtration experiments revealed that USP15 DU is monomeric in solution. Our data provide a framework to advance study of the structure and function of the DU subfamily. © 2011 American Chemical Society

Publicações sobre ensino em enfermagem na Revista da Escola de Enfermagem da USP Publicaciones sobre enseñanza en enfermería en la Revista da Escola de Enfermagem da USP Publications about nursing education in the Revista da Escola de Enfermagem da USP

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Elaine Emi Ito

2005-12-01

Full Text Available Produções científicas na área de ensino em enfermagem fornecem importantes contribuições para a formação de profissionais e exercício da enfermagem. Acreditando nesta perspectiva, foi realizado um estudo visando a identificar e analisar os artigos publicados na área de ensino em enfermagem, construindo categorias empíricas com base nos dados coletados. Trata-se de uma pesquisa bibliográfica, descritiva e quantitativa, tendo como fonte artigos publicados na Revista da Escola de Enfermagem da USP, durante o período de 1967 a 2002. Foram encontrados 1.137 artigos, sendo que 14,2% destes referem-se ao ensino de enfermagem, dos quais 89,0% dizem respeito à área de graduação. A análise descritiva dos artigos publicados, referentes ao ensino de enfermagem, desperta a preocupação sobre o desenvolvimento de pesquisas relacionadas ao tema e que acompanhem as mudanças das legislações e as exigências do mercado de trabalho.Producciones científicas en el área de la enseñanza en enfermería ofrecen importantes contribuciones para la formación de profesionales y el ejercicio de la enfermería. Creyendo en esta perspectiva, fue realizado un estudio visando identificar y analizar los artículos publicados en el área de la enseñanza en enfermería, construyendo categorías empíricas a partir de los datos recolectados. Se trata de una investigación bibliográfica, descriptiva y cuantitativa, teniendo como fuente artículos publicados en la Revista da Escola de Enfermagem da USP, durante el período de 1967 a 2002. Fueron encontrados 1,137 artículos de los cuales el 14,2% se refieren a la enseñanza de enfermería, y de éstos el 89,0% se refieren al área del pre grado. El análisis descriptivo de los artículos publicados, referentes a la enseñanza de enfermería, despierta preocupación sobre el desarrollo de investigaciones relacionadas al tema y que acompañen los cambios de las legislaciones y las exigencias del mercado de

Inhibitory properties of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives acting on glycogen metabolising enzymes.

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Díaz-Lobo, Mireia; Concia, Alda Lisa; Gómez, Livia; Clapés, Pere; Fita, Ignacio; Guinovart, Joan J; Ferrer, Joan C

2016-09-26

Glycogen synthase (GS) and glycogen phosphorylase (GP) are the key enzymes that control, respectively, the synthesis and degradation of glycogen, a multi-branched glucose polymer that serves as a form of energy storage in bacteria, fungi and animals. An abnormal glycogen metabolism is associated with several human diseases. Thus, GS and GP constitute adequate pharmacological targets to modulate cellular glycogen levels by means of their selective inhibition. The compound 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) is a known potent inhibitor of GP. We studied the inhibitory effect of DAB, its enantiomer LAB, and 29 DAB derivatives on the activity of rat muscle glycogen phosphorylase (RMGP) and E. coli glycogen synthase (EcGS). The isoform 4 of sucrose synthase (SuSy4) from Solanum tuberosum L. was also included in the study for comparative purposes. Although these three enzymes possess highly conserved catalytic site architectures, the DAB derivatives analysed showed extremely diverse inhibitory potential. Subtle changes in the positions of crucial residues in their active sites are sufficient to discriminate among the structural differences of the tested inhibitors. For the two Leloir-type enzymes, EcGS and SuSy4, which use sugar nucleotides as donors, the inhibitory potency of the compounds analysed was synergistically enhanced by more than three orders of magnitude in the presence of ADP and UDP, respectively. Our results are consistent with a model in which these compounds bind to the subsite in the active centre of the enzymes that is normally occupied by the glucosyl residue which is transferred between donor and acceptor substrates. The ability to selectively inhibit the catalytic activity of the key enzymes of the glycogen metabolism may represent a new approach for the treatment of disorders of the glycogen metabolism.

Uspešno vključevanje v socialno okolje

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Darka Podmenik

2002-12-01

Full Text Available Avtorica je v prispevku predstavila "aktivne" državljane in državljanke pri uspešnem vključevanju v družbeno okolje z delovanjem in učenjem. V prispevku navedeni podatki in primeri so bili obdelani v okviru mednarodnega raziskovalnega projekta Education and Training far Governance and Active Citizenship in Europe (ETGACE.

Development of USPS Laboratory and pilot-scale testing protocols

Science.gov (United States)

Carl Houtman; Nancy Ross Sutherland; David Bormett; Donald Donermeyer

2000-01-01

The ultimate goal of the US Postal Service (USPS) Environmentally Benign Stamp Program is to develop stamp adhesives that can be removed by unit operations found in recycling mills. The maintenance of final product quality specifications for a recycling mill while loading the feedstock with a significant quantity of adhesive is the criterion for success of this program...

Identification of a novel higher molecular weight isoform of USP7/HAUSP that interacts with the Herpes simplex virus type-1 immediate early protein ICP0.

Science.gov (United States)

Antrobus, Robin; Boutell, Chris

2008-10-01

The Herpes simplex virus type-1 (HSV-1) regulatory protein ICP0, a RING-finger E3 ubiquitin ligase, stimulates the onset of viral lytic replication and the reactivation of quiescent viral genomes from latency. Like many ubiquitin ligases ICP0 induces its own ubiquitination, a process that can lead to its proteasome-dependent degradation. ICP0 counteracts this activity by recruiting the cellular ubiquitin-specific protease USP7/HAUSP. Here we show that ICP0 can also interact with a previously unidentified isoform of USP7 (termed here USP7(beta)). This isoform is not a predominantly ubiquitinated, SUMO-modified, or phosphorylated species of USP7 but is constitutively expressed in a number of different cell types. Like USP7, USP7(beta) binds specifically to an electrophilic ubiquitin probe, indicating that it contains an accessible catalytic core with potential ubiquitin-protease activity. The interaction formed between ICP0 and USP7(beta) requires ICP0 to have an intact USP7-binding domain and results in its susceptibility to ICP0-mediated degradation during HSV-1 infection.

SIA - Sistema de Inventário Automatizado para as bibliotecas do SIBi/USP

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Rosa Maria Fischi Zani

Full Text Available Descreve a criação e implantação do Sistema de Inventário Automatizado - SAI - para o Sistema Integrado de Bibliotecas da Universidade de São Paulo - SIBi/USP -, ressaltando as vantagens do emprego da tecnologia para tornar mais ágil, eficaz e confiável o processo. O modelo mostrou-se flexível às particularidades dos acervos e das bibliotecas. Enumera os principais requisitos para automação do procedimento, bem como as tecnologias envolvidas e disponíveis no mercado. Após aplicação de teste piloto, o modelo foi aprovado pelas bibliotecas da USP.

Active site-targeted covalent irreversible inhibitors of USP7 impair the functions of Foxp3+ T-regulatory cells by promoting ubiquitination of Tip60.

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Feng Wang

Full Text Available Accumulation of Foxp3+ T-regulatory (Treg cells in the tumor microenvironment is associated with tumor immune evasion and poor patient outcome in the case of many solid tumors. Current therapeutic strategies for blocking Treg functions are not Treg-specific, and display only modest and transient efficacy. Recent studies revealed that ubiquitin-specific protease 7 (USP7 is essential for Treg functions by stabilizing expression of Tip60 and Foxp3, which together are central to the development and maintenance of the Treg cell lineage. Pharmacological inhibition of USP7 is therefore a promising strategy for suppressing Treg functions and promoting anti-tumor immunity. Previously, we reported the P5091 series of small molecule USP7 inhibitors and demonstrated their direct anti-tumor activity in vivo using xenograft models. However, the precise mechanism of action of these compounds was not well defined. In this study, we report the development and characterization of P217564, a second-generation USP7 inhibitor with improved potency and selectivity. P217564 selectively targets the catalytic cleft of USP7 and modifies its active site cysteine (C223 by forming a covalent adduct. Irreversible inhibition of USP7 results in durable downstream biological responses in cells, including down-regulation of Tip60 and consequent impairment of Treg suppressive function. In addition, we demonstrate that both USP7 and various USP7 substrates are subjected to Lys48-mediated ubiquitin modification, consistent with increased proteasomal degradation of these proteins because of USP7 inhibition.

HPLC method validation for modernization of the tetracycline hydrochloride capsule USP monograph

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Emad M. Hussien

2014-12-01

Full Text Available This paper is a continuation to our previous work aiming at development and validation of a reversed-phase HPLC for modernization of tetracycline-related USP monographs and the USP general chapter . Previous results showed that the method is accurate and precise for the assay of tetracycline hydrochloride and the limit of 4-epianhydrotetracycline impurity in the drug substance and oral suspension monographs. The aim of the current paper is to examine the feasibility of the method for modernization of USP tetracycline hydrochloride capsule monograph. Specificity, linearity, accuracy and precision were examined for tetracycline hydrochloride assay and 4-epianhydrotetracycline limit. The method was linear in the concentration range from 80% to 160% (r>0.9998 of the assay concentration (0.1 mg/mL for tetracycline hydrochloride and from 50% to 150% (r>0.997 of the acceptance criteria specified in tetracycline hydrochloride capsule monograph for 4-epianhydrotetracycline (NMT 3.0%. The recovery at three concentration levels for tetracycline hydrochloride assay was between 99% and 101% and the RSD from six preparations at the concentration 0.1 mg/mL is less than 0.6%. The recovery for 4-epianhydrotetracycline limit procedure over the concentration range from 50% to 150% is between 96% and 102% with RSD less than 5%. The results met the specified acceptance criteria.

Ecdysone receptor (EcR) and ultraspiracle (USP) genes from the cyclopoid copepod Paracyclopina nana: Identification and expression in response to water accommodated fractions (WAFs).

Science.gov (United States)

Puthumana, Jayesh; Lee, Min-Chul; Han, Jeonghoon; Kim, Hui-Su; Hwang, Dae-Sik; Lee, Jae-Seong

2017-02-01

Ecdysteroid hormones are pivotal in the development, growth, and molting of arthropods, and the hormone pathway is triggered by binding ecdysteroid to a heterodimer of the two nuclear receptors; ecdysone receptors (EcR) and ultraspiracle (USP). We have characterized EcR and USP genes, and their 5'-untranslated region (5'-UTR) from the copepod Paracyclopina nana, and studied mRNA transcription levels in post-embryonic stages and in response to water accommodated fractions (WAFs) of crude oil. The open reading frames (ORF) of EcR and USP were 1470 and 1287bp that encoded 490 and 429 amino acids with molecular weight of 121.18 and 105.03kDa, respectively. Also, a well conserved DNA-binding domain (DBD) and ligand-binding domain (LBD) were identified which confirmed by phylogenetic analysis. Messenger RNA transcriptional levels of EcR and USP were developmental stage-specific in early post-embryonic stages (N3-4). However, an evoked expression of USP was observed throughout copepodid stage and in adult females. WAFs (40 and 80%) were acted as an ecdysone agonist in P. nana, and elicited the mRNA transcription levels in adults. Developmental stage-specific transcriptional activation of EcR and USP in response to WAFs was observed. USP gene was down-regulated in the nauplius in response to WAF, whereas up-regulation of USP was observed in the adults. This study represents the first data of molecular elucidation of EcR and USP genes and their regulatory elements from P. nana and the developmental stage specific expression in response to WAFs, which can be used as potential biomarkers for environmental stressors with ecotoxicological evaluations in copepods. Copyright © 2016 Elsevier Inc. All rights reserved.

Thermostable amylolytic enzymes from a cellulolytic fungus Myceliophthora thermophila D14 (ATCC 48 104)

Energy Technology Data Exchange (ETDEWEB)

Sadhukhan, R K; Manna, S; Roy, S K; Chakrabarty, S L [Bose Research Inst., Calcutta (India). Dept. of Microbiology

1990-09-01

The production of amylolytic enzymes by a thermophilic cellulolytic fungus, Myceliophthora thermophila D14 was investigated by batch cultivation in Czapek-Dox medium at 45deg C. Among various nitrogenous compounds used, NaNO{sub 3} and KNO{sub 3} were found to be the best for amylase production. Starch, cellobiose and maltose induced the synthesis of amylase while glucose, fructose, galactose, lactose, arabinose, xylose, sorbitol, mesoinositol and sucrose did not. Calcium ions had the most stimulating effect on enzyme formation amongst many ions investigated. The synthesis of amylolytic enzymes was dependent on growth and occurred predominantly in the mid-stationary phase. The enzyme was active in a broad temperature range (50deg C-60deg C) and displayed activity optima at 60deg C and pH 5.6. (orig.).

USP e UFRJ: a influência das concepções alemã e francesa em suas fundações USP and UFRJ: the influence of the German and French conceptions upon their foundation

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Maria de Fátima Costa de Paula

2002-10-01

Full Text Available O artigo pretende demonstrar a influência das concepções alemã e francesa do século XIX sobre a Universidade de São Paulo (USP e a Universidade do Rio de Janeiro (URJ, no momento das suas respectivas fundações e em suas primeiras décadas de funcionamento, na primeira metade do século XX. Para tal, relacionamos as características da concepção alemã com a concepção da USP, no que se refere à idéia de universidade e à organização institucional da mesma. Fizemos um percurso semelhante para demonstrar a influência do modelo francês napoleônico sobre a URJ. Nas considerações finais, sugerimos a hegemonia da concepção norte-americana sobre os casos brasileiros investigados, a partir, sobretudo, da década de 1960, substituindo progressivamente as influências alemã e francesa.The present study investigates the influence of the XIXth century German and French thought on the establishment of the universities of São Paulo (USP and Rio de Janeiro (URJ, in Brazil. This research also shows how this influence remains during the first decades of the XXth century lasting until the 50's. In order to do that we related specific traits of the German conception with the USP conception and emphasized the definition od university and its organization. The same methodology was applied to analyse the napoleonic model´s influence upon the university of Rio de Janeiro. Finally we verify the growing and dominant influence of the North-American model in the cases above mentionned, mainly after the 60's. As a result we concluded that the North-American model gradually replaced the French and German models.

Rehabilitative treatment of cleft lip and palate: experience of the Hospital for Rehabilitation of Craniofacial Anomalies/USP (HRAC/USP - Part 1: overall aspects

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José Alberto de Souza Freitas

2012-02-01

Full Text Available Cleft lip and palate is the most common among craniofacial malformations and causes several esthetic and functional implications that require rehabilitation. This paper aims to generally describe the several aspects related to this complex pathology and the treatment protocol used by the Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo (HRAC-USP along 40 years of experience in the treatment of individuals with cleft lip and palate.

CSN-associated USP48 confers stability to nuclear NF-κB/RelA by trimming K48-linked Ub-chains.

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Schweitzer, Katrin; Naumann, Michael

2015-02-01

Diligent balance of nuclear factor kappa B (NF-κB) activity is essential owing to NF-κB's decisive role in cellular processes including inflammation, immunity and cell survival. Ubiquitin/proteasome-system (UPS)-dependent degradation of activated NF-κB/RelA involves the cullin-RING-ubiquitin-ligase (CRL) ECS(SOCS1). The COP9 signalosome (CSN) controls ubiquitin (Ub) ligation by CRLs through the removal of the CRL-activating Ub-like modifier NEDD8 from their cullin subunits and through deubiquitinase (DUB) activity of associated DUBs. However, knowledge about DUBs involved in the regulation of NF-κB activity within the nucleus is scarce. In this study we observed that USP48, a DUB of hitherto ill-defined function identified through a siRNA screen, associates with the CSN and RelA in the nucleus. We show that USP48 trims rather than completely disassembles long K48-linked free and substrate-anchored Ub-chains, a catalytic property only shared with ataxin-3 (Atx3) and otubain-1 (OTU1), and that USP48 Ub-chain-trimming activity is regulated by casein-kinase-2 (CK2)-mediated phosphorylation in response to cytokine-stimulation. Functionally, we demonstrate for the first time the CSN and USP48 to cooperatively stabilize the nuclear pool of RelA, thereby facilitating timely induction and shutoff of NF-κB target genes. In summary, this study demonstrates that USP48, a nuclear DUB regulated by CK2, controls the UPS-dependent turnover of activated NF-κB/RelA in the nucleus together with the CSN. Thereby USP48 contributes to a timely control of immune responses. Copyright © 2014 Elsevier B.V. All rights reserved.

USP Marion: A Few Prisoners Summon the Courage to Speak

OpenAIRE

Stephen C. Richards

2015-01-01

USP Marion is the first supermax federal penitentiary. Marionization refers to the experimental control program used at this prison. The prisoners speaking in this article suffered many years of solitary confinement. This research brief discusses some of what they experienced in their own words. These are the recollections of a few Marion prisoners that have summoned the courage to speak out and share their darkest memories.

Tools to Ensure Safe Medicines: New Monograph Tests in USP-NF

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Catherine Sheehan

2010-06-01

Full Text Available This paper describes USP-NF compendial updates to six ‘high-priority” excipient monographs: Glycerin, Propylene Glycol, Sorbitol Solution, Sorbitol Sorbitan Solution, Noncrystallizing Sorbitol Solution and Maltitol Solution. The USP-NF revisions arose from the Food and Drug Administration’s (FDA’s requests to include, as part of each monograph’s Identification test, a limit test to detect the presence of Diethylene Glycol (DEG, a toxic adulterant. These revisions align with the 2007 FDA Guidance for Industry: Testing of Glycerin for Diethylene Glycol (1, that drug product manufacturers perform a specific identity test for DEG on all containers of all lots of glycerin before glycerin is used in the manufacture and preparation of drug products. This paper describes several risk factors due to a complex global excipient supply chain, nonspecific specifications, inadequate supply chain qualification, and poor understanding of regulations. Strengthening and conformance to compendial specifications is one of the tools necessary to mitigate risk and help prevent the next DEG adulteration that is part of USP’s efforts to ensure safe medicines.

Overexpression of a Cytosolic Abiotic Stress Responsive Universal Stress Protein (SbUSP) Mitigates Salt and Osmotic Stress in Transgenic Tobacco Plants

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Udawat, Pushpika; Jha, Rajesh K.; Sinha, Dinkar; Mishra, Avinash; Jha, Bhavanath

2016-01-01

The universal stress protein (USP) is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologs of intron less SbUSP gene which encodes for salt and osmotic responsive USP. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control [wild-type (WT) and vector control (VC)] plants under different abiotic stress condition. Transgenic lines (T1) exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability, and lower electrolyte leakage and lipid peroxidation (malondialdehyde content) under stress treatments than control (WT and VC) plants. Lower accumulation of H2O2 and O2− radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant. PMID:27148338

Mass spectrometry analyses of normal and polyglutamine expanded ataxin-3 reveal novel interaction partners involved in mitochondrial function

DEFF Research Database (Denmark)

Kristensen, Line V.; Oppermann, Felix S.; Rauen, Matthias J.

2018-01-01

Deubiquitinating enzymes (DUBs) play important roles in a variety of cellular processes, including regulation of protein homeostasis. The DUB ataxin-3 is an enzyme implicated in protein quality control mechanisms. In the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3), ataxin-3...

Ubiquitin specific peptidase 5 mediates Histidine-rich protein Hpn induced cell apoptosis in hepatocellular carcinoma through P14-P53 signaling.

Science.gov (United States)

Liu, Yi; Wang, Wei-Mao; Zou, Li-Yi; Li, Li; Feng, Lu; Pan, Ming-Zhu; Lv, Min-Yi; Cao, Ying; Wang, Hua; Kung, Hsiang-Fu; Pang, Jian-Xin; Fu, Wei-Ming; Zhang, Jin-Fang

2017-06-01

Hpn is a small histidine-rich cytoplasmic protein from Helicobacter pylori and has been recognized as a high-risk factor for several cancers including gastric cancer, colorectal cancer, and MALT lymphoma. However, the relationship between Hpn and cancers remains elusive. In this study, we discovered that Hpn protein effectively suppressed cell growth and induced apoptosis in hepatocellular carcinoma (HCC). A two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics was performed to find the molecular targets of Hpn in HCC cells. It was identified that twelve proteins were differentially expressed, with USP5 being one of the most significantly downregulated protein. The P14 ARF -P53 signaling was activated by USP5 knockdown in HCC cells. Furthermore, USP5 overexpression significantly rescued the suppressive effect of Hpn on the viability of HCC cells. In conclusion, our study suggests that Hpn plays apoptosis-inducing roles through suppressing USP5 expression and activating the P14 ARF -P53 signaling. Therefore, Hpn may be a potential candidate for developing novel anti-HCC drugs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

USP Marion: A Few Prisoners Summon the Courage to Speak

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Stephen C. Richards

2015-02-01

Full Text Available USP Marion is the first supermax federal penitentiary. Marionization refers to the experimental control program used at this prison. The prisoners speaking in this article suffered many years of solitary confinement. This research brief discusses some of what they experienced in their own words. These are the recollections of a few Marion prisoners that have summoned the courage to speak out and share their darkest memories.

Proteasome Failure Promotes Positioning of Lysosomes around the Aggresome via Local Block of Microtubule-Dependent Transport

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Zaarur, Nava; Meriin, Anatoli B.; Bejarano, Eloy; Xu, Xiaobin; Gabai, Vladimir L.; Cuervo, Ana Maria

2014-01-01

Ubiquitinated proteins aggregate upon proteasome failure, and the aggregates are transported to the aggresome. In aggresomes, protein aggregates are actively degraded by the autophagy-lysosome pathway, but why targeting the aggresome promotes degradation of aggregated species is currently unknown. Here we report that the important factor in this process is clustering of lysosomes around the aggresome via a novel mechanism. Proteasome inhibition causes formation of a zone around the centrosome where microtubular transport of lysosomes is suppressed, resulting in their entrapment and accumulation. Microtubule-dependent transport of other organelles, including autophagosomes, mitochondria, and endosomes, is also blocked in this entrapment zone (E-zone), while movement of organelles at the cell periphery remains unaffected. Following the whole-genome small interfering RNA (siRNA) screen for proteins involved in aggresome formation, we defined the pathway that regulates formation of the E-zone, including the Stk11 protein kinase, the Usp9x deubiquitinating enzyme, and their substrate kinase MARK4. Therefore, upon proteasome failure, targeting of aggregated proteins of the aggresome is coordinated with lysosome positioning around this body to facilitate degradation of the abnormal species. PMID:24469403

40Ar/39Ar geochronology at the Instituto de Geociências, USP: instrumentation, analytical procedures, and calibration

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PAULO M. VASCONCELOS

2002-06-01

Full Text Available Laser heating 40Ar/39Ar geochronology provides high analytical precision and accuracy, mum-scale spatial resolution, and statistically significant data sets for the study of geological and planetary processes. A newly commissioned 40Ar/39Ar laboratory at CPGeo/USP, São Paulo, Brazil, equips the Brazilian scientific community with a new powerful tool applicable to the study of geological and cosmochemical processes. Detailed information about laboratory layout, environmental conditions, and instrumentation provides the necessary parameters for the evaluation of the CPGeo/USP 40Ar/39Ar suitability to a diverse range of applications. Details about analytical procedures, including mineral separation, irradiation at the IPEN/CNEN reactor at USP, and mass spectrometric analysis enable potential researchers to design the necessary sampling and sample preparation program suitable to the objectives of their study. Finally, the results of calibration tests using Ca and K salts and glasses, international mineral standards, and in-house mineral standards show that the accuracy and precision obtained at the 40Ar/39Ar laboratory at CPGeo/USP are comparable to results obtained in the most respected laboratories internationally. The extensive calibration and standardization procedures undertaken ensure that the results of analytical studies carried out in our laboratories will gain immediate international credibility, enabling Brazilian students and scientists to conduct forefront research in earth and planetary sciences.A geocronologia de 40Ar/39Ar por aquecimento a laser permite alta precisão e acurácia analítica, tem resolução espacial em escala micrométrica, e fornece um número de dados estatisticamente significantes para o estudo de processos geológicos e planetários. Um recém construído laboratório de 40Ar/39Ar no CPGeo/USP, São Paulo, Brazil, mune a sociedade científica brasileira com uma técnica eficaz aplicável aos estudos geol

Can laboratory and pilot recycling trials predict adhesive removal in commercial recycling systems? : results from the USPS environmentally benign stamp project

Science.gov (United States)

Carl Houtman; Daniel Seiter; Nancy Ross Sutherland; Donald Donermeyer

2002-01-01

The ultimate goal of the US Postal Service (USPS) Environmentally Benign Stamp Program is to develop stamp laminates, i.e., face paper, adhesive and siliconized liner, that do not cause difficulties in recycling mills. The criterion for success, and the USPS definition of benignity, is the avoidance of process and product quality hardships when such PSA laminates are...

A study of the fine structure, enzyme activities and pattern of 14CO2 ...

African Journals Online (AJOL)

A detailed study of selected grasses has been made with respect to fine structures characteristics, enzyme activities associated with C-4 and C-3 pathway photosynthesis, and short term carbon dioxide-14 incorporation experiments. A good correlation was obtained between the fine structure, the carbon pathway and the ...

Mineralisation of 14C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi.

Science.gov (United States)

Steffen, K T; Hofrichter, M; Hatakka, A

2000-12-01

Within a screening program, 27 soil litter-decomposing basidiomycetes were tested for ligninolytic enzyme activities using agar-media containing 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate), a humic acid or Mn2+ ions as indicator substrates. Most active species were found within the family Strophariaceae (Agrocybe praecox, Stropharia coronilla, S. rugosoannulata) and used for mineralisation experiments with a 14C-ring-labelled synthetic lignin (14C-DHP). The fungi mineralised around 25% of the lignin to 14CO2 within 12 weeks of incubation in a straw environment; about 20% of the lignin was converted to water-soluble fragments. Mn-peroxidase was found to be the predominant ligninolytic enzyme of all three fungi in liquid culture and its production was strongly enhanced in the presence of Mn2+ ions. The results of this study demonstrate that certain ubiquitous litter-decomposing basidiomycetes possess ligninolytic activities similar to the wood-decaying white-rot fungi, the most efficient lignin degraders in nature.

Detection of a Usp-like gene in Calotropis procera plant from the de novo assembled genome contigs of the high-throughput sequencing dataset

KAUST Repository

Shokry, Ahmed M.

2014-02-01

The wild plant species Calotropis procera (C. procera) has many potential applications and beneficial uses in medicine, industry and ornamental field. It also represents an excellent source of genes for drought and salt tolerance. Genes encoding proteins that contain the conserved universal stress protein (USP) domain are known to provide organisms like bacteria, archaea, fungi, protozoa and plants with the ability to respond to a plethora of environmental stresses. However, information on the possible occurrence of Usp in C. procera is not available. In this study, we uncovered and characterized a one-class A Usp-like (UspA-like, NCBI accession No. KC954274) gene in this medicinal plant from the de novo assembled genome contigs of the high-throughput sequencing dataset. A number of GenBank accessions for Usp sequences were blasted with the recovered de novo assembled contigs. Homology modelling of the deduced amino acids (NCBI accession No. AGT02387) was further carried out using Swiss-Model, accessible via the EXPASY. Superimposition of C. procera USPA-like full sequence model on Thermus thermophilus USP UniProt protein (PDB accession No. Q5SJV7) was constructed using RasMol and Deep-View programs. The functional domains of the novel USPA-like amino acids sequence were identified from the NCBI conserved domain database (CDD) that provide insights into sequence structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM). © 2014 Académie des sciences.

Evaluation of preservative effectiveness of gallic acid derivatives in aluminum hydroxide gel-USP

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Anurag Khatkar

2013-01-01

Full Text Available Background: Preservatives are added to most of the pharmaceutical preparations to prevent them from deterioration throughout their shelf life. Literature reveals that the common synthetic preservatives have many limitations, such as development of microbial resistance (in due course of time and several serious side-effects. Aim: The aim of this study is to find out new preservatives synthesized from natural sources, which may have better efficiency than the existing synthetic preservatives. The derivatives of naturally occurring gallic acid were subjected for their preservative efficacy study. Their preservative efficiency was evaluated and compared with the standard parabens. Materials and Methods: The selected amide, anilide and ester derivatives of gallic acid were subjected to preservative efficacy testing in an official antacid preparation, {aluminum hydroxide gel-USP (United States Pharmacopoeia} against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Candida albicans, and Aspergillus niger as representative challenging microorganisms as per USP 2004 guidelines. Results: The selected derivatives were found to be effective against all selected strains and showed preservative efficacy comparable to that of standard and even better in case E. coli, C. albicans and A. niger. The 8-hydroxy quinoline ester derivative showed better preservative efficacy than standard as well as other derivatives. Conclusion: The newly synthesized gallic acid preservatives were found to be effective in the proposed pharmaceutical preparation (Aluminium Hydroxide Gel - USP. Also, the synthesized preservatives have shown comparative and even better efficacy than the existing parabens and hence they have potential for use in pharmaceutical preparations.

Two females with mutations in USP9X highlight the variable expressivity of the intellectual disability syndrome.

Science.gov (United States)

Au, P Y B; Huang, L; Broley, S; Gallagher, L; Creede, E; Lahey, D; Ordorica, S; Mina, K; Boycott, K M; Baynam, G; Dyment, D A

2017-07-01

The genetic causes of intellectual disability (ID) are heterogeneous and include both chromosomal and monogenic etiologies. The X-chromosome is known to contain many ID-related genes and males show a marked predominance for intellectual disability. Here we report two females with syndromic intellectual disability. The first individual was relatively mild in her presentation with mild-moderate intellectual disability, hydronephrosis and altered pigmentation along the lines of Blaschko without additional congenital anomalies. A second female presented shortly after birth with dysmorphic facial features, post-axial polydactyly and, on follow-up assessment, demonstrated moderate intellectual disability. Chromosomal studies for Individual 1 identified an X-chromosome deletion due to a de novo pericentric inversion; the inversion breakpoint was associated with deletion of the 5'UTR of the USP9X, a gene which has been implicated in a syndromic intellectual disability affecting females. The second individual had a de novo frameshift mutation detected by whole-exome sequencing that was predicted to be deleterious, NM_001039590.2 (USP9X): c.4104_4105del (p.(Arg1368Serfs*2)). Haploinsufficiency of USP9X in females has been associated with ID and congenital malformations that include heart defects, scoliosis, dental abnormalities, anal atresia, polydactyly, Dandy Walker malformation and hypoplastic corpus callosum. The extent of the congenital malformations observed in Individual 1 was less striking than Individual 2 and other individuals previously reported in the literature, and suggests that USP9X mutations in females can have a wider spectrum of presentation than previously appreciated. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Insights into the Mechanism of Deubiquitination by JAMM Deubiquitinases from Cocrystal Structures of the Enzyme with the Substrate and Product

Science.gov (United States)

2015-01-01

AMSH, a conserved zinc metallo deubiquitinase, controls downregulation and degradation of cell-surface receptors mediated by the endosomal sorting complexes required for transport (ESCRT) machinery. It displays high specificity toward the Lys63-linked polyubiquitin chain, which is used as a signal for ESCRT-mediated endosomal–lysosomal sorting of receptors. Herein, we report the crystal structures of the catalytic domain of AMSH orthologue Sst2 from fission yeast, its ubiquitin (product)-bound form, and its Lys63-linked diubiquitin (substrate)-bound form at 1.45, 1.7, and 2.3 Å, respectively. The structures reveal that the P-side product fragment maintains nearly all the contacts with the enzyme as seen with the P portion (distal ubiquitin) of the Lys63-linked diubiquitin substrate, with additional coordination of the Gly76 carboxylate group of the product with the active-site Zn2+. One of the product-bound structures described herein is the result of an attempt to cocrystallize the diubiquitin substrate bound to an active site mutant presumed to render the enzyme inactive, instead yielding a cocrystal structure of the enzyme bound to the P-side ubiquitin fragment of the substrate (distal ubiquitin). This fragment was generated in situ from the residual activity of the mutant enzyme. In this structure, the catalytic water is seen placed between the active-site Zn2+ and the carboxylate group of Gly76 of ubiquitin, providing what appears to be a snapshot of the active site when the product is about to depart. Comparison of this structure with that of the substrate-bound form suggests the importance of dynamics of a flexible flap near the active site in catalysis. The crystal structure of the Thr319Ile mutant of the catalytic domain of Sst2 provides insight into structural basis of microcephaly capillary malformation syndrome. Isothermal titration calorimetry yields a dissociation constant (KD) of 10.2 ± 0.6 μM for the binding of ubiquitin to the enzyme, a value

A dose-response study in animals to evaluate the anticoagulant effect of the stage 2 unfractionated heparin USP monograph change.

Science.gov (United States)

Honchel, R; Carraway, J; Gopee, N; Callicott, R; Chen, J; Patton, R; Xu, Q; Zalkkar, J; Laniyonu, A; Krefting, I; Cato, M; Robie-Suh, K; Rieves, R

2011-08-01

The United States Pharmacopeia (USP) monograph for unfractionated heparin (UFH) was revised in October 2009. This revision was anticipated, based upon in vitro tests, to reduce UFH potency by approximately 10%. To study the potential in vivo consequences of the monograph change, we evaluated activated partial thromboplastin time (aPTT) and activated clotting time (ACT) responses in animals. Female mini-pigs and monkeys (n=8/species) were administered intravenously 60, 54, 48, or 42 U/kg and 50, 45, 40, or 35 U/kg "old" (pre-USP revision) UFH, respectively, in a Williams 4×4 crossover design. Blood samples for aPTT and ACT were collected at 15 min after dosing. The same study design was then repeated using "new" (post-USP revision) UFH. Mean "new" UFH aPTT and ACT values were generally lower than those for "old" UFH although individual animal responses varied considerably. The aPTT and ACT response was generally dose-proportional for both "old" and "new" UFH. These studies indicate that the USP monograph alteration for UFH may result in a modest reduction in the anticoagulant response across a population, but the variability in animal responses underscores the importance of individualization of clinical UFH dosing and the importance of anticoagulant test monitoring. Published by Elsevier Inc.

Overexpression of a cytosolic abiotic stress responsive universal stress protein (SbUSP mitigates salt and osmotic stress in transgenic tobacco plants

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Pushpika eUdawat

2016-04-01

Full Text Available The Universal Stress Protein (USP is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologues of intron less SbUSP gene which encodes for salt and osmotic responsive universal stress protein. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control (wild type and vector control plants under different abiotic stress condition. Transgenic lines (T1 exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability and lower electrolyte leakage and lipid peroxidation (malondialdehyde content under stress treatments than control (WT and VC plants. Lower accumulation of H2O2 and O2- radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis (PCA exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant.

Uspešnost obrtno-zborničnega sistema v Sloveniji

OpenAIRE

Štrekelj, Andreja

2016-01-01

Organizacije morajo zaradi vse večje konkurence in prostega nastopa na trgu stremeti k čim večji tržni prednosti oziroma prepoznavnosti in uspešnosti, ne glede na področje poslovanja. To velja tudi za neprofitne organizacije, ki svojemu neprofitnemu poslanstvu profitno sledijo in ga neprofitno uresničujejo. Med neprofitne organizacije spada tudi Obrtno-podjetniška zbornica Slovenije, ki v slovenskem prostoru deluje že 47 let in je nastala na podlagi skupnih interesov takratnih obrtnikov zarad...

Rehabilitative treatment of cleft lip and palate: experience of the Hospital for Rehabilitation of Craniofacial Anomalies/USP (HRAC/USP) - Part 4: oral rehabilitation.

Science.gov (United States)

Freitas, José Alberto de Souza; Almeida, Ana Lúcia Pompéia Fraga de; Soares, Simone; Neves, Lucimara Teixeira das; Garib, Daniela Gamba; Trindade-Suedam, Ivy Kiemle; Yaedú, Renato Yassutaka Faria; Lauris, Rita de Cássia Moura Carvalho; Oliveira, Thais Marchini; Pinto, João Henrique Nogueira

2013-01-01

Treatment of patients with cleft lip and palate is completed with fixed prostheses, removable, total, implants and aims to restore aesthetics, phonetics and function and should be guided by the basic principles of oral rehabilitation, such as physiology, stability, aesthetics, hygiene and the expectations of the patient. In order to obtain longevity of a prosthetic rehabilitation, the periodontal and dental tissue as well as the biomechanics of the prosthesis are to be respected. The purpose of this article is to describe the types of prosthetics treatment, which are performed at HRAC/USP for the rehabilitation of cleft area in adult patients.

USP2 Regulates the Intracellular Localization of PER1 and Circadian Gene Expression

DEFF Research Database (Denmark)

Yang, Yaoming; Duguay, David; Fahrenkrug, Jan

2014-01-01

. Although Per1 mRNA expression rhythm remained intact in the Usp2 KO MEFs, the expression profiles of other core clock genes were altered. This was also true for the expression of clock-controlled genes (e.g., Dbp, Tef, Hlf, E4bp4). A similar phase advance of PER1 nuclear localization rhythm and alteration...

Rehabilitative treatment of cleft lip and palate: experience of the Hospital for Rehabilitation of Craniofacial Anomalies/USP (HRAC/USP Part 4: Oral Rehabilitation

Directory of Open Access Journals (Sweden)

Jose Alberto de Souza FREITAS

2013-06-01

Full Text Available Treatment of patients with cleft lip and palate is completed with fixed prostheses, removable, total, implants and aims to restore aesthetics, phonetics and function and should be guided by the basic principles of oral rehabilitation, such as physiology, stability, aesthetics, hygiene and the expectations of the patient. In order to obtain longevity of a prosthetic rehabilitation, the periodontal and dental tissue as well as the biomechanics of the prosthesis are to be respected. The purpose of this article is to describe the types of prosthetics treatment, which are performed at HRAC/USP for the rehabilitation of cleft area in adult patients.

Frequency of USP6 rearrangements in myositis ossificans, brown tumor, and cherubism: molecular cytogenetic evidence that a subset of ''myositis ossificans-like lesions'' are the early phases in the formation of soft-tissue aneurysmal bone cyst

International Nuclear Information System (INIS)

Sukov, William R.; Erickson-Johnson, Michele; Unni, K.K.; Wang, Xiaoke; Oliveira, Andre M.; Franco, Marcello F.; Chou, Margaret M.; Wenger, Doris E.

2008-01-01

USP6 rearrangements with several partner genes have been identified recently in primary but not in secondary aneurysmal bone cysts (ABCs). Several lesions show histologic features that may overlap with ABC, including myositis ossificans (MO), brown tumor, and cherubism. The objective of this study was to assess whether these lesions harbored USP6 rearrangements. Twelve patients with classic radiologic and histologic features of MO, 6 with brown tumors, and 5 with cherubism diagnosed at our institution were studied for the presence of USP6 rearrangements using fluorescence in situ hybridization with probes flanking the USP6 locus on chromosome 17p13. In addition, conventional cytogenetic analysis was performed in 2 patients with cherubism. USP6 rearrangements were identified in 2 patients with radiologic and histologic features consistent with MO. None of the patients with brown tumor or cherubism demonstrated USP6 rearrangements. Cytogenetic analysis of the cherubism patients demonstrated normal karyotypes. These findings indicate that a subset of cases with apparent classic histologic and imaging features of MO are rather better classified as being soft-tissue ABC with clonal USP6 rearrangements. In contrast, no USP6 rearrangements were found in patients with cherubism or brown tumor, supporting the prevailing view that these lesions are distinct biologic entities. (orig.)

Saccharification of ozonated sugarcane bagasse using enzymes from Myceliophthora thermophila JCP 1-4 for sugars release and ethanol production.

Science.gov (United States)

de Cassia Pereira, Josiani; Travaini, Rodolfo; Paganini Marques, Natalia; Bolado-Rodríguez, Silvia; Bocchini Martins, Daniela Alonso

2016-03-01

The saccharification of ozonated sugarcane bagasse (SCB) by enzymes from Myceliophthora thermophila JCP 1-4 was studied. Fungal enzymes provided slightly higher sugar release than commercial enzymes, working at 50°C. Sugar release increased with temperature increase. Kinetic studies showed remarkable glucose release (4.99 g/L, 3%w/w dry matter) at 60°C, 8 h of hydrolysis, using an enzyme load of 10 FPU (filter paper unit). FPase and β-glucosidase activities increased during saccharification (284% and 270%, respectively). No further significant improvement on glucose release was observed increasing the enzyme load above 7.5 FPU per g of cellulose. Higher dry matter contents increased sugars release, but not yields. The fermentation of hydrolysates by Saccharomyces cerevisiae provided glucose-to-ethanol conversions around to 63%. Copyright © 2015 Elsevier Ltd. All rights reserved.

A mathematical analysis of drug dissolution in the USP flow through apparatus

Science.gov (United States)

McDonnell, David; D'Arcy, D. M.; Crane, L. J.; Redmond, Brendan

2018-03-01

This paper applies boundary layer theory to the process of drug dissolution in the USP (United States Pharmacopeia) Flow Through Apparatus. The mass transfer rate from the vertical planar surface of a compact within the device is examined. The theoretical results obtained are then compared with those of experiment. The paper also examines the effect on the dissolution process caused by the interaction between natural and forced convection within the apparatus and the introduction of additional boundaries.

CONTRASTANDO AS PRODUÇÕES DA REVISTA CONTABILIDADE & FINANÇAS (FEA-USP E REVISTA BASE (UNISINOS

Directory of Open Access Journals (Sweden)

FLÁVIO DONIZETE BATISTELLA

2008-01-01

Full Text Available La reestructuración del sistema de evaluación de publicaciones de la Funda-ción Coordinación de Perfeccionamiento de Personal de Nivel Superior (CAPES, el Sistema Qualis, produjo un fuerte incentivo a la publicación de artículos en periódicos. Aprehender la fase de la producción en dos periódicos nacionales es el foco de esta averiguación. Estos periódicos son: Revista Contabilidade & Finanças (FEA-USP e Revista Base (UNISINOS. El período de análisis engloba las publicaciones ocurridas en 2005 y en 2006. Los aspectos observados por la CAPES en su proceso de eva-luación de periódicos son eminentemente orientados en la edición y compaginación del periódico. En este sentido, el análisis adoptado en esta pesquisa posee enfoque en la forma de los periódicos, siendo que la metodología central es fundamentada en el modelo propuesto por Krzyzanowski y Ferreira (1998. Los resultados logrados sugieren que la Revista Contabilidad & Finanzas (FEA-USP presenta, con relación al apartado forma, un desempeño general clasificado como bueno, mientras que la Revista Base (Unisinos presenta un desempeño mediano. Se observó también una mayor concentración de autores en las publicaciones de la Revista Contabilidad & Finanzas (FEA-Usp (recurrencia con relación a la Revista Base (UNISINOS.

Solubilization of low-rank coal by Trichoderma atroviride: Evidence for the involvement of hydrolytic and oxidative enzymes by using C-14-labelled lignite

Energy Technology Data Exchange (ETDEWEB)

Holker, U.; Schmiers, H.; Grosse, S.; Winkelhofer, M.; Polsakiewicz, M.; Ludwig, S.; Dohse, J.; Hofer, M. [University of Bonn, Bonn (Germany). Inst. of Botany

2002-04-01

The deuteromycete Trichoderma atroviride is able to solubilize lignite in dependence on a given carbon source for growth. When cultivated on media containing glutamate, this mold excreted a set of different enzymes with hydrolytic activity. Addition of lignite to the growth media induced the synthesis of extracellular lignite-specific esterase activity but no evidence has been provided for its direct involvement in the process of lignite solubilization. Hence, the basic capability of T. atroviride enzymes to degrade a variety of ester and ether bonds at the surface or within the bulky lignite structure was tested using coal following its direct labelling with C-14-alkyl iodide. The participation of hydrolytic and oxidative enzymes in lignite degradation was assessed by measuring the release of C-14 radioactivity from selectively alkylated carboxylic and phenolic OH groups. T. atroviride cleaved both carboxylic esters using esterases and the phenolic ether bonds by using oxidative enzymes, most likely laccases.

Method of preparing uridine-diphospho-/sup 14/C-D-glucose of high molar activity and radiochemical purity by enzyme synthesis

Energy Technology Data Exchange (ETDEWEB)

Farkas, V; Biely, P; Koelbl, J

1980-06-15

A new method is described of enzyme synthesis of uridine-diphospho-/sup 14/C-D-glucose of high molar activity. After conversion of the initial /sup 14/C-D-glucose, the accompanying sugar phosphates and nucleoside-5'-phosphates are selectively hydrolyzed by the action of alkaline phosphatase in the presence of phenolphthalein as an indicator. Uridine-diphospho-/sup 14/C-D-glucose is separated by paper chromatography from the reaction mixture thus modified. Synthesis yields range within 75% and 85% relative to the starting /sup 14/C-D-glucose.

Frequency of USP6 rearrangements in myositis ossificans, brown tumor, and cherubism: molecular cytogenetic evidence that a subset of ''myositis ossificans-like lesions'' are the early phases in the formation of soft-tissue aneurysmal bone cyst

Energy Technology Data Exchange (ETDEWEB)

Sukov, William R.; Erickson-Johnson, Michele; Unni, K.K.; Wang, Xiaoke; Oliveira, Andre M. [Mayo Clinic, Department of Laboratory Medicine and Pathology, Rochester, MN (United States); Franco, Marcello F. [Universidade Federal do Estado de Sao Paulo (UNEFESP), Departamento de Patologia, Sao Paulo (Brazil); Chou, Margaret M. [University of Pennsylvania School of Medicine, Philadelphia, PA (United States); Wenger, Doris E. [Mayo Clinic, Department of Radiology, Rochester, MN (United States)

2008-04-15

USP6 rearrangements with several partner genes have been identified recently in primary but not in secondary aneurysmal bone cysts (ABCs). Several lesions show histologic features that may overlap with ABC, including myositis ossificans (MO), brown tumor, and cherubism. The objective of this study was to assess whether these lesions harbored USP6 rearrangements. Twelve patients with classic radiologic and histologic features of MO, 6 with brown tumors, and 5 with cherubism diagnosed at our institution were studied for the presence of USP6 rearrangements using fluorescence in situ hybridization with probes flanking the USP6 locus on chromosome 17p13. In addition, conventional cytogenetic analysis was performed in 2 patients with cherubism. USP6 rearrangements were identified in 2 patients with radiologic and histologic features consistent with MO. None of the patients with brown tumor or cherubism demonstrated USP6 rearrangements. Cytogenetic analysis of the cherubism patients demonstrated normal karyotypes. These findings indicate that a subset of cases with apparent classic histologic and imaging features of MO are rather better classified as being soft-tissue ABC with clonal USP6 rearrangements. In contrast, no USP6 rearrangements were found in patients with cherubism or brown tumor, supporting the prevailing view that these lesions are distinct biologic entities. (orig.)

O Instituto de Estudos Avançados da USP

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João Steiner

2004-04-01

Full Text Available COMENTA-SE a missão especial do Instituto de Estudos Avançados da USP a abrangente agenda de debates sobre problemas mundiais, nacionais e institucionais. A sobrevivência da biosfera do Planeta Terra está ameaçada pelas mudanças globais induzidas pela atividade humana. A par disto, as relações internacionais estão, hoje, marcadas pelo unilateralismo político e protecionismo comercial dos paises desenvolvidos. A inserção do Brasil neste mundo complexo traz no se bojo extraordinários desafios políticos e comerciais. No cenário nacional ressalta-se a Política de Ciência e Tecnologia com seu sucesso no que diz respeito à pós-graduação e produção científica a par do insucesso no uso do conhecimento e falta de inovação que ameaçam nossa competitividade internacional, desenvolvimento econômico e bem-estar social. Conclui-se chamando a atenção para a grande responsabilidade da USP e do IEA no debate dos problemas nacionais e internacionais.THE SPECIAL mission of the University of São Paulo's Institute of Advanced Studies is here outlined, with focus on a broad agenda involving global, national and institutional problems. The survival of the biosphere of the Planet Earth is endangered by global changes caused by human activities. In addition, the international environment is poisoned by the political unilateralism and commercial protectionism of the developed countries. The role of Brazil in this complex world holds extraordinary political and commercial challenges. Our national Science and Technology Policy has been successful in terms of postgraduate human resources and scientific production, but has lagged behind in applying knowledge and promoting technological innovation, thus endangering our international competitiveness, economical development and social welfare. The University of São Paulo and its Institute of Advanced Studies have great responsibilities in the discussion of national as well as international

Development and validation of an HPLC method for tetracycline-related USP monographs.

Science.gov (United States)

Hussien, Emad M

2014-09-01

A novel reversed-phase HPLC method was developed and validated for the assay of tetracycline hydrochloride and the limit of 4-epianhydrotetracycline hydrochloride impurity in tetracycline hydrochloride commercial bulk and pharmaceutical products. The method employed L1 (3 µm, 150 × 4.6 mm) columns, a mobile phase of 0.1% phosphoric acid and acetonitrile at a flow rate of 1.0 mL/min, and detection at 280 nm. The separation was performed in HPLC gradient mode. Forced degradation studies showed that tetracycline eluted as a spectrally pure peak and was well resolved from its degradation products. The fast degradation of tetracycline hydrochloride and 4-epianhydrotetracycline hydrochloride in solution was retarded by controlling the autosampler temperature at 4 °C and using 0.1% H3 PO4 as diluent. The robustness of the method was tested starting with the maximum variations allowed in the US Pharmacopeia (USP) general chapter Chromatography . The method was linear over the range 80-120% of the assay concentration (0.1 mg/mL) for tetracycline hydrochloride and 50-150% of the acceptance criteria specified in the individual USP monographs for 4-epianhydrotetracycline hydrochloride. The limit of quantification for 4-epianhydrotetracycline hydrochloride was 0.1 µg/mL, 20 times lower than the acceptance criteria. The method was specific, precise, accurate and robust. Copyright © 2014 John Wiley & Sons, Ltd.

Yeast Sgf73/Ataxin-7 serves to anchor the deubiquitination module into both SAGA and Slik(SALSA HAT complexes

Directory of Open Access Journals (Sweden)

Lee Kenneth K

2009-02-01

Full Text Available Abstract Spinocerebellar ataxia (SCA is a physically devastating, genetically inherited disorder characterized by abnormal brain function that results in the progressive loss of the ability to coordinate movements. There are many types of SCAs as there are various gene mutations that can cause this disease. SCA types 1–3, 6–10, 12, and 17 result from a trinucleotide repeat expansion in the DNA-coding sequence. Intriguingly, recent work has demonstrated that increased trinucleotde expansions in the SCA7 gene result in defect in the function of the SAGA histone acetyltransferase complex. The SCA7 gene encodes a subunit of the SAGA complex. This subunit is conserved in yeast as the SGF73 gene. We demonstrate that Sgf73 is required to recruit the histone deubiquitination module into both SAGA and the related SliK(SALSA complex, and to maintain levels of histone ubiquitination, which is necessary for regulation of transcription at a number of genes.

AVALIAÇÃO DE FONTES DE CARBONO PARA A PRODUÇÃO DE INIBIDOR DE CRESCIMENTO DE Aspergillus fumigatus USP2 por Corynebacterium sp.

Directory of Open Access Journals (Sweden)

Gabrielle Fernanda Zimmer

2013-07-01

Full Text Available O aumento significativo na incidência de infecções fúngicas invasivas e a resistência natural de agentes etiológicos a antifúngicos existentes têm motivado a constante pesquisa por novos agentes antifúngicos nos ultimos anos. Neste sentido, foi selcionada uma cepa de Corynebacterium sp. com potencial antagonista frente à Aspergilus fumigatus USP2. A cepa foi cultivada em fase submersa e em fase sólida, avaliando-se a variação das fontes de glicose, sacarose e glicerol em presença de peptona, bem como o meio sintético Czapek. Os caldos de cultivo submerso foram utilizados para o ensaio de antagonismo microbiano com o fungo Aspergillus fumigatus USP2. Os resultados apontam que o cultivo em fase sólida utilizando glicose como fonte de carbono apresenta maior potencial inibitório da cepa de Corynebacterium sp. sobre o fungo Aspergillus fumigatus USP2.

Biallelic Variants in OTUD6B Cause an Intellectual Disability Syndrome Associated with Seizures and Dysmorphic Features

DEFF Research Database (Denmark)

Santiago-Sim, Teresa; Burrage, Lindsay C; Ebstein, Frédéric

2017-01-01

regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated...

Interference of aldehyde metabolizing enzyme with diamine oxidase/histaminase/activity as determined by 14C putrescine method

International Nuclear Information System (INIS)

Fogel, W.A.; Bieganski, T.; Wozniak, J.; Maslinski, C.

1978-01-01

The Δ 1 pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi 14 C putrescine test (1961, Archs Biochem. Biophys., vol.95, 242), has been investigated in several tissue homogenates. When guinea pig liver homogenate was used as a source of enzyme in the presence of aldehyde dehydrogenase inhibitors chlorate hydrate and acetaldehyde the level of formation Δ 1 pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced Δ 1 pyrroline formation, but to a lesser degree. In other tissues, with very high initial diamine oxidase activity (rat intestine, dog kidney) or with very low diamine oxidase activity (guinea pig skin, dog liver) the influence of these inhibitors was only slight. Pyrazole, an inhibitor of alcohol dehydrogenase exerted only a small effect on Δ 1 pyrroline formation. All aldehyde-metabolizing enzymes inhibitors, except pyrazole, were without effect on purified pea seddling and hog kidney diamine oxidases. The use of aldehyde-metabolizing enzymes inhibitors may help to reveal the real values of diamine oxidase activity, when tissues homogenates are used as a source of enzyme. (author)

Mario Sironi nas chamadas Coleções Matarazzo do MAC USP

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Andrea Ronqui

Full Text Available O acervo de arte moderna italiana do MAC-USP pode ser considerado a maior coleção desse gênero fora da Itália e vem sendo objeto de estudo nos últimos anos. Das 71 obras italianas, seis são de autoria de Mario Sironi. Este artigo propõe uma apresentação do percurso do artista, com breve análise de sua evolução pictórica, a partir dos estudos já existentes, a fim de situar as obras sironianas do acervo do MAC no contexto de sua produção.

Terminating protein ubiquitination: Hasta la vista, ubiquitin.

Science.gov (United States)

Stringer, Daniel K; Piper, Robert C

2011-09-15

Ubiquitination is a post-translational modification that generally directs proteins for degradation by the proteasome or by lysosomes. However, ubiquitination has been implicated in many other cellular processes, including transcriptional regulation, DNA repair, regulation of protein-protein interactions and association with ubiquitin-binding scaffolds. Ubiquitination is a dynamic process. Ubiquitin is added to proteins by E3 ubiquitin ligases as a covalent modification to one or multiple lysine residues as well as non-lysine amino acids. Ubiquitin itself contains seven lysines, each of which can also be ubiquitinated, leading to polyubiquitin chains that are best characterized for linkages occurring through K48 and K63. Ubiquitination can also be reversed by the action of deubiquitination enzymes (DUbs). Like E3 ligases, DUbs play diverse and critical roles in cells. ( 1) Ubiquitin is expressed as a fusion protein, as a linear repeat or as a fusion to ribosomal subunits, and DUbs are necessary to liberate free ubiquitin, making them the first enzyme of the ubiquitin cascade. Proteins destined for degradation by the proteasome or by lysosomes are deubiquitinated prior to their degradation, which allows ubiquitin to be recycled by the cell, contributing to the steady-state pool of free ubiquitin. Proteins destined for degradation by lysosomes are also acted upon by both ligases and DUbs. Deubiquitination can also act as a means to prevent protein degradation, and many proteins are thought to undergo rounds of ubiquitination and deubiquitination, ultimately resulting in either the degradation or stabilization of those proteins. Despite years of study, examining the effects of the ubiquitination of proteins remains quite challenging. This is because the methods that are currently being employed to study ubiquitination are limiting. Here, we briefly examine current strategies to study the effects of ubiquitination and describe an additional novel approach that we have

Autophagy-Related Deubiquitinating Enzymes Involved in Health and Disease

OpenAIRE

El Magraoui, Fouzi; Reidick, Christina; Meyer, Hemut E.; Platta, Harald W.

2015-01-01

Autophagy is an evolutionarily-conserved process that delivers diverse cytoplasmic components to the lysosomal compartment for either recycling or degradation. This involves the removal of protein aggregates, the turnover of organelles, as well as the elimination of intracellular pathogens. In this situation, when only specific cargoes should be targeted to the lysosome, the potential targets can be selectively marked by the attachment of ubiquitin in order to be recognized by autophagy-recep...

COMMITTEES: SQM2009 - 14th International Conference on Strangeness in Quark Matter SQM2009 - 14th International Conference on Strangeness in Quark Matter

Science.gov (United States)

2008-04-01

Local Organizing Committee Takeshi Kodama Chair, UFRJ Jun Takahashi Co-chair, UNICAMP Ignácio Bediaga e Hickman CBPF Eduardo Fraga UFRJ Frederique Grassi USP Yogiro Hama USP Gastão Krein IFT Erasmo Madureira Ferreira UFRJ Marcelo G. Munhoz USP Fernando Navarra USP Sandra Padula IFT Alejandro Szanto de Toledo USP César Augusto Zen Vasconcellos UFRGS International Advisory Committee Jörg Aichelin Nantes Federico Antinori Padova Tamás Biró Budapest Peter Braun-Munzinger GSI Jean Cleymans Cape Town Láaszló Csernai Bergen Timothy Hallman BNL Huan Zhong Huang UCLA Takeshi Kodama Rio de Janeiro Yu-Gang Ma Shanghai Jes Madsen Aarhus Ágnes Mócsy Pratt University Berndt Müller Duke University Grazyna Odyniec LBNL Helmut Oeschler Darmstadt Johann Rafelski Arizona Hans Georg Ritter LBNL Gunther Rolland MIT Karel Šafařík CERN Ladislav Sandor Kosice University Jack Sandweiss Yale University George S F Stephans MIT Horst Stöcker Frankfurt Larry McLerranBNL Helmut Satz Universitä Bielefeld Nu Xu LBNL Fuqiang Wang Purdue University William A. Zajc Columbia University Pengfei Zhuang Tsinghua University

Rehabilitative treatment of cleft lip and palate: experience of the Hospital for Rehabilitation of Craniofacial Anomalies - USP (HRAC-USP - part 3: Oral and Maxillofacial Surgery

Directory of Open Access Journals (Sweden)

José Alberto de Souza Freitas

2012-12-01

Full Text Available This paper presents the treatment protocol of maxillofacial surgery in the rehabilitation process of cleft lip and palate patients adopted at HRAC-USP. Maxillofacial surgeons are responsible for the accomplishment of two main procedures, alveolar bone graft surgery and orthognathic surgery. The primary objective of alveolar bone graft is to provide bone tissue for the cleft site and then allow orthodontic movements for the establishment of an an adequate occlusion. When performed before the eruption of the maxillary permanent canine, it presents high rates of success. Orthognathic surgery aims at correcting maxillomandibular discrepancies, especially anteroposterior maxillary deficiencies, commonly observed in cleft lip and palate patients, for the achievement of a functional occlusion combined with a balanced face.

Enzymes in biogenesis of plant cell wall polysaccharides. Enzyme characterization using tracer techniques

International Nuclear Information System (INIS)

Dickinson, D.B.

1975-01-01

Enzymes and metabolic pathways, by which starch and cell wall polysaccharides are formed, were investigated in order to learn how these processes are regulated and to identify the enzymatic regulatory mechanisms involved. Germinating lily pollen was used for studies of cell wall formation, and pollen and maize endosperm for studies of starch biosynthesis. Hexokinase being the first step in conversion of hexoses to starch, wall polysaccharides and respiratory substrates, maize endosperm enzyme was assayed by its conversion of 14 C-hexose to 14 C-hexose-6-P, and rapid separation of the two labelled compounds on anion-exchange paper. This enzyme did not appear to be under tight regulation by feed-back inhibition or activation, nor to be severely inhibited by glucose-6-P or activated by citrate. ADP-glucose pyrophosphorylase and other pyrophosphorylases were assayed radiochemically with 14 C-glucose-1-P (forward direction) or 32-PPsub(i) (reverse direction). They showed that the maize endosperm enzyme was activated by the glycolytic intermediates fructose-6-P and 3-phosphoglycerate, and that low levels of the enzyme were present in the high sucrose-low starch mutant named shrunken-2. Under optimal in-vitro assay conditions, the pollen enzyme reacted four times faster than the observed in-vivo rate of starch accumulation. Biogenesis of plant cell wall polysaccharides requires the conversion of hexose phosphates to various sugar nucleotides and utilization of the latter by the appropriate polysaccharide synthetases. Lily pollen possesses a β-1,3-glucan synthetase which is activated up to six-fold by β-linked oligosaccharides. Hence, the in-vivo activity of this enzyme may be modulated by such effector molecules

Supplement to the paper 'Quadratic Sagnac effect — the influence of the gravitational potential of the Coriolis force on the phase difference between the arms of a rotating Michelson interferometer (an explanation of D C Miller's experimental results, 1921 – 1926)' (Usp. Fiz. Nauk185 431 (2015) [Phys. Usp.58 398 (2015)])

International Nuclear Information System (INIS)

Malykin, G B; Pozdnyakova, V I

2015-01-01

The paper 'Quadratic Sagnac effect — the influence of the gravitational potential of the Coriolis force on the phase difference between the arms of a rotating Michelson interferometer (an explanation of D C Miller's experimental results, 1921 – 1926)' (Usp. Fiz. Nauk 185 431 (2015) [Phys. Usp. 58 398 (2015)]) is amended and supplemented with information concerning earlier work on the influence of rotation on Michelson – Morley's nonzero results. (letters to the editors)

Synthesis and enzyme inhibitory studies of some new N-alkylated/aralkylated N-(4-ethoxyphenyl)-2,3-drobenzo-(1,4)-dioxin-6-sulfonamides

International Nuclear Information System (INIS)

Abbasi, M.A.; Islam, M.; Rehman, A.U.; Siddiqui, S.Z.

2016-01-01

The research endeavor was aimed to synthesize N-alkyl/aralkylated-N-(4-ethoxyphenyl)-2,3-dihydrobenzo-(1,4)-dioxine-6 sulfonamides and to evaluate their enzyme inhibitory potential. The target molecules were synthesized in two steps. The first step involved the reaction of 4-ethoxyaniline (1) with N-2,3-dihydrobenzo(1,4)-dioxin-6-sulfonyl chloride (2) under dynamic pH control maintained by 10% aqueous Na/sub 2/CO/sub 3/ to yield N-(4-ethoxyphenyl)-2,3-dihydrobenzo-(1,4)-dioxine-6-sulfonamide (3). In second step parent compound 3 was reacted with various alkyl/aralkyl halides (4a-l) in N,N'-dimethylformamide and catalytic amount of lithium hydride to accomplish some new N-alkyl/aralkylated-N-(4-ethoxyphenyl)-2,3-dihydrobenzo-(1,4)-dioxine-6 sulfonamides (5a-l). Probable structures of the synthesized compounds were characterized by contemporary spectral techniques i.e. IR, 1H-NMR and EIMS and were finally evaluated for enzyme inhibitory potential against a-glucosidase and urease. The synthesized compounds exhibited moderate to weak therapeutic potential throughout the series. (author)

The 14C-monomethylamino-antipyrine breath test as in vivo parameter for characterizing the induction of the drug catabolizing enzyme system in the guinea pig

International Nuclear Information System (INIS)

Gramatzki, S.

1981-01-01

The aim of these investigations was to help clarify the following questions: 1) Does MAAP, following 14 C labelling of the exocyclic aminomethyl group, offer a suitable substrate for a breath test in guinea pigs. 2) Which procedures for evaluating the 14 C exhalation curves of the breath test are especially valid. 3) Can an induction of the drug catabolizing enzyme system following pre-treatment with various inducing substances be detected by the 14 C-MAAP breath test. 4) Do inducer-specific differences arise in response to the 14 C-MAAP breath test by which the inducers can be characterized. 5) Is monomethylamino-antipyrine similar to amidopyrine in that it is a suitable independent in vivo parameter for the drug metasbolizing enzyme system in the liver of guinea pigs. (orig./MG) [de

Interference of aldehyde metabolizing enzyme with diamine oxidase/histaminase/activity as determined by /sup 14/C putrescine method

Energy Technology Data Exchange (ETDEWEB)

Fogel, W A [Polish Academy of Sciences, Cracow (Poland). Inst. of Pharmacology; Bieganski, T; Wozniak, J; Maslinski, C

1978-01-01

The ..delta../sup 1/ pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi /sup 14/C putrescine test (1961, Archs Biochem. Biophys., vol.95, 242), has been investigated in several tissue homogenates. When guinea pig liver homogenate was used as a source of enzyme in the presence of aldehyde dehydrogenase inhibitors chlorate hydrate and acetaldehyde the level of formation ..delta../sup 1/ pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced ..delta../sup 1/ pyrroline formation, but to a lesser degree. In other tissues, with very high initial diamine oxidase activity (rat intestine, dog kidney) or with very low diamine oxidase activity (guinea pig skin, dog liver) the influence of these inhibitors was only slight. Pyrazole, an inhibitor of alcohol dehydrogenase exerted only a small effect on ..delta../sup 1/ pyrroline formation. All aldehyde-metabolizing enzymes inhibitors, except pyrazole, were without effect on purified pea seddling and hog kidney diamine oxidases. The use of aldehyde-metabolizing enzymes inhibitors may help to reveal the real values of diamine oxidase activity, when tissues homogenates are used as a source of enzyme.

Eucalyptus ESTs involved in the production of 9-cis epoxycarotenoid dioxygenase, a regulatory enzyme of abscisic acid production

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Iraê A. Guerrini

2005-01-01

Full Text Available Abscisic acid (ABA regulates stress responses in plants, and genomic tools can help us to understand the mechanisms involved in that process. FAPESP, a Brazilian research foundation, in association with four private forestry companies, has established the FORESTs database (https://forests.esalq.usp.br. A search was carried out in the Eucalyptus expressed sequence tag database to find ESTs involved with 9-cis epoxycarotenoid dioxygenase (NCED, the regulatory enzyme for ABA biosynthesis, using the basic local BLAST alignment tool. We found four clusters (EGEZLV2206B11.g, EGJMWD2252H08.g, EGBFRT3107F10.g, and EGEQFB1200H10.g, which represent similar sequences of the gene that produces NCED. Data showed that the EGBFRT3107F10.g cluster was similar to the maize (Zea mays NCED enzyme, while EGEZLV2206B11.g and EGJMWD2252H08.g clusters were similar to the avocado (Persea americana NCED enzyme. All Eucalyptus clusters were expressed in several tissues, especially in flower buds, where ABA has a special participation during the floral development process.

Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

Energy Technology Data Exchange (ETDEWEB)

Byrne, Ann; McLaren, Rajashree P.; Mason, Paul; Chai, Lilly; Dufault, Michael R.; Huang, Yinyin; Liang, Beirong; Gans, Joseph D.; Zhang, Mindy; Carter, Kara; Gladysheva, Tatiana B.; Teicher, Beverly A.; Biemann, Hans-Peter N.; Booker, Michael; Goldberg, Mark A.; Klinger, Katherine W.; Lillie, James [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Madden, Stephen L., E-mail: steve.madden@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Jiang, Yide, E-mail: yide.jiang@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States)

2010-01-15

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21{sup /Cip} and p27{sup /Kip1}. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

Synthesis and analysis of the opioid analgesic [14C]-fentanyl

International Nuclear Information System (INIS)

Bagley, J.R.; Wilhelm, J.A.

1992-01-01

The synthesis of [ 14 C]-fentanyl, the radiolabelled congener of the potent opioid analgesic chosen for utilization in drug disposition studies, is described. [ 14 C]-Labelling was achieved in the first of two steps, a room temperature reduction of the in situ generated Schiff base from 1-phenylethyl-4-piperidone and [UL- 14 C]-aniline hydrochloride with sodium triacetoxyborohydride. A nearly instantaneous production of fentanyl was accomplished at room temperature with the addition of propionyl chloride. The overall radiochemical yield was 18%. The method described is efficiently adaptable for submicromolar scale while yielding a product of sufficient specific activity for in vivo studies. Our solvent system for thin layer chromatography was superior to the USP system reported for chromatographic analysis of fentanyl. This is the first reported preparation of [ 14 C]-fentanyl with the radiolabel in the aniline benzene ring. (author)

Structure and function of α-glucan debranching enzymes

DEFF Research Database (Denmark)

Møller, Marie Sofie; Henriksen, Anette; Svensson, Birte

2016-01-01

α-Glucan debranching enzymes hydrolyse α-1,6-linkages in starch/glycogen, thereby, playing a central role in energy metabolism in all living organisms. They belong to glycoside hydrolase families GH13 and GH57 and several of these enzymes are industrially important. Nine GH13 subfamilies include α......-glucan debranching enzymes; isoamylase and glycogen debranching enzymes (GH13_11); pullulanase type I/limit dextrinase (GH13_12–14); pullulan hydrolase (GH13_20); bifunctional glycogen debranching enzyme (GH13_25); oligo-1 and glucan-1,6-α-glucosidases (GH13_31); pullulanase type II (GH13_39); and α-amylase domains......_39 enzymes could represent a “missing link” between the strictly α-1,6-specific debranching enzymes and the enzymes with dual specificity and α-1,4-linkage preference....

TRANSPLANTE RENAL EM BLOCO DE DOADORES-CADÁVERES INFANTIS: EXPERIÊNCIA DO HCFMRP-USP IN BLOC KIDNEY TRANSPLANTATION FROM PEDIATRIC DONORS THE HCFMRP-USP EXPERIENCE

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A.A.Jr Rodrigues

2001-01-01

Full Text Available A grande procura de órgãos para transplante tornou necessária a aceitação de potenciais doadores pediátricos a fim de aumentar a oferta. Trabalhos anteriores relatando menor sobrevida do enxerto e taxas maiores de complicação fizeram com que cirurgiões considerassem o assunto com cautela. Esse é um estudo retrospectivo com o objetivo de analisar os resultados de transplantes renais em bloco de doadores infantis (The high demand for organs for transplantation has made it necessary to consider using the youngest of potential donors in order to increase the organ supply. Previous reports of decreased graft survival and increased complication rates have made surgeons wary of using such kidneys. This is a chart review with the objective to analyse the results achieved with transplantation of children kidneys (< 2 years in the HCFMRP-USP from 1998 to 2000. A total of 5 pacients received en bloc renal transplants from donors aged 9 months to 2 years old, weighthing between 8 and 14 kg. Results: Only one patient lost the allograft within 8 days of transplantation due to allograft trombosis. Two patients had ureteral complications, one with ureteral necrosis and urinary fistula and the other with total ureterovesical anastomosis disrupture. Both were successfully repaired. Intra operatory complication was experienced in one pacient, with arterial anastomosis rupture. In loco reperfusion and repair was made. Another patient had inferior renal pole rupture, treated with polar nefrectomy at the 4º PO. Despite such problems, mean follow up of 20,7 months showed serum creatinine ranging from 0,9 to 2 mg/100ml, with no serious complications. Conclusion: Our data support the use of en bloc child cadaveric kidneys, even from infant donors less than two year of age, when the organ necessity supplant the higher rates of complications.

Comparison of anesthetic efficacy between lidocaine with and without magnesium sulfate USP 50% for inferior alveolar nerve blocks in patients with symptomatic irreversible pulpitis.

Science.gov (United States)

Shetty, Krishna Prasad; Satish, Sarvepalli Venkata; Kilaru, Krishna Rao; Sardar, Poonam; Luke, Alexander M

2015-04-01

The purpose of this prospective, randomized, double-blind, placebo-controlled study was to compare the anesthetic efficacy between lidocaine with and without magnesium sulfate USP 50% for inferior alveolar nerve (IAN) blocks in patients with symptomatic irreversible pulpitis. One hundred patients with symptomatic irreversible pulpitis of mandibular posterior teeth were selected for the study. The patients received 1 mL magnesium sulfate USP 50% or distilled water (placebo) 1 hour before administration of conventional IAN block. Endodontic access cavity preparation was initiated 15 minutes after the IAN block injection. Lip numbness was recorded for all the patients. Success of IAN block was defined as no or mild pain on the visual analogue scale during access cavity preparation and initial instrumentation. The success rate for the IAN block was 58% for magnesium sulfate group and 32% for the placebo group, with statistically significant difference between the 2 groups (P = .016). In mandibular posterior teeth diagnosed with symptomatic irreversible pulpitis, preoperative administration of 1 mL magnesium sulfate USP 50% resulted in statistically significant increase in success of IAN block compared with placebo. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Padronização da coleta de dados nas bibliotecas do SIBi/USP

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Dorotéa Maris Estela Fill

Full Text Available A coleta de dados estatísticos no Sistema Integrado de Bibliotecas da USP começou a ser realizada de forma padronizada e sistêmica a partir de 1987, utilizando como ferramenta o formulário denominado Relatório Individual por Bibliotecas - RIBi. Em 2002, foi criado um projeto específico para analisar e aprimorar o RIBi cuja proposta foi avaliada e validada pelo Sistema. Apresentam-se as ações empreendidas pela equipe, entre elas a revisão do Guia de Preenchimento do RIBi e a plataforma web.

GMP production of [18F]FDOPA and issues concerning its quality analyses as in USP 'Fluorodopa F 18 Injection'

International Nuclear Information System (INIS)

Kao, C.K.; Hsu Wenlin; Xie Hengli; Lin Mingchi; Lan Wenchun; Chao Haoyu

2011-01-01

Lately, 6-[ 18 F]fluoro-L-DOPA (FDOPA) has found increase in its clinical demand for whole-body positron emission tomography (PET) scans, and two key issues in fulfilling this demand are the difficulties in producing FDOPA under the recently imposed PET drug good manufacturing practice (GMP) regulations and in providing it in the quality meeting the terms of major compendia. This paper describes the approaches for the GMP production of FDOPA and for the product testing to meet the standard of United States Pharmacopeia (USP) 'Fluorodopa F 18 Injection.' FDOPA was produced by the carrier-added electrophilic aromatic substitution reaction in the facility complying Pharmaceutical Inspection Cooperation Scheme clean room standard. The special aseptic handling technique was applied to minimize the bioburden. The product quality control followed all testing items and procedures, including three different settings of high performance liquid chromatography (HPLC). The process yielded FDOPA average 2.60±0.26 GBq (N=22) in every batch. All qualities of the product were within the specifications described in the USP 'Fluorodopa F 18 Injection.' The entire production was audited by the government authority and certified to comply with the latest PET drug GMP regulation. Our efforts in producing FDOPA following all aspects of GMP requirements have resulted in a product with the USP quality and certified as GMP complied. The routine production yields enough doses for three to four whole-body scans in each batch. The issues discussed in the report provide good reference for producers planning in routine production for PET drugs that are not commonly produced or with complicated compendial quality control tests. (author)

Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

Science.gov (United States)

Pek, Han Bin; Lim, Pei Yu; Liu, Chengcheng; Lee, Dong-Yup; Bi, Xuezhi; Wong, Fong Tian; Ow, Dave Siak-Wei

2017-05-01

To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively. Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

Azole affinity of sterol 14α-demethylase (CYP51) enzymes from Candida albicans and Homo sapiens.

Science.gov (United States)

Warrilow, Andrew G; Parker, Josie E; Kelly, Diane E; Kelly, Steven L

2013-03-01

Candida albicans CYP51 (CaCYP51) (Erg11), full-length Homo sapiens CYP51 (HsCYP51), and truncated Δ60HsCYP51 were expressed in Escherichia coli and purified to homogeneity. CaCYP51 and both HsCYP51 enzymes bound lanosterol (K(s), 14 to 18 μM) and catalyzed the 14α-demethylation of lanosterol using Homo sapiens cytochrome P450 reductase and NADPH as redox partners. Both HsCYP51 enzymes bound clotrimazole, itraconazole, and ketoconazole tightly (dissociation constants [K(d)s], 42 to 131 nM) but bound fluconazole (K(d), ~30,500 nM) and voriconazole (K(d), ~2,300 nM) weakly, whereas CaCYP51 bound all five medical azole drugs tightly (K(d)s, 10 to 56 nM). Selectivity for CaCYP51 over HsCYP51 ranged from 2-fold (clotrimazole) to 540-fold (fluconazole) among the medical azoles. In contrast, selectivity for CaCYP51 over Δ60HsCYP51 with agricultural azoles ranged from 3-fold (tebuconazole) to 9-fold (propiconazole). Prothioconazole bound extremely weakly to CaCYP51 and Δ60HsCYP51, producing atypical type I UV-visible difference spectra (K(d)s, 6,100 and 910 nM, respectively), indicating that binding was not accomplished through direct coordination with the heme ferric ion. Prothioconazole-desthio (the intracellular derivative of prothioconazole) bound tightly to both CaCYP51 and Δ60HsCYP51 (K(d), ~40 nM). These differences in binding affinities were reflected in the observed 50% inhibitory concentration (IC(50)) values, which were 9- to 2,000-fold higher for Δ60HsCYP51 than for CaCYP51, with the exception of tebuconazole, which strongly inhibited both CYP51 enzymes. In contrast, prothioconazole weakly inhibited CaCYP51 (IC(50), ~150 μM) and did not significantly inhibit Δ60HsCYP51.

Estudo de compatibilização de linguagens documentárias na área odontológica para o banco de dados bibliográficos da USP - DEDALUSStudy of compatibility of docummentary languages in dentistry for the bibliographic data base of USP Dedalus

Directory of Open Access Journals (Sweden)

Suely Cafazzi Prati

1997-11-01

Full Text Available A partir da criação do Banco de Dados Bibliográficos da USP - DEDALUS, em 1981, pelo Sistema Integrado de Bibliotecas da USP (SIBi-USP e, considerando ainda a crescente especificidade das áreas de conhecimento, bem como o aumento da produtividade científica, houve a preocupação com a necessidade de se estabelecer critérios para a formação de linguagens documentárias como garantia da qualidade no acesso à informação pelos usuários. Criou-se uma Comissão de Estudos (META 1 com essa finalidade. Tal Comissão foi subdividida em diferentes grupos de acordo com as suas áreas de especialidade. Desse modo, para a Odontologia, foi formado um grupo de trabalho envolvendo as bibliotecas das Faculdades de Odontologia de São Paulo e Bauru, para desenvolver os seguintes procedimentos metodológicos: 1 compatibilizar os termos de odontologia, com base nas Fontes de Referência existentes na área; 2 estabelecer uma única estrutura temática; 3 incluir nesta estrutura os blocos de assuntos gerados pelo programa META 01; 4 analisar os assuntos bloco a bloco, procurando definir as relações lógico-semânticas; 5 conceituar os termos que forem necessários; 6 obter feedback, dos usuários, bibliotecários e especialistas, como avaliação de acompanhamento e final. Os resultados parciais até o momento com relação à compatibilização dos termos foram: estabelecimento do critério da linguagem adotada segundo o perfil de interesse do usuário da área odontológica e definição de indicadores de qualidade frente à limitação dos instrumentos de trabalho existentes.

The energy rational and alternative sources use at USP: results analysis; O uso racional de energia e fontes alternatives na USP: analise de resultados

Energy Technology Data Exchange (ETDEWEB)

Saidel, Marco Antonio; Gimenes, Andre Luiz Veiga; Castello Branco, Paula Luiz [Universidade de Sao Paulo (USP), SP (Brazil). Dept. de Engenharia de Energia e Automacao Eletricas. Grupo de Energia]. E-mails: saidel@pea.usp.br; gimenes@gmail.com; paulacastello@pea.usp.br

2006-07-01

This paper presents results from the PUREFA - Programa de Uso Racional de Energia e Fontes Alternativas da Universidade de Sao Paulo -, in accordance with actions originated on the 13 physical goals and respective impacts for the University. The actions comprehend: the extension of the distributed energy generation of USP through implantation of 9 kWp photovoltaic and 15 kW of biogas generation; implantation of permanent incentives to the efficient and rational use of energy; creation of regulations for the energy efficiency on buildings; replacement of public and internal lighting systems and air-conditioning; installation of solar heating systems at Sao Carlos, Piracicaba and Sao Paulo, replacing the electric shower; installation of water heating solar system at central restaurant at campus CUASO, at Sao Paulo. The results show the importance of destination resources for the energy efficiency of public sector, the fast return of the investments performed and the potentialization capacity and the dissemination of that actions when applied to the University.

Effect of Probiotic Bacillus megaterium PTB 1.4 on the Population of Intestinal Microflora, Digestive Enzyme Activity and the Growth of Catfish (Clarias sp.

Directory of Open Access Journals (Sweden)

Wahyu Afrilasari

2016-10-01

Full Text Available This study aimed to analyze the effect of Bacillus megaterium PTB 1.4 on the population of intestinal microflora, digestive enzyme activity, and the growth of catfish. Gnotobiotic and normal fish were used. Treatment using gnotobiotic was divided into gnoto (with feed and 100 μg/mL rifampicin and gnotoplus (with feed, 100 μg/mL rifampicin, and 1% probiotic; whereas treatment using normal fish was divided into normalplus (with feed and 1% probiotic and normal (only feed. The amount of bacteria on gastrointestinal tract was measured 30 days after treatments using the total plate count method. The results indicated no significant difference in bacterial growth between gnotobiotic and normal fish. The total amount of probiotic bacteria with normalplus treatment was significantly different with gnotoplus. The activity of protease and amylase enzymes, and specific growth rate in normalplus treatment were significantly higher (p < 0.05 than other treatments. Bacillus megaterium PTB 1.4 increased the activity of digestive enzymes and the growth of catfish.

Synthesis and analysis of the opioid analgesic [[sup 14]C]-fentanyl

Energy Technology Data Exchange (ETDEWEB)

Bagley, J.R.; Wilhelm, J.A. (Anaquest Inc., Murray Hill, NJ (United States))

1992-11-01

The synthesis of [[sup 14]C]-fentanyl, the radiolabelled congener of the potent opioid analgesic chosen for utilization in drug disposition studies, is described. [[sup 14]C]-Labelling was achieved in the first of two steps, a room temperature reduction of the in situ generated Schiff base from 1-phenylethyl-4-piperidone and [UL-[sup 14]C]-aniline hydrochloride with sodium triacetoxyborohydride. A nearly instantaneous production of fentanyl was accomplished at room temperature with the addition of propionyl chloride. The overall radiochemical yield was 18%. The method described is efficiently adaptable for submicromolar scale while yielding a product of sufficient specific activity for in vivo studies. Our solvent system for thin layer chromatography was superior to the USP system reported for chromatographic analysis of fentanyl. This is the first reported preparation of [[sup 14]C]-fentanyl with the radiolabel in the aniline benzene ring. (author).

Development and validation of an ICP-MS method for the determination of elemental impurities in TP-6076 active pharmaceutical ingredient (API) according to USP 〈232〉/〈233〉.

Science.gov (United States)

Chahrour, Osama; Malone, John; Collins, Mark; Salmon, Vrushali; Greenan, Catherine; Bombardier, Amy; Ma, Zhongze; Dunwoody, Nick

2017-10-25

The new guidelines of the United States pharmacopeia (USP), European pharmacopeia (EP) and international conference on harmonization (ICH) regulating elemental impurities limits in pharmaceuticals signify the end of unspecific analysis of metals as outlined in USP 〈231〉. The new guidelines specify both daily doses and concentration/limits of elemental impurities in pharmaceutical final products, active pharmaceutical ingredients (API) and excipients. In chapter USP 〈233〉 method implementation, validation and quality control during the analytical process are described. We herein report the use of a stabilising matrix that overcomes low spike recovery problem encountered with Os and allows the determination of all USP required elemental impurities (As, Cd, Hg, Pb, V, Cr, Ni, Mo, Cu, Pt, Pd, Ru, Rh, Os and Ir) in a single analysis. The matrix was used in the validation of a method to determine elemental impurities in TP-6076 active pharmaceutical ingredient (API) by ICP-MS according to the procedures defined in USP〈233〉 and to GMP requirements. This validation will support the regulatory submission of TP-6076 which is a novel tetracycline analogue effective against the most urgent multidrug-resistant gram-negative bacteria. Evaluation of TP-6076 in IND-enabling toxicology studies has led to the initiation of a phase 1 clinical trial. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and application of a biorelevant dissolution method using USP apparatus 4 in early phase formulation development.

Science.gov (United States)

Fang, Jiang B; Robertson, Vivian K; Rawat, Archana; Flick, Tawnya; Tang, Zhe J; Cauchon, Nina S; McElvain, James S

2010-10-04

Dissolution testing is frequently used to determine the rate and extent at which a drug is released from a dosage form, and it plays many important roles throughout drug product development. However, the traditional dissolution approach often emphasizes its application in quality control testing and usually strives to obtain 100% drug release. As a result, dissolution methods are not necessarily biorelevant and meaningful application of traditional dissolution methods in the early phases of drug product development can be very limited. This article will describe the development of a biorelevant in vitro dissolution method using USP apparatus 4, biorelevant media, and real-time online UV analysis. Several case studies in the areas of formulation selection, lot-to-lot variability, and food effect will be presented to demonstrate the application of this method in early phase formulation development. This biorelevant dissolution method using USP apparatus 4 provides a valuable tool to predict certain aspects of the in vivo drug release. It can be used to facilitate the formulation development/selection for pharmacokinetic (PK) and clinical studies. It may also potentially be used to minimize the number of PK studies, and to aid in the design of more efficient PK and clinical studies.

Heparin sodium compliance to USP monograph: structural elucidation of an atypical 2.18 ppm NMR signal.

Science.gov (United States)

Mourier, Pierre A J; Guichard, Olivier Y; Herman, Fréderic; Viskov, Christian

2012-01-01

The ¹H nuclear magnetic resonance (NMR) acceptance criteria in the new heparin US Pharmacopeia (USP) monograph do not take into account potential structural modifications responsible for any extra signals observed in ¹H NMR spectra, some purified heparins may be non-compliant under the proposed new USP guidelines and incorrectly classified as unsuitable for pharmaceutical use. Heparins from the "ES" source, containing an extra signal at 2.18 ppm, were depolymerized under controlled conditions using heparinases I, II, and III. The oligosaccharides responsible for the 2.18 ppm signal were enriched using orthogonal chromatographic techniques. After multiple purification steps, we obtained an oligosaccharide mixture containing a highly enriched octasaccharide bearing the structural modification responsible for the extra signal. Following heparinase I depolymerization, a pure tetrasaccharide containing the fingerprint structural modification was isolated for full structural determination. Using 1D and 2D ¹H NMR spectroscopy, the structural moiety responsible for the extra signal at 2.18 ppm was identified as an acetyl group on the heparin backbone, most likely resulting from a very minor manufacturing process side reaction that esterifies the uronic acid at position 3. Such analytical peculiarity has always been present in this heparin source and it was used safety over the years. Copyright © 2012 Elsevier B.V. All rights reserved.

Computational hydrodynamic comparison of a mini vessel and a USP 2 dissolution testing system to predict the dynamic operating conditions for similarity of dissolution performance.

Science.gov (United States)

Wang, Bing; Bredael, Gerard; Armenante, Piero M

2018-03-25

The hydrodynamic characteristics of a mini vessel and a USP 2 dissolution testing system were obtained and compared to predict the tablet-liquid mass transfer coefficient from velocity distributions near the tablet and establish the dynamic operating conditions under which dissolution in mini vessels could be conducted to generate concentration profiles similar to those in the USP 2. Velocity profiles were obtained experimentally using Particle Image Velocimetry (PIV). Computational Fluid Dynamics (CFD) was used to predict the velocity distribution and strain rate around a model tablet. A CFD-based mass transfer model was also developed. When plotted against strain rate, the predicted tablet-liquid mass transfer coefficient was found to be independent of the system where it was obtained, implying that a tablet would dissolve at the same rate in both systems provided that the concentration gradient between the tablet surface and the bulk is the same, the tablet surface area per unit liquid volume is identical, and the two systems are operated at the appropriate agitation speeds specified in this work. The results of this work will help dissolution scientists operate mini vessels so as to predict the dissolution profiles in the USP 2, especially during the early stages of drug development. Copyright © 2018 Elsevier B.V. All rights reserved.

Dynamic relationships between microbial biomass, respiration, inorganic nutrients and enzyme activities: informing enzyme based decomposition models

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Daryl L Moorhead

2013-08-01

Full Text Available We re-examined data from a recent litter decay study to determine if additional insights could be gained to inform decomposition modeling. Rinkes et al. (2013 conducted 14-day laboratory incubations of sugar maple (Acer saccharum or white oak (Quercus alba leaves, mixed with sand (0.4% organic C content or loam (4.1% organic C. They measured microbial biomass C, carbon dioxide efflux, soil ammonium, nitrate, and phosphate concentrations, and β-glucosidase (BG, β-N-acetyl-glucosaminidase (NAG, and acid phosphatase (AP activities on days 1, 3, and 14. Analyses of relationships among variables yielded different insights than original analyses of individual variables. For example, although respiration rates per g soil were higher for loam than sand, rates per g soil C were actually higher for sand than loam, and rates per g microbial C showed little difference between treatments. Microbial biomass C peaked on day 3 when biomass-specific activities of enzymes were lowest, suggesting uptake of litter C without extracellular hydrolysis. This result refuted a common model assumption that all enzyme production is constitutive and thus proportional to biomass, and/or indicated that part of litter decay is independent of enzyme activity. The length and angle of vectors defined by ratios of enzyme activities (BG/NAG versus BG/AP represent relative microbial investments in C (length, and N and P (angle acquiring enzymes. Shorter lengths on day 3 suggested low C limitation, whereas greater lengths on day 14 suggested an increase in C limitation with decay. The soils and litter in this study generally had stronger P limitation (angles > 45˚. Reductions in vector angles to < 45˚ for sand by day 14 suggested a shift to N limitation. These relational variables inform enzyme-based models, and are usually much less ambiguous when obtained from a single study in which measurements were made on the same samples than when extrapolated from separate studies.

HBK-14 and HBK-15 Do Not Influence Blood Pressure, Lipid Profile, Glucose Level, or Liver Enzymes Activity after Chronic Treatment in Rats.

Science.gov (United States)

Pytka, Karolina; Głuch-Lutwin, Monika; Knutelska, Joanna; Jakubczyk, Magdalena; Waszkielewicz, Anna; Kotańska, Magdalena

2016-01-01

Older and even new antidepressants cause adverse effects, such as orthostatic hypotension, hyper- or hypoglycemia, liver injury or lipid disorders. In our previous experiments we showed significant antidepressant- and anxiolytic-like activities of dual 5-HT1A and 5-HT7 antagonists with α1-adrenolitic properties i.e. 1-[(2,6-dimethylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-14) and 1-[(2-chloro-6-methylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-15). Here, we evaluated the influence of chronic administration of HBK-14 and HBK-15 on blood pressure (non-invasive blood pressure measurement system for rodents), lipid profile (total cholesterol, low density lipoproteins-LDL, high density lipoproteins-HDL, triglycerides), glucose level, and liver enzymes activity (aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase). We determined potential antihistaminic (isolated guinea pig ileum) and antioxidant properties (ferric reducing ability of plasma-FRAP, non-protein thiols-NPSH, stable free radical diphenylpicrylhydrazyl-DPPH) cytotoxicity. Our experiments revealed that HBK-14 and HBK-15 did not influence blood pressure, lipid profile, glucose level or liver enzymes activity in rats after 2-week treatment. We also showed that none of the compounds possessed antioxidant or cytotoxic properties at antidepressant- and anxiolytic-like doses. HBK-14 and HBK-15 very weakly blocked H1 receptors in guinea pig ileum. Positive results of our preliminary experiments on the safety of HBK-14 and HBK-15 encourage further studies concerning their effectiveness in the treatment of depression and/or anxiety disorders.

HBK-14 and HBK-15 Do Not Influence Blood Pressure, Lipid Profile, Glucose Level, or Liver Enzymes Activity after Chronic Treatment in Rats.

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Karolina Pytka

Full Text Available Older and even new antidepressants cause adverse effects, such as orthostatic hypotension, hyper- or hypoglycemia, liver injury or lipid disorders. In our previous experiments we showed significant antidepressant- and anxiolytic-like activities of dual 5-HT1A and 5-HT7 antagonists with α1-adrenolitic properties i.e. 1-[(2,6-dimethylphenoxyethoxyethyl]-4-(2-methoxyphenylpiperazine hydrochloride (HBK-14 and 1-[(2-chloro-6-methylphenoxyethoxyethyl]-4-(2-methoxyphenylpiperazine hydrochloride (HBK-15. Here, we evaluated the influence of chronic administration of HBK-14 and HBK-15 on blood pressure (non-invasive blood pressure measurement system for rodents, lipid profile (total cholesterol, low density lipoproteins-LDL, high density lipoproteins-HDL, triglycerides, glucose level, and liver enzymes activity (aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase. We determined potential antihistaminic (isolated guinea pig ileum and antioxidant properties (ferric reducing ability of plasma-FRAP, non-protein thiols-NPSH, stable free radical diphenylpicrylhydrazyl-DPPH cytotoxicity. Our experiments revealed that HBK-14 and HBK-15 did not influence blood pressure, lipid profile, glucose level or liver enzymes activity in rats after 2-week treatment. We also showed that none of the compounds possessed antioxidant or cytotoxic properties at antidepressant- and anxiolytic-like doses. HBK-14 and HBK-15 very weakly blocked H1 receptors in guinea pig ileum. Positive results of our preliminary experiments on the safety of HBK-14 and HBK-15 encourage further studies concerning their effectiveness in the treatment of depression and/or anxiety disorders.

RESULTADOS DO 13º CONTECSI USP – CONGRESSO INTERNACIONAL DE GESTÃO DA TECNOLOGIA E SISTEMAS DE INFORMAÇÃO; 36th WCARS – WORLD CONTINUOUS AUDITING AND REPORTING SYSTEMS SYMPOSIUM; 2º TOI ECA USP – 2º CONGRESSO INTERNACIONAL EM TECNOLOGIA E ORGANIZAÇÃO DA INFORMAÇÃO; 2nd GAESI POLI USP - INTERNATIONAL IT MEETING

OpenAIRE

Riccio, Edson Luiz; Sakata, Marici Cristine Gramacho

2016-01-01

RESUMO Procuramos relatar neste documento as estatísticas, os dados e a importância do 13º CONTECSI – International Conference on Information Systems and Technology Management - Congresso Internacional de Gestão da Tecnologia e Sistemas de Informação, realizado nos dias 1, 2 e 3 de junho de 2016 pelo TECSI/EAC/FEA/USP/ECA/POLI na Universidade de São Paulo, Brasil. Este relatório apresenta: Estatísticas do 13º CONTECSI, Justificativas e Objetivos do evento, Programa, Sessões Plenárias, Consórc...

35 ANOS DE PUBLICAÇÕES ACADÊMICAS DA REVISTA DE ADMINISTRAÇÃO DA USP

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Henrique César Melo Ribeiro

2016-06-01

Full Text Available Este artigo mapeia a produção científica da Revista de Administração da USP no período de 1977 a 2011. Metodologicamente, o estudo se baseia em técnicas de pesquisa bibliométrica e sociométrica, utilizando estatística descritiva e multivariada em 979 artigos identificados. Os principais resultados encontrados foram: predominância de artigos de autoria conjunta; os autores Sbragia e Vasconcellos foram os que mais publicaram artigos; os pesquisadores Porter, M. E. e Kotler, P. foram os mais citados nas referências nos 35 anos de pesquisa; a Universidade de São Paulo foi a Instituição de Ensino Superior (IES que mais publicou; alta centralidade de grau nas redes de coautoria e das IES; a abordagem quantitativa se destacou nos 979 artigos investigados; os temas Cultura Organizacional, Governança Corporativa, Estratégias Empresariais, Mercado de Capitais, Estrutura Organizacional e Desempenho foram os mais evidenciados no período de 1977 a 2011. Concluiu-se de forma geral, que o acervo da Revista de Administração da USP reflete o universo da produção acadêmica da área de administração, ajudando assim a desenvolver, disseminar e socializar a área por meio de artigos científicos publicados na literatura acadêmica nacional.

A academia e participação social no Brasil: teses e dissertações sobre movimentos sociais produzidas na Unicamp e na Usp entre 1970 e 1995

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Samira Kauchakje

2002-01-01

Full Text Available Este artigo apresenta algumas reflexões suscitadas por uma pesquisa de doutoramento que versava sobre teses e dissertações acerca de movimentos sociais produzidas na USP e UNICAMP. A partir de alguns dados deste estudo, este artigo desenvolve-se tendo como pressuposto que há ligação entre interesse acadêmico e realidade socialThis article to present some meditation to one research of graduat about thesis and dissertation about social movement to make in USP and UNICAMP. To breack some consideration in that study, this article developing to support to have a alliance between academic interest and social reality.

H2A-DUBbing the mammalian epigenome: expanding frontiers for histone H2A deubiquitinating enzymes in cell biology and physiology.

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Belle, Jad I; Nijnik, Anastasia

2014-05-01

Posttranslational modifications of histone H2A through the attachment of ubiquitin or poly-ubiquitin conjugates are common in mammalian genomes and play an important role in the regulation of chromatin structure, gene expression, and DNA repair. Histone H2A deubiquitinases (H2A-DUBs) are a group of structurally diverse enzymes that catalyze the removal ubiquitin from histone H2A. In this review we provide a concise summary of the mechanisms that mediate histone H2A ubiquitination in mammalian cells, and review our current knowledge of mammalian H2A-DUBs, their biochemical activities, and recent developments in our understanding of their functions in mammalian physiology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Enzyme Vs. Extremozyme -32 ...

Indian Academy of Sciences (India)

Enzymes are biocatalytic protein molecules that enhance the rates of ... to physical forces (hydrogen bonds, hydrophobic 1, electrostatic and Van der ... conformation. In 1995 ... surface against 14.7% in Klenow poll (some of the hydrophobic.

Mafai e Scipione: a Scuola Romana di Pittura no MAC-USP

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Benjamim Saviani

2012-01-01

Full Text Available Estudo crítico sobre obras de dois pintores italianos, Mario Mafai e Gino Bonichi (dito Scipione, pertencentes à Scuola Romana di Pittura, presentes no Museu de Arte Contemporânea da Universidade de São Paulo (MAC-USP: Rapaz e Natureza-morta (Mafai e Oceano Indiano (Scipione integram o primeiro acervo do atual museu, e são advindas da antiga coleção do Museu de Arte Moderna de São Paulo (MAM-SP, formada por Ciccillo Matarazzo. Além de estudo iconográfico das obras em si, vem adjunto um estudo sobre sua relação com os acervos de arte moderna paulistanos, em formação em meados do século XX, de forte relação com a arte moderna italiana e os projetos político-culturais de ítalo-descendentes presentes em São Paulo, traço marcante do cenário cultural da cidade.

Rehabilitative treatment of cleft lip and palate: experience of the Hospital for Rehabilitation of Craniofacial Anomalies - USP (HRAC-USP) - Part 2: Pediatric Dentistry and Orthodontics

Science.gov (United States)

FREITAS, José Alberto de Souza; GARIB, Daniela Gamba; OLIVEIRA, Thais Marchini; LAURIS, Rita de Cássia Moura Carvalho; de ALMEIDA, Ana Lúcia Pompéia Fraga; NEVES, Lucimara Teixeira; TRINDADE-SUEDAM, Ivy Kiemle; YAEDÚ, Renato Yassutaka Faria; SOARES, Simone; PINTO, João Henrique Nogueira

2012-01-01

The aim of this article is to present the pediatric dentistry and orthodontic treatment protocol of rehabilitation of cleft lip and palate patients performed at the Hospital for Rehabilitation of Craniofacial Anomalies - University of São Paulo (HRAC-USP). Pediatric dentistry provides oral health information and should be able to follow the child with cleft lip and palate since the first months of life until establishment of the mixed dentition, craniofacial growth and dentition development. Orthodontic intervention starts in the mixed dentition, at 8-9 years of age, for preparing the maxillary arch for secondary bone graft procedure (SBGP). At this stage, rapid maxillary expansion is performed and a fixed palatal retainer is delivered before SBGP. When the permanent dentition is completed, comprehensive orthodontic treatment is initiated aiming tooth alignment and space closure. Maxillary permanent canines are commonly moved mesially in order to substitute absent maxillary lateral incisors. Patients with complete cleft lip and palate and poor midface growth will require orthognatic surgery for reaching adequate anteroposterior interarch relationship and good facial esthetics. PMID:22666849

Rehabilitative treatment of cleft lip and palate: experience of the Hospital for Rehabilitation of Craniofacial Anomalies - USP (HRAC-USP - Part 2: Pediatric Dentistry and Orthodontics

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José Alberto de Souza Freitas

2012-04-01

Full Text Available The aim of this article is to present the pediatric dentistry and orthodontic treatment protocol of rehabilitation of cleft lip and palate patients performed at the Hospital for Rehabilitation of Craniofacial Anomalies - University of São Paulo (HRAC-USP. Pediatric dentistry provides oral health information and should be able to follow the child with cleft lip and palate since the first months of life until establishment of the mixed dentition, craniofacial growth and dentition development. Orthodontic intervention starts in the mixed dentition, at 8-9 years of age, for preparing the maxillary arch for secondary bone graft procedure (SBGP. At this stage, rapid maxillary expansion is performed and a fixed palatal retainer is delivered before SBGP. When the permanent dentition is completed, comprehensive orthodontic treatment is initiated aiming tooth alignment and space closure. Maxillary permanent canines are commonly moved mesially in order to substitute absent maxillary lateral incisors. Patients with complete cleft lip and palate and poor midface growth will require orthognatic surgery for reaching adequate anteroposterior interarch relationship and good facial esthetics.

Rehabilitative treatment of cleft lip and palate: experience of the Hospital for Rehabilitation of Craniofacial Anomalies-USP (HRAC-USP)--part 2: pediatric dentistry and orthodontics.

Science.gov (United States)

Freitas, José Alberto de Souza; Garib, Daniela Gamba; Oliveira, Marchini; Lauris, Rita de Cássia Moura Carvalho; Almeida, Ana Lúcia Pompéia Fraga de; Neves, Lucimara Teixeira; Trindade-Suedam, Ivy Kiemle; Yaedú, Renato Yassutaka Faria; Soares, Simone; Pinto, João Henrique Nogueira

2012-01-01

The aim of this article is to present the pediatric dentistry and orthodontic treatment protocol of rehabilitation of cleft lip and palate patients performed at the Hospital for Rehabilitation of Craniofacial Anomalies-University of São Paulo (HRAC-USP). Pediatric dentistry provides oral health information and should be able to follow the child with cleft lip and palate since the first months of life until establishment of the mixed dentition, craniofacial growth and dentition development. Orthodontic intervention starts in the mixed dentition, at 8-9 years of age, for preparing the maxillary arch for secondary bone graft procedure (SBGP). At this stage, rapid maxillary expansion is performed and a fixed palatal retainer is delivered before SBGP. When the permanent dentition is completed, comprehensive orthodontic treatment is initiated aiming tooth alignment and space closure. Maxillary permanent canines are commonly moved mesially in order to substitute absent maxillary lateral incisors. Patients with complete cleft lip and palate and poor midface growth will require orthognatic surgery for reaching adequate anteroposterior interarch relationship and good facial esthetics.

Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30

Energy Technology Data Exchange (ETDEWEB)

Anderson, Lindsey N.; Culley, David E.; Hofstad, Beth A.; Chauvigne-Hines, Lacie M.; Zink, Erika M.; Purvine, Samuel O.; Smith, Richard D.; Callister, Stephen J.; Magnuson, Jon M.; Wright, Aaron T.

2013-12-01

Development of alternative, non-petroleum based sources of bioenergy that can be applied in the short-term find great promise in the use of highly abundant and renewable lignocellulosic plant biomass.1 This material obtained from different feedstocks, such as forest litter or agricultural residues, can yield liquid fuels and other chemical products through biorefinery processes.2 Biofuels are obtained from lignocellulosic materials by chemical pretreatment of the biomass, followed by enzymatic decomposition of cellulosic and hemicellulosic compounds into soluble sugars that are converted to desired chemical products via microbial metabolism and fermentation.3, 4 To release soluble sugars from polymeric cellulose multiple enzymes are required, including endoglucanase, exoglucanase, and β-glucosidase.5, 6 However, the enzymatic hydrolysis of cellulose into soluble sugars remains a significant limiting factor to the efficient and economically viable utilization of lignocellulosic biomass for transport fuels.7, 8 The primary industrial source of cellulose and hemicellulases is the mesophilic soft-rot fungus Trichoderma reesei,9 having widespread applications in food, feed, textile, pulp, and paper industries.10 The genome encodes 200 glycoside hydrolases, including 10 cellulolytic and 16 hemicellulolytic enzymes.11 The hypercellulolytic catabolite derepressed strain RUT-C30 was obtained through a three-step UV and chemical mutagenesis of the original T. reesei strain QM6a,12, 13 in which strains M7 and NG14 were intermediate, having higher cellulolytic activity than the parent strain but less activity and higher catabolite repression than RUT-C30.14 Numerous methods have been employed to optimize the secreted enzyme cocktail of T. reesei including cultivation conditions, operational parameters, and mutagenesis.3 However, creating an optimal and economical enzyme mixture for production-scale biofuels synthesis may take thousands of experiments to identify.

Governance Assessment Corporate Financial Industries in the Core PEIEX POLI - USP - between 2012-2013

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José Flávio Messias

2015-06-01

Full Text Available This present article is an applied research based on micro, small and medium enterprises - SMEs, assisted by Export Industrial Extension Project - PEIEX, APEX Brazil, in partnership with the Center Vanzolini - POLI / USP - SP. In this sense, we tried to identify the adoption of corporate governance practices and financial controls as a management tool in business, since some of them had been met and trained in the previous period. We use the information obtained from the questionnaires applied in PEIEX Project participating companies, as well as information obtained specifically in the visits, trying to identify the importance attached by them to financial controls and assess improvement in the level of corporate governance gathered by these companies

Revista USP em foco: Percorrendo o sertão

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Lucia de Oliveira Almeida

2008-10-01

Full Text Available http://dx.doi.org/10.5007/1984-784x.2008v8n12p146 Em 1989, o reitor José Goldemberg assina o editorial de lançamento da Revista USP, no qual delineia um periódico cuja principal atribuição seria constituir-se em veículo para as pesquisas realizadas por professores e alunos da própria Universidade de São Paulo. No conjunto formado pelos quarenta primeiros fascículos, a tentativa de realização de uma leitura do presente se destaca na seção dossiê, apontada pelo próprio reitor como o espaço principal da revista. Nesses ensaios, em boa medida, o foco está concentrado na criação artística que emerge na década de oitenta e no início dos anos noventa. São textos de peculiar interesse, pois o periódico tenta olhar para o quadro enquanto a tinta ainda está fresca, visando promover um balanço no qual a música, o teatro e o cinema são analisados sem a rede de segurança que a longa distância dos anos oferece aos analistas.

Inovação: uma análise do papel da agência USP de inovação na geração de propriedade intelectual e nos depósitos de patentes da Universidade de São Paulo Analysis of the role of USP agency for innovation in generation of intellectual property and patent deposits of University of São Paulo

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Emerson Antonio Maccari

2011-12-01

Full Text Available A concorrência entre as empresas que atuam no mercado, tanto no âmbito nacional como internacional, vem se intensificando nos últimos anos, em consequência da liberalização comercial e econômica ocorrida na década de 1990. A inovação tecnológica nas empresas tem se configurado como fator fundamental para a manutenção de suas atividades e para o crescimento e desenvolvimento do país. Nesse contexto, as patentes, principalmente as acadêmicas, têm ganhado notoriedade no estabelecimento de políticas públicas em um ambiente caracterizado por essas inovações. Sabendo da importância das patentes, este artigo buscou aprofundar as discussões teóricas no que concerne à investigação do papel da Agência USP de Inovação na geração de propriedade intelectual e nos depósitos de patentes da Universidade de São Paulo. Para tanto, adotou-se como metodologia uma formulação do tipo pesquisa exploratória descritiva, por meio de uma entrevista em profundidade com o diretor geral da Agência USP de Inovação. Os dados coletados na entrevista foram analisados por meio de técnicas qualitativas de análise de conteúdo, permitindo fazer uma descrição detalhada sobre o papel da Agência USP de Inovação na geração de propriedade intelectual e nos depósitos de patentes realizados pela Universidade de São Paulo no contexto brasileiro após a sua criação.The concurrency intensification among the companies that act at the market, both at national and international sphere, has been intensified lately as consequence of commercial and economic liberalization occurred at the nineties years. Companies' technologic innovation has been understood as the essential factor for the maintenance of their activities and for the increase and development of their countries. In this context, the patents, mainly the academic ones, have acquired notoriety at the establishment of public policies in an environment characterized by these innovations

Noncommutative o*(N) and usp*(2N) algebras and the corresponding gauge field theories

International Nuclear Information System (INIS)

Bars, I.; Sheikh-Jabbari, M.M.; Vasiliev, M.A.

2001-03-01

The extension of the noncommutative u * (N) Lie algebra to noncommutative orthogonal and symplectic Lie algebras is studied. Using an anti-automorphism of the star-matrix algebra, we show that the u * (N) can consistently be restricted to o * (N) and usp * (N) algebras that have new mathematical structures. We give explicit fundamental matrix representations of these algebras, through which the formulation for the corresponding noncommutative gauge field theories are obtained. In addition, we present a D-brane configuration with an orientifold which realizes geometrically our algebraic construction, thus embedding the new noncommutative gauge theories in superstring theory in the presence of a constant background magnetic field. Some algebraic generalizations that may have applications in other areas of physics are also discussed. (author)

The golden age of Drosophila research at the Universidade de São Paulo (USP): A testimonial on the decades 1940-1950.

Science.gov (United States)

de Magalhães, Luiz Edmundo; Vilela, Carlos Ribeiro

2014-03-01

This article is a testimonial written by the first author regarding the research work performed with Drosophila between 1943 and 1959, at the Departamento de Biologia Geral of the Universidade de São Paulo (USP), which permitted the building of a nucleus of excellence. This research work, focused on the systematics and population genetics of the Neotropical species, began during the first of Dobzhansky's six visits to USP. Special attention was given to the multinational megaprojects conducted during his longer stays, from August 1948 to July 1949 and from June 1955 to July 1956. The role played by the Rockefeller Foundation is duly remembered, and so is the undeniable contribution brought by Dobzhansky, to the establishment of several laboratories dedicated to research in the field of natural population genetics and to the qualification of human resources. On the other hand, important "backstage" episodes are retrieved which were omitted in the official history and occurred from the planning to the execution of the project, carried out on the Angra dos Reis islands. Special attention was given to the relationship problems which resulted from Dobzhansky's, the leader's, difficult personality and contributed to the failure of the second and last multinational project.

Antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response.

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Jeffrey W Perry

Full Text Available Ubiquitin (Ub is a vital regulatory component in various cellular processes, including cellular responses to viral infection. As obligate intracellular pathogens, viruses have the capacity to manipulate the ubiquitin (Ub cycle to their advantage by encoding Ub-modifying proteins including deubiquitinases (DUBs. However, how cellular DUBs modulate specific viral infections, such as norovirus, is poorly understood. To examine the role of DUBs during norovirus infection, we used WP1130, a small molecule inhibitor of a subset of cellular DUBs. Replication of murine norovirus in murine macrophages and the human norovirus Norwalk virus in a replicon system were significantly inhibited by WP1130. Chemical proteomics identified the cellular DUB USP14 as a target of WP1130 in murine macrophages, and pharmacologic inhibition or siRNA-mediated knockdown of USP14 inhibited murine norovirus infection. USP14 is a proteasome-associated DUB that also binds to inositol-requiring enzyme 1 (IRE1, a critical mediator of the unfolded protein response (UPR. WP1130 treatment of murine macrophages did not alter proteasome activity but activated the X-box binding protein-1 (XBP-1 through an IRE1-dependent mechanism. In addition, WP1130 treatment or induction of the UPR also reduced infection of other RNA viruses including encephalomyocarditis virus, Sindbis virus, and La Crosse virus but not vesicular stomatitis virus. Pharmacologic inhibition of the IRE1 endonuclease activity partially rescued the antiviral effect of WP1130. Taken together, our studies support a model whereby induction of the UPR through cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and the UPR represent novel targets for future development of broad spectrum antiviral therapies.

Gradientes térmicos em whitetopping ultradelgado na pista experimental instrumentada na USP

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Deividi da Silva Pereira

2009-12-01

Full Text Available

Este trabalho apresenta, primeiramente, uma sucinta descrição da execução de um trecho experimental de Whitetopping Ultradelgado (WTUD no campus da Universidade de São Paulo (USP bem como de sua instrumentação. São apresentados e discutidos os resultados referentes a temperaturas obtidos por esta instrumentação durante o outono, analisando as diferenças encontradas nos diferenciais térmicos para placas de diferentes dimensões, obtidos por termoresistores instalados nos cantos, bordas e centro das mesmas. Com base em medidas de deformação obtidas por strain-gages são avaliados valores típicos das tensões induzidas no WTUD pelo efeito isolado do gradiente térmico sobre as placas de concreto de cimento Portland (CCP.

"Tabela Brasileira de Composição de Alimentos - USP: banco de dados de alimentos industrializados"

OpenAIRE

Maria Stella Bonin Ito

2003-01-01

Informações sobre composição de alimentos são de grande importância em qualquer estudo sobre nutrição humana. A Rede Brasileira de Dados de Composição de Alimentos (BRASILFOODS) coordena as atividades nacionais sobre composição de alimentos e está ligada a Rede Latino-Americana de Dados de Composição de Alimentos (LATINFOODS), responsável pela elaboração da Tabela de Composição da América Latina. A Tabela Brasileira de Composição de Alimentos – Universidade de São Paulo (TBCA–USP) foi desenvo...

A decrease in ubiquitination and resulting prolonged life-span of KIT underlies the KIT overexpression-mediated imatinib resistance of KIT mutation-driven canine mast cell tumor cells.

Science.gov (United States)

Kobayashi, Masato; Kuroki, Shiori; Kurita, Sena; Miyamoto, Ryo; Tani, Hiroyuki; Tamura, Kyoichi; Bonkobara, Makoto

2017-10-01

Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by re‑treatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.

Regulation of NF-kappaB activity and inducible nitric oxide synthase by regulatory particle non-ATPase subunit 13 (Rpn13)

DEFF Research Database (Denmark)

Mazumdar, Tuhina; Gorgun, F Murat; Sha, Youbao

2010-01-01

Human Rpn13, also known as adhesion regulating molecule 1 (ADRM1), was recently identified as a novel 19S proteasome cap-associated protein, which recruits the deubiquitinating enzyme UCH37 to the 26S proteasome. Knockdown of Rpn13 by siRNA does not lead to global accumulation of ubiquitinated ce......, this study defines two substrates, with important roles in inflammation and host defense for the Rpn13/UCH37 pathway....

Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes.

Science.gov (United States)

Mattossovich, Rosanna; Iacono, Roberta; Cangiano, Giuseppina; Cobucci-Ponzano, Beatrice; Isticato, Rachele; Moracci, Marco; Ricca, Ezio

2017-11-28

The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-D-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-D-xylans to remove successive D-xylose residues from the non-reducing termini. We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation. Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes

Probiotic activity of lignocellulosic enzyme as bioactivator for rice husk degradation

Science.gov (United States)

Lamid, Mirni; Al-Arif, Anam; Warsito, Sunaryo Hadi

2017-02-01

The utilization of lignocellulosic enzyme will increase nutritional value of rice husk. Cellulase consists of C1 (β-1, 4-glucan cellobiohydrolase or exo-β-1,4glucanase), Cc (endo-β-1,4-glucanase) and component and cellobiose (β-glucocidase). Hemicellulase enzyme consists of endo-β-1,4-xilanase, β-xilosidase, α-L arabinofuranosidase, α-D-glukuronidaseand asetil xilan esterase. This research aimed to study the activity of lignocellulosic enzyme, produced by cows in their rumen, which can be used as a bioactivator in rice husk degradation. This research resulted G6 and G7 bacteria, producing xylanase and cellulase with the activity of 0.004 U mL-1 and 0.021 U mL-1; 0.003 ( U mL-1) and 0.026 (U mL-1) respectively.

Particle diffusional layer thickness in a USP dissolution apparatus II: a combined function of particle size and paddle speed.

Science.gov (United States)

Sheng, Jennifer J; Sirois, Paul J; Dressman, Jennifer B; Amidon, Gordon L

2008-11-01

This work was to investigate the effects of particle size and paddle speed on the particle diffusional layer thickness h(app) in a USP dissolution apparatus II. After the determination of the powder dissolution rates of five size fractions of fenofibrate, including <20, 20-32, 32-45, 63-75, and 90-106 microm, the present work shows that the dependence of h(app) on particle size follows different functions in accordance with the paddle speed. At 50 rpm, the function of h(app) is best described by a linear plot of h{app} = 9.91sqrt d-23.31 (R(2) = 0.98) throughout the particle diameter, d, from 6.8 to 106 microm. In contrast, at 100 rpm a transitional particle radius, r, of 23.7 microm exists, under which linear relationship h(app) = 1.59r (R(2) = 0.98) occurs, but above which h(app) becomes a constant of 43.5 microm. Thus, h(app) changes not only with particle size, but also with the hydrodynamics under standard USP configurations, which has been overlooked in the past. Further, the effects of particle size and paddle speed on h(app) were combined using dimensionless analysis. Within certain fluid velocity/particle regime, linear correlation of h(app)/d with the square-root of Reynolds number (d\\varpi/upsilon){1/2}, that is, h{app}/d = 1.5207 - 9.25 x 10{- 4} (d\\varpi/n){1/2} (R(2) = 0.9875), was observed.

Preparation of D-[U-14C]galactose and α-D-[U-14C]galactose-1-phosphate

International Nuclear Information System (INIS)

Kolina, J.; Hromadkova, B.

1989-01-01

Optically pure D-[U- 14 C]galactose was prepared on a preparatory scale using the galactokinase enzyme. The suggested procedure allows to also prepare a α-D-[U- 14 C]galactose-1-phosphate and L-[U- 14 ]galactose giving good yield. The experiments proved that the raw fraction isolated from yeast of the Kluyveromyces fragilis strain or the Kluyveromyces lactis strain shows sufficient activity. Phosphorylation of D-[U- 14 C]galactose practically terminates after 30 mins of incubation. DL-[U- 14 C]galactose isolated using preparatory paper chromatography from the acid hydrolyzate of [U- 14 C] polysaccharide is a satisfactory radioactive precursor. The developed preparation procedure theoretically contributed towards the further elucidation of the problem of the proportional representation of galactose stereo-isomers in extracellular polysaccharide isolated from red algae. In this respect data in the literature differ and some sources state a significantly higher propertion of L-galactose. The experiments showed that [U- 14 C] polysaccharide isolated from the red algae Porphyridium cruentum prevalently contains D-[U- 14 C]galactose, which confirms the process of enzyme reaction. (author). 1 tab., 4 refs

Impact of vibration and agitation speed on dissolution of USP prednisone tablets RS and various IR tablet formulations.

Science.gov (United States)

Seeger, Nicole; Lange, Sigrid; Klein, Sandra

2015-08-01

Dissolution testing is an in vitro procedure which is widely used in quality control (QC) of solid oral dosage forms and, given that real biorelevant test conditions are applied, can also be used as a predictive tool for the in vivo performance of such formulations. However, if a dissolution method is intended to be used for such purposes, it has to deliver results that are only determined by the quality of the test product, but not by other variables. In the recent past, more and more questions were arising on how to address the effects of vibration on dissolution test results. The present study was performed to screen for the correlation of prednisone dissolution of USP Prednisone Tablets RS with vibration caused by a commercially available vibration source as well as to investigate how drug release from a range of immediate release formulations containing class 1-4 drugs of the biopharmaceutical classification scheme is affected by vibration when performing dissolution experiments at different agitation rates. Results of the present study show that the dissolution process of oral drug formulations can be affected by vibration. However, it also becomes clear that the degree of which a certain level of vibration impacts dissolution is strongly dependent on several factors such as drug properties, formulation parameters, and the design of the dissolution method. To ensure the establishment of robust and predictive dissolution test methods, the impact of variation should thus be considered in method design and validation.

Os primeiros tempos da pesquisa em sociologia da educação na USP The early period of research in sociology of education at USP

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Celso de Rui Beisiegel

2013-01-01

Full Text Available O artigo fundamenta-se na participação do autor como aluno de graduação e pós-graduação no curso de Ciências Sociais da antiga Faculdade de Filosofia, Ciências e Letras da Universidade de São Paulo (FFCL-USP. Considera também informações e experiências por ele acumuladas como pesquisador do Centro Regional de Pesquisas Educacionais (CRPE de São Paulo (a partir de 1957 e como professor da disciplina Sociologia da Educação, após a criação da Faculdade de Educação no âmbito da reforma universitária de 1970. Situa a importância da visão de conjunto dos problemas colocados à disciplina no livro Sociologia educacional, concluído em 1940 por Fernando de Azevedo, e encontra em Florestan Fernandes e, especialmente, em Antonio Candido os pontos de partida para a investigação sistemática da educação sob uma perspectiva sociológica. Nesse sentido, o autor apresenta os primeiros trabalhos de Luiz Pereira, hoje clássicos na disciplina, como admiravelmente embasados nas orientações então encontradas em seus mestres Antonio Candido e Florestan Fernandes. Em seguida, examina a crescente ampliação do número de pesquisadores e das bases institucionais de promoção da pesquisa em sociologia da educação, ampliação esta que sugere amplo elenco de questões sobre a situação atual da investigação sociológica no campo da educação.The article draws from the author’s experience as an undergraduate and later as a graduate student of the course on Social Sciences at the old Faculty of Philosophy, Sciences and Letters of the University of São Paulo (FFCL-USP. It also incorporates information and experiences accumulated as a researcher at the Regional Center for Education Research (CRPE in São Paulo (from 1957 onwards and as a professor in the discipline of Sociology of Education after the establishment of the Faculty of Education within the scope of the 1970 university reform. It situates the importance of the group

The predominant molecular state of bound enzyme determines the strength and type of product inhibition in the hydrolysis of recalcitrant polysaccharides by processive enzymes.

Science.gov (United States)

Kuusk, Silja; Sørlie, Morten; Väljamäe, Priit

2015-05-01

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the (14)C-labeled crystalline polymeric substrates (14)C chitin nanowhiskers and (14)C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed Ki (obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the Ki (obs) of Cel7A was an order of magnitude higher than the true Ki value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The golden age of Drosophila research at the Universidade de São Paulo (USP): A testimonial on the decades 1940–1950

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de Magalhães, Luiz Edmundo; Vilela, Carlos Ribeiro

2014-01-01

This article is a testimonial written by the first author regarding the research work performed with Drosophila between 1943 and 1959, at the Departamento de Biologia Geral of the Universidade de São Paulo (USP), which permitted the building of a nucleus of excellence. This research work, focused on the systematics and population genetics of the Neotropical species, began during the first of Dobzhansky’s six visits to USP. Special attention was given to the multinational megaprojects conducted during his longer stays, from August 1948 to July 1949 and from June 1955 to July 1956. The role played by the Rockefeller Foundation is duly remembered, and so is the undeniable contribution brought by Dobzhansky, to the establishment of several laboratories dedicated to research in the field of natural population genetics and to the qualification of human resources. On the other hand, important “backstage” episodes are retrieved which were omitted in the official history and occurred from the planning to the execution of the project, carried out on the Angra dos Reis islands. Special attention was given to the relationship problems which resulted from Dobzhansky’s, the leader’s, difficult personality and contributed to the failure of the second and last multinational project. PMID:24688301

mRNA decapping enzyme from ribosomes of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Stevens, A.

1980-01-01

By use of [ 3 H]methyl-5'-capped [ 14 C]mRNA from yeast as a substrate, a decapping enzyme activity has been detected in enzyme fractions derived from a high salt wash of ribosomes of Saccharomyces cerevisiae. The product of the decapping reaction is [ 3 H]m 7 GDP. That the enzyme is not a non-specific pyrophosphatase is suggested by the finding that the diphosphate product, m 7 GpppA(G), and UDP-glucose are not hydrolyzed

Revealing hidden effect of earthworm on C distribution and enzyme activity

Science.gov (United States)

Razavi, Bahar S.; Hoang, Duyen; Kuzyakov, Yakov

2017-04-01

Despite its importance for terrestrial nutrient and carbon cycling, the spatial organization and localization of microbial activity in soil and in biopores is poorly understood. We hypothesized that biopores created by earthworm play a critical role in reducing the gap of SOM input and microbial activities between topsoil and subsoil. Accordingly, Carbon (C) allocation by earthworms was related to enzyme distribution along soil profile. For the first time we visualized spatial distribution of enzyme activities (β-glucosidase, chitinase and acid phosphatase) and C allocation (by 14C imaging) in earthworm biopores in topsoil and subsoil. Soil zymography (an in situ method for the analysis of the two-dimensional distribution of enzyme activity in soil) was accompanied with 14C imaging (a method that enables to trace distribution of litter and C in soil profile) to visualize change of enzyme activities along with SOM incorporation by earthworms from topsoil to subsoil. Experiment was set up acquiring rhizoboxes (9×1×50 cm) filled up with fresh soil and 3 earthworms (L. terrestris), which were then layered with 14C-labeled plant-litter of 0.3 MBq on the soil surface. 14C imaging and zymography have been carried out after one month. Activities of all enzymes regardless of their nutrient involvement (C, N, P) were higher in the biopores than in bulk soil, but the differences were larger in topsoil compared to subsoil. Among three enzymes, Phosphatase activity was 4-times higher in the biopore than in the bulk soil. Phosphatase activity was closely associated with edge of burrows and correlate positively with 14C activity. These results emphasized especial contribution of hotspheres such as biopores to C allocation in subsoil - which is limited in C input and nutrients - and in stimulation of microbial and enzymatic activity by input of organic residues, e.g. by earthworms. In conclusion, biopore increased enzymatic mobilization of nutrients (e.g. P) inducing allocation

Study of vitamin D serum level in patients with epilepsy treated with enzyme-inducing and non enzyme-inducing medications

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sima Hashemipour

2014-01-01

Full Text Available Background : Changes of serum minerals and vitamin D have been reported in anticonvulsant drugs user patients. The present study aimed at comparing the changes of serum minerals and vitamin D among two groups of enzyme-inducing and non enzyme-inducing anticonvulsant drug users. Methods: In this study 22 patients treated with enzyme-inducing drugs (carbamazepin, phenytoin, phenobarbital were compared to 25 patients of matched sex, age, and BMI treated with non enzyme-inducing drugs (sodium evaporate, lamotrigine. Serum calcium, phosphate, parathormone, and 25-hydroxy vitamin D were calculated in both groups. Calcium was measured by Calorimetery method. Parathormone and vitamin D were measured using ELISA method. Results: The mean serum vitamin D level was lower in enzyme-inducing than non enzyme-inducing drugs users (15.9±8.3 and 24.2±14.8, P=0.02. Frequency of vitamin D deficiency was higher in enzyme-inducing compared to non enzyme-inducing drugs users, 84% and 48% , respectively (P=0.016. The mean serum calcium level was significantly lower in enzyme-inducing drugs users. (8.7±0.2 vs. 9.0± 0.7, p= 0.05. Four percent in enzyme-inducing group compared to twenty four percent of non enzyme-inducing group had secondary hyperparathyroidism (P=0.016. Conclusion: While vitamin D deficiency is more frequent in enzyme-inducing drug users, secondary hyperparathyroidism is less frequent.

Structural Basis of Competitive Recognition of p53 and MDM2 by HAUSP/USP7: Implications for the Regulation of the p53-MDM2 Pathway.

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2006-01-01

Full Text Available Herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7, a deubiquitylating enzyme of the ubiquitin-specific processing protease family, specifically deubiquitylates both p53 and MDM2, hence playing an important yet enigmatic role in the p53-MDM2 pathway. Here we demonstrate that both p53 and MDM2 specifically recognize the N-terminal tumor necrosis factor-receptor associated factor (TRAF-like domain of HAUSP in a mutually exclusive manner. HAUSP preferentially forms a stable HAUSP-MDM2 complex even in the presence of excess p53. The HAUSP-binding elements were mapped to a peptide fragment in the carboxy-terminus of p53 and to a short-peptide region preceding the acidic domain of MDM2. The crystal structures of the HAUSP TRAF-like domain in complex with p53 and MDM2 peptides, determined at 2.3-A and 1.7-A resolutions, respectively, reveal that the MDM2 peptide recognizes the same surface groove in HAUSP as that recognized by p53 but mediates more extensive interactions. Structural comparison led to the identification of a consensus peptide-recognition sequence by HAUSP. These results, together with the structure of a combined substrate-binding-and-deubiquitylation domain of HAUSP, provide important insights into regulation of the p53-MDM2 pathway by HAUSP.

Enzymic saccharification of some pretreated agricultural wastes

Energy Technology Data Exchange (ETDEWEB)

El-Gammal, S.M.A.; Sadek, M.A.

1988-01-01

Cellulosie wastes, artichoke leaves and stalks, sugar-cane bagasse and fennel seeds after extraction of essential oils were treated with various concentrations of peracetic acid at 100/sup 0/C, 60/sup 0/C and room temperature several times, washed with water and ethanol and air dried. The degree of enzymatic solubilization of each treated cellulosic waste was measured with Aspergillus niger cellulase (Endo-1,4-B-Glucanase; 1,4-(1,3; 1,4)-..beta..-D-glucan 4-glucanohydrolase; EC 3. 2.1.4). Artichoke waste and sugar-cane bagasse were solubilized more efectively by the enzymethan fennel waste. Data are presented describing the effect of time, enzyme and substrate concentration on the rate of enzymic hydrolysis. Infrared spectra of the treated and untreated cellulosic materials were recorded.

Chemical and radiological characterization of meals served by the COSEAS (USP-SP); Caracterizacao quimica e radiologica de refeicoes servidas pelo COSEAS/USP-SP

Energy Technology Data Exchange (ETDEWEB)

Favaro, Deborah Ines Teixeira [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), SP (Brazil). Centro do Reator de Pesquisas. Lab. de Analise por Ativacao Neutronica], e-mail: defavaro@ipen.br; Chioccola, Gabriella Saitta; Bortoli, Maritsa; Cozzolino, Silvia Maria Franciscato [Universidade de Sao Paulo (USP), SP (Brazil). Faculdade de Ciencias Farmaceuticas. Lab. de Alimentos e Nutricao Experimental; Taddei, Maria Helena [Comissao Nacional de Energia Nuclear (CNEN), Pocos de Caldas, MG (Brazil). Coordenacao do Laboratrio de Pocos de Caldas (COLAB); Mazzilli, Barbara Paci [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), SP (Brazil). Div. de Radiometria Ambiental

2009-01-15

This study presents the results obtained for the chemical and radiological analyses of lunch meals served in the cafeteria at the University of Sao Paulo-USP (COSEAS restaurant) in Brazil, on 5 non-consecutive days. On each of the five days, the meals were collected in triplicate and then freeze-dried for analysis totalizing 15 samples. The determination of the proximate composition complied with the AOAC standardized methodologies. The content of Ca, Fe, K, Na, Se, and Zn was determined by instrumental neutron activation analysis and the method validation was performed by certified reference materials analyses. Based on the concentration data, the daily intake of each mineral was calculated (corresponding to 40% of the total daily intake) and compared to the new recommendations set by the National Research Council (USA) considering the 19-30 year-old women range group. Comparing the average values obtained in this study with the recommended values, it was possible to verify that for macronutrients and the micronutrients Fe, Se, and Zn the recommendations were followed; for Ca and K they were deficient and exceeded the recommendation value for Na. The radionuclides {sup 40}K, {sup 60}Co, {sup 137}Cs, and {sup 131}I were determined by gamma spectrometry; {sup 90}Sr by liquid scintillation and {sup 210}Po, {sup 234}U, {sup 232}Th, {sup 238}U, {sup 235}U, {sup 228}Th, {sup 230}Th, and {sup 232}Th by alpha spectrometry. All radionuclides are present in concentrations below the limits set by the Food and Agriculture Organization (FAO). (author)

FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2.

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Maria Castella

2015-10-01

Full Text Available The Fanconi anemia (FA-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex. However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.

MRI studies of the hydrodynamics in a USP 4 dissolution testing cell.

Science.gov (United States)

Shiko, G; Gladden, L F; Sederman, A J; Connolly, P C; Butler, J M

2011-03-01

We present a detailed study of hydrodynamics inside the flow-through dissolution apparatus when operated according to USP recommendations. The pulsatile flow inside the flow-through cell was measured quantitatively using magnetic resonance imaging (MRI) at a spatial resolution of 234 × 234 μm(2) and slice thickness of 1 mm. We report the experimental protocols developed for in situ MRI studies and the effect that the operating conditions and tablet orientation have on the hydrodynamics inside commercial flow cells. It was found that the flow field inside the dissolution cells was, at most operating conditions, heterogeneous, rather than fully developed laminar flow, and characterised by re-circulation and backward flow. A model tablet was shown to be contacted by a wide distribution of local velocities as a function of position and orientation in the flow cell. The use of 1 mm beads acted as a distributor of the flow but did not suffice to ensure a fully developed laminar flow profile. These results emphasise the necessity to understand the influence of test conditions on dissolution behaviour in defining robust flow-through dissolution methods. Copyright © 2010 Wiley-Liss, Inc.

Influence of dissolution media pH and USP1 basket speed on erosion and disintegration characteristics of immediate release metformin hydrochloride tablets.

Science.gov (United States)

Desai, Divyakant; Wong, Benjamin; Huang, Yande; Tang, Dan; Hemenway, Jeffrey; Paruchuri, Srinivasa; Guo, Hang; Hsieh, Daniel; Timmins, Peter

2015-01-01

To investigate the influence of the pH of the dissolution medium on immediate release 850 mg metformin hydrochloride tablets. A traditional wet granulation method was used to manufacture metformin hydrochloride tablets with or without a disintegrant. Tablet dissolution was conducted using the USP apparatus I at 100 rpm. In spite of its pH-independent high solubility, metformin hydrochloride tablets dissolved significantly slower in 0.1 N HCl (pH 1.2) and 50 mM pH 4.5 acetate buffer compared with 50 mM pH 6.8 phosphate buffer, the dissolution medium in the USP. Metformin hydrochloride API compressed into a round 1200 mg disk showed a similar trend. When basket rotation speed was increased from 100 to 250 rpm, the dissolution of metformin hydrochloride tablets was similar in all three media. Incorporation of 2% w/w crospovidone in the tablet formulation improved the dissolution although the pH-dependent trend was still evident, but incorporation of 2% w/w croscarmellose sodium resulted in rapid pH-independent tablet dissolution. In absence of a disintegrant in the tablet formulation, the dissolution was governed by the erosion-diffusion process. Even for a highly soluble drug, a super-disintegrant was needed in the formulation to overcome the diffusion layer limitation and change the dissolution mechanism from erosion-diffusion to disintegration.

Dynamics of ligninolytic enzyme production in Ganoderma applanatum depending on cultivation type

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Ćilerdžić Jasmina Lj.

2011-01-01

Full Text Available Ganoderma applanatum belongs to the group of white-rot fungi, due to a well-developed ligninolytic enzyme system. White-rot fungi have attracted great scientific attention in recent years, especially with respect to their enzymatic potential for the bioremediation of persistent pollutants. Contrary to G. lucidum, which medicinal properties, as well as ligninolytic enzyme system have been extensively studied, enzymatic system of G. applanatum has not been studied yet. Thus, the aim of this study was to analyze the dynamics of laccase, Mn-dependent peroxidase, and versatile peroxidase activity during submerged and solid state cultivation on two selected plant raw materials. Enzyme activity was determined spectrophotometrically after 7, 10 and 14 days of cultivation. The peak of laccase activity (220.14 Ul-1 was noted after 14 days of submerged wheat straw fermentation. Maximum level of Mn-dependent peroxidase (110.91 Ul-1 and versatile peroxidase (116.20 Ul-1 activity was obtained in the medium with oak sawdust after 14 days of submerged cultivation.

Dibasic calcium phosphate dihydrate, USP material compatibility with gamma radiation

Science.gov (United States)

Betancourt Quiles, Maritza

Gamma radiation is a commonly used method to reduce the microbial bioburden in compatible materials when it is applied at appropriate dose levels. Gamma irradiation kills bacteria and mold by breaking down the organism’s DNA and inhibiting cell division. The purpose of this study is to determine the radiation dosage to be used to treat Dibasic Calcium Phosphate Dihydrate, USP (DCPD) and to evaluate its physicochemical effects if any, on this material. This material will be submitted to various doses of gamma radiation that were selected based on literature review and existing regulations that demonstrate that this method is effective to reduce or eliminate microbial bioburden in natural source and synthetic materials. Analytical testing was conducted to the DCPD exposed material in order to demonstrate that gamma radiation does not alter the physicochemical properties and material still acceptable for use in the manufacture of pharmaceutical products. The results obtained through this study were satisfactory and demonstrated that the gamma irradiation dosages from 5 to 30 kGy can be applied to DCPD without altering its physicochemical properties. These are supported by the Assay test data evaluation of lots tested before and after gamma irradiation implementation that show no significant statistical difference between irradiated and non irradiated assay results. The results of this study represent an achievement for the industry since they provide as an alternative the use of Gamma irradiation technology to control the microbial growth in DCPD.

Changes in growth, survival and digestive enzyme activities of Asian ...

African Journals Online (AJOL)

A study was conducted to determine the effects of different dietary treatments on the growth, survival and digestive enzyme activities of Mystus nemurus larvae. Newly hatched larvae were reared for 14 days in twelve 15 L glass aquaria (for growth and survival) and eight 300 L fiberglass tanks (for enzyme samples) at a ...

Silica-Immobilized Enzyme Reactors

Science.gov (United States)

2007-08-01

Silica-IMERs 14 implicated in neurological disorders such as Schizophrenia and Parkinson’s disease.[86] Drug discovery for targets that can alter the...primarily the activation of prodrugs and proantibiotics for cancer treatments or antibiotic therapy , respectively.[87] Nitrobenzene nitroreductase was...BuChE) Monolith disks* Packed Silica Biosilica Epoxide- Silica Silica-gel Enzyme Human AChE Human AChE Human AChE Equine BuChE Human

RNF20 and USP44 regulate stem cell differentiation by modulating H2B monoubiquitylation

Science.gov (United States)

Fuchs, Gilad; Shema, Efrat; Vesterman, Rita; Kotler, Eran; Wolchinsky, Zohar; Wilder, Sylvia; Golomb, Lior; Pribluda, Ariel; Zhang, Feng; Haj-Yahya, Mahmood; Feldmesser, Ester; Brik, Ashraf; Yu, Xiaochun; Hanna, Jacob; Aberdam, Daniel; Domany, Eytan; Oren, Moshe

2012-01-01

Summary Embryonic stem cells (ESC) maintain high genomic plasticity, essential for their capacity to enter diverse differentiation pathways. Post-transcriptional modifications of chromatin histones play a pivotal role in maintaining this plasticity. We now report that one such modification, monoubiquitylation of histone H2B on lysine 120 (H2Bub1), catalyzed by the E3 ligase RNF20, increases during ESC differentiation and is required for efficient execution of this process. This increase is particularly important for the transcriptional induction of relatively long genes during ESC differentiation. Furthermore, we identify the deubiquitinase USP44 as a negative regulator of H2B ubiquitylation, whose downregulation during ESC differentiation contributes to the increase in H2Bub1. Our findings suggest that optimal ESC differentiation requires dynamic changes in H2B ubiquitylation patterns, which must occur in a timely and well-coordinated manner. PMID:22681888

Recent advances in understanding Cushing disease: resistance to glucocorticoid negative feedback and somatic USP8 mutations [version 1; referees: 2 approved

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Eleni Daniel

2017-05-01

Full Text Available Cushing’s disease is a rare disease with a characteristic phenotype due to significant hypercortisolism driven by over-secretion of adrenocorticotropic hormone and to high morbidity and mortality if untreated. It is caused by a corticotroph adenoma of the pituitary, but the exact mechanisms leading to tumorigenesis are not clear. Recent advances in molecular biology such as the discovery of somatic mutations of the ubiquitin-specific peptidase 8 (USP8 gene allow new insights into the pathogenesis, which could be translated into exciting and much-needed therapeutic applications.

Rapid optimization of enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock combinations

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Walton Jonathan D

2010-10-01

Full Text Available Abstract Background Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]. A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP at the same protein loadings. Results When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3 and a lower proportion of endo-β1,4-glucanase (EG1 compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, �

Ultra-performance liquid chromatography-tandem mass spectrometry-based multiplex enzyme assay for six enzymes associated with hereditary hemolytic anemia.

Science.gov (United States)

Park, Chul Min; Lee, Kyunghoon; Jun, Sun-Hee; Song, Sang Hoon; Song, Junghan

2017-08-15

Deficiencies in erythrocyte metabolic enzymes are associated with hereditary hemolytic anemia. Here, we report the development of a novel multiplex enzyme assay for six major enzymes, namely glucose-6-phosphate dehydrogenase, pyruvate kinase, pyrimidine 5'-nucleotidase, hexokinase, triosephosphate isomerase, and adenosine deaminase, deficiencies in which are implicated in erythrocyte enzymopathies. To overcome the drawbacks of traditional spectrophotometric enzyme assays, the present assay was based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The products of the six enzymes were directly measured by using ion pairing UPLC-MS/MS, and the precision, linearity, ion suppression, optimal sample amounts, and incubation times were evaluated. Eighty-three normal individuals and 13 patients with suspected enzymopathy were analyzed. The UPLC running time was within 5min. No ion suppression was observed at the retention time for the products or internal standards. We selected an optimal dilution factor and incubation time for each enzyme system. The intra- and inter-assay imprecision values (CVs) were 2.5-12.1% and 2.9-14.3%, respectively. The linearity of each system was good, with R 2 values >0.97. Patient samples showed consistently lower enzyme activities than those from normal individuals. The present ion paring UPLC-MS/MS assay enables facile and reproducible multiplex evaluation of the activity of enzymes implicated in enzymopathy-associated hemolytic anemia. Copyright © 2017 Elsevier B.V. All rights reserved.

Ferredoxin-linked chloreplast enzymes. Progress report, August 15, 1990--August 14, 1993

Energy Technology Data Exchange (ETDEWEB)

NONE

1996-01-01

Progress has clearly been made on all of the goals set forth in the original proposal. Although the monoclonal antibodies raised against FNR turned out no to be useful for mapping the FNR/ferredoxin or FNR/NADP+ interaction domains, good progress has been made on mapping the FNR/ferredoxin interaction domains by an alternative technique, differential chemical modification. Furthermore, the techniques developed for differential chemical modifications of these two proteins - taurine modification of aspartate and glutamate residues and biotin modification of lysine residues - should be useful for mapping the interaction domains of many proteins that associate through electrostatic interactions. Finally, progress has also been made with respect to another ferredoxin-dependent enzyme involved in the earliest steps of plant nitrogen metabolism - nitrite reductase. Questions concerning the subunit composition and heme content of the enzyme have been resolved and evidence demonstrating the involvement of lysine and arginine residues in binding ferredoxin has been obtained for the first time.

Redes de Pesquisa Entre os Egressos do Doutorado em Ciências Contábeis da FEA/USP

Directory of Open Access Journals (Sweden)

Ilse Maria Beuren

2010-02-01

Full Text Available O estudo objetiva analisar as redes de pesquisa dos egressos do curso de Doutorado em Ciências Contábeis da FEA/USP. A pesquisa documental foi realizada nos currículos Lattes dos doutores em controladoria e contabilidade, formados pela Universidade de São Paulo, no período de 1962 a 2008. Da população de 185 doutores do Doutorado de Ciências Contábeis da FEA/USP, uma amostra de 158 foi selecionada, pois 27 currículos Lattes não estavam disponíveis. Na análise dos dados, utilizou-se estatística descritiva para quantificação da produção científica dos doutores, conforme informações disponíveis nos currículos Lattes. Para análise das redes de pesquisa entre os egressos, utilizou-se o software UNICET®. Os resultados da pesquisa evidenciam as instituições que os egressos estão vinculados atualmente, as funções que exercem nestas instituições, a produção científica dos egressos no período de 1998 a 2008 e as redes de pesquisa predominantes entre os egressos. Constatou-se que a Universidade de São Paulo concentra o maior número de docentes egressos e que a produção científica dos doutores pesquisados está predominantemente publicada em anais de congressos. Quanto às redes, observou-se que estabelecem redes de cooperação com predomínio de ligações fracas em livros, capítulos de livros e textos de revistas/jornais, porém relações de cooperação fortes na publicação de artigos em periódicos. Conclui-se que há ainda necessidade de um fortalecimento nas redes estabelecidas entre os egressos analisados para proporcionar um aprimoramento científico na área contábil.

25. Steenbock symposium -- Biosynthesis and function of metal clusters for enzymes: Proceedings

Energy Technology Data Exchange (ETDEWEB)

NONE

1997-12-31

This symposium was held June 10--14, 1997 in Madison, Wisconsin. The purpose of this conference was to provide a multidisciplinary forum for exchange of state-of-the-art information on biochemistry of enzymes that have an affinity for metal clusters. Attention is focused on the following: metal clusters involved in energy conservation and remediation; tungsten, molybdenum, and cobalt-containing enzymes; Fe proteins, and Mo-binding proteins; nickel enzymes; and nitrogenase.

Effect of irradiation on lysosomal enzyme activation in cultured macrophages

International Nuclear Information System (INIS)

Clarke, C.; Wills, E.D.

1980-01-01

The effect of γrays on lysosomal enzyme activity of normal and immune macrophages of DBA/2 mice cultured in vitro has been studied. A dose of 500 rad did not significantly affect lysosomal enzyme activity 3 hours after irradiation but caused the activity to increase to 1.4 times the control value 22.5 hours after irradiation. 22.5 hours after a dose of 3000 rad the enzyme activity increased to 2.5 times the control. Lysosomal enzyme activity of the macrophages was also markedly increased by immunization of the mice with D lymphoma cells, before culture in vitro, but irradiation of these cells with a dose of 500 rad caused a further increase in lysosomal enzyme activity. The results indicate that immunization and irradiation both cause stimulation of lysosomal enzyme activity in macrophages but that the mechanisms of activation are unlikely to be identical. (author)

Electron beam technology for production of preparations of immobilized enzymes

International Nuclear Information System (INIS)

Gonchar, A.M.; Auslender, V.L.; Polyakov, V.A.

1995-01-01

Possibility of electron beam usage for proteases immobilization on 1,4-polyalkylene oxide (1,4-PAO) was studied to obtain biologically active complex for multi-purpose usage. It is shown that immobilization of Bacillus Subtilis protease is done due to free-radical linking of enzyme and carrier with formation of mycelium-like structures. Immobilization improves heat resistance of enzyme up to 60 centigrade without substrate and up to 80 centigrade in presence of substrate, widens range pH activity in comparison with non-immobilized forms. Immobilized proteases does not contain peroxides and long-live radicals. Our results permitted to create technologies for production of medical and veterinary preparations, active components for wool washing agents and leather fabrication technology

Gclust Server: 30235 [Gclust Server

Lifescience Database Archive (English)

Full Text Available ase cofactor, forms deubiquitination complex with Ubp3p that coregulates anterograde and retrograde transpor...tin protease cofactor, forms deubiquitination complex with Ubp3p that coregulates anterograde and retrograde

O desempenho terminológico dos descritores em Ciência da Informação do Vocabulário Controlado do SIBi/USP nos processos de indexação manual, automática e semi-automática

Directory of Open Access Journals (Sweden)

Vania Mara Alves Lima

Full Text Available Avaliou-se o desempenho terminológico, nos processos de indexação manual, automática e semi-automática, dos descritores, do Vocabulário Controlado do SIBi/USP, que representam o domínio da Ciência da Informação. Concluiu-se que os atuais descritores em Ciência da Informação do Vocabulário Controlado do SIBi/USP para representar adequadamente o conteúdo do corpus indexado devem ser ampliados e contextualizados através de definições terminológicas, de maneira a atender as necessidades de informação de seus usuários.

Evaluation of the Activities of Antioxidant Enzyme and Lysosomal Enzymes of the Longissimus dorsi Muscle from Hanwoo (Korean Cattle) in Various Freezing Conditions

OpenAIRE

Kang, Sun Moon; Kang, Geunho; Seong, Pil-Nam; Park, Beomyoung; Kim, Donghun; Cho, Soohyun

2014-01-01

This study was conducted to evaluate the activities of antioxidant enzyme (glutathione peroxidase (GSH-Px)) and lysosomal enzymes (alpha-glucopyranosidase (AGP) and beta-N-acetyl-glucosaminidase (BNAG)) of the longissimus dorsi (LD) muscle from Hanwoo (Korean cattle) in three freezing conditions. Following freezing at -20, -60, and -196? (liquid nitrogen), LD samples (48 h post-slaughter) were treated as follows: 1) freezing for 14 d, 2) 1 to 4 freeze-thaw cycles (2 d of freezing in each cycl...

Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth*

Science.gov (United States)

Mertz, Joseph; Tan, Haiyan; Pagala, Vishwajeeth; Bai, Bing; Chen, Ping-Chung; Li, Yuxin; Cho, Ji-Hoon; Shaw, Timothy; Wang, Xusheng; Peng, Junmin

2015-01-01

The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 down-regulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interactomes; and Mib1 analysis provides a comprehensive interactome for investigating its role in signaling networks and neuronal development. PMID:25931508

HPRT Enzyme Activity of Blood Cells From Patients With Downs Syndrome

International Nuclear Information System (INIS)

Sbubber, E.K.; Abdul-Rahman, M.H.; Sultan, A.F.; Hamamy, H.A.

1998-01-01

Hypoxanthine phosphoribosyl transferase (HPRT) enzyme activity was determined in erythrocytes from 16 children (aged below one year to 11 year) with down s syndrome using 8-C 14 Hypoxanthine and radioeleelrophorsis techniques. Significant (P<0.01) reduction in HPRT enzyme activity was seen in D S children compared to that of 18 (age and sex matched) healthy children. Pure 21 - trisomic erythrocytes expressed lower enzyme activity than mosaic cell. Mothers of D S children showed significantly (P<0.01) lower enzyme activity than mothers of normal children . Reduced activity of HPRT enzyme was also observed in PHA-stimulated lymphocytes of DS children and their mothers. These results indicated that deficiency of HPRT in D S patients may contribute to the abnormal purine metabolism associated with the symptomatology of this syndrome

Production and assay of cellulolytic enzyme activity of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya abbatoir, Indonesia

Directory of Open Access Journals (Sweden)

W. P. Lokapirnasari

2015-03-01

Full Text Available Aim: This study aims to produce and assay cellulolytic enzyme activity (endo-(1,4-β-D-glucanase, exo-(1,4-β-Dglucanase, and β-glucosidase, at optimum temperature and optimum pH of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya Abbatoir, Indonesia. Materials and Methods: To produce enzyme from a single colony of E. cloacae WPL 214, 98 × 1010 CFU/ml of isolates was put into 20 ml of liquid medium and incubated in a shaker incubator for 16 h at 35°C in accordance with growth time and optimum temperature of E. cloacae WPL 214. Further on, culture was centrifuged at 6000 rpm at 4°C for 15 min. Pellet was discarded while supernatant containing cellulose enzyme activity was withdrawn to assay endo-(1,4-β-D-glucanase, exo-(1,4-β-D-glucanase, and β-glucosidase. Results: Cellulase enzyme of E. cloacae WPL 214 isolates had endoglucanase activity of 0.09 U/ml, exoglucanase of 0.13 U/ml, and cellobiase of 0.10 U/ml at optimum temperature 35°C and optimum pH 5. Conclusion: E. cloacae WPL 214 isolated from bovine rumen fluid waste produced cellulose enzyme with activity as cellulolytic enzyme of endo-(1,4-β-D-glucanase, exo-(1,4-β-D-glucanase and β-glucosidase.

Abordagens da Controladoria nos Trabalhos do Congresso USP de Controladoria e Contabilidade: Um Enfoque nas Perspectivas Conceitual, Procedimental e Organizacional

Directory of Open Access Journals (Sweden)

Ilse Maria Beuren

2012-01-01

Full Text Available The study identifies the approach of comptrollership on papers published in the annals of the USP Congress on Comptrollership and Accounting from 2001 to 2006, based on documentary research and descriptive denotation. The results show that in the conceptual perspective there is no concern in defining the term comptrollership and neither its relationship with other areas of knowledge. In the procedural perspective, there is no consensus about the typical activities but it stands out the one related to information systems. In the organizational perspective, the comptrollership is not seen as a formally established organizational unit with power of decision, but as an administrative unit that supports the decision-making.

Gerenciamento de resíduos químicos e águas servidas no laboratório de isótopos estáveis do CENA/USP Chemistry waste and residues waters management at the stable isotope laboratory of the CENA/USP

Directory of Open Access Journals (Sweden)

José Albertino Bendassolli

2003-08-01

Full Text Available A system for disposal and recovery of the main effluents and chemical waist from isotope separation plants and enriched compounds-15N and 34S production has been carried out at the Stable Isotope Laboratory (LIE of the CENA/USP. Around four hundred thousand liters of effluents has been recovered yearly. Among the recovered chemical wastes, the more relevant are: ammonia; brome; ammonium and sodium sulfate; sodium hydroxide; sulfur dioxide; and hydrochloric acid. Chemical wastes containg recoverable heavy metals (Ag, Cr and Cu and solvents (methanol, ethanol and acetone are processed and recovered. Gaseous emissions, mainly H2S are used for recovery of heavy metals solutions. The minimization of the residues waters, as well the reduction of electric energy consume was established using a water deionization system. A cost/effect balance of the process is reported.

Effect of whole-body X-irradiation on lysosomal enzymes

Energy Technology Data Exchange (ETDEWEB)

D' souza, D W; Vakil, U K; Srinivasan, A [Bhabha Atomic Research Centre, Bombay (India). Biochemistry and Food Technology Div.

1974-06-01

Effects of whole-body x irradiation with sublethal dose (400 rad) on three intestinal lysosomal enzymes, namely, arylsulphatase, cathepsin and acid phosphatases, have been studied. They are almost equally distributed throughout the entire small intestine region. X irradiation adversely affects the integrity of lysosomal membranes. ''Free'' and ''total'' lysosomal enzyme activities exhibit maxima on 6th day. These activities return to normal level on 14th day when there is rapid generation of villi, indicating that lysosomal activities correlate with the progression of injury and of repair mechanism after sublethal dose of x irradiation. The increase in total lysosomal activity may be due to its decreased breakdown, since the rate of protein synthesis in intestinal mucosa is reduced. This is evidenced by reduced incorporation of orally fed /sup 14/C leucine into acid insoluble proteins. (auth)

Enzyme

Science.gov (United States)

Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

Interaction between chitosan and its related enzymes: A review.

Science.gov (United States)

Shinya, Shoko; Fukamizo, Tamo

2017-11-01

Chitosan-related enzymes including chitosanases, exo-β-glucosaminidases, and enzymes having chitosan-binding modules recognize ligands through electrostatic interactions between the acidic amino acids in proteins and amino groups of chitosan polysaccharides. However, in GH8 chitosanases, several aromatic residues are also involved in substrate recognition through stacking interactions, and these enzymes consequently hydrolyze β-1,4-glucan as well as chitosan. The binding grooves of these chitosanases are extended and opened at both ends of the grooves, so that the enzymes can clamp a long chitosan polysaccharide. The association/dissociation of positively charged glucosamine residues to/from the binding pocket of a GH2 exo-β-glucosaminidase controls the p K a of the catalytic acid, thereby maintaining the high catalytic potency of the enzyme. In contrast to chitosanases, chitosan-binding modules only accommodate a couple of glucosamine residues, predominantly recognizing the non-reducing end glucosamine residue of chitosan by electrostatic interactions and a hydrogen-bonding network. These structural findings on chitosan-related enzymes may contribute to future applications for the efficient conversion of the chitin/chitosan biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

Assay of glutamine phosphoribosylpyrophosphate amidotransferase using [1-14C]phosphoribosylpyrophosphate

International Nuclear Information System (INIS)

Ross, G.R.; Idriss, S.D.; Willis, R.C.; Seegmiller, J.E.

1983-01-01

Glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) catalyzes the transfer of the amide group of glutamine to 5-phospho-α-D-ribose-1-pyrophosphate. It is the first enzyme committed to the synthesis of purines by the de novo pathway. Previous assays of enzyme activity have either measured the phosphoribosylpyrophosphate-dependent disappearance of radioactive glutamine or have linked this reaction to subsequent steps in the purine pathway. A new assay for activity of the enzyme by directly measuring the synthesis of the product of the reaction, 5-β-phosphoribosyl-1-amine, using [1- 14 C]phosphoribosylpyrophosphate as substrate is described. Substrate and product are separated by thin-layer chromatography and identified by autoradiography. Glutamine or ammonia may be used as substrates; the apparent K/sub m/ values of the human lymphoblast enzyme are 0.46 mM for glutamine and 0.71 mM for ammonia. GMP is a considerably more potent inhibitor of the human lymphoblast enzyme than is AMP; 6-diazo-5-oxo-L-norleucine inhibits only glutamine-dependent activity and has no effect on ammonia-dependent activity

La USP como paradigma de la publicidad racionalista. Auge y declive del mecanismo estratégico más popular de la publicidad moderna

OpenAIRE

Fernández Gómez, Jorge David; Sanz Marcos, Paloma

2016-01-01

La teoría de la USP (unique selling proposition), se ha configurado a lo largo de la historia publicitaria como referencia en lo que a estrategia publicitaria se refiere. Este artículo trata de ofrecer por un lado, una perspectiva recapituladora de los orígenes y circunstancias que dieron popularidad al concepto y por otro, un análisis de aquellos errores de planteamiento que agilizaron su debilitamiento con la llegada de nuevas teorías como la de la «Imagen de Marca» The USP’s Theory (un...

Effect of modification with 1,4-α-glucan branching enzyme on the rheological properties of cassava starch.

Science.gov (United States)

Li, Yadi; Li, Caiming; Gu, Zhengbiao; Hong, Yan; Cheng, Li; Li, Zhaofeng

2017-10-01

Steady and dynamic shear measurements were used to investigate the rheological properties of cassava starches modified using the 1,4-α-glucan branching enzyme (GBE) from Geobacillus thermoglucosidans STB02. GBE treatment lowered the hysteresis loop areas, the activation energy (E a ) values and the parameters in rheological models of cassava starch pastes. Moreover, GBE treatment increased its storage (G') and loss (G″) moduli, and decreased their tan δ (ratio of G″/G') values and frequency-dependencies. Scanning electron microscopic studies showed the selective and particular attack of GBE on starch granules, and X-ray diffraction analyses showed that GBE treatment produces significant structural changes in amylose and amylopectin. These changes demonstrate that GBE modification produces cassava starch with a more structured network and improved stability towards mechanical processing. Differential scanning calorimetric analysis and temperature sweeps indicated greater resistance to granule rupture, higher gel rigidity, and a large decrease in the rate of initial conformational ordering with increasing GBE treatment time. Pronounced changes in rheological parameters revealed that GBE modification enhances the stability of cassava starch and its applicability in the food processing industry. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization and mode of action of xylanases ␁and some accessory enzymes

NARCIS (Netherlands)

Kormelink, F.J.M.

1992-01-01

Three endo-(l,4)-β-D-xylanases; (Endo I, Endo II, and Endo III), a (1,4)-β-xylosidase and an (1,4)-β-D-arabinoxylan arabinofuranohydrolase (AXH) were purified from a culture filtrate produced by Aspergillus awamori CMI 142717. In addition to these enzymes, an acetyl

Chemical and radiological characterization of meals served by the COSEAS (USP-SP)

International Nuclear Information System (INIS)

Favaro, Deborah Ines Teixeira; Mazzilli, Barbara Paci

2009-01-01

This study presents the results obtained for the chemical and radiological analyses of lunch meals served in the cafeteria at the University of Sao Paulo-USP (COSEAS restaurant) in Brazil, on 5 non-consecutive days. On each of the five days, the meals were collected in triplicate and then freeze-dried for analysis totalizing 15 samples. The determination of the proximate composition complied with the AOAC standardized methodologies. The content of Ca, Fe, K, Na, Se, and Zn was determined by instrumental neutron activation analysis and the method validation was performed by certified reference materials analyses. Based on the concentration data, the daily intake of each mineral was calculated (corresponding to 40% of the total daily intake) and compared to the new recommendations set by the National Research Council (USA) considering the 19-30 year-old women range group. Comparing the average values obtained in this study with the recommended values, it was possible to verify that for macronutrients and the micronutrients Fe, Se, and Zn the recommendations were followed; for Ca and K they were deficient and exceeded the recommendation value for Na. The radionuclides 40 K, 60 Co, 137 Cs, and 131 I were determined by gamma spectrometry; 90 Sr by liquid scintillation and 210 Po, 234 U, 232 Th, 238 U, 235 U, 228 Th, 230 Th, and 232 Th by alpha spectrometry. All radionuclides are present in concentrations below the limits set by the Food and Agriculture Organization (FAO). (author)

Implantação de um programa de gerenciamento de resíduos químicos e águas servidas nos laboratórios de ensino e pesquisa no CENA/USP Establishment of a management program for chemical residues and waste water, generated in laboratories of the centro de energia nuclear na agricultura (CENA/USP

Directory of Open Access Journals (Sweden)

Glauco Arnold Tavares

2005-08-01

Full Text Available The aim of this work is to establish a program for the treatment of chemical residues and waste waters at the Centro de Energia Nuclear na Agricultura (CENA/USP, for environmental preservation and training of staff. Five tons of stored residues and the ones currently generated in the laboratories have to be treated. Rational use of water is also part of the program. The traditional purification by distillation has been replaced by purification with ion exchange resins. Lower energy consumption and better water quality were achieved.

Silica Sol-Gel Entrapment of the Enzyme Chloro peroxidase

International Nuclear Information System (INIS)

Le, T.; Chan, S.; Ebaid, B.; Sommerhalter, M.

2015-01-01

The enzyme chloro peroxidase (CPO) was immobilized in silica sol-gel beads prepared from tetramethoxysilane. The average pore diameter of the silica host structure (∼3 nm) was smaller than the globular CPO diameter (∼6 nm) and the enzyme remained entrapped after sol-gel maturation. The catalytic performance of the entrapped enzyme was assessed via the pyrogallol peroxidation reaction. Sol-gel beads loaded with 4 μg CPO per mL sol solution reached 9-12% relative activity compared to free CPO in solution. Enzyme kinetic analysis revealed a decrease in K_cat but no changes in K_M or K_I . Product release or enzyme damage might thus limit catalytic performance. Yet circular dichroism and visible absorption spectra of transparent CPO sol-gel sheets did not indicate enzyme damage. Activity decline due to methanol exposure was shown to be reversible in solution. To improve catalytic performance the sol-gel protocol was modified. The incorporation of 5, 20, or 40% methyltrimethoxysilane resulted in more brittle sol-gel beads but the catalytic performance increased to 14% relative to free CPO in solution. The use of more acidic casting buffers (ph 4.5 or 5.5 instead of 6.5) resulted in a more porous silica host reaching up to 18% relative activity

Ultrasound-assisted extraction and characterization of hydrolytic and oxidative enzymes produced by solid state fermentation.

Science.gov (United States)

Szabo, Orsolya Erzsebet; Csiszar, Emilia; Toth, Karolina; Szakacs, George; Koczka, Bela

2015-01-01

Ligninolytic and hydrolytic enzymes were produced with six selected fungi on flax substrate by solid state fermentation (SSF). The extracellular enzyme production of the organisms in two SSF media was evaluated by measuring the soluble protein concentration and the filter paper, endoxylanase, 1,4-β-d-glucosidase, 1,4-β-d-endoglucanase, polygalacturonase, lignin peroxidase, manganese peroxidase and laccase activities of the clear culture solutions produced by conventional extraction from the SSF materials. The SSF material of the best enzyme producer (Trichoderma virens TUB F-498) was further investigated to enhance the enzyme recovery by low frequency ultrasound treatment. Performance of both the original and ultrasound macerated crude enzyme mixtures was evaluated in degradation of the colored lignin-containing and waxy materials of raw linen fabric. Results proved that sonication (at 40%, 60% and 80% amplitudes, for 60min) did not result in reduction in the filter paper, lignin peroxidase and laccase activities of the crude enzyme solution, but has a significant positive effect on the efficiency of enzyme extraction from the SSF material. Depending on the parameters of sonication, the enzyme activities in the extracts obtained can be increased up to 129-413% of the original activities measured in the control extracts recovered by a common magnetic stirrer. Sonication also has an effect on both the enzymatic removal of the lignin-containing color materials and hydrophobic surface layer from the raw linen. Copyright © 2014 Elsevier B.V. All rights reserved.

Preparation and HPLC isolation of L-[U-14C]tryptophan from enzyme hydrolysate labelled with 14C

International Nuclear Information System (INIS)

Novak, J.; Tintera, S.; Hromadkova, B.

1990-01-01

Tryptophan was obtained from biomass of the blue-green alga Synechococcus elongatus cultivated under 14 CO 2 . After partial purification, the protein fraction was subjected to enzymatic hydrolysis using pronase. Semipreparative isolation of L-[U- 14 C]tryptophan was accomplished on a HPLC column of Separon S Hema 1000 CM, 2% ethanol were added to the eluent, and a precolumn packed with the basic anion exchanger Spheron 1000 DEAE was used. Always after the passage of L-[U- 14 C]tryptophan, the precolumn was decoupled. The substance was collected in 96% ethanol. After removing the solvent by vacuum evaporation, the sample was analyzed on a column packed with Separon SIX C 18 in the eluent of 0.1M-NaH 2 PO 4 , 2% methanol. When the desired radiochemical purity was not attained, the sample was purified on Separon SIX C 18 using 2% methanol. The final radiochemical purity achieved by using this method is 98%. (P.A.). 5 figs., 2 tabs., 4 refs

Enzyme Informatics

Science.gov (United States)

Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

2012-01-01

Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

Atenção Integrada às Doenças Prevalentes na Infância (AIDPI na prática de enfermeiros egressos da USP Atención Integrada a las Enfermedades Prevalentes de la Infancia (AIEPI en la práctica de enfermeros graduados en USP Integrated Management of Childhood Illness (IMCI in the practice of nurses graduated from USP

Directory of Open Access Journals (Sweden)

Cinthia Hiroko Higuchi

2011-06-01

Full Text Available Descreveu-se a incorporação da estratégia Atenção Integrada às Doenças Prevalentes na Infância (AIDPI na prática de enfermeiros egressos da Escola de Enfermagem da Universidade de São Paulo (EE-USP. Desenvolveu-se estudo de caso de abordagem qualitativa, com coleta de dados em grupo focal e análise de conteúdo do tipo temática. A estratégia AIDPI foi considerada ferramenta importante na atenção à saúde infantil, porém apenas o módulo de avaliação fazia parte da prática profissional. Destacam-se como dificuldades para sua utilização: não implantação nos serviços, desconhecimento por parte de colegas e barreiras institucionais. Ainda que com uso restrito e não sistematizado, a AIDPI possibilita ao enfermeiro prestar atenção integrada e integral à criança o que justifica sua abordagem na graduação. Manutenção do vídeo didático, ampliação da prática, integração das disciplinas e otimização dos conteúdos e da carga horária foram apontadas como relevantes para o aperfeiçoamento do ensino da AIDPI na graduação.Se describió la incorporación de la estrategia Atención Integrada a las Enfermedades Prevalentes de la Infancia (AIEPI en la práctica de enfermeros formados en la Escuela de Enfermería de la Universidad de São Paulo (EE-USP. Estudio de caso de carácter cualitativo, con recogida de datos en grupos focales y análisis de contenido temático. AIEPI fue considerada importante herramienta en el cuidado del niño, pero sólo el módulo de evaluación hacía parte de la práctica profesional. Dificultades en el uso de AIEPI fueron: no implantación en los servicios, desconocimiento de la estrategia por parte de colegas, barreras institucionales. Aunque con uso restringido y no sistematizado, cuando utilizado AIEPI permite a los enfermeros prestar atención integrada y integral al niño, lo que justifica su contenido en la graduación. Mantenimiento de vídeo educativo, expansión de pr

System precision assessment ExacTrac 6D® BrainLab of the Hospital das Clinicas da Faculdade de Medicina da USP; Avaliacao da precisao do sistema Exactrac 6D® BrainLab do Hospital das Clinicas da Faculdade de Medicina da USP

Energy Technology Data Exchange (ETDEWEB)

Maistro, Carlos E.B., E-mail: carloseduardo.bravinmaistro@gmail.com [Programa de Residencia Multiprofissional em Fisica Medica, Faculdade de Medicina de Sao Paulo, SP (Brazil); Nakandakari, Marcos V.N.; Ribeiro, Victor A.B.; Sales, Camila P. de; Rodrigues, Laura N. [Universidade de Sao Paulo (USP), SP (Brazil). Instituto de Radiologia. Servico de Radioterapia. Hospital das Clinicas

2015-08-15

The goal of this study was to evaluate the precision of ExacTrac 6D® Brainlab system, installed at Hospital das Clinicas da Faculdade de Medicina da USP, in frameless radiosurgery treatments. Four sets of tests were performed for different purposes in order to assess the following parameters: the accuracy of location through infrared system; evaluation of the reproducibility of fusion algorithm; evaluation of the X-ray system; and the end-to-end test with the goal of assess the overall accuracy of the system. It was found that the infrared system showed a maximum deviation of 0.5 mm in terms of positioning and the X-ray system showed a precision of 0.15 mm and 0.6°. The reproducibility of fusion algorithms provided a maximum deviation in position which was less than 0.5 mm and 0.5° and the quantitative analysis of the results for end-to-end test showed an overall accuracy of the system better than 0.8 mm. (author)

Temperature dependence of desoxyribosylation of pyrimidine derivatives labelled with carbon-14 and tritium

International Nuclear Information System (INIS)

Pritasil, L.; Filip, J.

1977-01-01

The effect of the temperature and concentration of the enzyme preparation from Escherichia coli B on the reaction of pyrimidine bases with 2-desoxy-α-D-riboso-l-phosphate or α-D-riboso-l-phosphate was studied. It was found that at +2 deg C and low enzyme concentration higher yields of nucleosides are obtained than at the commonly used temperature of 37 deg C. The reaction time, however, must be protracted. The prepared [2- 14 C] uridine, 2'-deoxy[2- 14 C] uridine and [2- 14 C] thymidine had a high specific activity and radiochemical purity

Embryonic demise caused by targeted disruption of a cysteine protease Dub-2.

Science.gov (United States)

Baek, Kwang-Hyun; Lee, Heyjin; Yang, Sunmee; Lim, Soo-Bin; Lee, Wonwoo; Lee, Jeoung Eun; Lim, Jung-Jin; Jun, Kisun; Lee, Dong-Ryul; Chung, Young

2012-01-01

A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet. To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/- heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2-/- homozygous alleles could not survive. In addition, Dub-2-/- embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2-/- embryos could not be established. Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.

Embryonic demise caused by targeted disruption of a cysteine protease Dub-2.

Directory of Open Access Journals (Sweden)

Kwang-Hyun Baek

Full Text Available BACKGROUND: A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/- heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2-/- homozygous alleles could not survive. In addition, Dub-2-/- embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2-/- embryos could not be established. CONCLUSIONS: Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.

Structure, function and regulation of the enzymes in the starch biosynthetic pathway.

Energy Technology Data Exchange (ETDEWEB)

Geiger, Jim

2013-11-30

Starch is the major reserve polysaccharide in nature and accounts for the majority of the caloric intact of humans. It is also gaining importance as a renewable and biodegradable industrial material. There is burgeoning interest in increasing the amount and altering the properties of the plant starches by plant genetic modification. A rational approach to this effort will require a detailed, atomic-level understanding of the enzymatic processes that produce the starch granule. The starch granule is a complex particle made up of alternating layers of crystalline and amorphous lamellae. It consists of two types of polymer, amylose, a polymer of relatively long chains of α-1,4-linked glucans that contain virtually no branches, and amylopectin, which is highly branched and contains much shorter chains. This complex structure is synthesized by the coordinate activities of the starch synthases (SS), which elongate the polysaccharide chain by addition of glucose units via α-1,4 linkages using ADP- glucose as a donor, and branching enzymes (BE), which branch the polysaccharide chain by cleavage of α₋1,4 linkages and subsequent re-attachment via α₋1,6 linkages. Several isoforms of both starch synthase (SS) and branching enzyme (BE) are found in plants, including SSI, SSII, SSIII and granule- bound SS (GBSS), and SBEI, SBEIIa and SBEIIb. These isoforms have different activities and substrate and product specificities and play different roles in creating the granule and determining the properties of the resulting starch. The overarching goal of this proposal is to begin to understand the regulation and specificities of these enzymes at the atomic level. High-resolution X-ray structures of these enzymes bound to substrates and products will be determined to visualize the molecular interactions responsible for the properties of the enzymes. Hypotheses regarding these issues will then be tested using mutagenesis and enzyme assays. To date, we have determined the

Pancreatic Enzymes

Science.gov (United States)

... Contact Us DONATE NOW GENERAL DONATION PURPLESTRIDE Pancreatic enzymes Home Facing Pancreatic Cancer Living with Pancreatic Cancer ... and see a registered dietitian. What are pancreatic enzymes? Pancreatic enzymes help break down fats, proteins and ...

Dgroup: DG00882 [KEGG MEDICUS

Lifescience Database Archive (English)

Full Text Available oridazine hydrochloride (JP17/USP) ... Neuropsychiatric agent ... DG01905 ... Phenothiazine antipsychotics ... DG01478...p inhibitor ... DG01645 ... CYP2D6 inhibitor ATC code: N05AC02 Phenothiazine antipsychotics DRD2 [HSA:1813] [KO:K04145] Enzyme: CYP2D6 [HSA:1565] ...

The Infrared Fixed Points of 3d $\\mathcal{N}=4$ $USp(2N)$ SQCD Theories arXiv

CERN Document Server

Assel, Benjamin

We derive the algebraic description of the Coulomb branch of 3d $\\mathcal{N}=4$ $USp(2N)$ SQCD theories with $N_f$ fundamental hypermultiplets and determine their low energy physics in any vacuum from the local geometry of the moduli space, identifying the interacting SCFTs which arise at singularities and possible extra free sectors. The SCFT with the largest moduli space arises at the most singular locus on the Coulomb branch. For $N_f>2N$ (good theories) it sits at the origin of the conical variety as expected. For $N_f =2N$ we find two separate most singular points, from which the two isomorphic components of the Higgs branch of the UV theory emanate. The SCFTs sitting at any of these two vacua have only odd dimensional Coulomb branch generators, which transform under an accidental $SU(2)$ global symmetry. We provide a direct derivation of their moduli spaces of vacua, and propose a Lagrangian mirror theory for these fixed points. For $2 \\le N_f < 2N$ the most singular locus has one or two extended com...

Analysis of corrosion products in some metallic statuettes of the Museum of Archaeology and Ethnology (MAE-USP)

International Nuclear Information System (INIS)

Rizzutto, Marcia A.; Tabacniks, Manfredo H.; Added, Nemitala; Barbosa, Marcel D.L.; Lima, Silvia Cunha; Melo, Hercilio G.; Neiva, Augusto C.

2005-01-01

The recent acquisition of a sealed chamber with controlled humidity by the Museum of Archaeology and Ethnology of the University of Sao Paulo (MAE-USP) requires new methods for conservation and restoration of metallic objects in its collection. To establish new procedures for the identification of corrosion mechanisms and agents in the exhibition environment, and to set up new standards for conservation of the museum's collection, Proton Induced X-Ray Emission (PIXE) elementary analysis of some metallic objects is in progress, using the external beam facility at LAMFI. The first analysis involved metallic objects from the collection of MAE, two African statuettes 'male Edans' from the Ogboni Secret Society, of the Ilobu-Ioruba ethnic group, one pectoral adornment from the Chimu culture, Peru and one anthropomorphic pendant from the Tairona culture, Colombia. The in air non destructive PIXE analysis allowed identifying major and some secondary components in the alloys and in the corrosion products on the samples, data that were used to identify the corrosion sources and to set up the exhibition environment. (author)

Cyclopentenone prostaglandins as potential inducers of phase II detoxification enzymes. 15-deoxy-delta(12,14)-prostaglandin j2-induced expression of glutathione S-transferases.

Science.gov (United States)

Kawamoto, Y; Nakamura, Y; Naito, Y; Torii, Y; Kumagai, T; Osawa, T; Ohigashi, H; Satoh, K; Imagawa, M; Uchida, K

2000-04-14

Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes, such as glutathione S-transferases (GSTs). In the present study, we developed a cell culture system that potently responds to phenolic antioxidants and found that antitumor prostaglandins (PGs) are potential inducers of GSTs. We screened primary hepatocytes and multiple cell lines for inducing GST activity upon incubation with the phenolic antioxidant (tert-butylhydroquinone) and found that rat liver epithelial RL34 cells most potently responded. Based on an extensive screening of diverse chemical agents on the induction of GST activity in RL34 cells, the J2 series of PGs, 15-deoxy-Delta(12,14)-prostaglandin J2 (15-deoxy-Delta(12,14)-PGJ2) in particular, were found to be potential inducers of GST. Enhanced gene expression of Class pi GST isozyme (GSTP1) by 15-deoxy-Delta(12,14)-PGJ2 was evident as a drastic elevation of the mRNA level. Hence, we examined the molecular mechanism underlying the 15-deoxy-Delta(12, 14)-PGJ2-induced GSTP1 gene expression. From functional analysis of various deletion mutant genes, we found that the 15-deoxy-Delta(12, 14)-PGJ2 reponse element was localized in a region containing a GSTP1 enhancer I (GPEI) that consists of two imperfect phorbol 12-O-tetradecanoylphorbol-13-acetate response elements. When the GPEI was combined with the minimum GSTP1 promoter, the element indeed showed an enhancer activity in response to 15-deoxy-Delta(12, 14)-PGJ2. Point mutations of either of the two imperfect 12-O-tetradecanoylphorbol-13-acetate response elements in GPEI completely abolished the enhancer activity. Gel mobility shift assays demonstrated that 15-deoxy-Delta(12,14)-PGJ2 specifically stimulated the binding of nuclear proteins including the transcription factor c-Jun, but not Nrf2, to GPEI. These results

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

Science.gov (United States)

Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

2017-04-01

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

The use of enzymes for beer brewing: Thermodynamic comparison on resource use

International Nuclear Information System (INIS)

Donkelaar, Laura H.G. van; Mostert, Joost; Zisopoulos, Filippos K.; Boom, Remko M.; Goot, Atze-Jan van der

2016-01-01

The exergetic performance of beer produced by the conventional malting and brewing process is compared with that of beer produced using an enzyme-assisted process. The aim is to estimate if the use of an exogenous enzyme formulation reduces the environmental impact of the overall brewing process. The exergy efficiency of malting was 77%. The main exergy losses stem from the use of natural gas for kilning and from starch loss during germination. The exergy efficiency of the enzyme production process ranges between 20% and 42% depending on if the by-product was considered useful. The main exergy loss was due to high power requirement for fermentation. The total exergy input in the enzyme production process was 30 times the standard chemical exergy of the enzyme, which makes it exergetically expensive. Nevertheless, the total exergy input for the production of 100 kg beer was larger for the conventional process (441 MJ) than for the enzyme-assisted process (354 MJ). Moreover, beer produced using enzymes reduced the use of water, raw materials and natural gas by 7%, 14% and 78% respectively. Consequently, the exergy loss in the enzyme production process is compensated by the prevention of exergy loss in the total beer brewing process. - Highlights: • The exergetic production costs of enzymes are ±30 times their standard chemical exergy. • These costs of enzymes should be taken into account in exergy analysis. • Enzyme-assisted brewing is more exergy efficient than brewing with malted barley. • Enzyme-assisted brewing saves raw material, water and energy.

Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

Science.gov (United States)

Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

2016-01-01

Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

Candidate coffee reference material for element content: production and certification schemes adopted at CENA/USP

Energy Technology Data Exchange (ETDEWEB)

Tagliaferro, Fabio Sileno; Fernandes, Elisabete A. de Nadai; Bacchi, Marcio Arruda; Franca, Elvis Joacir de [Centro de Energia Nuclear na Agricultura (CENA/USP), Piracicaba, SP (Brazil). Lab. de Radioisotopos], e-mail: fabiotag@cena.usp.br, e-mail: lis@cena.usp.br, e-mail: mabacchi@cena.usp.br, e-mail: ejfranca@cena.usp.br; Bode, Peter; Bacchi, Marcio Arruda; Franca, Elvis Joacir de [Delft University of Technology, Delft (Netherlands). Interfaculty Reactor Inst.], e-mail: P.Bode@iri.tudelft.nl

2003-07-01

Certified reference materials (CRMs) play a fundamental role in analytical chemistry establishing the traceability of measurement results and assuring accuracy and reliability. In spite of the huge importance of measurements in the food sector, Brazil does not produce CRMs to supply the demand. Consequently the acquisition of CRMs depends on imports at high costs. The coffee sector needs CRMs, however there is no material that represents the coffee composition. Since 1998, the Laboratorio de Radioisotopos (LRi) of CENA/USP has been involved in analysis of coffee. During this period, knowledge has been accumulated about several aspects of coffee, such as system of cultivation, elemental composition, homogeneity of the material, possible contaminants and physical properties of beans. Concomitantly, LRi has concentrated efforts in the field of metrology in chemistry, and now all this expertise is being used as the basis for the production of a coffee certified reference material (CRM) for inorganic element content. The scheme developed for the preparation and certification of coffee RM relies on the ISO Guides 34 and 35. The approaches for selection, collection and preparation of the material, moisture determination method, homogeneity testing, certification and long-term stability testing are discussed and a time frame for the expected accomplishments is provided. (author)

Candidate coffee reference material for element content: production and certification schemes adopted at CENA/USP

International Nuclear Information System (INIS)

Tagliaferro, Fabio Sileno; Fernandes, Elisabete A. de Nadai; Bacchi, Marcio Arruda; Franca, Elvis Joacir de; Bode, Peter; Bacchi, Marcio Arruda; Franca, Elvis Joacir de

2003-01-01

Certified reference materials (CRMs) play a fundamental role in analytical chemistry establishing the traceability of measurement results and assuring accuracy and reliability. In spite of the huge importance of measurements in the food sector, Brazil does not produce CRMs to supply the demand. Consequently the acquisition of CRMs depends on imports at high costs. The coffee sector needs CRMs, however there is no material that represents the coffee composition. Since 1998, the Laboratorio de Radioisotopos (LRi) of CENA/USP has been involved in analysis of coffee. During this period, knowledge has been accumulated about several aspects of coffee, such as system of cultivation, elemental composition, homogeneity of the material, possible contaminants and physical properties of beans. Concomitantly, LRi has concentrated efforts in the field of metrology in chemistry, and now all this expertise is being used as the basis for the production of a coffee certified reference material (CRM) for inorganic element content. The scheme developed for the preparation and certification of coffee RM relies on the ISO Guides 34 and 35. The approaches for selection, collection and preparation of the material, moisture determination method, homogeneity testing, certification and long-term stability testing are discussed and a time frame for the expected accomplishments is provided. (author)

High-throughput screening for industrial enzyme production hosts by droplet microfluidics

DEFF Research Database (Denmark)

Sjostrom, Staffan L.; Bai, Yunpeng; Huang, Mingtao

2014-01-01

A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its α......-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in α-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host...

Biosynthesis of cellulolytic enzymes by Aspergillus niger A. n. 33 and its selectants

Energy Technology Data Exchange (ETDEWEB)

Czajkowska, D.; Hornecka, D.; Ilnicka-Olejniczak, O.

1988-01-01

The aim of the investigations was to obtain - from the parent strain Aspergillus niger A.n. 33 - selectants with an increased ability of cellulolytic enzymes biosynthesis. Own selection methods allowed to receive two selectants A.n. 33/2 and A.n. 33/20 characterized by enhanced activities of saccharifying cellulase (respectively 0.11 and 0.14 FPU/cm/sup 3/), endo-beta-1,4-glucanase (15.4 and 21.8 U/cm/sup 3/) and cellobiase (0.6 and 1.4 IU/cm/sup 3/) as compared with the parent strain (FPA - 0.09 IU, CMC - 8.2 U and CB - 0.1 IU/cm/sup 3/). Moreover, the selectants differed in shape and size of conidial heads, in shape and colour of conidia, as well as in structure and shape of hyphase. Enzyme preparations obtained after ultrafiltration of liquid cultures were characterized by following activities: FPA-4-16 IU, CMC-900-1800 U, CB-60-120 IU and xylanase-250-280 IU/cm/sup 3/.

Angiotensin-converting enzyme inhibition in diabetic nephropathy

DEFF Research Database (Denmark)

Parving, H H; Rossing, P; Hommel, E

1995-01-01

The aim of our prospective study was to evaluate putative progression promoters, kidney function, and prognosis during long-term treatment with angiotensin-converting enzyme inhibition in insulin-dependent diabetes mellitus patients suffering from diabetic nephropathy. Eighteen consecutive......, albuminuria (geometric mean +/- antilog SE) 982 +/- 1.2 micrograms/min, and GFR 98 +/- 5 mL/min/1.73 m2. Angiotensin-converting enzyme inhibition induced a significant reduction during the whole treatment period of blood pressure (137/85 +/- 3/1 mm Hg; P ....01), and the rate of decline in GFR was 4.4 +/- 0.7 mL/min/yr, in contrast to previous reports of 10 to 14 mL/min/yr (natural history). Univariate analysis revealed a significant correlation between the rate of decline in GFR and mean arterial blood pressure (r = 0.58, P = 0.01), albuminuria (r = 0.67, P

The secreted L-arabinose isomerase displays anti-hyperglycemic effects in mice.

Science.gov (United States)

Rhimi, Moez; Bermudez-Humaran, Luis G; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, Héla; Langella, Philippe; Maguin, Emmanuelle

2015-12-21

The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food

Assessing cellulolysis in passive treatment systems for mine drainage: a modified enzyme assay.

Science.gov (United States)

McDonald, Corina M; Gould, W Douglas; Lindsay, Matthew B J; Blowes, David W; Ptacek, Carol J; Condon, Peter D

2013-01-01

A modified cellulase enzyme assay was developed to monitor organic matter degradation in passive treatment systems for mine drainage. This fluorogenic substrate method facilitates assessment of exo-(1,4)-β-D-glucanase, endo-(1,4)-β-D-glucanase, and β-glucosidase, which compose an important cellulase enzyme system. The modified method was developed and refined using samples of organic carbon-amended mine tailings from field experiments where sulfate reduction was induced as a strategy for managing water quality. Sample masses (3 g) and the number of replicates ( ≥ 3) were optimized. Matrix interferences within these metal-rich samples were found to be insignificant. Application of this modified cellulase assay method provided insight into the availability and degradation of organic carbon within the amended tailings. Results of this study indicate that cellulase enzyme assays can be applied to passive treatment systems for mine drainage, which commonly contain elevated concentrations of metals. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes.

Science.gov (United States)

Wei, Hui; Wang, Erkang

2013-07-21

Over the past few decades, researchers have established artificial enzymes as highly stable and low-cost alternatives to natural enzymes in a wide range of applications. A variety of materials including cyclodextrins, metal complexes, porphyrins, polymers, dendrimers and biomolecules have been extensively explored to mimic the structures and functions of naturally occurring enzymes. Recently, some nanomaterials have been found to exhibit unexpected enzyme-like activities, and great advances have been made in this area due to the tremendous progress in nano-research and the unique characteristics of nanomaterials. To highlight the progress in the field of nanomaterial-based artificial enzymes (nanozymes), this review discusses various nanomaterials that have been explored to mimic different kinds of enzymes. We cover their kinetics, mechanisms and applications in numerous fields, from biosensing and immunoassays, to stem cell growth and pollutant removal. We also summarize several approaches to tune the activities of nanozymes. Finally, we make comparisons between nanozymes and other catalytic materials (other artificial enzymes, natural enzymes, organic catalysts and nanomaterial-based catalysts) and address the current challenges and future directions (302 references).

Evaluation of the Discrepancy between the European Pharmacopoeia Test and an Adopted United States Pharmacopoeia Test Regarding the Weight Uniformity of Scored Tablet Halves: Is Harmonization Required?

Science.gov (United States)

Zaid, Abdel Naser; Ghoush, Abeer Abu; Al-Ramahi, Rowa'; Are'r, Mohammed

2012-01-01

The aim of this study was to evaluate whether there exists any discrepancy between the European Pharmacopoeia (Ph. Eur.) and adopted United States Pharmacopeia (USP) tests concerning the weight uniformity measurements of tablet halves after splitting. The USP method does not contain provisions to evaluate split tablets, so here we adopt their whole tablet weight uniformity method. Twenty-nine different commercial scored tablets (local and imported) were divided. The split units were individually weighed and the relative standard deviation (RSD) for each product was calculated and then evaluated according to both the adopted USP and the Ph. Eur. tests of weight uniformity. Twenty out of the 29 products tested failed the USP test, while 14 of them failed the Ph. Eur. test. Nine products passed both the USP and Ph. Eur. tests. Six products passed the Ph. Eur. test but failed the USP test, with all of these products having an RSD greater than 6%. The correlation coefficient between the weight and content of split halves for three randomly selected products-corotenol 100 mg, corotenol 50 mg, and lorazepam 2.5 mg-was found to be 0.986, 0.998, and 0.72, respectively. A clear difference can be seen between outcomes obtained by the two compendial tablet splitting methods with regard to weight uniformity. Results from the USP test showed that tighter measures are needed to pass the test. Our results argue that the Ph. Eur. should revise the existing weight uniformity test on scored tablets to include the RSD parameter in it. The USP should include this adopted test as a specific test for scored tablet halves, not just whole tablets. Manufacturers in some cases will need to improve the quality of the produced scored tablets in order to pass the USP test, especially those with low therapeutic indices. Finally, harmonization between the pharmacopoeias regarding the weight uniformity testing of split tablets is warranted. The aim of this study was to evaluate whether there

Enzymes involved in cholesterol homeostasis in outer vs inner cortices of the guinea pig adrenal

International Nuclear Information System (INIS)

Brody, R.I.

1988-01-01

Adrenocortical cells require cholesterol for steroid hormone synthesis. Intracellular free cholesterol levels are maintained by the actions of three key enzymes: HMG CoA reductase, a rate limiting enzyme of cholesterol biosynthesis, acyl CoA:cholesterol acyltransferase (ACAT), which esterifies cholesterol to fatty acids, and cholesterol ester hydrolase (CEH), which releases stored cholesterol by clearing the ester bond. The guinea pig adrenal cortex, which can be separated into a lipid-rich outer zone and a lipid-poor inner zone, provides a good model in which to determine whether the morphological differences in these regions correlate with functional distinctions in enzymes of cholesterol homeostasis. These studies have shown that there are great differences in these enzymes in the outer and inner zones of the guinea pig adrenal cortex. The cholesterol-rich outer zone possesses greater activities of ACAT and CEH than the inner zone, and, in untreated animals, these enzymes are nearly maximally stimulated. Both zones had substantial levels of HMG CoA reductase, as measured by enzyme assay and ELISA, and these levels increased following ACTH stimulation. However, only the outer zone incorporated 14 C-acetate into steroids and cholesterol to any great degree in vitro, and only in this zone was incorporation increased following incubation of cultures with ACTH. The discrepancies between HMG CoA reductase levels and 14 C-acetate incorporation in the inner zone indicate that cholesterol synthesis must be regulated differently in this zone

[Advances on enzymes and enzyme inhibitors research based on microfluidic devices].

Science.gov (United States)

Hou, Feng-Hua; Ye, Jian-Qing; Chen, Zuan-Guang; Cheng, Zhi-Yi

2010-06-01

With the continuous development in microfluidic fabrication technology, microfluidic analysis has evolved from a concept to one of research frontiers in last twenty years. The research of enzymes and enzyme inhibitors based on microfluidic devices has also made great progress. Microfluidic technology improved greatly the analytical performance of the research of enzymes and enzyme inhibitors by reducing the consumption of reagents, decreasing the analysis time, and developing automation. This review focuses on the development and classification of enzymes and enzyme inhibitors research based on microfluidic devices.

Lithium-induced neuroprotection in stroke involves increased miR-124 expression, reduced RE1-silencing transcription factor abundance and decreased protein deubiquitination by GSK3β inhibition-independent pathways.

Science.gov (United States)

Doeppner, Thorsten R; Kaltwasser, Britta; Sanchez-Mendoza, Eduardo H; Caglayan, Ahmet B; Bähr, Mathias; Hermann, Dirk M

2017-03-01

Lithium promotes acute poststroke neuronal survival, which includes mechanisms that are not limited to GSK3β inhibition. However, whether lithium induces long-term neuroprotection and enhanced brain remodeling is unclear. Therefore, mice were exposed to transient middle cerebral artery occlusion and lithium (1 mg/kg bolus followed by 2 mg/kg/day over up to 7 days) was intraperitoneally administered starting 0-9 h after reperfusion onset. Delivery of lithium no later than 6 h reduced infarct volume on day 2 and decreased brain edema, leukocyte infiltration, and microglial activation, as shown by histochemistry and flow cytometry. Lithium-induced neuroprotection persisted throughout the observation period of 56 days and was associated with enhanced neurological recovery. Poststroke angioneurogenesis and axonal plasticity were also enhanced by lithium. On the molecular level, lithium increased miR-124 expression, reduced RE1-silencing transcription factor abundance, and decreased protein deubiquitination in cultivated cortical neurons exposed to oxygen-glucose deprivation and in brains of mice exposed to cerebral ischemia. Notably, this effect was not mimicked by pharmacological GSK3β inhibition. This study for the first time provides efficacy data for lithium in the postacute ischemic phase, reporting a novel mechanism of action, i.e. increased miR-124 expression facilitating REST degradation by which lithium promotes postischemic neuroplasticity and angiogenesis.

Direct comparison of enzyme histochemical and immunohistochemical methods to localize an enzyme

NARCIS (Netherlands)

van Noorden, Cornelis J. F.

2002-01-01

Immunohistochemical localization of enzymes is compared directly with localization of enzyme activity with (catalytic) enzyme histochemical methods. The two approaches demonstrate principally different aspects of an enzyme. The immunohistochemical method localizes the enzyme protein whether it is

Computational enzyme design: transitioning from catalytic proteins to enzymes.

Science.gov (United States)

Mak, Wai Shun; Siegel, Justin B

2014-08-01

The widespread interest in enzymes stem from their ability to catalyze chemical reactions under mild and ecologically friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biological catalysts capable of performing the desired chemical transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chemistry and force-field based protein molecular modeling have led to the design of novel proteins capable of catalyzing chemical reactions not catalyzed by naturally occurring enzymes. Here we discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward. Published by Elsevier Ltd.

Identification of over producer strain of endo-ß-1,4-glucanase in ...

African Journals Online (AJOL)

Cellulases are a group of hydrolytic enzymes capable of degrading cellulose to smaller sugar components like glucose units. These enzymes are produced by fungi and bacteria. The aim of this research was to identify a Aspergillus species with over production of endo-β-1,4-glucanase. Properties of endo-β-1 ...

Stabilization of enzymes in ionic liquids via modification of enzyme charge.

Science.gov (United States)

Nordwald, Erik M; Kaar, Joel L

2013-09-01

Due to the propensity of ionic liquids (ILs) to inactivate enzymes, the development of strategies to improve enzyme utility in these solvents is critical to fully exploit ILs for biocatalysis. We have developed a strategy to broadly improve enzyme utility in ILs based on elucidating the effect of charge modifications on the function of enzymes in IL environments. Results of stability studies in aqueous-IL mixtures indicated a clear connection between the ratio of enzyme-containing positive-to-negative sites and enzyme stability in ILs. Stability studies of the effect of [BMIM][Cl] and [EMIM][EtSO4 ] on chymotrypsin specifically found an optimum ratio of positively-charged amine-to-negatively-charged acid groups (0.39). At this ratio, the half-life of chymotrypsin was increased 1.6- and 4.3-fold relative to wild-type chymotrypsin in [BMIM][Cl] and [EMIM][EtSO4 ], respectively. The half-lives of lipase and papain were similarly increased as much as 4.0 and 2.4-fold, respectively, in [BMIM][Cl] by modifying the ratio of positive-to-negative sites of each enzyme. More generally, the results of stability studies found that modifications that reduce the ratio of enzyme-containing positive-to-negative sites improve enzyme stability in ILs. Understanding the impact of charge modification on enzyme stability in ILs may ultimately be exploited to rationally engineer enzymes for improved function in IL environments. Copyright © 2013 Wiley Periodicals, Inc.

Artificial Enzymes, "Chemzymes"

DEFF Research Database (Denmark)

Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M

2008-01-01

Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successf......Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models...... that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called...... "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well...

Comparative gene expression of intestinal metabolizing enzymes.

Science.gov (United States)

Shin, Ho-Chul; Kim, Hye-Ryoung; Cho, Hee-Jung; Yi, Hee; Cho, Soo-Min; Lee, Dong-Goo; Abd El-Aty, A M; Kim, Jin-Suk; Sun, Duxin; Amidon, Gordon L

2009-11-01

The purpose of this study was to compare the expression profiles of drug-metabolizing enzymes in the intestine of mouse, rat and human. Total RNA was isolated from the duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mouse, rat and human were ca. 60% of 22690 sequences, 40% of 8739 and 47% of 12559, respectively. Total genes of metabolizing enzymes subjected in this study were 95, 33 and 68 genes in mouse, rat and human, respectively. Of phase I enzymes, the mouse exhibited abundant gene expressions for Cyp3a25, Cyp4v3, Cyp2d26, followed by Cyp2b20, Cyp2c65 and Cyp4f14, whereas, the rat showed higher expression profiles of Cyp3a9, Cyp2b19, Cyp4f1, Cyp17a1, Cyp2d18, Cyp27a1 and Cyp4f6. However, the highly expressed P450 enzymes were CYP3A4, CYP3A5, CYP4F3, CYP2C18, CYP2C9, CYP2D6, CYP3A7, CYP11B1 and CYP2B6 in the human. For phase II enzymes, glucuronosyltransferase Ugt1a6, glutathione S-transferases Gstp1, Gstm3 and Gsta2, sulfotransferase Sult1b1 and acyltransferase Dgat1 were highly expressed in the mouse. The rat revealed predominant expression of glucuronosyltransferases Ugt1a1 and Ugt1a7, sulfotransferase Sult1b1, acetyltransferase Dlat and acyltransferase Dgat1. On the other hand, in human, glucuronosyltransferases UGT2B15 and UGT2B17, glutathione S-transferases MGST3, GSTP1, GSTA2 and GSTM4, sulfotransferases ST1A3 and SULT1A2, acetyltransferases SAT1 and CRAT, and acyltransferase AGPAT2 were dominantly detected. Therefore, current data indicated substantial interspecies differences in the pattern of intestinal gene expression both for P450 enzymes and phase II drug-metabolizing enzymes. This genomic database is expected to improve our understanding of interspecies variations in estimating intestinal prehepatic clearance of oral drugs.

Dgroup: DG00874 [KEGG MEDICUS

Lifescience Database Archive (English)

Full Text Available AN/USP) ... D02163 ... Fluphenazine maleate (JAN) ... Neuropsychiatric agent ... DG01905 ... Phenothiazine antipsychoti...ubstrate ... DG01644 ... CYP2D6 substrate ATC code: N05AB02 Antipsychotic, Dopamine D2 receptor antagonist Phenothiazine derivative DRD2 [HSA:1813] [KO:K04145] Enzyme: CYP2D6 [HSA:1565] ...

Hepatoprotective effects of Nigella sativa L and Urtica dioica L on lipid peroxidation, antioxidant enzyme systems and liver enzymes in carbon tetrachloride-treated rats

Science.gov (United States)

Kanter, Mehmet; Coskun, Omer; Budancamanak, Mustafa

2005-01-01

AIM: To investigate the effects of Nigella sativa L (NS) and Urtica dioica L (UD) on lipid peroxidation, antioxidant enzyme systems and liver enzymes in CCl4-treated rats. METHODS: Fifty-six healthy male Wistar albino rats were used in this study. The rats were randomly allotted into one of the four experimental groups: A (CCl4-only treated), B (CCl4+UD treated), C (CCl4+NS treated) and D (CCl4+UD+NS treated), each containing 14 animals. All groups received CCl4 (0.8 mL/kg of body weight, sc, twice a week for 60 d). In addition, B, C and D groups also received daily i.p. injections of 0.2 mL/kg NS or/and 2 mL/kg UD oils for 60 d. Group A, on the other hand, received only 2 mL/kg normal saline solution for 60 d. Blood samples for the biochemical analysis were taken by cardiac puncture from randomly chosen-seven rats in each treatment group at beginning and on the 60th d of the experiment. RESULTS: The CCl4 treatment for 60 d increased the lipid peroxidation and liver enzymes, and also decreased the antioxidant enzyme levels. NS or UD treatment (alone or combination) for 60 d decreased the elevated lipid peroxidation and liver enzyme levels and also increased the reduced antioxidant enzyme levels. The weight of rats decreased in group A, and increased in groups B, C and D. CONCLUSION: NS and UD decrease the lipid per-oxidation and liver enzymes, and increase the anti-oxidant defense system activity in the CCl4-treated rats. PMID:16425366

Recycle of enzymes and substrate following enzymatic hydrolysis of steam-pretreated aspenwood

Energy Technology Data Exchange (ETDEWEB)

Mes-Hartree, M.; Hogan, C.M.; Saddler, J.N.

1987-09-01

The commercial production of chemicals and fuels from lignocellulosic residues by enzymatic means still requires considerable research on both the technical and economic aspects. Two technical problems that have been identified as requiring further research are the recycle of the enzymes used in hydrolysis and the reuse of the recalcitrant cellulose remaining after incomplete hydrolysis. Enzyme recycle is required to lower the cost of the enzymes, while the reuse of the spent cellulose will lower the feedstock cost. The conversion process studied was a combined enzymatic hydrolysis and fermentation (CHF) procedure that utilized the cellulolytic enzymes derived from the fungus Trichoderma harzianum E58 and the yeast Saccharomyces cerevisiae. The rate and extent of hydrolysis and ethanol production was monitored as was the activity and hydrolytic potential of the enzymes remaining in the filtrate after the hydrolysis period. When a commercial cellulose was used as the substrate for a routine 2-day CHF process, 60% of the original filter paper activity could be recovered. When steam-treated, water-extracted aspenwood was used as the substrate, only 13% of the original filter paper activity was detected after a similar procedure. The combination of 60% spent enzymes with 40% fresh enzymes resulted in the production of 30% less reducing sugars than the original enzyme mixture. Since 100% hydrolysis of the cellulose portion is seldom accomplished in an enzymatic hydrolysis process, the residual cellulose was used as a substrate for the growth of T. harzianum E58 and production of cellulolytic enzymes. The residue remaining after the CHF process was used as a substrate for the production of the cellulolytic enzymes. The production of enzymes from the residue of the Solka Floc hydrolysis was greater than the production of enzymes from the original Solka Floc. (Refs. 14).

Distribution of enzyme activity hotspots induced by earthworms in top- and subsoil

Science.gov (United States)

Hoang, D. T. T.

2016-12-01

Earthworms (Lumbricus terrestris L.) not only affect soil physics, but they also boost microbial activities and consequently create important hotspots of microbial mediated carbon and nutrient turnover through their burrowing activity. However, it is still unknown to which extend earthworms change the enzyme distribution and activity inside their burrows in top- and subsoil horizons. We hypothesized that earthworm burrows, which are enriched in available substrates, have higher percentage of enzyme activity hotspots than soil without earthworms, and that enzyme activities decreased with increasing depth because of the increasing recalcitrance of organic matter in subsoil. We visualized enzyme distribution inside and outside of worm burrows (biopores) by in situ soil zymography and measured enzyme kinetics of 6 enzymes - β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) - in pore and bulk soil material up to 105 cm. Zymography showed a heterogeneous distribution of hotspots in worm burrows. The hotspot areas was 2.4 to 14 times larger in the burrows than in soil without earthworms. However, the dispersion index of hotspot distribution showed more aggregated hotspots in soil without earthworms than in soil with earthworms and burrow wall. Enzyme activities decreased with depth, by a factor of 2 to 8 due to fresh C input from the soil surface. Compared to bulk soil, enzyme activities in topsoil biopores were up to 11 times higher for all enzymes, but in the subsoil activities of XYL, NAG and APT were lower in earthworm biopores than bulk soil. In conclusion, hotspots were twice as concentrated close to earthworm burrows as in surrounding soil. Earthworms exerted stronger effects on enzyme activities in biopores in the topsoil than in subsoil. Keywords: Earthworms, hotspots, enzyme activities, enzyme distribution, subsoil

Study of boron detection limit using the in-air PIGE set-up at LAMFI-USP

International Nuclear Information System (INIS)

Moro, M. V.; Silva, T. F.; Trindade, G. F.; Added, N.; Tabacniks, M. H.

2014-01-01

The quantification of small amounts of boron in materials is of extreme importance in different areas of materials science. Boron is an important contaminant and also a silicon dopant in the semiconductor industry. Boron is also extensively used in nuclear power plants, either for neutron shielding or for safety control and boron is an essential nutrient for life, either vegetable or animal. The production of silicon solar cells, by refining metallurgical-grade silicon (MG-Si) requires the control and reduction of several silicon contaminants to very low concentration levels. Boron is one of the contaminants of solar-grade silicon (SG-Si) that must be controlled and quantified at sub-ppm levels. In the metallurgical purification, boron quantification is usually made by Inductive Coupled Plasma Mass Spectrometry, (ICP-MS) but the results need to be verified by an independent analytical method. In this work we present the results of the analysis of silicon samples by Particle Induced Gamma-Ray Emission (PIGE) aiming the quantification of low concentrations of boron. PIGE analysis was carried out using the in-air external beam line of the Laboratory for Materials Analysis with Ion Beams (LAMFI-USP) by the 10 B(p,αγ( 7 Be nuclear reaction, and measuring the 429 keV γ-ray. The in-air PIGE measurements at LAMFI have a quantification limit of the order of 10 16 at/cm 2

Study of boron detection limit using the in-air PIGE set-up at LAMFI-USP

Science.gov (United States)

Moro, M. V.; Silva, T. F.; Trindade, G. F.; Added, N.; Tabacniks, M. H.

2014-11-01

The quantification of small amounts of boron in materials is of extreme importance in different areas of materials science. Boron is an important contaminant and also a silicon dopant in the semiconductor industry. Boron is also extensively used in nuclear power plants, either for neutron shielding or for safety control and boron is an essential nutrient for life, either vegetable or animal. The production of silicon solar cells, by refining metallurgical-grade silicon (MG-Si) requires the control and reduction of several silicon contaminants to very low concentration levels. Boron is one of the contaminants of solar-grade silicon (SG-Si) that must be controlled and quantified at sub-ppm levels. In the metallurgical purification, boron quantification is usually made by Inductive Coupled Plasma Mass Spectrometry, (ICP-MS) but the results need to be verified by an independent analytical method. In this work we present the results of the analysis of silicon samples by Particle Induced Gamma-Ray Emission (PIGE) aiming the quantification of low concentrations of boron. PIGE analysis was carried out using the in-air external beam line of the Laboratory for Materials Analysis with Ion Beans (LAMFI-USP) by the 10B ( p ,αγ(7Be nuclear reaction, and measuring the 429 keV γ-ray. The in-air PIGE measurements at LAMFI have a quantification limit of the order of 1016 at/cm2.

Drying of enzyme immobilized on eco-friendly supports | Costa-Silva ...

African Journals Online (AJOL)

Immobilized derivatives obtained had decreased enzyme activity (≈ 30.0%) during a storage period of six months; and retained an average of 50.0% of the initial activity after five reuse cycles. Water content in immobilized derivatives varied between 4.2 and 6.1% and the water activities ranged from 0.14 to 0.30. Key words: ...

A procedure for batch separation of 14C-hexose from 14C-sucrose

International Nuclear Information System (INIS)

Tarpley, L.; Vietor, D.M.

1991-01-01

This presentation describes a method for separating 14 C-hexose from 14 C-sucrose in extracts of plant tissue. Portions of ethanol extracts are treated with activated charcoal in microcentrifuge tubes. Aliquots are removed, ethanol evaporated and replaced with reaction mixture that phosphorylates hexose (HEPPS, K 2 HPO 4 , Mg(C 2 H 3 O 2 ) 2 , ovalbumen, Na 2 ATP, yeast hexokinase). After a time course, the hexokinase reaction is stopped (slowed considerably) to minimize effects of contamination enzyme activities. The stopping agent used is lyxose, a nonphosphorylable analogue of glucose. The strong anionic charge of phosphate introduced through the hexokinase action results in binding (> 95%) of hexose-phosphate to anion-exchange resin. Sucrose remains unbound (> 95%) in solution. This batch ion-exchange is performed in microcentrifuge tubes to allow many samples to be processed simultaneously. Recovery of radiolabel in extracts is complete (99%), and determinations are repeatable (cv = 23%). This method for routinely separating and quantifying 14 C-hexose and 14 C-sucrose in plant tissue extracts can contribute to the economy and feasibility of studies of 14 C-photoassimilate partitioning to soluble sugars within and among plant tissues

Enzyme detection by microfluidics

DEFF Research Database (Denmark)

2013-01-01

Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

The regulation and catalytic mechanism of the NADP-malic enzyme from tobacco leaves

Czech Academy of Sciences Publication Activity Database

Doubnerová, V.; Potůčková, L.; Müller, Karel; Ryšlavá, H.

2009-01-01

Roč. 14, 8-9 (2009), s. 893-906 ISSN 0352-5139 Institutional research plan: CEZ:AV0Z50380511 Keywords : NADP-malic enzyme * macroergic compounds * Nicotiana tabacum L. Subject RIV: ED - Physiology Impact factor: 0.820, year: 2009

Cloning, Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris

Directory of Open Access Journals (Sweden)

Pichapak Sriyapai

2015-06-01

Full Text Available A thermostable esterase gene (hydS14 was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG and catalytic triad (Ser88-Asp208-His235 of the esterase/lipase superfamily. The HydS14 sequence shows 46%–64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases, has three conserved regions, and contains the novel motif (GY(FSLG, which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14 was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0–8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The Km and Vmax values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2–C6, displaying optimal activity with p-nitrophenyl-C4 (Kcat/Km = 11.74 mM−1·S−1. In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.

Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger.

Science.gov (United States)

van der Kaaij, R M; Yuan, X-L; Franken, A; Ram, A F J; Punt, P J; van der Maarel, M J E C; Dijkhuizen, L

2007-07-01

In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the alpha-amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with alpha-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new alpha-(1,4)-glycosidic bonds and therefore belong to the group of the 4-alpha-glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing alpha-(1,3)-glycosidic bonds as acceptor substrate. An agtA knockout of A. niger showed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with alpha-glucans in their cell walls, but not in yeast species lacking cell wall alpha-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed.

Drug: D01772 [KEGG MEDICUS

Lifescience Database Archive (English)

Full Text Available agent ... DG02030 ... Anesthetics ... DG02027 ... General anesthetics ... DG02028 ... Inhalational anaesthetics ... DG02027 ... General anesthetics... ... DG02028 ... Inhalational anaesthetics Same as: C13240 ATC code: N01AA01 ... Enzyme: CYP2E... D01772 Drug Ether (JP17/USP); Diethyl ether ... C4H10O D01772.gif ... Neuropsychiatric

7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

Science.gov (United States)

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

Dicty_cDB: Contig-U10276-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available 1_1( AB019491 |pid:none) Homo sapiens IDN4-GGTR8 mRNA, part... 39 0.52 ( Q92738 ) RecName: Full=USP6 N-terminal-li...em... 40 0.18 BC051184_1( BC051184 |pid:none) Mus musculus USP6 N-terminal like,... 39 0.31 (Q66K14) RecName...: Full=TBC1 domain family member 9B; &AB449893_... 39 0.31 (Q80XC3) RecName: Full=USP6 N-terminal-like prote...7489 ) 1095460264250 Global-Ocean-Sampling_GS-31-01-01-1... 44 0.91 2 ( EA427602 ) Sequence 77 from patent U...ns chromosome 12 clone ABC7-42389800N19... 46 3.3 1 ( ER597226 ) 1093016226857 Global-Ocean-Sampling_GS-3

Molecular imprinting of enzymes with water-insoluble ligands for nonaqueous biocatalysis.

Science.gov (United States)

Rich, Joseph O; Mozhaev, Vadim V; Dordick, Jonathan S; Clark, Douglas S; Khmelnitsky, Yuri L

2002-05-15

Attaining higher levels of catalytic activity of enzymes in organic solvents is one of the major challenges in nonaqueous enzymology. One of the most successful strategies for enhancing enzyme activity in organic solvents involves tuning the enzyme active site by molecular imprinting with substrates or their analogues. Unfortunately, numerous imprinters of potential importance are poorly soluble in water, which significantly limits the utility of this method. In the present study, we have developed strategies that overcome this limitation of the molecular-imprinting technique and that thus expand its applicability beyond water-soluble ligands. The solubility problem can be addressed either by converting the ligands into a water-soluble form or by adding relatively high concentrations of organic cosolvents, such as tert-butyl alcohol and 1,4-dioxane, to increase their solubility in the lyophilization medium. We have succeeded in applying both of these strategies to produce imprinted thermolysin, subtilisin, and lipase TL possessing up to 26-fold higher catalytic activity in the acylation of paclitaxel and 17beta-estradiol compared to nonimprinted enzymes. Furthermore, we have demonstrated for the first time that molecular imprinting and salt activation, applied in combination, produce a strong additive activation effect (up to 110-fold), suggesting different mechanisms of action involved in these enzyme activation techniques.

Elevated Liver Enzymes

Science.gov (United States)

Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

Stability of Enzymes in Granular Enzyme Products for Laundry Detergents

DEFF Research Database (Denmark)

Biran, Suzan; Bach, Poul; Simonsen, Ole

Enzymes have long been of interest to the detergent industry due to their ability to improve the cleaning efficiency of synthetic detergents, contribute to shortening washing times, and reduce energy and water consumption, provision of environmentally friendlier wash water effluents and fabric care....... However, incorporating enzymes in detergent formulations gives rise to numerous practical problems due to their incompatibility with and stability against various detergent components. In powdered detergent formulations, these issues can be partly overcome by physically isolating the enzymes in separate...... particles. However, enzymes may loose a significant part of their activity over a time period of several weeks. Possible causes of inactivation of enzymes in a granule may be related to the release of hydrogen peroxide from the bleaching chemicals in a moisture-containing atmosphere, humidity, autolysis...

High-throughput functional screening of steroid substrates with wild-type and chimeric P450 enzymes.

Science.gov (United States)

Urban, Philippe; Truan, Gilles; Pompon, Denis

2014-01-01

The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

Structural complex of sterol 14[alpha]-demethylase (CYP51) with 14[alpha]-methylenecyclopropyl-[delta]7-24, 25-dihydrolanosterol[S

Energy Technology Data Exchange (ETDEWEB)

Hargrove, Tatiana Y.; Wawrzak, Zdzislaw; Liu, Jialin; Waterman, Michael R.; Nes, W. David; Lepesheva, Galina I. (Vanderbilt); (TTU); (NWU)

2012-06-28

Sterol 14{alpha}-demethylase (CYP51) that catalyzes the removal of the 14{alpha}-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14{alpha}-methylenecyclopropyl-{Delta}7-24,25-dihydrolanosterol (MCP). This sterol binds tightly to all protozoan CYP51s and acts as a competitive inhibitor of F105-containing (plant-like) T. brucei and Leishmania (L) infantum orthologs, but it has a much stronger, mechanism-based inhibitory effect on I105-containing (animal/fungi-like) T. cruzi CYP51. Depicting substrate orientation in the conserved CYP51 binding cavity, the complex specifies the roles of the contact amino acid residues and sheds new light on CYP51 substrate specificity. It also provides an explanation for the effect of MCP on T. cruzi CYP51. Comparison with the ligand-free and azole-bound structures supports the notion of structural rigidity as the characteristic feature of the CYP51 substrate binding cavity, confirming the enzyme as an excellent candidate for structure-directed design of new drugs, including mechanism-based substrate analog inhibitors.

Enzyme structure, enzyme function and allozyme diversity in ...

African Journals Online (AJOL)

In estimates of population genetic diversity based on allozyme heterozygosity, some enzymes are regularly more variable than others. Evolutionary theory suggests that functionally less important molecules, or parts of molecules, evolve more rapidly than more important ones; the latter enzymes should then theoretically be ...

UUCD: a family-based database of ubiquitin and ubiquitin-like conjugation.

Science.gov (United States)

Gao, Tianshun; Liu, Zexian; Wang, Yongbo; Cheng, Han; Yang, Qing; Guo, Anyuan; Ren, Jian; Xue, Yu

2013-01-01

In this work, we developed a family-based database of UUCD (http://uucd.biocuckoo.org) for ubiquitin and ubiquitin-like conjugation, which is one of the most important post-translational modifications responsible for regulating a variety of cellular processes, through a similar E1 (ubiquitin-activating enzyme)-E2 (ubiquitin-conjugating enzyme)-E3 (ubiquitin-protein ligase) enzyme thioester cascade. Although extensive experimental efforts have been taken, an integrative data resource is still not available. From the scientific literature, 26 E1s, 105 E2s, 1003 E3s and 148 deubiquitination enzymes (DUBs) were collected and classified into 1, 3, 19 and 7 families, respectively. To computationally characterize potential enzymes in eukaryotes, we constructed 1, 1, 15 and 6 hidden Markov model (HMM) profiles for E1s, E2s, E3s and DUBs at the family level, separately. Moreover, the ortholog searches were conducted for E3 and DUB families without HMM profiles. Then the UUCD database was developed with 738 E1s, 2937 E2s, 46 631 E3s and 6647 DUBs of 70 eukaryotic species. The detailed annotations and classifications were also provided. The online service of UUCD was implemented in PHP + MySQL + JavaScript + Perl.

Method for enzyme synthesis of radioactive thymine 5'-deoxyribonucleotides

International Nuclear Information System (INIS)

Nejedly, Z.; Ekl, J.; Hybs, K.; Kolina, J.; Filip, J.; Votruba, I.; Skoda, J.

1978-01-01

The enzyme synthesis is described for thymidine-5'-monophosphate, thymidine-5'-diphosphate and thymidine-5'-triphosphate specifically or nonspecifically labelled with 14 C or 3 H. The anabolic transformation of radioactive thymine to radioactive thymine 5'-deoxyribonucleotides is catalyzed by the action of enzyme preparations separated from Escherichia coli bacteria. It is achieved by the action of nonpurified cell-free extracts on special auxotrophic mutants of the thymine-dependent Escherichia coli SPT - strain in the presence of deoxyriboso-1-phosphate and adenosine-5'-triphosphate. The radioactive thymidine-5'-monophosphate may further be phosphorylated. In reaction mixtures, radioactive thymine, deoxyriboso-1-phosphate and adenosine-5'-triphosphate are used in molar ratios of 1:1:2 to 1:10:100, the optimum molar ratio being 1:5:10. (B.S.)

Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

Science.gov (United States)

Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

2017-11-22

Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes

Directory of Open Access Journals (Sweden)

Philippe Urban

2014-01-01

Full Text Available The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining.

Science.gov (United States)

Schallmey, Marcus; Koopmeiners, Julia; Wells, Elizabeth; Wardenga, Rainer; Schallmey, Anett

2014-12-01

Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Study of boron detection limit using the in-air PIGE set-up at LAMFI-USP

Energy Technology Data Exchange (ETDEWEB)

Moro, M. V.; Silva, T. F.; Trindade, G. F.; Added, N.; Tabacniks, M. H. [Institute of Physics, University of São Paulo, SP (Brazil)

2014-11-11

The quantification of small amounts of boron in materials is of extreme importance in different areas of materials science. Boron is an important contaminant and also a silicon dopant in the semiconductor industry. Boron is also extensively used in nuclear power plants, either for neutron shielding or for safety control and boron is an essential nutrient for life, either vegetable or animal. The production of silicon solar cells, by refining metallurgical-grade silicon (MG-Si) requires the control and reduction of several silicon contaminants to very low concentration levels. Boron is one of the contaminants of solar-grade silicon (SG-Si) that must be controlled and quantified at sub-ppm levels. In the metallurgical purification, boron quantification is usually made by Inductive Coupled Plasma Mass Spectrometry, (ICP-MS) but the results need to be verified by an independent analytical method. In this work we present the results of the analysis of silicon samples by Particle Induced Gamma-Ray Emission (PIGE) aiming the quantification of low concentrations of boron. PIGE analysis was carried out using the in-air external beam line of the Laboratory for Materials Analysis with Ion Beams (LAMFI-USP) by the {sup 10}B(p,αγ({sup 7}Be nuclear reaction, and measuring the 429 keV γ-ray. The in-air PIGE measurements at LAMFI have a quantification limit of the order of 10{sup 16} at/cm{sup 2}.

Optimization of parameters for enhanced oil recovery from enzyme treated wild apricot kernels.

Science.gov (United States)

Rajaram, Mahatre R; Kumbhar, Baburao K; Singh, Anupama; Lohani, Umesh Chandra; Shahi, Navin C

2012-08-01

Present investigation was undertaken with the overall objective of optimizing the enzymatic parameters i.e. moisture content during hydrolysis, enzyme concentration, enzyme ratio and incubation period on wild apricot kernel processing for better oil extractability and increased oil recovery. Response surface methodology was adopted in the experimental design. A central composite rotatable design of four variables at five levels was chosen. The parameters and their range for the experiments were moisture content during hydrolysis (20-32%, w.b.), enzyme concentration (12-16% v/w of sample), combination of pectolytic and cellulolytic enzyme i.e. enzyme ratio (30:70-70:30) and incubation period (12-16 h). Aspergillus foetidus and Trichoderma viride was used for production of crude enzyme i.e. pectolytic and cellulolytic enzyme respectively. A complete second order model for increased oil recovery as the function of enzymatic parameters fitted the data well. The best fit model for oil recovery was also developed. The effect of various parameters on increased oil recovery was determined at linear, quadric and interaction level. The increased oil recovery ranged from 0.14 to 2.53%. The corresponding conditions for maximum oil recovery were 23% (w.b.), 15 v/w of the sample, 60:40 (pectolytic:cellulolytic), 13 h. Results of the study indicated that incubation period during enzymatic hydrolysis is the most important factor affecting oil yield followed by enzyme ratio, moisture content and enzyme concentration in the decreasing order. Enzyme ratio, incubation period and moisture content had insignificant effect on oil recovery. Second order model for increased oil recovery as a function of enzymatic hydrolysis parameters predicted the data adequately.

Beta-D-xylosidase from Selenomonas ruminantium: thermodynamics of enzyme-catalyzed and noncatalyzed reactions

Science.gov (United States)

Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-beta-D-xylopyranoside (4NPX), 4-nitrophenyl-alpha-L-arabi...

Mechanism of action of clostridial glycine reductase: Isolation and characterization of a covalent acetyl enzyme intermediate

International Nuclear Information System (INIS)

Arkowitz, R.A.; Abeles, R.H.

1991-01-01

Clostridial glycine reductase consists of proteins A, B, and C and catalyzes the reaction glycine + P i + 2e - → acetyl phosphate + NH 4 + . Evidence was previously obtained that is consistent with the involvement of an acyl enzyme intermediate in this reaction. The authors now demonstrate that protein C catalyzes exchange of [ 32 P]P i into acetyl phosphate, providing additional support for an acetyl enzyme intermediate on protein C. Furthermore, they have isolated acetyl protein C and shown that it is qualitatively, catalytically competent. Acetyl protein C can be obtained through the forward reaction from protein C and Se-(carboxymethyl)selenocysteine-protein A, which is generated by the reaction of glycine with proteins A and B. Acetyl protein C can also be generated through the reverse reaction by the addition of acetyl phosphate to protein C. Both procedures lead to the same acetyl enzyme. The acetyl enzyme reacts with P i to give acetyl phosphate. When [ 14 C]acetyl protein C is denaturated with TCA and redissolved with urea, radioactivity remained associated with the protein. Treatment with KBH 4 removes all the radioactivity associated with protein C, resulting in the formation of [ 14 C]ethanol. They conclude that a thiol group on protein C is acetylated. Proteins A and C together catalyze the exchange of tritium atoms from [ 3 H]H 2 O into acetyl phosphate. This exchange reaction supports the proposal that an enol of the acetyl enzyme is an intermediate in the reaction sequence

BIOINSPIRED DESIGN AND DIRECTED EVOLUTION OF IRON CONTAINING ENZYMES FOR GREENSYNTHETIC PROCESSES AND BIOREMEDIATION

Science.gov (United States)

SU833912Title: Bioinspired Design and Directed Evolution of Iron Containing Enzymes for Green Synthetic Processes and BioremediationEdward I. Solomon, Shaun D. Wong, Lei Liu, Caleb B. Bell, IIICynthia Nolt-HelmsProject Period: August 15, 2008 - August 14,...

Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

Science.gov (United States)

Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

2015-01-01

This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.

Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses.

Science.gov (United States)

Azad, Kimi; Banerjee, Manidipa; Johnson, John E

2017-09-29

Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

PHGDH Defines a Metabolic Subtype in Lung Adenocarcinomas with Poor Prognosis

Directory of Open Access Journals (Sweden)

Boxi Zhang

2017-06-01

Full Text Available Molecular signatures are emerging determinants of choice of therapy for lung adenocarcinomas. An evolving therapeutic approach includes targeting metabolic dependencies in cancers. Here, using an integrative approach, we have dissected the metabolic fingerprints of lung adenocarcinomas, and we show that Phosphoglycerate dehydrogenase (PHGDH, the rate-limiting enzyme in serine biosynthesis, is highly expressed in a adenocarcinoma subset with poor prognosis. This subset harbors a gene signature for DNA replication and proliferation. Accordingly, models with high levels of PHGDH display rapid proliferation, migration, and selective channeling of serine-derived carbons to glutathione and pyrimidines, while depletion of PHGDH shows potent and selective toxicity to this subset. Differential PHGDH protein levels were defined by its degradation, and the deubiquitinating enzyme JOSD2 is a regulator of its protein stability. Our study provides evidence that a unique metabolic program is activated in a lung adenocarcinoma subset, described by PHGDH, which confers growth and survival and may have therapeutic implications.

Purification and characterization of sheep platelet cyclo-oxygenase. Acetylation by aspirin prevents haemin binding to the enzyme.

Science.gov (United States)

Boopathy, R; Balasubramanian, A S

1986-01-01

Arachidonate cyclo-oxygenase (prostaglandin synthetase; prostaglandin endoperoxide synthetase; EC 1.14.99.1) was purified from sheep platelets. The purification procedure involved hydrophobic column chromatography using either Ibuprofen-Sepharose, phenyl-Sepharose or arachidic acid-Sepharose as the first step followed by metal-chelate Sepharose and haemin-Sepharose affinity chromatography. The purified enzyme (Mr approximately 65,000) was homogeneous as observed by SDS/polyacrylamide-gel electrophoresis and silver staining. The enzyme was a glycoprotein with mannose as the neutral sugar. Haemin or haemoglobin was essential for activity. The purified enzyme could bind haemin exhibiting a characteristic absorption maximum at 410 nm. The enzyme after metal-chelate column chromatography could undergo acetylation by [acetyl-3H]aspirin. The labelled acetylated enzyme could not bind to haemin-Sepharose, presumably due to acetylation of a serine residue involved in the binding to haemin. The acetylated enzyme also failed to show its characteristic absorption maximum at 410 nm when allowed to bind haemin. Images Fig. 1. Fig. 4. PMID:3101664

Effect of low concentrations of ozone on the enzymes catalase, peroxidase, papain and urease

Energy Technology Data Exchange (ETDEWEB)

Todd, G W

1958-01-01

The enzymes catalase, peroxidase, papain and urease were treated in vitro with low concentrations of ozone gas. Wide variations were found in the sensitivity of the enzymes to the inhibitory action of the gas. Papain showed the greatest sensitivity; the rest required a much greater amount of ozone for inactivation. Comparisons of ozone and hydrogen peroxide as inhibitors of papain and urease showed ozone to be 30 times as effective as hydrogen peroxide on papain and 3 times as effective on urease. 14 references, 2 figures, 3 tables.

Anksioznost učencev 6. razreda osnovne šole pri pouku angleščine kot tujega jezika v povezavi z njihovo učno uspešnostjo

OpenAIRE

Praček, Anja

2017-01-01

Magistrsko delo obravnava anksioznost šestošolcev pri pouku angleščine kot tujega jezika v povezavi z njihovo učno uspešnostjo pri pouku angleščine. Pouk angleščine se v marsičem razlikuje od drugih šolskih predmetov in prav ta posebna učna situacija lahko pri učencih izzove anksioznost. Magistrsko delo je sestavljeno iz teoretičnega in empiričnega dela. V teoretičnem delu je s pomočjo literature na kratko predstavljena trenutna situacija poučevanja in učenja angleščine v slovenskih javnih...

The surface science of enzymes

DEFF Research Database (Denmark)

Rod, Thomas Holm; Nørskov, Jens Kehlet

2002-01-01

One of the largest challenges to science in the coming years is to find the relation between enzyme structure and function. Can we predict which reactions an enzyme catalyzes from knowledge of its structure-or from its amino acid sequence? Can we use that knowledge to modify enzyme function......? To solve these problems we must understand in some detail how enzymes interact with reactants from its surroundings. These interactions take place at the surface of the enzyme and the question of enzyme function can be viewed as the surface science of enzymes. In this article we discuss how to describe...... catalysis by enzymes, and in particular the analogies between enzyme catalyzed reactions and surface catalyzed reactions. We do this by discussing two concrete examples of reactions catalyzed both in nature (by enzymes) and in industrial reactors (by inorganic materials), and show that although analogies...

Patterns of functional enzyme activity in fungus farming ambrosia beetles.

Science.gov (United States)

De Fine Licht, Henrik H; Biedermann, Peter H W

2012-06-06

In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray

Magnetically responsive enzyme powders

Energy Technology Data Exchange (ETDEWEB)

Pospiskova, Kristyna, E-mail: kristyna.pospiskova@upol.cz [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Safarik, Ivo, E-mail: ivosaf@yahoo.com [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

2015-04-15

Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction.

Enzymes in Fermented Fish.

Science.gov (United States)

Giyatmi; Irianto, H E

Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

Effect of aspirin and prostaglandins on the carbohydrate metabolism in albino rats.: glucose oxidation through different pathways and glycolytic enzymes

International Nuclear Information System (INIS)

Balasubramanian, A.; Ramakrishnan, S.

1980-01-01

The effect of chronic and acute doses of aspirin and prostaglandins F2α and E2 individually on the oxidation of glucose through Embden Meyerhof-TCA cycle and pentose phosphate pathways and some key glycolytic enzymes of liver were studied in male albino rats. Studies were extended to find the combined effect of PGF2α and E2 with an acute dose of aspirin. There was increased utilisation of both 1- 14 C glucose and 6- 14 C glucose on aspirin treatment. However, the metabolism through the EM-TCA pathway was more pronounced as shown by a reduced ratio of 14 CO 2 from 1- 14 C and 6- 14 C glucose. Two hepatic key glycolytic enzymes viz. hexokinase and pyruvate kinase were increased due to aspirin treatment. Withdrawal of aspirin corrected the above impaired carbohydrate metabolism in liver. Prostaglandin F2α also caused a reduction in the utilisation of 1- 14 C glucose, while PGE2 recorded an increase in the utilisation of both 1- 14 C and 6- 14 C glucose when compared to controls, indicating that different members of prostaglandins could affect metabolisms and differently. Administration of the PGs and aspirin together showed an increase in the utilisation of 6- 14 C glucose. (auth.)

Thermodynamic activity-based intrinsic enzyme kinetic sheds light on enzyme-solvent interactions.

Science.gov (United States)

Grosch, Jan-Hendrik; Wagner, David; Nistelkas, Vasilios; Spieß, Antje C

2017-01-01

The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics, and enzyme-solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert-butyl ether (MTBE) mixtures as a model solvent. Substrate concentrations and substrate thermodynamic activities were varied separately to identify the individual thermodynamic and kinetic effects on the enzyme activity. Microkinetic parameters based on concentration and thermodynamic activity were derived to successfully identify a positive effect of MTBE on the availability of the substrate to the enzyme, but a negative effect on the enzyme performance. In conclusion, thermodynamic activity-based kinetic modeling might be a suitable tool to initially curtail the type of enzyme-solvent interactions and thus, a powerful first step to potentially understand the phenomena that occur in nonconventional media in more detail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:96-103, 2017. © 2016 American Institute of Chemical Engineers.

Profiling the orphan enzymes

Science.gov (United States)

2014-01-01

The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new

Dgroup: DG00792 [KEGG MEDICUS

Lifescience Database Archive (English)

Full Text Available hydrochloride (USP) ... Neuropsychiatric agent ... DG02030 ... Anesthetics ... DG02027 ... General anesthetics ... DG02026 ... Opioid anesthetics... ... DG02027 ... General anesthetics ... DG02026 ... Opioid anesthetics ... DG01564 ... Opioid receptor ...agonist ... DG01563 ... mu-Opioid receptor agonist ATC code: N01AH02 General anesthetics OPRM1 [HSA:4988] [KO:K04215] Enzyme: CYP3A [HSA:1576 1577 1551] ...

Effect of irradiation on immobilized enzymes compared with that on enzymes in solution

International Nuclear Information System (INIS)

Schachinger, L.; Schippel, C.; Altmann, E.; Diepold, B.; Yang, C.; Jaenike, M.; Hochhaeuser, E.

1985-01-01

Glucose oxidase and catalase were immobilized by attaching them to nylon fibers that had been treated with triethyloxonium-tetrafluoroborate, diaminohexane and glutaraldialdehyde according to Morris, Campell and Hornby (1975). This method assures that the enzymes are bound to a side chain of the polyamide structure. Enzyme activity (as measured by the O 2 -uptake and by microcalorimetry) was found to be unchanged after 2 years. The apparent Ksub(m)-constants of the immobilized enzymes with glucose were the same as those for enzymes in solution. GOD and catalase immobilized in poly(acrylamide) gel had the same Ksub(m)-value. Despite the high stability during storage, the radiation induced inactivation of enzymes immobilized on gel or chromosorb, an inorganic carrier, was of the same order of magnitude as that of the dissolved enzymes. The enzymes bound to nylon fibers showed a higher radiation sensitivity. This might have been caused by an additional attack on the binding site of the carrier. (orig.)

Enzyme-driven Bacillus spore coat degradation leading to spore killing.

Science.gov (United States)

Mundra, Ruchir V; Mehta, Krunal K; Wu, Xia; Paskaleva, Elena E; Kane, Ravi S; Dordick, Jonathan S

2014-04-01

The bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment. However these enzymes are poorly characterized. Nonetheless given the potential for proteases to catalyze coat degradation, we screened 10 commercially available proteases for their ability to degrade the spore coats of B. cereus and B. anthracis. Proteinase K and subtilisin Carlsberg, for B. cereus and B. anthracis spore coats, respectively, led to a morphological change in the otherwise impregnable coat structure, increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating germination. Specifically in the presence of lysozyme, proteinase K resulted in 14-fold faster enzyme induced germination and exhibited significantly shorter lag times, than spores without protease pretreatment. Furthermore, the germinated spores were shown to be vulnerable to a lytic enzyme (PlyPH) resulting in effective spore killing. The spore surface in response to proteolytic degradation was probed using scanning electron microscopy (SEM), which provided key insights regarding coat degradation. The extent of coat degradation and spore killing using this enzyme-based pretreatment approach is similar to traditional, yet far harsher, chemical decoating methods that employ detergents and strong denaturants. Thus the enzymatic route reduces the environmental burden of chemically mediated spore killing, and demonstrates that a mild and environmentally benign biocatalytic spore killing is achievable. © 2013 Wiley Periodicals, Inc.

Molecular engineering of fungal GH5 and GH26 beta-(1,4-mannanases toward improvement of enzyme activity.

Directory of Open Access Journals (Sweden)

Marie Couturier

Full Text Available Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in kcat /KM due to a significant decrease of KM, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in kcat /KM compared to the parental enzyme. It displayed a mutation in the linker region (P140L that may confer more flexibility to the linker and another mutation (D416G located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (kcat/KM.

Direct Electron Transfer of Enzymes in a Biologically Assembled Conductive Nanomesh Enzyme Platform.

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Lee, Seung-Woo; Lee, Ki-Young; Song, Yong-Won; Choi, Won Kook; Chang, Joonyeon; Yi, Hyunjung

2016-02-24

Nondestructive assembly of a nanostructured enzyme platform is developed in combination of the specific biomolecular attraction and electrostatic coupling for highly efficient direct electron transfer (DET) of enzymes with unprecedented applicability and versatility. The biologically assembled conductive nanomesh enzyme platform enables DET-based flexible integrated biosensors and DET of eight different enzyme with various catalytic activities. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of enzyme inducing anticonvulsants on ethosuximide pharmacokinetics in epileptic patients

Science.gov (United States)

GIACCONE, M.; BARTOLI, A.; GATTI, G.; MARCHISELLI, R.; PISANI, F.; LATELLA, M.A.; PERUCCA, E.

1996-01-01

1To assess the effect of enzyme inducing anticonvulsants on ethosuximide pharmacokinetics, plasma ethosuximide concentrations after a single oral dose (500 mg) of the drug were compared in 12 healthy control subjects and 10 epileptic patients receiving chronic therapy with phenobarbitone, phenytoin and/or carbamazepine. 2Compared with controls, epileptic patients showed markedly shorter ethosuximide half-lives (29.0±7.8 vs 53.7±14.3 h, means±s.d., Panticonvulsants, the effect probably being mediated by stimulation of cytochrome CYP3A activity. 4The enhancement of ethosuximide clearance in patients comedicated with enzyme inducing anticonvulsants is likely to be clinically relevant. Higher ethosuximide dosages will be required to achieve therapeutic drug concentrations in these patients. PMID:8799524

Site-saturation mutagenesis of Glomerella cingulata cutinase gene for enhanced enzyme thermostability

Science.gov (United States)

Hanapi, Wan Nurhidayah Wan; Iuan-Sheau, Chin; Mahadi, Nor Muhammad; Murad, Abdul Munir Abdul; Bakar, Farah Diba Abu

2015-09-01

Cutinase is an important biocatalyst for various industrial applications. This enzyme which has dual functionality comparable to esterases and lipases, is efficient in the hydrolysis of soluble esters and emulsified triacylglycerols. Naturally-occurring enzymes usually have disadvantages when applied in non-natural catalysis due to Glomerella cingulata cutinase enzyme thermostability. It is postulated that by increasing the rigidity at certain amino acid positions showing high mobility based on the three-dimensional structure of G. cingulata cutinase, the improvement in thermostability will be achieved. The amino acid N82 of G. cingulata cutinase was selected based on its high B-factor value determined via the B-FITTER program. Megaprimer PCR was employed to introduce mutations at the chosen site by randomization using NNK degenerate primers. About 300 transformants were selected for screening of positive cutinase variants. The N82_V14 cutinase variant was observed to be more thermostable at an almost 2-fold increase when exposed at 50°C for 1 hr as compared to the wild-type enzyme. This study may provide valuable information regarding thermal stability of cutinases denaturation at high temperatures.

[Utilizing the ultraintense JanUSP laser at LLNL]. 99-ERD-049 Final LDRD Report

International Nuclear Information System (INIS)

Patel, P K; Price, D F; Mackinnon, A J; Springer, P T

2002-01-01

Recent advances in laser and optical technologies have now enabled the current generation of high intensity, ultrashort-pulse lasers to achieve focal intensities of 10 20 -10 21 W/cm 2 in pulse durations of 100-500fs. These ultraintense laser pulses are capable of producing highly relativistic plasma states with densities, temperatures, and pressures rivaling those found in the interiors of stars and nuclear weapons. Utilizing the ultraintense 100TW JanUSP laser at LLNL we have explored the possibility of ion shock heating small micron-sized plasmas to extremely high energy densities approaching 1GJ/g on timescales of a few hundred femtoseconds. The JanUSP laser delivers 10 Joules of energy in a 100fs pulse in a near diffraction-limited beam, producing intensities on target of up to 10 21 W/cm 2 . The electric field of the laser at this intensity ionizes and accelerates electrons to relativistic MeV energies. The sudden ejection of electrons from the focal region produces tremendous electrostatic forces which in turn accelerate heavier ions to MeV energies. The predicted ion flux of 1 MJ/cm 2 is sufficient to achieve thermal equilibrium conditions at high temperature in solid density targets. Our initial experiments were carried out at the available laser contrast of 10 -7 (i.e. the contrast of the amplified spontaneous emission (ASE), and of the pre-pules produced in the regenerative amplifier). We used the nuclear photoactivation of Au-197 samples to measure the gamma production above 12MeV-corresponding to the threshold for the Au-197(y,n) reaction. Since the predominant mechanism for gamma production is through the bremsstrahlung emission of energetic electrons as they pass through the solid target we were able to infer a conversion yield of several percent of the incident laser energy into electrons with energies >12MeV. This result is consistent with the interaction of the main pulse with a large pre-formed plasma. The contrast of the laser was improved to

Enzymes for improved biomass conversion

Science.gov (United States)

Brunecky, Roman; Himmel, Michael E.

2016-02-02

Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

Deduction of kinetic mechanism in multisubstrate enzyme reactions from tritium isotope effects. Application to dopamine beta-hydroxylase

International Nuclear Information System (INIS)

Klinman, J.P.; Humphries, H.; Voet, J.G.

1980-01-01

Primary tritium isotope effects have been measured for the hydroxylation of [2-3H] dopamine catalyzed by dopamine beta-hydroxylase. Experimental values vary from 8.8 +/- 1.4 at 0.02 mM oxygen to 4.1 +/- 0.6 at 1.0 mM oxygen. It is shown that the observed dependence of the isotope effect on oxygen concentration provides unequivocal evidence for a kinetically significant dissociation of both dopamine and oxygen from enzyme, ternary complex. This approach, which is applicable to any multisubstrate enzyme characterized by detectable kinetic isotope effects, provides an alternate to classical methods for the elucidation of kinetic order in enzyme-catalyzed reactions

Magnetic resonance imaging and image analysis for assessment of HPMC matrix tablets structural evolution in USP Apparatus 4.

Science.gov (United States)

Kulinowski, Piotr; Dorożyński, Przemysław; Młynarczyk, Anna; Węglarz, Władysław P

2011-05-01

The purpose of the study was to present a methodology for the processing of Magnetic Resonance Imaging (MRI) data for the quantification of the dosage form matrix evolution during drug dissolution. The results of the study were verified by comparison with other approaches presented in literature. A commercially available, HPMC-based quetiapine fumarate tablet was studied with a 4.7T MR system. Imaging was performed inside an MRI probe-head coupled with a flow-through cell for 12 h in circulating water. The images were segmented into three regions using threshold-based segmentation algorithms due to trimodal structure of the image intensity histograms. Temporal evolution of dry glassy, swollen glassy and gel regions was monitored. The characteristic features were observed: initial high expansion rate of the swollen glassy and gel layers due to initial water uptake, dry glassy core disappearance and maximum area of swollen glassy region at 4 h, and subsequent gel layer thickness increase at the expense of swollen glassy layer. The temporal evolution of an HPMC-based tablet by means of noninvasive MRI integrated with USP Apparatus 4 was found to be consistent with both the theoretical model based on polymer disentanglement concentration and experimental VIS/FTIR studies.

Quantitative iTRAQ secretome analysis of Aspergillus niger reveals novel hydrolytic enzymes.

Science.gov (United States)

Adav, Sunil S; Li, An A; Manavalan, Arulmani; Punt, Peter; Sze, Siu Kwan

2010-08-06

The natural lifestyle of Aspergillus niger made them more effective secretors of hydrolytic proteins and becomes critical when this species were exploited as hosts for the commercial secretion of heterologous proteins. The protein secretion profile of A. niger and its mutant at different pH was explored using iTRAQ-based quantitative proteomics approach coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study characterized 102 highly confident unique proteins in the secretome with zero false discovery rate based on decoy strategy. The iTRAQ technique identified and relatively quantified many hydrolyzing enzymes such as cellulases, hemicellulases, glycoside hydrolases, proteases, peroxidases, and protein translocating transporter proteins during fermentation. The enzymes have potential application in lignocellulosic biomass hydrolysis for biofuel production, for example, the cellulolytic and hemicellulolytic enzymes glucan 1,4-alpha-glucosidase, alpha-glucosidase C, endoglucanase, alpha l-arabinofuranosidase, beta-mannosidase, glycosyl hydrolase; proteases such as tripeptidyl-peptidase, aspergillopepsin, and other enzymes including cytochrome c oxidase, cytochrome c oxidase, glucose oxidase were highly expressed in A. niger and its mutant secretion. In addition, specific enzyme production can be stimulated by controlling pH of the culture medium. Our results showed comprehensive unique secretory protein profile of A. niger, its regulation at different pH, and the potential application of iTRAQ-based quantitative proteomics for the microbial secretome analysis.

An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

Science.gov (United States)

Mohamad, Nur Royhaila; Marzuki, Nur Haziqah Che; Buang, Nor Aziah; Huyop, Fahrul; Wahab, Roswanira Abdul

2015-01-01

The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies. PMID:26019635

Enzyme inhibition by iminosugars

DEFF Research Database (Denmark)

López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus

2013-01-01

Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes...

Targeted enzyme prodrug therapies.

Science.gov (United States)

Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

2010-09-01

The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

Enzyme-MOF (metal-organic framework) composites.

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Lian, Xizhen; Fang, Yu; Joseph, Elizabeth; Wang, Qi; Li, Jialuo; Banerjee, Sayan; Lollar, Christina; Wang, Xuan; Zhou, Hong-Cai

2017-06-06

The ex vivo application of enzymes in various processes, especially via enzyme immobilization techniques, has been extensively studied in recent years in order to enhance the recyclability of enzymes, to minimize enzyme contamination in the product, and to explore novel horizons for enzymes in biomedical applications. Possessing remarkable amenability in structural design of the frameworks as well as almost unparalelled surface tunability, Metal-Organic Frameworks (MOFs) have been gaining popularity as candidates for enzyme immobilization platforms. Many MOF-enzyme composites have achieved unprecedented results, far outperforming free enzymes in many aspects. This review summarizes recent developments of MOF-enzyme composites with special emphasis on preparative techniques and the synergistic effects of enzymes and MOFs. The applications of MOF-enzyme composites, primarily in transferation, catalysis and sensing, are presented as well. The enhancement of enzymatic activity of the composites over free enzymes in biologically incompatible conditions is emphasized in many cases.

From 20th century metabolic wall charts to 21st century systems biology: database of mammalian metabolic enzymes.

Science.gov (United States)

Corcoran, Callan C; Grady, Cameron R; Pisitkun, Trairak; Parulekar, Jaya; Knepper, Mark A

2017-03-01

The organization of the mammalian genome into gene subsets corresponding to specific functional classes has provided key tools for systems biology research. Here, we have created a web-accessible resource called the Mammalian Metabolic Enzyme Database ( https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/MetabolicEnzymeDatabase.html) keyed to the biochemical reactions represented on iconic metabolic pathway wall charts created in the previous century. Overall, we have mapped 1,647 genes to these pathways, representing ~7 percent of the protein-coding genome. To illustrate the use of the database, we apply it to the area of kidney physiology. In so doing, we have created an additional database ( Database of Metabolic Enzymes in Kidney Tubule Segments: https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/), mapping mRNA abundance measurements (mined from RNA-Seq studies) for all metabolic enzymes to each of 14 renal tubule segments. We carry out bioinformatics analysis of the enzyme expression pattern among renal tubule segments and mine various data sources to identify vasopressin-regulated metabolic enzymes in the renal collecting duct. Copyright © 2017 the American Physiological Society.

Determination of the quantity of acetyl CoA carboxylase by [14C]methyl avidin binding

International Nuclear Information System (INIS)

Roman-Lopez, C.R.; Goodson, J.; Allred, J.B.

1987-01-01

Conditions are described under which monomeric [ 14 C]methyl avidin binds to SDS-denatured biotin enzymes and remains bound through polyacrylamide gel electrophoresis. The location of radioactive proteins on the dried gel was determined by fluorography and their identity was established by subunit molecular weight. The relative quantity of bound radioactive avidin, stoichiometrically equivalent to the molar quantity of biotin protein, can be determined by scanning the fluorograph with a soft laser densitometer. To determine the absolute quantity of biotin protein, the radioactive areas of the dried gel were cut out, resolubilized, and assayed for radioactivity. Since the specific radioactivity of the [ 14 C]methyl avidin was known, the quantity of avidin bound and therefore the quantity of biotin enzyme could be calculated. The method is illustrated by the analysis of purified acetyl CoA carboxylase and is applied to the analysis of biotin enzymes in isolated rat liver mitochondria

Use of Proteolytic Enzymes in the Treatment of Proteinaceous Esophageal Food Impaction.

Science.gov (United States)

Morse, Christopher R; Wang, Howard; Donahue, Dean M; Garrity, Julie M; Allan, James S

2016-01-01

Proteinaceous esophageal food impaction typically requires endoscopic intervention. An alternative approach is the use of proteolytic enzymes. Concerns regarding the use of proteolytic enzymes include the risk of perforation and aspiration pneumonitis. We retrospectively reviewed our series of 69 patients treated with papain to determine the safety and efficacy of proteolytic enzymes. Patients were retrospectively reviewed if treated for an esophageal food impaction from 1999 through 2008. Median age was 56 years (range 19-91 years), with 46 male and 23 female patients. In 27 patients (39%) this was their first presentation, in 14 (20%) it was the second, and 28 (41%) had multiple previous episodes. Meat was the cause in 49 (71%), chicken in 6 (9%), fish in 3 (4%), and unspecified in 11 (16%). All patients presented with dysphagia for solids, 56 (81%) could not tolerate liquids. Papain solution, 1 tsp in 8 oz of water, was given to patients in an unlimited quantity. Papain was successful in relieving the obstruction in 60 patients (87%). The remaining 9 patients (13%) underwent endoscopy with successful retrieval. No patient suffered a perforation, either with papain ingestion or endoscopy. There were no episodes of pneumonitis or pneumonia. We have used proteolytic enzymes with a high success rate and with minimal complication. Further, if proteolytic enzymes fail, endoscopy can be performed safely and effectively. We recommend the use of proteolytic enzymes as the initial management in all patients with proteinaceous food impaction of the esophagus. Published by Elsevier Inc.

Patterns of functional enzyme activity in fungus farming ambrosia beetles

Directory of Open Access Journals (Sweden)

De Fine Licht Henrik H

2012-06-01

Full Text Available Abstract Introduction In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae, wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. Results We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Conclusion Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily

Structural differences of matrix metalloproteinases. Homology modeling and energy minimization of enzyme-substrate complexes

DEFF Research Database (Denmark)

Terp, G E; Christensen, I T; Jørgensen, Flemming Steen

2000-01-01

Matrix metalloproteinases are extracellular enzymes taking part in the remodeling of extracellular matrix. The structures of the catalytic domain of MMP1, MMP3, MMP7 and MMP8 are known, but structures of enzymes belonging to this family still remain to be determined. A general approach...... to the homology modeling of matrix metalloproteinases, exemplified by the modeling of MMP2, MMP9, MMP12 and MMP14 is described. The models were refined using an energy minimization procedure developed for matrix metalloproteinases. This procedure includes incorporation of parameters for zinc and calcium ions...... in the AMBER 4.1 force field, applying a non-bonded approach and a full ion charge representation. Energy minimization of the apoenzymes yielded structures with distorted active sites, while reliable three-dimensional structures of the enzymes containing a substrate in active site were obtained. The structural...

Enzymes and membrane proteins of ADSOL-preserved red blood cells

Directory of Open Access Journals (Sweden)

Maria Sueli Soares Leonart

2000-03-01

Full Text Available CONTEXT: The preservative solution ADSOL (adenine, dextrose, sorbitol, sodium chloride and mannitol maintains red cell viability for blood trans-fusion for 6 weeks. It would be useful to know about its preservation qualities over longer periods. OBJECTIVE: To determine some red cell biochemical parameters for peri-ods of up to 14 weeks in order to determine whether the red cell metabo-lism integrity would justify further studies aiming at increasing red cell preservation and viability. DESIGN: Biochemical evaluation designed to study red cell preservation. SETTING: São Paulo University erythrocyte metabolism referral center. SAMPLE: Six normal blood donors from the University Hospital of the Universidade Federal do Paraná, Curitiba, Brazil. MAIN MEASUREMENTS: Weekly assay of erythrocyte adenosine-5´-triphosphate (ATP, 2,3-diphosphoglycerate (2,3DPG, hexokinase (HX, phosphofructokinase (PFK, pyruvate kinase (PK, glucose-6-phosphate dehydrogenase (G-6-PD, 6-phosphogluconic dehydrogenase (6-PGD, glyceraldehyde-3-phosphate dehydrogenase (GAPD, glutathione reduc-tase (GR, glutathione peroxidase (GSHPx, plasma sodium and potas-sium, blood pH, and membrane proteins of red cells preserved in ADSOL were studied during storage for 14 weeks storage. RESULTS: During ADSOL preservation, erythrocyte ATP concentration decreased 60% after 5 weeks, and 90% after 10 weeks; the pH fell from 6.8 to 6.4 by the 14th week. 2,3-DPG concentration was stable during the first week, but fell 90% after 3 weeks and was exhausted after 5 weeks. By the end of the 5th week, an activity decrease of 16-30% for Hx, GAPD, GR, G-6-PD and 6-PGD, 35% for PFK and GSHPx, and 45% for PK were observed. Thereafter, a uniform 10% decay was observed for all enzymes up to the 14th week. The red blood cell membrane pro-teins did not show significant alterations in polyacrylamide gel electro-phoresis (SDS-PAGE during the 14 weeks. CONCLUSION: Although the blood viability was shown to be poor

Enzyme recycling in lignocellulosic biorefineries

DEFF Research Database (Denmark)

Jørgensen, Henning; Pinelo, Manuel

2017-01-01

platform. Cellulases are the most important enzymes required in this process, but the complex nature of lignocellulose requires several other enzymes (hemicellulases and auxiliary enzymes) for efficient hydrolysis. Enzyme recycling increases the catalytic productivity of the enzymes by reusing them...... for several batches of hydrolysis, and thereby reduces the overall cost associated with the hydrolysis. Research on this subject has been ongoing for many years and several promising technologies and methods have been developed and demonstrated. But only in a very few cases have these technologies been...... upscaled and tested in industrial settings, mainly because of many difficulties with recycling of enzymes from the complex lignocellulose hydrolyzate at industrially relevant conditions, i.e., high solids loadings. The challenges are associated with the large number of different enzymes required...

Paroxysmal atrial fibrillation: dynamics of the main antioxidant enzymes--superoxide dismutase and catalase.

Science.gov (United States)

Negreva, Mariya N; Penev, Atanas P; Georgiev, Svetoslav Zh; Aleksandrova, Albena A

2014-01-01

Researchers have a particularly strong interest in the mechanisms implicated in the clinical manifestation of atrial fibrillation. To examine dynamically the activity of the antioxidant enzymes, superoxide dismutase and catalase in patients with paroxysmal atrial fibrillation (duration enzyme activity was determined by a spectrophotometric method. The average duration of atrial fibrillation episodes until the time of hospitalization was 8.14 hours (from 2 to 24 hours). During patient hospitalization the activity of superoxide dismutase and catalase was considerably higher compared to that of the controls (8.46 +/- 0.26 vs 5.81 +/- 0.14 U/mg Hb; 7.36 +/- 0.25 vs 4.76 +/- 0.12 E240/min/mg Hb; P catalase remained increased (5.11 +/- 0.08 vs 4.76 +/- 0.12 E240/min/mg Hb, p catalase even in the early hours of clinical manifestation of the disorder, which then slowly decreased with the restoration of sinus rhythm. Therefore, we can conclude that changes in oxidative status are closely related to the disease and are probably a part of the intimate mechanisms related to its initiation and clinical course.

Enzymes of industrial purpose - review of the market of enzyme preparations and prospects for its development

Directory of Open Access Journals (Sweden)

A. A. Tolkacheva

2017-01-01

Full Text Available Microbial enzyme preparations are increasingly replacing conventional chemical catalysts in a number of industrial processes. Such drugs, in addition to environmental friendliness and high activity, have a number of advantages over enzyme preparations of vegetable and animal origin, namely: the production of microbial enzymes in bioreactors is easily controlled and predictable; excreted microbiological enzymes are more stable than intracellular animals and plant enzymes; the genetic diversity of microorganisms makes it possible to produce enzyme preparations with a wide range of specificity; microbiological enzymes can be synthesized year-round, in contrast to the production of plant enzymes, which is often seasonal. The leaders of the world market of enzymes are proteases and amylases, which account for 25% and 15%, respectively. Over the past five years, the world market for carbohydrases, including mainly amylases, cellulases and xylanases, has been the fastest growing segment of the enzyme market with an aggregate annual growth rate of more than 7.0%. Another major product of the industrial enzyme market, which has a great potential for growth, is lipases. From the point of view of designation, the main part is represented by food and food enzymes. The Russian market continues to be unsaturated - the current supply is not able to meet the needs of the Russian feed and food industry in enzyme preparations. Enzyme preparations of domestic producers are in demand in forage production, while food industrial enterprises prefer imported products. The most significant enterprises in the enzymatic industry in Russia at the moment are Sibbiofarm, AgroSistema, Agroferment. In the light of the Russian policy of increasing food security, the development of the domestic enzyme industry is an extremely topical task.

Continuous enzyme reactions with immobilized enzyme tubes prepared by radiation cast-polymerization

International Nuclear Information System (INIS)

Kumakura, Minoru; Kaetsu, Isao

1986-01-01

Immobilized glucose oxidase tubes were prepared by radiation cast-polymerization of 2-hydroxyethyl methacrylate and tetraethyleneglycol diacrylate monomer at low temperatures. The immobilized enzyme tubes which were spirally set in a water bath were used as reactor, in which the enzyme activity varied with tube size and flow rate of the substrate. The conversion yield of the substrate in continuous enzyme reaction was about 80%. (author)

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillus oryzae.

Science.gov (United States)

Tanaka, Mizuki; Hiramoto, Tetsuya; Tada, Hinako; Shintani, Takahiro; Gomi, Katsuya

2017-07-01

Aspergillus oryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the homolog of E6-AP carboxyl terminus ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucose-induced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by Western blotting, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although α-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD had a negligible effect on α-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by disruption of the deubiquitinating enzyme-encoding gene creB The α-amylase activity of the creB mutant was 1.6-fold higher than that of the wild type, and the dephosphorylation mimic of CreD further improved the α-amylase activity by 2.6-fold. These results indicate that a combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzae IMPORTANCE In eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucose-induced endocytosis of transporters is mediated by

Non-homologous isofunctional enzymes: a systematic analysis of alternative solutions in enzyme evolution.

Science.gov (United States)

Omelchenko, Marina V; Galperin, Michael Y; Wolf, Yuri I; Koonin, Eugene V

2010-04-30

Evolutionarily unrelated proteins that catalyze the same biochemical reactions are often referred to as analogous - as opposed to homologous - enzymes. The existence of numerous alternative, non-homologous enzyme isoforms presents an interesting evolutionary problem; it also complicates genome-based reconstruction of the metabolic pathways in a variety of organisms. In 1998, a systematic search for analogous enzymes resulted in the identification of 105 Enzyme Commission (EC) numbers that included two or more proteins without detectable sequence similarity to each other, including 34 EC nodes where proteins were known (or predicted) to have distinct structural folds, indicating independent evolutionary origins. In the past 12 years, many putative non-homologous isofunctional enzymes were identified in newly sequenced genomes. In addition, efforts in structural genomics resulted in a vastly improved structural coverage of proteomes, providing for definitive assessment of (non)homologous relationships between proteins. We report the results of a comprehensive search for non-homologous isofunctional enzymes (NISE) that yielded 185 EC nodes with two or more experimentally characterized - or predicted - structurally unrelated proteins. Of these NISE sets, only 74 were from the original 1998 list. Structural assignments of the NISE show over-representation of proteins with the TIM barrel fold and the nucleotide-binding Rossmann fold. From the functional perspective, the set of NISE is enriched in hydrolases, particularly carbohydrate hydrolases, and in enzymes involved in defense against oxidative stress. These results indicate that at least some of the non-homologous isofunctional enzymes were recruited relatively recently from enzyme families that are active against related substrates and are sufficiently flexible to accommodate changes in substrate specificity.

A Prospective Randomized, Double-Blind, Two-Period Crossover Pharmacokinetic Trial Comparing Green Coffee Bean Extract-A Botanically Sourced Caffeine-With a Synthetic USP Control.

Science.gov (United States)

Morton, Kayce; Knight, Katelin; Kalman, Douglas; Hewlings, Susan

2018-04-16

Coffee is a primary dietary source of the chlorogenic acids (CGAs) of phenolic compounds. Coffee contains caffeine and other phytonutrients, including CGAs. Caffeine on its own has been well characterized and descried pharmacokinetically in the literature, less so for CGAs. The purpose of this double-blind crossover study was to determine the comparative pharmacokinetics of CGAs with caffeine (natural extract) with synthetic caffeine (US Pharmacopeia [USP] standard). Sixteen healthy male subjects were randomly assigned to take 1 dose of product 1, 60 mg of botanically sourced caffeine from 480 mg of green coffee bean extract, or product 2, 60 mg of synthetic USP caffeine, with 5 days between. Blood analysis was done to determine the levels of CGA compounds, more specifically 3-, 4-, and 5-caffeoylquinic acid (CQA), and serum caffeine. The natural caffeine extract exhibited mean peak concentrations (C max ) of 3-CQA (11.4 ng/mL), 4-CQA (6.84 ng/mL), and 5-CQA (7.20 ng/mL). The mean systemic 4-hour exposure (AUC 0-4 h ) was 3-CQA (27.3 ng·h/mL), 4-CQA (16.1 ng·h/mL), and 5-CQA (15.7 ng·h/mL). The median t max was 3-CQA (1.00 hour), 4-CQA (1.00 hour), and 5-CQA (1.50 hours). The t max of caffeine was 0.75 hours (natural extract) and 0.63 hours (synthetic caffeine). C max and AUC 0-4 h of serum caffeine were statistically equivalent between products. The geometric least-squares mean ratios (GMRs) of C max and AUC 0-4 h of caffeine were 97.77% (natural extract) and 98.33% (synthetic caffeine). It would appear that CGA compounds from the natural caffeine extract are bioavailable, and 3-CGA may be the compound most absorbed. In addition, caffeine sourced from natural extract versus synthetic were statistically similar for pharmacokinetic parameters. There were no adverse events or safety concerns. © 2018 The Authors. Clinical Pharmacology in Drug Development Published by Wiley Periodicals, Inc. on behalf of The American College of Clinical Pharmacology.

Characterization of phospholipase C gamma enzymes with gain-of-function mutations.

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Everett, Katy L; Bunney, Tom D; Yoon, Youngdae; Rodrigues-Lima, Fernando; Harris, Richard; Driscoll, Paul C; Abe, Koichiro; Fuchs, Helmut; de Angelis, Martin Hrabé; Yu, Philipp; Cho, Wohnwa; Katan, Matilda

2009-08-21

Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

Two Strategies for Microbial Production of an Industrial Enzyme-Alpha-Amylase

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Bernhardsdotter, Eva C. M. J.; Garriott, Owen; Pusey, Marc L.; Ng, Joseph D.

2003-01-01

Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments including hot springs, soda lakes and arctic water. This ability of survival at extreme conditions has rendered extremophiles to be of interest in astrobiology, evolutionary biology as well as in industrial applications. Of particular interest to the biotechnology industry are the biological catalysts of the extremophiles, the extremozymes, whose unique stabilities at extreme conditions make them potential sources of novel enzymes in industrial applications. There are two major approaches to microbial enzyme production. This entails enzyme isolation directly from the natural host or creating a recombinant expression system whereby the targeted enzyme can be overexpressed in a mesophilic host. We are employing both methods in the effort to produce alpha-amylases from a hyperthermophilic archaeon (Thermococcus) isolated from a hydrothermal vent in the Atlantic Ocean, as well as from alkaliphilic bacteria (Bacillus) isolated from a soda lake in Tanzania. Alpha-amylases catalyze the hydrolysis of internal alpha-1,4-glycosidic linkages in starch to produce smaller sugars. Thermostable alpha-amylases are used in the liquefaction of starch for production of fructose and glucose syrups, whereas alpha-amylases stable at high pH have potential as detergent additives. The alpha-amylase encoding gene from Thermococcus was PCR amplified using carefully designed primers and analyzed using bioinformatics tools such as BLAST and Multiple Sequence Alignment for cloning and expression in E.coli. Four strains of Bacillus were grown in alkaline starch-enriched medium of which the culture supernatant was used as enzyme source. Amylolytic activity was detected using the starch-iodine method.

Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

Science.gov (United States)

Bi, Xiaodong; Liu, Zhen

2014-12-16

Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

Enzymic lactose hydrolysis

Energy Technology Data Exchange (ETDEWEB)

Miller, J J; Brand, J C

1980-01-01

Acid or enzymic hydrolysis can be used to hydrolyze lactose. Advantages of both are compared and details of enzymic hydrolysis using yeast or fungal enzymes given. The new scheme outlined involves recycling lactase. Because lactose and lactase react to ultrafiltration (UF) membranes differently separation is possible. Milk or milk products are ultrafiltered to separate a concentrate from a lactose-rich permeate which is treated with lactase in a reactor until hydrolysis reaches a required level. The lactase can be removed by UF as it does not permeate the membrane, and it is recycled back to the reactor. Permeate from the second UF stage may or may not be recombined with the concentrate from the first stage to produce a low lactose product (analysis of a typical low-lactose dried whole milk is given). Batch or continuous processes are explained and a batch process without enzyme recovery is discussed. (Refs. 4).

Enzyme Mimics: Advances and Applications.

Science.gov (United States)

Kuah, Evelyn; Toh, Seraphina; Yee, Jessica; Ma, Qian; Gao, Zhiqiang

2016-06-13

Enzyme mimics or artificial enzymes are a class of catalysts that have been actively pursued for decades and have heralded much interest as potentially viable alternatives to natural enzymes. Aside from having catalytic activities similar to their natural counterparts, enzyme mimics have the desired advantages of tunable structures and catalytic efficiencies, excellent tolerance to experimental conditions, lower cost, and purely synthetic routes to their preparation. Although still in the midst of development, impressive advances have already been made. Enzyme mimics have shown immense potential in the catalysis of a wide range of chemical and biological reactions, the development of chemical and biological sensing and anti-biofouling systems, and the production of pharmaceuticals and clean fuels. This Review concerns the development of various types of enzyme mimics, namely polymeric and dendrimeric, supramolecular, nanoparticulate and proteinic enzyme mimics, with an emphasis on their synthesis, catalytic properties and technical applications. It provides an introduction to enzyme mimics and a comprehensive summary of the advances and current standings of their applications, and seeks to inspire researchers to perfect the design and synthesis of enzyme mimics and to tailor their functionality for a much wider range of applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

USP38, FREM3, SDC1, DDC, and LOC727982 Gene Polymorphisms and Differential Susceptibility to Severe Malaria in Tanzania.

Science.gov (United States)

Manjurano, Alphaxard; Sepúlveda, Nuno; Nadjm, Behzad; Mtove, George; Wangai, Hannah; Maxwell, Caroline; Olomi, Raimos; Reyburn, Hugh; Drakeley, Christopher J; Riley, Eleanor M; Clark, Taane G

2015-10-01

Populations exposed to Plasmodium falciparum infection develop genetic mechanisms of protection against severe malarial disease. Despite decades of genetic epidemiological research, the sickle cell trait (HbAS) sickle cell polymorphism, ABO blood group, and other hemoglobinopathies remain the few major determinants in severe malaria to be replicated across different African populations and study designs. Within a case-control study in a region of high transmission in Tanzania (n = 983), we investigated the role of 40 new loci identified in recent genome-wide studies. In 32 loci passing quality control procedures, we found polymorphisms in USP38, FREM3, SDC1, DDC, and LOC727982 genes to be putatively associated with differential susceptibility to severe malaria. Established candidates explained 7.4% of variation in severe malaria risk (HbAS polymorphism, 6.3%; α-thalassemia, 0.3%; ABO group, 0.3%; and glucose-6-phosphate dehydrogenase deficiency, 0.5%) and the new polymorphisms, another 4.3%. The regions encompassing the loci identified are promising targets for the design of future treatment and control interventions. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates.

Science.gov (United States)

Várnai, Anikó; Viikari, Liisa; Marjamaa, Kaisa; Siika-aho, Matti

2011-01-01

The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger β-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance. Copyright © 2010 Elsevier Ltd. All rights reserved.

Hydrolytic enzyme activities in shiitake mushroom (Lentinula edodes) strains cultivated on coffee pulp.

Science.gov (United States)

Mata, Gerardo; Salmones, Dulce; Pérez-Merlo, Rosalía

Hydrolytic enzyme production (cellulases, laminarinases and xylanases) was studied in cultures of Lentinula edodes on sterilized coffee pulp. Samples of substrate colonized by mycelia were taken after 7, 14, 21, 28 and 35 days of incubation at 25°C (W1 to W5) and during the fruiting period at different stages: formation of primordia (PF), first harvest (H) and one week after the first harvest (PH). The enzymatic activity was lower during the early mycelial growth and showed higher levels during the formation and development of fruiting bodies. During the reproductive stage of the fungus, the samples were subjected to a soaking treatment; however, it was not possible to relate this soaking treatment to the increase in enzyme production. The levels of enzymatic activity suggest that secretion of the studied enzymes does not influence the adaptability of the strains to the substrate. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Alginate-modifying enzymes: Biological roles and biotechnological uses

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Helga eErtesvåg

2015-05-01

Full Text Available Alginate denotes a group of industrially important 1-4-linked biopolymers composed of the C-5-epimers β-D-mannuronic acid (M and α-L-guluronic acid (G. The polysaccharide is manufactured from brown algae where it constitutes the main structural cell wall polymer. The physical properties of a given alginate molecule, e.g. gel-strength, water-binding capacity, viscosity and biocompatibility, are determined by polymer length, the relative amount and distribution of G residues and the acetyl content, all of which are controlled by alginate modifying enzymes. Alginate has also been isolated from some bacteria belonging to the genera Pseudomonas and Azotobacter, and bacterially synthesized alginate may be O-acetylated at O-2 and/or O-3. Initially, alginate is synthesized as polymannuronic acid, and some M residues are subsequently epimerized to G residues. In bacteria a mannuronan C-5-epimerase (AlgG and an alginate acetylase (AlgX are integral parts of the protein complex necessary for alginate polymerisation and export. All alginate-producing bacteria use periplasmic alginate lyases to remove alginate molecules aberrantly released to the periplasm. Alginate lyases are also produced by organisms that utilize alginate as carbon source. Most alginate-producing organisms encode more than one mannuronan C-5 epimerase, each introducing its specific pattern of G residues. Acetylation protects against further epimerization and from most alginate lyases. One enzyme with alginate deacetylase activity from Pseudomonas syringae has been reported. Functional and structural studies reveal that alginate lyases and epimerases have related enzyme mechanisms and catalytic sites. Alginate lyases are now utilized as tools for alginate characterization. Secreted epimerases have been shown to function well in vitro, and have been engineered further in order to obtain enzymes that can provide alginates with new and desired properties for use in medical and

Chondroitin / dermatan sulfate modification enzymes in zebrafish development.

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Judith Habicher

Full Text Available Chondroitin/dermatan sulfate (CS/DS proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS. Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.

Characterising Complex Enzyme Reaction Data.

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Handan Melike Dönertaş

Full Text Available The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG. Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution.

Enzyme Immobilization: An Overview on Methods, Support Material, and Applications of Immobilized Enzymes.

Science.gov (United States)

Sirisha, V L; Jain, Ankita; Jain, Amita

Immobilized enzymes can be used in a wide range of processes. In recent years, a variety of new approaches have emerged for the immobilization of enzymes that have greater efficiency and wider usage. During the course of the last two decades, this area has rapidly expanded into a multidisciplinary field. This current study is a comprehensive review of a variety of literature produced on the different enzymes that have been immobilized on various supporting materials. These immobilized enzymes have a wide range of applications. These include applications in the sugar, fish, and wine industries, where they are used for removing organic compounds from waste water. This study also reviews their use in sophisticated biosensors for metabolite control and in situ measurements of environmental pollutants. Immobilized enzymes also find significant application in drug metabolism, biodiesel and antibiotic production, bioremediation, and the food industry. The widespread usage of immobilized enzymes is largely due to the fact that they are cheaper, environment friendly, and much easier to use when compared to equivalent technologies. © 2016 Elsevier Inc. All rights reserved.

Crystallization and preliminary X-ray analysis of ZHE1, a hatching enzyme from the zebrafish Danio rerio

International Nuclear Information System (INIS)

Okada, Akitoshi; Nagata, Koji; Sano, Kaori; Yasumasu, Shigeki; Kubota, Keiko; Ohtsuka, Jun; Iuchi, Ichiro; Tanokura, Masaru

2009-01-01

The hatching enzyme of zebrafish, ZHE1, was expressed, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belonged to space group P2 1 2 1 2 1 and diffracted X-rays to a resolution of 1.14 Å. The hatching enzyme of the zebrafish, ZHE1 (29.3 kDa), is a zinc metalloprotease that catalyzes digestion of the egg envelope (chorion). ZHE1 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. Two diffraction data sets with resolution ranges 50.0–1.80 and 50.0–1.14 Å were independently collected from two crystals and were merged to give a highly complete data set over the full resolution range 50.0–1.14 Å. The space group was assigned as primitive orthorhombic P2 1 2 1 2 1 , with unit-cell parameters a = 32.9, b = 62.5, c = 87.4 Å. The crystal contained one ZHE1 molecule in the asymmetric unit

Random-walk enzymes

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Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

2015-09-01

Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C →U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

Variation in levels of non-starch polysaccharides and endogenous endo-1,4-β-xylanases affects the nutritive value of wheat for poultry.

Science.gov (United States)

Cardoso, V; Fernandes, E A; Santos, H M M; Maçãs, B; Lordelo, M M; Telo da Gama, Luis; Ferreira, L M A; Fontes, C M G A; Ribeiro, T

2018-04-01

1. Endo-1,4-β-xylanase is known to improve the nutritive value of wheat-based diets for poultry by degrading dietary arabinoxylans. However, broilers' response to supplementation of wheat-based diets with exogenous endo-1,4-β-xylanase is not always observed. 2. In this study, 108 different wheat lots were analysed for levels of extract viscosity as well as for endogenous endo-1,4-β-xylanase activity, and the impact of these two variables in animal performance was tested. 3. Results revealed that endogenous endo-1,4-β-xylanase activity and extract viscosity content varied widely among different wheat lots. Thus, a trial was conducted to evaluate the efficacy of exogenous enzyme supplementation in broiler diets using wheats with different levels of extract viscosity and endogenous endo-1,4-β-xylanase activity. 4. The data revealed that exogenous enzyme supplementation was only effective when the wheat present in the diet had high levels of extract viscosity (14.8 cP) with low endogenous endo-1,4-β-xylanase activity (347.0 U/kg). Nevertheless, it is apparent that exogenous microbial xylanases reduce digesta extract viscosity and feed conversion ratio independently of the endogenous properties presented by different wheat lots. 5. The data suggest that extract viscosity and/or endogenous endo-1,4-β-xylanase activity affect the response to enzyme supplementation by poultry fed on wheat-based diets.

Enzyme stabilization for pesticide degradation

Energy Technology Data Exchange (ETDEWEB)

Rivers, D.B.; Frazer, F.R. III; Mason, D.W.; Tice, T.R.

1988-01-01

Enzymes offer inherent advantages and limitations as active components of formulations used to decontaminate soil and equipment contaminated with toxic materials such as pesticides. Because of the catalytic nature of enzymes, each molecule of enzyme has the potential to destroy countless molecules of a contaminating toxic compound. This degradation takes place under mild environmental conditions of pH, temperature, pressure, and solvent. The basic limitation of enzymes is their degree of stability during storage and application conditions. Stabilizing methods such as the use of additives, covalent crosslinking, covalent attachment, gel entrapment, and microencapsulation have been directed developing an enzyme preparation that is stable under extremes of pH, temperature, and exposure to organic solvents. Initial studies were conducted using the model enzymes subtilisin and horseradish peroxidase.

Enzyme Molecules in Solitary Confinement

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Raphaela B. Liebherr

2014-09-01

Full Text Available Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.

Hyper production of cellulose degrading endo (1,4 β-d-glucanase from Bacillus licheniformis KIBGE-IB2

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Asad Karim

2015-04-01

Full Text Available Cellulase hydrolyzes β (1,4 glycosidic linkages of cellulose polymer to soluble sugar. An extracellular enzyme production by Bacillus licheniformis KIBGE-IB2 (GenBank accession No. GU216259 was studied under various environmental conditions. Maximum enzyme production was measured in the liquid fermentation medium after 48 h, containing (gL−1, CMC, 5.0; peptone, 15.0; yeast extract, 15.0; CaCl2·2H2O, 0.001; FeSO4·7H2O, 0.001; K2HPO4, 5.0; NaH2PO4, 5.0 and MgSO4·7H2O, 1.0. The optimal pH and temperature for enzyme production was found to be 6.0 and 37°C, respectively. It was also found that beside soluble sugars, a significant amount of enzyme production was obtained when biomass (wheat bran and orange peel were examined as a sole carbon source. The current findings indicate that endo (1,4 β-d-glucanase from B. licheniformis KIBGE-IB2 can be beneficial for commercial purpose.

Enzyme technology: Key to selective biorefining

DEFF Research Database (Denmark)

Meyer, Anne S.

2014-01-01

to the reaction is a unique trait of enzyme catalysis. Since enzyme selectivity means that a specific reaction is catalysed between particular species to produce definite products, enzymes are particularly fit for converting specific compounds in mixed biomass streams. Since enzymes are protein molecules...... their rational use in biorefinery processes requires an understanding of the basic features of enzymes and reaction traits with respect to specificity, kinetics, reaction optima, stability and structure-function relations – we are now at a stage where it is possible to use nature’s enzyme structures as starting...... point and then improve the functional traits by targeted mutation of the protein. The talk will display some of our recent hypotheses related to enzyme action, recently obtained results within knowledge-based enzyme improvements as well as cast light on research methods used in optimizing enzyme...

Efficient immobilization of AGE and NAL enzymes onto functional amino resin as recyclable and high-performance biocatalyst.

Science.gov (United States)

Cheng, Jian; Zhuang, Wei; Tang, Chenglun; Chen, Yong; Wu, Jinglan; Guo, Ting; Ying, Hanjie

2017-03-01

N-Acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid lyase (NAL) were immobilized for synthesis of N-acetylneuraminic acid (Neu5Ac) on three resins: Amberzyme oxirane resin (AOR), poly (styrene-co-DVB)-Br resin (PBR) and amino resin (AR). The loading capacity and immobilized enzyme activity showed that AR was the best carrier. Three methods of glutaraldehyde cross-linking were tested and simultaneous cross-linking and immobilization was demonstrated to be the best method. The functional properties of immobilized AGE and NAL were studied and compared to those of the free enzyme. The highest enzyme activities of free and immobilized AGE were obtained in 0.1 M potassium phosphate buffer at pH 7.5 and a temperature of 37 °C. Comparatively, the highest NAL activities were at pH 8.5. Meanwhile, an increase in K m (from 1.14 to 1.31 mg·mL -1 for AGE and from 1.05 to 1.25 mg·mL -1 for NAL) and a decrease in V max (from 177.53 to 106.37 µg·min -1 mL -1 for AGE and from 126.41 to 95.96 µg·min -1 mL -1 for NAL) were recorded after immobilization. The AR-glutaraldehyde-enzyme system exhibited better thermal stability than the free enzyme, and retained 72% of its initial activity even after eight repeated runs. The apparent activation energy (E a ) of the free and immobilized AGE (NAL) was 117.14 kJ·mol -1 (124.21 kJ·mol -1 ) and 78.45 kJ·mol -1 (66.64 kJ·mol -1 ), respectively, implying that the catalytic efficiency of the immobilized enzyme was restricted by mass-transfer rather than kinetic limit. Subsequently, Neu5Ac production from GlcNAc using immobilized enzymes in one reactor was carried out resulting 101.45 g·L -1 of Neu5Ac and the highest conversion ratio of 82%. This method of enzyme immobilization may have a promising future for Neu5Ac production in industry.

Suitability of a liquid chromatography assay of neomycin sulfate to replace the microbiological assay for neomycin in USP Monographs.

Science.gov (United States)

Hanko, Valoran P; Rohrer, Jeffrey S

2010-01-05

The current USP National Formulary contains 65 Monographs for drug formulations containing neomycin. All 65 Monographs prescribe a bioassay for neomycin assay. This bioassay, based on cell culture, is labor intensive, has poor precision, and cannot be adapted for purity or identification. High-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAE-IPAD), a liquid chromatography technique, has been shown to be suitable for neomycin purity analysis and neomycin assay of an over-the-counter first aid cream (Hanko and Rohrer [17]). Here we propose that an HPAE-IPAD assay can replace the bioassay in the 65 neomycin-containing Monographs. We applied the HPAE-IPAD assay to four neomycin-containing drug products representing the four classes of formulations found in the 65 Monographs, liquid, solid, suspension, and cream. Each drug was analyzed with two chromatography systems, and on 3 separate days. For all products, HPAE-IPAD measurements were precise and accurate with respect to the label concentrations. There was also high accuracy for spike recovery of neomycin from the four drug products throughout 70-150% of the labeled concentration. These results suggest that an HPAE-IPAD assay would be an accurate assay for neomycin, and would be faster and more precise than the current bioassay.

The graduate as a tool of institutional assessment: an analysis of academic knowledge and employability with FEARP/USP graduates

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Claudio de Souza Miranda

2015-02-01

Full Text Available The assessment process in education has been the target of many studies, including higher education. The assessment process can occur during several levels; this study is done with the graduates. The focus of this study was the Faculty of Economics, Business and Accouting of Ribeirão Preto – USP, in order to answer the following research question: How is the integration of graduate students in the market? The faculty had 17 graduate groups that was composed of 1,520 graduates in its three courses. The researchers were able to connect 1,185, and they obtained 725 answers. Most of the graduates are in the Southeast, especially in São Paulo (84.7%. In terms of post-graduation courses, 32.1% were at MBA courses and 28.1% were at Mastering courses. Regarding employability was observed 91.8% are employed, 5.0% are looking for jobs and only 3.2% are inactive. Among the employed, 80.9% acts at their graduation area. These employees are at public and private sector, and they have an average income of R$ 9,6313. About the graduation course they scored 8.2 out of 10, despite some criticism of undergraduate learning and the market.

Phage lytic enzymes: a history.

Science.gov (United States)

Trudil, David

2015-02-01

There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of 'bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

Nanoarmored Enzymes for Organic Enzymology: Synthesis and Characterization of Poly(2-Alkyloxazoline)-Enzyme Conjugates.

Science.gov (United States)

Leurs, Melanie; Tiller, Joerg C

2017-01-01

The properties of enzymes can be altered significantly by modification with polymers. Numerous different methods are known to obtain such polymer-enzyme conjugates (PECs). However, there is no universal method to render enzymes into PECs that are fully soluble in organic solvents. Here, we present a method, which achieves such high degree of modification of proteins that the majority of modified enzymes will be soluble in organic solvents. This is achieved by preparing poly(2-alkyloxazoline)s (POx) with an NH 2 end group and coupling this functional polymer via pyromellitic acid dianhydride onto the amino groups of the respective protein. The resulting PECs are capable of serving as surfactants for unmodified proteins, rendering the whole mixture organosoluble. Depending on the nature of the POx and the molecular weight and the nature of the enzyme, the PECs are soluble in chloroform or even toluene. Another advantage of this method is that the poly(2-alkyloxazoline) can be activated with the coupling agent and used for the enzyme conjugation without further purification. The POx-enzyme conjugates generated by this modification strategy show modulated catalytic activity in both, aqueous and organic, systems. © 2017 Elsevier Inc. All rights reserved.

Bioaccumulation of glyphosate and its formulation Roundup Ultra in Lumbriculus variegatus and its effects on biotransformation and antioxidant enzymes

Energy Technology Data Exchange (ETDEWEB)

Contardo-Jara, Valeska [Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Department of Inland Fisheries, Biochemical Regulation, Mueggelseedamm 301, 12587 Berlin (Germany)], E-mail: contardo@igb-berlin.de; Klingelmann, Eva [Technische Universitaet Berlin/Berlin Institute of Technology, Department of Ecology, Chair of Soil Protection, Salzufer 12, 10587 Berlin (Germany)], E-mail: eva.klingelmann@TU-Berlin.de; Wiegand, Claudia [Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Department of Inland Fisheries, Biochemical Regulation, Mueggelseedamm 301, 12587 Berlin (Germany); Humboldt University Berlin, Faculty of Biology, Unter den Linden 6, 10099 Berlin (Germany)], E-mail: cwiegand@igb-berlin.de

2009-01-15

The bioaccumulation potential of glyphosate and the formulation Roundup Ultra, as well as possible effects on biotransformation and antioxidant enzymes in Lumbriculus variegatus were compared by four days exposure to concentrations between 0.05 and 5 mg L{sup -1} pure glyphosate and its formulation. Bioaccumulation was determined using {sup 14}C labeled glyphosate. The bioaccumulation factor (BCF) varied between 1.4 and 5.9 for the different concentrations, and was higher than estimated from log P{sub ow}. Glyphosate and its surfactant POEA caused elevation of biotransformation enzyme soluble glutathione S-transferase at non-toxic concentrations. Membrane bound glutathione S-transferase activity was significantly elevated in Roundup Ultra exposed worms, compared to treatment with equal glyphosate concentrations, but did not significantly differ from the control. Antioxidant enzyme superoxide dismutase was significantly increased by glyphosate but in particular by Roundup Ultra exposure indicating oxidative stress. The results show that the formulation Roundup Ultra is of more ecotoxicological relevance than the glyphosate itself. - Roundup Ultra is of more ecotoxicological relevance than the active ingredient, glyphosate, to Lumbriculus variegatus regarding accumulation potential and enzymatic responses.

Bioaccumulation of glyphosate and its formulation Roundup Ultra in Lumbriculus variegatus and its effects on biotransformation and antioxidant enzymes

International Nuclear Information System (INIS)

Contardo-Jara, Valeska; Klingelmann, Eva; Wiegand, Claudia

2009-01-01

The bioaccumulation potential of glyphosate and the formulation Roundup Ultra, as well as possible effects on biotransformation and antioxidant enzymes in Lumbriculus variegatus were compared by four days exposure to concentrations between 0.05 and 5 mg L -1 pure glyphosate and its formulation. Bioaccumulation was determined using 14 C labeled glyphosate. The bioaccumulation factor (BCF) varied between 1.4 and 5.9 for the different concentrations, and was higher than estimated from log P ow . Glyphosate and its surfactant POEA caused elevation of biotransformation enzyme soluble glutathione S-transferase at non-toxic concentrations. Membrane bound glutathione S-transferase activity was significantly elevated in Roundup Ultra exposed worms, compared to treatment with equal glyphosate concentrations, but did not significantly differ from the control. Antioxidant enzyme superoxide dismutase was significantly increased by glyphosate but in particular by Roundup Ultra exposure indicating oxidative stress. The results show that the formulation Roundup Ultra is of more ecotoxicological relevance than the glyphosate itself. - Roundup Ultra is of more ecotoxicological relevance than the active ingredient, glyphosate, to Lumbriculus variegatus regarding accumulation potential and enzymatic responses

Engineering Cellulase Enzymes for Bioenergy

Science.gov (United States)

Atreya, Meera Elizabeth

Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

Screening and Mutation Breeding of the Celloulonmonas flavigena CR-14 and Optimization of Its Fermentation Conditions

Directory of Open Access Journals (Sweden)

YANG Ji-ye

2016-05-01

Full Text Available The Celloulonmonas flavigena CR-14 was screened from the rumen of cattle, which had the ability of producting cellulose and was stored in our laboratory. In order to improve the cellulose activity of the CR-14, it was treated with nitrite, ultraviolet(UV and complex mutagenesis. Initial selection identified 6 cellulase-producing strains, as demonstrated by their ability to turn Congo red plates yellow. These 6 strains were then rescreened by determining the cellulase activity of broth. Finally, through nitrite and UV mutagenesis,Y-UA-18 strain with the high cellulose yield and stable characteristics, which enzymatic activity was 10.57 U, 1.67 times of starting strain CR-14, was screened out. In order to provide a theoretical reference to the industrial production of the strain Y-UA-18, we conducted the single factor test and orthogonal test to the fermentation medium components and the fermentation conditions. The results showed that the best medium of strain Y-UA-18 enzyme production was as followed:straw powder 1.0%, nitrogen source 0.6%, MgSO4 0.1%, KH2PO4 0.1%, NaCl 0.08%. The optimum fermentation conditions were:initial pH 7.0, incubation temperature 30 ℃, fermentation time 3 d, the effect of ventilation for strain Y-UA-18 enzyme production was not obvious, but under the condition of anaerobic, the enzyme activity of fermentation liquor was reduced, 0.1% TW-80 had no effect for enzyme production.

Impact of enzyme loading on the efficacy and recovery of cellulolytic enzymes immobilized on enzymogel nanoparticles.

Science.gov (United States)

Samaratunga, Ashani; Kudina, Olena; Nahar, Nurun; Zakharchenko, Andrey; Minko, Sergiy; Voronov, Andriy; Pryor, Scott W

2015-03-01

Cellulase and β-glucosidase were adsorbed on a polyacrylic acid polymer brush grafted on silica nanoparticles to produce enzymogels as a form of enzyme immobilization. Enzyme loading on the enzymogels was increased to a saturation level of approximately 110 μg (protein) mg(-1) (particle) for each enzyme. Enzymogels with varied enzyme loadings were then used to determine the impact on hydrolysis rate and enzyme recovery. Soluble sugar concentrations during the hydrolysis of filter paper and Solka-Floc with the enzymogels were 45 and 53%, respectively, of concentrations when using free cellulase. β-Glucosidase enzymogels showed lower performance; hydrolyzate glucose concentrations were just 38% of those using free enzymes. Increasing enzyme loading on the enzymogels did not reduce net efficacy for cellulase and improved efficacy for β-glucosidase. The use of free cellulases and cellulase enzymogels resulted in hydrolyzates with different proportions of cellobiose and glucose, suggesting differential attachment or efficacy of endoglucanases, exoglucanases, and β-glucosidases present in cellulase mixtures. When loading β-glucosidase individually, higher enzyme loadings on the enzymogels produced higher hydrolyzate glucose concentrations. Approximately 96% of cellulase and 66 % of β-glucosidase were recovered on the enzymogels, while enzyme loading level did not impact recovery for either enzyme.

[Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

Science.gov (United States)

Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

2015-09-01

Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

Effect of pH 5 enzyme from liver on the protein synthesis by mammary gland subcellular fractions in vitro

International Nuclear Information System (INIS)

Singh, Jaspal; Singh, Ajit; Ganguli, N.C.

1976-01-01

The effect of pH 5 enzyme fraction of liver on the protein synthesizing activity of the subcellular fractions of the mammary gland has been investigated. Results indicate that (1) lactating liver pH 5 enzyme stimulates protein synthesis which is enhanced by the addition of ATP-generating system and (2) the enzyme fractions from the non-lactating liver inhibits the protein synthesis by mammary fractions, but in some cases like mitochondrial and supernatant fractions of mammary it elevates the synthesis when supplemented with ATP-generating system. Chlorella protein hydrolysate- 14 C was used as a tracer and rabits were used as experimental animals. (M.G.B.)

Functional performance and physical activity levels of subjects enrolled at an Open University of the Third Age at the School of Arts, Sciences and Humanities of the University of São Paulo (UnATI EACH-USP

Directory of Open Access Journals (Sweden)

Ruth Caldeira de Melo

2013-05-01

Full Text Available The aim of this study was to evaluate the functional performance, according to the physical activity levels, of the students of UnATI EACH-USP. The study included 269 subjects, of both sexes, aged between 50 and 80 years-old. The most active group showed better functional performance in balance, mobility and lower limb strength tests compared with the sedentary group. On the other hand, perform physical activities in an irregular fashion was not associated to better functional performance in middleaged/older subjects.

On-site hydrolytic enzymes production from fungal co-cultivation of Bermuda grass and corn cob.

Science.gov (United States)

Amaro-Reyes, Aldo; Gracida, Jorge; Huizache-Peña, Nelson; Elizondo-García, Norberto; Salazar-Martínez, José; García Almendárez, Blanca E; Regalado, Carlos

2016-07-01

Solid state fermentation (SSF) is used to produce industrial enzymes. The objective of this study was to use a co-culture of Aspergillus niger GS1 and Trichoderma reesei, grown on a mixture of Bermuda grass and corn cob to obtain fermented forage (FF) rich in hydrolytic enzymes, as a value added ingredient for animal feed. FPase, amylase and xylanase productivities (dry matter, DM) were 8.8, 181.4, and 42.1Ug(-1)h(-1), respectively (1U=reducing sugars released min(-1)), after 12-16h of SSF with C/N=60. Cellulose, hemicellulose and lignin decreased 1.6-, 2.7- and 1.9-fold (DM), respectively. In vitro ruminal and true digestibility of DM was improved 2.4- and 1.4-fold. Ruminal digestion of FF reduced 1.32-fold the acetate:propionate ratio, which may reduce the environmental impact of ruminants feeding. On-site hydrolytic enzymes productivity using SSF without enzymes extraction could be of economic potential for digestibility improvement in animal feed. Copyright © 2016 Elsevier Ltd. All rights reserved.

BAKERY ENZYMES IN CEREAL TECHNOLOGIES

Directory of Open Access Journals (Sweden)

Václav Koman

2012-10-01

Full Text Available Normal 0 21 false false false SK X-NONE X-NONE Bread is the most common and traditional food in the world. For years, enzymes such as malt and fungal alpha-amylase have been used in bread making. Due to the changes in the baking industry and the ever-increasing demand for more natural products, enzymes have gained real importance in bread-making. If an enzyme is added, it is often destroyed by the heat during the baking process. For generations, enzymes have been used for the improvement of texture and appearance, enhancement of nutritional values and generation of appealing flavours and aromas. Enzymes used in bakery industry constitute nearly one third of the market. The bakery products have undergone radical improvements in quality over the past years in terms of flavour, texture and shelf-life. The the biggest contributor for these improvementsis the usage of enzymes. Present work seeks to systematically describe bakery enzymes, their classification, benefits, usage and chemical reactions in the bread making process.doi:10.5219/193

Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization

Energy Technology Data Exchange (ETDEWEB)

Kim, Moon Il; Kim, Jungbae; Lee, Jinwoo; Jia, Hongfei; Na, Hyon Bin; Youn, Jongkyu; Kwak, Ja Hun; Dohnalkova, Alice; Grate, Jay W.; Wang, Ping; Hyeon, Taeghwan; Park, Hyun-Gyu; Chang, Ho Nam

2007-02-01

alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for two weeks under even rigorously shaking condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.

Immobilized enzymes and cells

Energy Technology Data Exchange (ETDEWEB)

Bucke, C; Wiseman, A

1981-04-04

This article reviews the current state of the art of enzyme and cell immobilization and suggests advances which might be made during the 1980's. Current uses of immobilized enzymes include the use of glucoamylase in the production of glucose syrups from starch and glucose isomerase in the production of high fructose corn syrup. Possibilities for future uses of immobilized enzymes and cells include the utilization of whey and the production of ethanol.

Nitrocellulose membrane-based enzyme-linked immunoassay for dengue serotype-1 IgM detection

International Nuclear Information System (INIS)

Leon, S.; Guevara, C.; Chunga, A.

1999-01-01

To evaluate the sensitivity and specifity of a nitrocellulose membrane-based immunoassay for dengue IgM, with respect to capture enzyme immunoassay, for the diagnosis of dengue virus infection. 101 serum samples were processed and divided into 2 groups: 53 from dengue serotype 1 (DEN1) infected patients, and 48 from healthy subjects. Both groups were tested with a nitrocellulose membrane-based IgM capture enzyme immunoassay (NMB-EIA) and also with an ELISA as referential pattern. NMB-EIA testing detected IgM anti-DEN1 in 94,34% of samples from infected patients, and in 14,58% of control samples, whereas ELISA fails to report false positive or false negative results: NMB-EIA appears to be a good alternative for dengue infection diagnosis. (authors)

Enzymatic Hydrolysis of Wheat Arabinoxylan by a Recombinant "Minimal" Enzyme Cocktail Containing beta-Xylosidase and Novel endo-1,4-beta-Xylanase and alpha-L-Arabinofuranosidase Activities

DEFF Research Database (Denmark)

Sørensen, Hanne R.; Pedersen, Sven; Jørgensen, Christel T.

2007-01-01

24 h at pH 5, 50 degrees C. A 10%:40%:50% mixture of Abf II, Abf III, and beta-xyl released 56 mg of arabinose and 91 mg of xylose per gram of vinasse dry matter after 24 h at pH 5, 50 degrees C. The optimal dosages of the "minimal" enzyme cocktails were determined to be 0.4, 0.3, and 0.2 g enzyme......This study describes the identification of the key enzyme activities required in a "minimal" enzyme cocktail able to catalyze hydrolysis of water-soluble and water-insoluble wheat arabinoxylan and whole vinasse, a fermentation effluent resulting from industrial ethanol manufacture from wheat...

Improvement in phytoremediation potential of Solanum nigrum under cadmium contamination through endophytic-assisted Serratia sp. RSC-14 inoculation.

Science.gov (United States)

Khan, Abdur Rahim; Ullah, Ihsan; Khan, Abdul Latif; Park, Gun-Seok; Waqas, Muhammad; Hong, Sung-Jun; Jung, Byung Kwon; Kwak, Yunyoung; Lee, In-Jung; Shin, Jae-Ho

2015-09-01

The growth of hyperaccumulator plants is often compromised by increased toxicity of metals like cadmium (Cd). However, extraction of such metals from the soil can be enhanced by endophytic microbial association. Present study was aimed to elucidate the potential of microbe-assisted Cd phytoextraction in hyperaccumulator Solanum nigrum plants and their interactions under varied Cd concentrations. An endophytic bacteria Serratia sp. RSC-14 was isolated from the roots of S. nigrum. In addition to Cd tolerance up to 4 mM, the RSC-14 exhibited phosphate solubilization and secreted plant growth-promoting phytohormones such as indole-3-acetic acid (54 μg/mL). S. nigrum plants were inoculated with RSC-14 and were grown in different concentrations of Cd (0, 10, and 30 mg Cd kg(-1) sand). Results revealed that Cd treatment caused significant cessation in plant growth, biomass, and chlorophyll content, whereas significantly higher malondialdehyde (MDA) and electrolyte production in leaves were observed in a dose-dependent manner. Conversely, RSC-14 inoculation relived the toxic effects of Cd-induced stress by significantly increasing root/shoot growth, biomass production, and chlorophyll content and decreasing MDA and electrolytes contents. Ameliorative effects on host growth were also observed by the regulation of metal-induced oxidative stress enzymes such as catalase, peroxidase, and polyphenol peroxidase. Activities of these enzymes were significantly reduced in RSC-14 inoculated plants as compared to control plants under Cd treatments. The lower activities of stress responsive enzymes suggest modulation of Cd stress by RSC-14. The current findings support the beneficial uses of Serratia sp. RSC-14 in improving the phytoextraction abilities of S. nigrum plants in Cd contamination.

Crystal structure of a cold-active protease (Pro21717) from the psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, at 1.4 Å resolution: Structural adaptations to cold and functional analysis of a laundry detergent enzyme.

Science.gov (United States)

Park, Ha Ju; Lee, Chang Woo; Kim, Dockyu; Do, Hackwon; Han, Se Jong; Kim, Jung Eun; Koo, Bon-Hun; Lee, Jun Hyuck; Yim, Joung Han

2018-01-01

Enzymes isolated from organisms found in cold habitats generally exhibit higher catalytic activity at low temperatures than their mesophilic homologs and are therefore known as cold-active enzymes. Cold-active proteases are very useful in a variety of biotechnological applications, particularly as active ingredients in laundry and dishwashing detergents, where they provide strong protein-degrading activity in cold water. We identified a cold-active protease (Pro21717) from a psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, and determined the crystal structure of its catalytic domain (CD) at a resolution of 1.4 Å. The Pro21717-CD structure shows a conserved subtilisin-like fold with a typical catalytic triad (Asp185, His244, and Ser425) and contains four calcium ions and three disulfide bonds. Interestingly, we observed an unexpected electron density at the substrate-binding site from a co-purified peptide. Although the sequence of this peptide is unknown, analysis of the peptide-complexed structure nonetheless provides some indication of the substrate recognition and binding mode of Pro21717. Moreover, various parameters, including a wide substrate pocket size, an abundant active-site loop content, and a flexible structure provide potential explanations for the cold-adapted properties of Pro21717. In conclusion, this is first structural characterization of a cold-adapted subtilisin-like protease, and these findings provide a structural and functional basis for industrial applications of Pro21717 as a cold-active laundry or dishwashing detergent enzyme.

Imbalance between pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity in acute respiratory distress syndrome

NARCIS (Netherlands)

Wösten-van Asperen, Roelie M.; Bos, Albert P.; Bem, Reinout A.; Dierdorp, Barbara S.; Dekker, Tamara; van Goor, Harry; Kamilic, Jelena; van der Loos, Chris M.; van den Berg, Elske; Bruijn, Martijn; van Woensel, Job B.; Lutter, René

2013-01-01

Angiotensin-converting enzyme and its effector peptide angiotensin II have been implicated in the pathogenesis of acute respiratory distress syndrome. Recently, angiotensin-converting enzyme 2 was identified as the counter-regulatory enzyme of angiotensin-converting enzyme that converts angiotensin

Imbalance between pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity in acute respiratory distress syndrome

NARCIS (Netherlands)

Wosten-van Asperen, Roelie M.; Bos, Albert; Bem, Reinout A.; Dierdorp, Barbara S.; Dekker, Tamara; van Goor, Harry; Kamilic, Jelena; van der Loos, Chris M.; van den Berg, Elske; Bruijn, Martijn; van Woensel, Job B.; Lutter, Rene

2013-01-01

Objective: Angiotensin-converting enzyme and its effector peptide angiotensin II have been implicated in the pathogenesis of acute respiratory distress syndrome. Recently, angiotensin-converting enzyme 2 was identified as the counter-regulatory enzyme of angiotensin-converting enzyme that converts

Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Surface and enzyme effects.

Science.gov (United States)

Ward, Keeran; Xi, Jingshu; Stuckey, David C

2015-12-01

The use of non-ionic colloidal liquid aphrons (CLAs) as a support for enzyme immobilisation was investigated. Formulation required the mixing of an aqueous-surfactant solution with a relatively non-polar solvent-surfactant solution, forming a solvent droplet surrounded by a thin stabilised aqueous film (soapy shell). Studies utilising anionic surfactants have showed increased retention, however, very little have been understood about the forces governing immobilisation. This study seeks to determine the effects of enzyme properties on CLA immobilisation by examining a non-ionic/non-polar solvent system comprised of two non-ionic surfactants, Tween 20 and 80, mineral oil and the enzymes lipase, aprotinin and α-chymotrypsin. From these results it was deduced that hydrophobic interactions strongly governed immobilisation. Confocal Scanning Laser Microscopy (CSLM) revealed that immobilisation was predominantly achieved by surface adsorption attributed to hydrophobic interactions between the enzyme and the CLA surface. Enzyme surface affinity was found to increase when added directly to the formulation (pre-manufacture addition), as opposed to the bulk continuous phase (post-manufacture addition), with α-chymotrypsin and aprotinin being the most perturbed, while lipase was relatively unaffected. The effect of zeta potential on immobilisation showed that enzymes adsorbed better closer to their pI, indicating that charge minimisation was necessary for immobilisation. Finally, the effect of increasing enzyme concentration in the aqueous phase resulted in an increase in adsorption for all enzymes due to cooperativity between protein molecules, with saturation occurring faster at higher adsorption rates. Copyright © 2015 Elsevier B.V. All rights reserved.

Synthesis of enzymes connected with mycoparasitism by ectomycorrhizal fungi.

Science.gov (United States)

Mucha, Joanna; Dahm, Hanna; Strzelczyk, Edmund; Werner, Antoni

2006-03-01

The production of enzymes involved in mycoparasitism by several strains of ectomycorrhizal fungi: Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), S. bovinus (15-3), S. luteus (14-7) on different substrates such as colloidal chitin, mycelia of Trichoderma harzianum, T. virens and Mucor hiemalis was examined. Chitinases and beta-1,3-glucanases were assayed spectrophotometrically by measuring the amount of reducing sugars releasing from suitable substrate by means of Miller's method. Beta-glucosidases were determined by measuring the amount of p-nitrophenol released from p-nitrophenyl-beta-D-glucopyranoside. It was observed that A. muscaria (16-3) and L. laccata (9-12) biosynthesized the highest activity of enzymes in contrast to the strains of S. bovinus and S. luteus. The mycelium of T. harzianum turned out to be the best substrate for the induction of beta-1,3-glucanases and beta-glucosidases for both strains of L. laccata, although the difference in the induction of chitinases in the presence of mycelia of different species of Trichoderma was not indicated.

Allosteric regulation of epigenetic modifying enzymes.

Science.gov (United States)

Zucconi, Beth E; Cole, Philip A

2017-08-01

Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings. Copyright © 2017 Elsevier Ltd. All rights reserved.

DGAT enzymes and triacylglycerol biosynthesis

Science.gov (United States)

Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.

2008-01-01

Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, and the use of molecular tools, including mice deficient in either enzyme, has shed light on their functions. Although DGAT enzymes are involved in TG synthesis, they have distinct protein sequences and differ in their biochemical, cellular, and physiological functions. Both enzymes may be useful as therapeutic targets for diseases. Here we review the current knowledge of DGAT enzymes, focusing on new advances since the cloning of their genes, including possible roles in human health and diseases. PMID:18757836

de novo computational enzyme design.

Science.gov (United States)

Zanghellini, Alexandre

2014-10-01

Recent advances in systems and synthetic biology as well as metabolic engineering are poised to transform industrial biotechnology by allowing us to design cell factories for the sustainable production of valuable fuels and chemicals. To deliver on their promises, such cell factories, as much as their brick-and-mortar counterparts, will require appropriate catalysts, especially for classes of reactions that are not known to be catalyzed by enzymes in natural organisms. A recently developed methodology, de novo computational enzyme design can be used to create enzymes catalyzing novel reactions. Here we review the different classes of chemical reactions for which active protein catalysts have been designed as well as the results of detailed biochemical and structural characterization studies. We also discuss how combining de novo computational enzyme design with more traditional protein engineering techniques can alleviate the shortcomings of state-of-the-art computational design techniques and create novel enzymes with catalytic proficiencies on par with natural enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Measurement of enzyme activity.

Science.gov (United States)

Harris, T K; Keshwani, M M

2009-01-01

To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.

Using guanidine-hydrochloride for fast and efficient protein digestion and single-step affinity-purification mass spectrometry

DEFF Research Database (Denmark)

Poulsen, Jon Wriedt; Madsen, Christian Toft; Young, Clifford

2013-01-01

be optimally completed within 30 min with endoprotease Lys-C. No chemical artifacts were introduced when samples were incubated in Gnd-HCl at 95 °C, making Gnd-HCl an appropriate digestion buffer for shotgun proteomics. Current methodologies for investigating protein-protein interactions (PPIs) often require......Protein digestion is an integral part of the "shotgun" proteomics approach and commonly requires overnight incubation prior to mass spectrometry analysis. Quadruplicate "shotgun" proteomic analysis of whole yeast lysate demonstrated that Guanidine-Hydrochloride (Gnd-HCl) protein digestion can....... To validate the Gnd-HCl approach, label-free PPI analysis of several GFP-tagged yeast deubiquitinating enzymes was performed. The identification of known interaction partners demonstrates the utility of the optimized Gnd-HCl protocol that is also scalable to the 96 well-plate format....

Gammaherpesviral Tegument Proteins, PML-Nuclear Bodies and the Ubiquitin-Proteasome System

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Florian Full

2017-10-01

Full Text Available Gammaherpesviruses like Epstein-Barr virus (EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV subvert the ubiquitin proteasome system for their own benefit in order to facilitate viral gene expression and replication. In particular, viral tegument proteins that share sequence homology to the formylglycineamide ribonucleotide amidotransferase (FGARAT, or PFAS, an enzyme in the cellular purine biosynthesis, are important for disrupting the intrinsic antiviral response associated with Promyelocytic Leukemia (PML protein-associated nuclear bodies (PML-NBs by proteasome-dependent and independent mechanisms. In addition, all herpesviruses encode for a potent ubiquitin protease that can efficiently remove ubiquitin chains from proteins and thereby interfere with several different cellular pathways. In this review, we discuss mechanisms and functional consequences of virus-induced ubiquitination and deubiquitination for early events in gammaherpesviral infection.

Digestive enzyme as benchmark for insecticide resistance development in Culex pipiens larvae to chemical and bacteriologic insecticides.

Science.gov (United States)

Kamel, Nashwa H; Bahgat, Iman M; El Kady, Gamal A

2013-04-01

This work monitored changes in some digestive enzymes (trypsin and aminopeptidase) associated with the building up of resistance in Cx. pipiens larvae to two chemical insecticides (methomyl and/or malathion) and one biological insecticide (Bacillus thuringiensis-H14 or B.t H 14). The LC50 value of methomyl for both field- and the 12th generation (F12) of the selected strain was 1.789 ppm and 8.925 ppm respectively. The LC50 value of malathion for both field and the F12 of the selected strain was 0.082 ppm and 0.156 ppm respectively, and those of B.t H14 of field strain and the F12 was 2.550ppm & 2.395ppm respectively. The specific activity of trypsin enzyme in control susceptible colony was 20.806 +/- 0.452micromol/min/mg protein; but at F4 and F8 for malathion and methomyl treated larvae were 10.810 +/- 0.860 & 15.616+/-0.408 micromol/min/mg protein, respectively. Trypsin activity of F12 in treated larvae with B.t.H14 was 2.097 +/- 0.587 microiol/min/mg protein. Aminopeptidase specific activity for susceptible control larvae was 173.05 +/- 1.3111 micromol/min/mg protein. This activity decreased to 145.15 +/- 4.12, 152.497 +/- 6.775 & 102.04 +/- 3.58a micromol/min/mg protein after larval (F 12) treatment with methomyl, malathion and B.t H 14 respectively.

Mutagenesis of the redox-active disulfide in mercuric ion reductase: Catalysis by mutant enzymes restricted to flavin redox chemistry

International Nuclear Information System (INIS)

Distefano, M.D.; Au, K.G.; Walsh, C.T.

1989-01-01

Mercuric reductase, a flavoenzyme that possesses a redox-active cystine, Cys 135 Cys 140 , catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, the authors have constructed mutants lacking a redox-active disulfide by eliminating Cys 135 (Ala 135 Cys 140 ), Cys 14 (Cys 135 Ala 140 ), or both (Ala 135 Ala 140 ). Additionally, they have made double mutants that lack Cys 135 (Ala 135 Cys 139 Cys 140 ) or Cys 140 (Cys 135 Cys 139 Ala 140 ) but introduce a new Cys in place of Gly 139 with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH 2 redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. Preliminary evidence for the Ala 135 Cys 139 Cys 14 mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala 135 Cys 140 protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. They conclude that the Cys 135 and Cys 140 thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate

Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media.

Science.gov (United States)

Taneja, Kapila; Bajaj, Bijender Kumar; Kumar, Sandeep; Dilbaghi, Neeraj

2017-07-01

Intravascular thrombosis is one of the major causes of variety of cardiovascular disorders leading to high mortality worldwide. Fibrinolytic enzymes from microbial sources possess ability to dissolve these clots and help to circumvent these problems in more efficient and safer way. In the present study, fibrinolytic protease with higher fibrinolytic activity than plasmin was obtained from Serratia sp. KG-2-1 isolated from garbage dump soil. Response surface methodology was used to study the interactive effect of concentration of maltose, yeast extract + peptone (1:1), incubation time, and pH on enzyme production and biomass. Maximum enzyme production was achieved at 33 °C after 24 h at neutral pH in media containing 1.5% Maltose, 4.0% yeast extract + peptone and other trace elements resulting in 1.82 folds increased production. The enzyme was purified from crude extract using ammonium sulfate precipitation and DEAE-Sephadex chromatography resulting in 12.9 fold purification with 14.9% yield. The purified enzyme belongs to metalloprotease class and had optimal activity in conditions similar to physiological environment with temperature optima of 40 °C and pH optima of 8. The enzyme was found to be stable in various solvents and its activity was enhanced in presence of Na + , K + , Ba 2+ , Cu 2+ , Mn 2+ , Hg 2+ but inhibited by Ca 2+ and Fe 3+ . Hence, the obtained enzyme may be used as potential therapeutic agent in combating various thrombolytic disorders.

Thermodynamics of Enzyme-Catalyzed Reactions Database

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SRD 74 Thermodynamics of Enzyme-Catalyzed Reactions Database (Web, free access)   The Thermodynamics of Enzyme-Catalyzed Reactions Database contains thermodynamic data on enzyme-catalyzed reactions that have been recently published in the Journal of Physical and Chemical Reference Data (JPCRD). For each reaction the following information is provided: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the data and an evaluation thereof.

Gestão de custos aplicada a hospitais universitários públicos: a experiência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP Cost management applied to public university hospitals

Directory of Open Access Journals (Sweden)

Carlos Alberto Grespan Bonacim

2010-08-01

Full Text Available Este artigo apresenta a experiência de implantação da metodologia de custos baseada em atividades (ABC do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, por meio de um estudo de caso, num período de cinco anos. As características dos hospitais universitários de alta tecnologia, tais como atividades docente-assistenciais e serviços básicos de saúde, fazem com que seus custos sejam mais elevados do que os de hospitais não universitários. Na sua trajetória institucional, o HCFMRP-USP não possuía uma metodologia consistente de mensuração e avaliação dos custos de sua atividade assistencial. No entanto, dificuldades de financiamento de suas atividades, associadas a uma completa reestruturação em seu modelo de atendimento vinculado ao Sistema Único de Saúde, impulsionaram a adoção do mapeamento de custos por atividades, para demonstrar, de forma adequada, como pode ser mensurado o impacto do ensino nos custos assistenciais, visto que a agenda democrática nacional tem reivindicado transparência, accountability e, fundamentalmente, eficiência na gestão dos recursos públicosThis paper presents the experience of implementing the methodology of cost-based activities (ABC at The Ribeirão Preto Medical School Clinics Hospital of the University of São Paulo (HCFMRP-USP, through a case study over a period of five years. Characteristics of high technology university hospitals, such as teaching and assistance, make their costs higher than those of non-university hospitals. In its institutional history, HCFMRP-USP lacked a consistent methodology for measuring and assessing the costs of its assistance activities. However, difficulties in financing these activities, associated with a complete restructuring in the hospital's healthcare model linked to the National Health System, have furthered the adoption of cost mapping, in order to assess adequately the impact of teaching activities

The management of isolated positive syphilis enzyme immunoassay results in HIV-negative patients attending a sexual health clinic.

Science.gov (United States)

Thorley, Nicola; Adebayo, Michael; Smit, Erasmus; Radcliffe, Keith

2016-08-01

An unconfirmed positive treponemal enzyme immunoassay (enzyme immunoassay positive, Treponema pallidum particle agglutination negative and rapid plasma reagin negative) presents a clinical challenge to distinguish early syphilis infection from false-positive results. These cases are referred for syphilis line assay (INNO-LIA) and recalled for repeat syphilis serology. We performed a retrospective audit to establish the proportion of HIV-negative cases with unconfirmed positive enzyme immunoassay results, the proportion of these cases that received an INNO-LIA test and repeat syphilis serology testing and reviewed the clinical outcomes; 0.35% (80/22687) cases had an unconfirmed positive treponemal enzyme immunoassay result. Repeat syphilis serology was performed in 80% (64/80) cases, but no additional cases of syphilis were identified. Eighty-eight per cent (70/80) received an INNO-LIA test; 14% (5/37) unconfirmed enzyme immunoassay-positive cases with no prior history of syphilis were confirmed on INNO-LIA assay, supporting a diagnosis of latent syphilis. As a confirmatory treponemal test, the INNO-LIA assay may be more useful than repeat syphilis serological testing. © The Author(s) 2016.

Action of amylolytic and pullulytic enzymes from various anaerobic thermophiles on linear and branched glucose polymers

Energy Technology Data Exchange (ETDEWEB)

Koch, R [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F.R.). Arbeitsbereich Biotechnologie 1

1990-10-01

A detailed study has been conducted on the action of starch hydrolyzing enzymes from thermophilic anaerobic bacteria belonging to the genera Clostridium, Thermoanaerobacter and Thermobacteroides. The appearance of multiple bands on polyacrylamide gels with amylolytic as well as pullulytic activities was shown to be a general feature of bacteria investigated. Analysis of the hydrolysis products of each protein band clearly demonstrated the capability of these organisms to hydrolyze {alpha}-1,4-glycosidic bonds in linear oligosaccharides and {alpha}-1,6-glycosidic linkages in pullulan. Furthermore, the enzyme system of thermophilic bacteria investigated was also capable of attacking in the {alpha}-1,6-linkages in branched oligosaccharides. Due to the action of these thermoactive enzymes with multiple specificity an almost complete hydrolysis of raw starch and maltodextrin could be achieved under the same conditions and in one step. (orig.).

Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic

Science.gov (United States)

Schoonen, Lise; Nolte, Roeland J. M.; van Hest, Jan C. M.

2016-07-01

The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions.The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. Electronic supplementary information (ESI) available: Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations. See DOI: 10.1039/c6nr04181g

Lipid peroxidation and antioxidant enzymes activity in Plasmodium vivax malaria patients evolving with cholestatic jaundice

Science.gov (United States)

2013-01-01

Background Plasmodium vivax infection has been considered a benign and self-limiting disease, however, recent studies highlight the association between vivax malaria and life-threatening manifestations. Increase in reactive oxygen species has already been described in vivax malaria, as a result of the increased metabolic rate triggered by the multiplying parasite, and large quantities of toxic redox-active byproducts generated. The present study aimed to study the oxidative stress responses in patients infected with P. vivax, who developed jaundice (hyperbilirubinaemia) in the course of the disease, a common clinical complication related to this species. Methods An evaluation of the lipid peroxidation and antioxidant enzymes profile was performed in 28 healthy individuals and compared with P. vivax infected patients with jaundice, i.e., bilirubin jaundice (34 patients), on day 1 (D1) and day 14 (D14) after anti-malarial therapy. Results Hyperbilirubinaemia was more frequent among women and patients experiencing their first malarial infection, and lower haemoglobin and higher lactate dehydrogenase levels were observed in this group. Malondialdehyde levels and activity of celuroplasmin and glutathione reductase were increased in the plasma from patients with P. vivax with jaundice compared to the control group on D1. However, the activity of thioredoxin reductase was decreased. The enzymes glutathione reductase, thioredoxin reductase, thiols and malondialdehyde also differed between jaundiced versus non-jaundiced patients. On D14 jaundice and parasitaemia had resolved and oxidative stress biomarkers were very similar to the control group. Conclusion Cholestatic hyperbilirubinaemia in vivax malaria cannot be totally disassociated from malaria-related haemolysis. However, significant increase of lipid peroxidation markers and changes in antioxidant enzymes in patients with P. vivax-related jaundice was observed. These results suggest oxidative processes contributing

Drug: D03365 [KEGG MEDICUS

Lifescience Database Archive (English)

Full Text Available D03365 Drug Nicotine (USP); Habitrol (TN) ... C10H14N2 D03365.gif ... Other ... DG01718 ... Drugs... for addictive disorder ... DG01715 ... Drugs for nicotine dependence Cyp substrate ... DG01892 ... CYP1A2 substra

Functional analyses of multiple lichenin-degrading enzymes from the rumen bacterium Ruminococcus albus 8.

Science.gov (United States)

Iakiviak, Michael; Mackie, Roderick I; Cann, Isaac K O

2011-11-01

Ruminococcus albus 8 is a fibrolytic ruminal bacterium capable of utilization of various plant cell wall polysaccharides. A bioinformatic analysis of a partial genome sequence of R. albus revealed several putative enzymes likely to hydrolyze glucans, including lichenin, a mixed-linkage polysaccharide of glucose linked together in β-1,3 and β-1,4 glycosidic bonds. In the present study, we demonstrate the capacity of four glycoside hydrolases (GHs), derived from R. albus, to hydrolyze lichenin. Two of the genes encoded GH family 5 enzymes (Ra0453 and Ra2830), one gene encoded a GH family 16 enzyme (Ra0505), and the last gene encoded a GH family 3 enzyme (Ra1595). Each gene was expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity. Upon screening on a wide range of substrates, Ra0453, Ra2830, and Ra0505 displayed different hydrolytic properties, as they released unique product profiles. The Ra1595 protein, predicted to function as a β-glucosidase, preferred cleavage of a nonreducing end glucose when linked by a β-1,3 glycosidic bond to the next glucose residue. The major product of Ra0505 hydrolysis of lichenin was predicted to be a glucotriose that was degraded only by Ra0453 to glucose and cellobiose. Most importantly, the four enzymes functioned synergistically to hydrolyze lichenin to glucose, cellobiose, and cellotriose. This lichenin-degrading enzyme mix should be of utility as an additive to feeds administered to monogastric animals, especially those high in fiber.

DNA-Based Enzyme Reactors and Systems

Directory of Open Access Journals (Sweden)

Veikko Linko

2016-07-01

Full Text Available During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications.

Cold-Adapted Enzymes

Science.gov (United States)

Georlette, D.; Bentahir, M.; Claverie, P.; Collins, T.; D'amico, S.; Delille, D.; Feller, G.; Gratia, E.; Hoyoux, A.; Lonhienne, T.; Meuwis, M.-a.; Zecchinon, L.; Gerday, Ch.

In the last few years, increased attention has been focused on enzymes produced by cold-adapted micro-organisms. It has emerged that psychrophilic enzymes represent an extremely powerful tool in both protein folding investigations and for biotechnological purposes. Such enzymes are characterised by an increased thermosensitivity and, most of them, by a higher catalytic efficiency at low and moderate temperatures, when compared to their mesophilic counterparts. The high thermosensitivity probably originates from an increased flexibility of either a selected area of the molecular edifice or the overall protein structure, providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Structure modelling and recent crystallographic data have allowed to elucidate the structural parameters that could be involved in this higher resilience. It was demonstrated that each psychrophilic enzyme adopts its own adaptive strategy. It appears, moreover, that there is a continuum in the strategy of protein adaptation to temperature, as the previously mentioned structural parameters are implicated in the stability of thermophilic proteins. Additional 3D crystal structures, site-directed and random mutagenesis experiments should now be undertaken to further investigate the stability-flexibility-activity relationship.

Enzymic construction of maltosaccharide chains on a heart protein

International Nuclear Information System (INIS)

Kay, M.J.; Kirkman, B.R.; Lomako, J.; Rodriguez, I.R.; Tandecarz, J.S.; Fliesler, S.J.; Whelan, W.J.

1987-01-01

The authors have reported that when 100,000 g pellets of rabbit-heart and rabbit-muscle homogenates are incubated with UDP( 14 C)glucose, the sugar is incorporated into a protein with Mr 40 KDa. They suggested that these in vitro observations corresponded to the initial stage in the synthesis of glycogen on a protein that they have named glycogenin and which in rabbit muscle appears to be covalently linked to the glycogen via tyrosine residues. The following new observations support the role of a protein as the precursor of glycogen and suggest that glycogen-free glycogenin is present in heart tissue. (1) The ( 14 C)glucose residues added to the heart protein can be removed with glycogenolytic enzymes that hydrolyse 1,4-alpha-glucosidic bonds and therefore constitute synthetic maltosaccharide chains. (2) The newly added glucose residues appear to be attached to pre-existing glucose residues on the protein. Chain elongation does not proceed beyond a few glucose residues. (3) The further relevance of these observations to glycogen synthesis shown by a Western blot in which the radioglucosylated heart protein was found to cross-react with polyclonal antibody to glycogenin obtained from rabbit-muscle glycogen

Quantitative proteomic study of Aspergillus Fumigatus secretome revealed deamidation of secretory enzymes.

Science.gov (United States)

Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

2015-04-24

Aspergillus sp. plays an essential role in lignocellulosic biomass recycling and is also exploited as cell factories for the production of industrial enzymes. This study profiled the secretome of Aspergillus fumigatus when grown with cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). Post translational modifications (PTMs) of proteins play a critical role in protein functions. However, our understanding of the PTMs in secretory proteins is limited. Here, we present the identification of PTMs such as deamidation of secreted proteins of A. fumigatus. This study quantified diverse groups of extracellular secreted enzymes and their functional classification revealed cellulases and glycoside hydrolases (32.9%), amylases (0.9%), hemicellulases (16.2%), lignin degrading enzymes (8.1%), peptidases and proteases (11.7%), chitinases, lipases and phosphatases (7.6%), and proteins with unknown function (22.5%). The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulates expression of specific cellulases and hemicellulases, and their abundance level as a function of substrate. In-depth data analysis revealed deamidation as a major PTM of key cellulose hydrolyzing enzymes like endoglucanases, cellobiohydrolases and glucosidases. Hemicellulose degrading endo-1,4-beta-xylanase, monosidases, xylosidases, lignin degrading laccase, isoamyl alcohol oxidase and oxidoreductases were also found to be deamidated. The filamentous fungi play an essential role in lignocellulosic biomass recycling and fungal strains belonging to Aspergillus were also exploited as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. In this study, extracellular proteins secreted by thermophilic A. fumigatus when grown with cellulose, xylan and starch were profiled using isobaric tags for relative and absolute quantification (iTRAQ) by

Increased serum levels of hyaluronic acid in pregnancies complicated by preeclampsia or hemolysis, elevated liver enzymes, and low platelets syndrome.

Science.gov (United States)

Osmers, R G; Schütz, E; Diedrich, F; Wehry, B; Krauss, T; Oellerich, M; Kuhn, W

1998-02-01

Fifteen percent of patients who later have hemolysis, elevated liver enzymes, and low platelets syndrome develop initially have nonspecific symptoms. Early diagnosis could ensure adequate obstetric management; however, prognostic biochemical tests are lacking. We hypothesized that elevated hyaluronic acid serum levels might be an early indicator of hemolysis, elevated liver enzymes, and low platelets syndrome because it is known to be a sensitive marker of liver cell function. Hyaluronic acid in serum was measured in patients with normal pregnancies (n = 109) and in those patients with pregnancies complicated by preeclampsia (n = 14) or hemolysis, elevated liver enzymes, and low platelets syndrome (n = 11). A significant increase in hyaluronic acid serum concentrations was observed in patients with hemolysis, elevated liver enzymes, and low platelets syndrome or with preeclampsia (p hyaluronic acid serum levels in hemolysis, elevated liver enzymes, and low platelets syndrome correlated with the clinical severity of the individual course of disease as measured by intensive care unit time (r = 0.72; p hyaluronic acid in preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome are significantly elevated and might play an important diagnostic and prognostic role in patients with hemolysis, elevated liver enzymes, and low platelets syndrome.

Enzyme Engineering for In Situ Immobilization.

Science.gov (United States)

Rehm, Fabian B H; Chen, Shuxiong; Rehm, Bernd H A

2016-10-14

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

[The rise of enzyme engineering in China].

Science.gov (United States)

Li, Gaoxiang

2015-06-01

Enzyme engineering is an important part of the modern biotechnology. Industrial biocatalysis is considered the third wave of biotechnology following pharmaceutical and agricultural waves. In 25 years, China has made a mighty advances in enzyme engineering research. This review focuses on enzyme genomics, enzyme proteomics, biosynthesis, microbial conversion and biosensors in the Chinese enzyme engineering symposiums and advances in enzyme preparation industry in China.

Catalysis by a de novo zinc-mediated protein interface: implications for natural enzyme evolution and rational enzyme engineering.

Science.gov (United States)

Der, Bryan S; Edwards, David R; Kuhlman, Brian

2012-05-08

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.

In vitro and comparative study on the extracellular enzyme activity of molds isolated from keratomycosis and soil

Directory of Open Access Journals (Sweden)

Arumugam Mythili

2014-10-01

Full Text Available AIM:To isolate and identify the molds involved in mycotic keratitis; to isolate corresponding species from soil samples; to compare the extracellular enzyme activity indices of the molds isolated from keratitis cases and the corresponding soil isolates.METHODS:The specimens were collected from the target patients attending the microbiology laboratory of tertiary eye hospital in Coimbatore, Tamilnadu state, India. The isolates were subjected for identification based on the growth on solid media, direct microscopy and lacto phenol cotton blue wet mount preparation. Extracellular enzymes such as lipase, deoxyribonuclease (DNase, α-amylase, protease, cellulase and pectinase produced by the fungalisolates were screened on solid media supplemented with the corresponding substrates. Based on growth and zone diameter, the enzyme activity indices were calculated and were compared with that of the soil fungalisolates.RESULTS:A total of 108 clinical samples were collected from a tertiary eye care hospital and out of which 60 fungal isolates were obtained. Among these, Fusarium spp. (n=30, non sporulating molds (n=9, Aspergillus flavus (n=6, Bipolaris spp. (n=6, Exserohilum spp. (n=4, Curvularia spp. (n=3, Alternaria spp. (n=1 and Exophiala spp. (n=1were identified and designated as FS1-30, NSM1-9, AF1-6, BS1-6, ES1-4, CS1-3, AS1 and EX1, respectively. For comparative analysis, soil samples were also collected from which, one isolate of each Fusarium spp., Aspergillus flavus, Bipolaris spp., Exserohilum spp., and Curvularia spp., respectively were selected. Highest lipase activity was seen in corneal isolate NSM2 (EAI= 2.14. The DNase activity was higher in NSM9 (EAI=1.88. In case of protease, Fusarium spp. (FS9 had prominent enzyme activity index of 1.38; α-amylase activity was also superior in corneal isolate FS13 with EAI of 1.63 when compared to other isolates. The enzyme activity index for cellulase was also noted to be higher in corneal isolates i

The Degradation of 14C-Glutamic Acid by L-Glutamic Acid Decarboxylase.

Science.gov (United States)

Dougherty, Charles M; Dayan, Jean

1982-01-01

Describes procedures and semi-micro reaction apparatus (carbon dioxide trap) to demonstrate how a particular enzyme (L-Glutamic acid decarboxylase) may be used to determine the site or sites of labeling in its substrate (carbon-14 labeled glutamic acid). Includes calculations, solutions, and reagents used. (Author/SK)

Accuracy of Cirrus HD-OCT and Topcon SP-3000P for measuring central corneal thickness.

Science.gov (United States)

Calvo-Sanz, Jorge A; Ruiz-Alcocer, Javier; Sánchez-Tena, Miguel A

2017-02-18

To compare and analyze the interchangeability of three measuring systems, each based on a different technique, for central corneal thickness (CCT) analysis. CCT measurements were measured using optical coherence tomography (OCT), non-contact specular microscopy (NCSM), and ultrasonic pachymetry (USP) in 60 eyes of 60 healthy patients with a mean age of 66.5±15.0 years and a mean spherical equivalent of 0.43±1.14 D. Analysis of variations in measurement concordance and correlation among the three different methods were performed. Comparison of CCT measurements were done using Bland-Altman plots (with bias and 95% confidence intervals), intraclass correlation coefficient (ICC), and paired t-student analysis. Mean CCT values were: 549.20±26.91μm for USP (range 503-618μm), 514.20±27.49μm for NCSM (range 456-586μm) and 542.80±25.56μm for OCT (range 486-605μm). CCT values obtained with NCMS were significantly lower than those obtained with OCT and USP methods. NCMS CCT value was 36.08±10.72μm lower than USP value (p<0.05), and NCMS CCT value was 7.88±8.86μm lower than OCT value (p<0.05). ICC between USP-NCSM pair was 0.488 and 0.909 between USP-OCT pair. OCT and UPS offered highly comparable results, whereas NCSM offered lower mean CCT values compared to the other two methods. Therefore, NCSM should not be considered a reliable method for measuring CCT and should rather be considered for assessing longitudinal changes in the same patient. Copyright © 2017 Spanish General Council of Optometry. Published by Elsevier España, S.L.U. All rights reserved.

Synergistic action of recombinant accessory hemicellulolytic and pectinolytic enzymes to Trichoderma reesei cellulase on rice straw degradation.

Science.gov (United States)

Laothanachareon, Thanaporn; Bunterngsook, Benjarat; Suwannarangsee, Surisa; Eurwilaichitr, Lily; Champreda, Verawat

2015-12-01

Synergism between core cellulases and accessory hydrolytic/non-hydrolytic enzymes is the basis of efficient hydrolysis of lignocelluloses. In this study, the synergistic action of three recombinant accessory enzymes, namely GH62 α-l-arabinofuranosidase (ARA), CE8 pectin esterase (PET), and GH10 endo-1,4-beta-xylanase (XYL) from Aspergillus aculeatus expressed in Pichia pastoris to a commercial Trichoderma reesei cellulase (Accellerase® 1500; ACR) on hydrolysis of alkaline pretreated rice straw was studied using a mixture design approach. Applying the full cubic model, the optimal ratio of quaternary enzyme mixture was predicted to be ACR:ARA:PET:XYL of 0.171:0.079:0.100:0.150, which showed a glucose releasing efficiency of 0.173 gglc/FPU, higher than the binary ACR:XYL mixture (0.122 gglc/FPU) and ACR alone (0.081 gglc/FPU) leading to a 47.3% increase in glucose yield compared with that from ACR at the same cellulase dosage. The result demonstrates the varying degree of synergism of accessory enzymes to cellulases useful for developing tailor-made enzyme systems for bio-industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enzyme structure and interaction with inhibitors

International Nuclear Information System (INIS)

London, R.E.

1983-01-01

This article reviews some of the results of studies on the 13 C-labeled enzyme dihydrofolate reductase (DHFR). Nuclear magnetic resonance (NMR) techniques are used in combination with isotopic labeling to learn about the structure and dynamics of this enzyme. 13 C-labeling is used for the purpose of studying enzyme/substrate and enzyme/inhibitor interactions. A second set of studies with DHFR was designed to investigate the basis for the high affinity between the inhibitor methotrexate and DHFR. The label was placed on the inhibitor, rather than the enzyme

[Hydrogen production and enzyme activity of acidophilic strain X-29 at different C/N ratio].

Science.gov (United States)

Li, Qiu-bo; Xing, De-feng; Ren, Nan-qi; Zhao, Li-hua; Song, Ye-ying

2006-04-01

Some fermentative bacteria can produce hydrogen by utilizing carbohydrate and other kinds of organic compounds as substrates. Hydrogen production was also determined by both the limiting of growth and related enzyme activity in energy metabolism. Carbon and nitrogen are needed for the growth and metabolism of microorganisms. In addition, the carbon/nitrogen (C/N) ratio can influence the material metabolized and the energy produced. In order to improve the hydrogen production efficiency of the bacteria, we analyzed the effect of different C/N ratios on hydrogen production and the related enzyme activities in the acidophilic strain X-29 using batch test. The results indicate that the differences in the metabolism level and enzyme activity are obvious at different C/N ratios. Although the difference in liquid fermentative products produced per unit of biomass is not obvious, hydrogen production is enhanced at a specifically determined ratio. At a C/N ratio of 14 the accumulative hydrogen yield of strain X-29 reaches the maximum, 2210.9 mL/g. At different C/N ratios, the expression of hydrogenase activity vary; the activity of hydrogenase decrease quickly after reaching a maximum along with the fermentation process, but the time of expression is short. The activity of alcohol dehydrogenase (ADH) tend to stabilize after reaching a peak along with the fermentation process, the difference in expression activity is little, and the expression period is long at different C/N ratios. At a C/N ratio of 14 hydrogenase and ADH reach the maximum 2.88 micromol x (min x mg)(-1) and 33.2 micromol x (min x mg)(-1), respectively. It is shown that the C/N ratio has an important effect on enhancing hydrogen production and enzyme activity.

[Study on soil enzyme activities and microbial biomass carbon in greenland irrigated with reclaimed water].

Science.gov (United States)

Pan, Neng; Hou, Zhen-An; Chen, Wei-Ping; Jiao, Wen-Tao; Peng, Chi; Liu, Wen

2012-12-01

The physicochemical properties of soils might be changed under the long-term reclaimed water irrigation. Its effects on soil biological activities have received great attentions. We collected surface soil samples from urban green spaces and suburban farmlands of Beijing. Soil microbial biomass carbon (SMBC), five types of soil enzyme activities (urease, alkaline phosphatase, invertase, dehydrogenase and catalase) and physicochemical indicators in soils were measured subsequently. SMBC and enzyme activities from green land soils irrigated with reclaimed water were higher than that of control treatments using drinking water, but the difference is not significant in farmland. The SMBC increased by 60.1% and 14.2% than those control treatments in 0-20 cm soil layer of green land and farmland, respectively. Compared with their respective controls, the activities of enzymes in 0-20 cm soil layer of green land and farmland were enhanced by an average of 36.7% and 7.4%, respectively. Investigation of SMBC and enzyme activities decreased with increasing of soil depth. Significantly difference was found between 0-10 cm and 10-20 cm soil layer in green land. Soil biological activities were improved with long-term reclaimed water irrigation in Beijing.

Positron emitter labeled enzyme inhibitors

International Nuclear Information System (INIS)

Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

1990-01-01

This invention involves a new strategy for imagining and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography

Discovery of novel hydroxamates as highly potent tumor necrosis factor-[alpha] converting enzyme inhibitors. Part II: Optimization of the S3′ pocket

Energy Technology Data Exchange (ETDEWEB)

Mazzola Jr., Robert D.; Zhu, Zhaoning; Sinning, Lisa; McKittrick, Brian; Lavey, Brian; Spitler, James; Kozlowski, Joseph; Neng-Yang, Shih; Zhou, Guowei; Guo, Zhuyan; Orth, Peter; Madison, Vincent; Sun, Jing; Lundell, Daniel; Niu, Xiaoda (SPRI)

2010-10-01

A series of cyclopropyl hydroxamic acids were prepared. Many of the compounds displayed picomolar affinity for the TACE enzyme while maintaining good to excellent selectivity profiles versus MMP-1, -2, -3, -7, -14, and ADAM-10. X-ray analysis of an inhibitor in the TACE active site indicated that the molecules bound to the enzyme in the S1{prime}-S3{prime} pocket.

GENPLAT: an automated platform for biomass enzyme discovery and cocktail optimization.

Science.gov (United States)

Walton, Jonathan; Banerjee, Goutami; Car, Suzana

2011-10-24

the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (β-glucosidase), EX3 (endo-β1,4-xylanase, GH10), and BX (β-xylosidase).

Compounds from silicones alter enzyme activity in curing barnacle glue and model enzymes.

Science.gov (United States)

Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H

2011-02-17

Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management.

Prediction of Wild-type Enzyme Characteristics

DEFF Research Database (Denmark)

Geertz-Hansen, Henrik Marcus

of biotechnology, including enzyme discovery and characterization. This work presents two articles on sequence-based discovery and functional annotation of enzymes in environmental samples, and two articles on analysis and prediction of enzyme thermostability and cofactor requirements. The first article presents...... a sequence-based approach to discovery of proteolytic enzymes in metagenomes obtained from the Polar oceans. We show that microorganisms living in these extreme environments of constant low temperature harbour genes encoding novel proteolytic enzymes with potential industrial relevance. The second article...... presents a web server for the processing and annotation of functional metagenomics sequencing data, tailored to meet the requirements of non-bioinformaticians. The third article presents analyses of the molecular determinants of enzyme thermostability, and a feature-based prediction method of the melting...

MODULATION OF THE INFLAMMATORY CYTOKINES AND CYTOPROTECTIVE ENZYME BY BILIRUBIN TREATMENT TO ENHANCE CUTANEOUS WOUND HEALING IN RATS

Directory of Open Access Journals (Sweden)

Raju Prasad

2017-06-01

Full Text Available Inflammation is the main process of wound healing where expression of certain cytokines likes Interleukin-10 (IL-10 and Tumour necrosis factor ∝ (TNF ∝ plays an important role. In view of the antioxidant potential of bilirubin, the present study was aimed to evaluate time-dependent (day 3, 7, 14 wound healing effects of bilirubin ointment (0.3% in excisional wound model in rats. Thirty-six acclimatized healthy male Wistar rats (120-150g were divided into control and treated groups containing 18 rats each. Each group was further sub- divided into three sub-groups (day 3, 7 and 14 days, n= 6. The ointment base (soft paraffin 90%, lanolin 5% and hard paraffin 5% and bilirubin ointment (0.3% were applied topically once daily for 14 days in control and treated group respectively. The wound area was determined on days 3, 7, and 14. The mRNA expression of TNF ∝ gene and IL-10 gene were determined on days 3, 7 and 14 by Real Time PCR and their protein levels by ELISA method. The protein expression of cyto-protective enzyme HO-1 (Heme oxygenage-1 and growth factor VEGF (Vascular growth factor was determined by western blotting method. The mRNA expression and protein level of TNF ∝ was significantly reduced and IL-10 was significantly increased whereas the expression of HO-1 enzyme and VEGF was significantly increased in treated group on days 3, 7 and 14. It may be concluded that the bilirubin has pro-healing potential.

Therapeutic Enzymes: Applications and Approaches to Pharmacological Improvement.

Science.gov (United States)

Yari, Maryam; Ghoshoon, Mohammad B; Vakili, Bahareh; Ghasemi, Younes

2017-01-01

Among therapeutic proteins, enzymes represent small and of course profitable market. They can be used to treat important, rare, and deadly diseases. Enzyme therapy is the only available treatment for certain disorders. Here, pharmaceutical enzymes are reviewed. They are categorized in four main groups, enzymes in replacement therapy, enzymes in cancer treatment, enzymes for fibrinolysis, and finally enzymes that are used topically for various treatments. Furthermore, enzyme gene therapy and future perspective of therapeutic enzymes are mentioned in brief. There are many important approved enzymes in pharmaceutical market. Several approaches such as point mutation, fusion protein designing, glycoengineering, and PEGylation were used to achieve improved enzymes. Although sometimes enzymes were engineered to facilitate production and purification process, appropriate delivery to target sites, extending half-life, and reducing immunogenicity are among the main goals of engineering approaches. Overall, enzymes play a critical role in treatment of common and rare diseases. Evaluation of new enzymes as well as improvement of approved enzymes are of the most important challenges in biotechnology. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases

International Nuclear Information System (INIS)

Besanger, Travis R.; Hodgson, Richard J.; Green, James R.A.; Brennan, John D.

2006-01-01

Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low μL/min range. Using the enzyme γ-glutamyl transpeptidase (γ-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in k cat and decreases in K M , switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography

Effect of inhibition of microsomal Ca(2+)-ATPase on cytoplasmic calcium and enzyme secretion in pancreatic acini.

Science.gov (United States)

Metz, D C; Pradhan, T K; Mrozinski, J E; Jensen, R T; Turner, R J; Patto, R J; Gardner, J D

1994-01-13

We used thapsigargin (TG), 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ) and cyclopiazonic acid (CPA), each of which inhibits microsomal Ca(2+)-ATPase, to evaluate the effects of this inhibition on cytoplasmic free calcium ([Ca2+]i) and secretagogue-stimulated enzyme secretion in rat pancreatic acini. Using single-cell microspectrofluorimetry of fura-2-loaded acini we found that all three agents caused a sustained increase in [Ca2+]i by mobilizing calcium from inositol-(1,4,5)-trisphosphate-sensitive intracellular calcium stores and by promoting influx of extracellular calcium. Concentrations of all three agents that increased [Ca2+]i potentiated the stimulation of enzyme secretion caused by secretagogues that activate adenylate cyclase but inhibited the stimulation of enzyme secretion caused by secretagogues that activate phospholipase C. With BHQ, potentiation of adenylate cyclase-mediated enzyme secretion occurred immediately whereas inhibition of phospholipase C-mediated enzyme secretion occurred only after several min of incubation. In addition, the effects of BHQ and CPA on both [Ca2+]i and secretagogue-stimulated enzyme secretion were reversed completely by washing whereas the actions of TG could not be reversed by washing. Concentrations of BHQ in excess of those that caused maximal changes in [Ca2+]i inhibited all modes of stimulated enzyme secretion by a mechanism that was apparently unrelated to changes in [Ca2+]i. Finally, in contrast to the findings with TG and BHQ, CPA inhibited bombesin-stimulated enzyme secretion over a range of concentrations that was at least 10-fold lower than the range of concentrations over which CPA potentiated VIP-stimulated enzyme secretion.

Warming increases hotspot areas of enzyme activity and shortens the duration of hot moments in the detritusphere

Science.gov (United States)

Ma, Xiaomin; Razavi, Bahar S.; Holz, Maire; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2017-04-01

Temperature effects on enzyme kinetics and on the spatial distribution of microbial hotspots are important because of their potential feedback to climate change. We used direct zymography to study the spatial distributions of enzymes responsible for P (phosphatase), C (cellobiohydrolase) and N (leucine-aminopeptidase) cycles in the rhizosphere (living roots of maize) and detritusphere (7 and 14 days after cutting shoots). Soil zymography was coupled with enzyme kinetics to test temperature effects (10, 20, 30 and 40 °C) on the dynamics and localization of these three enzymes in the detritusphere. Total hotspot areas of enzyme activity were 1.9-7.9 times larger and their extension was broader in the detritusphere compared to rhizosphere. From 10 to 30 °C, the hotspot areas enlarged by a factor of 2-24 and Vmax increased by 1.5-6.6 times; both, however, decreased at 40 °C. For the first time, we found a close positive correlation between Vmax and the areas of enzyme activity hotspots, indicating that maximum reaction rate is coupled with hotspot formation. The substrate turnover time at 30 °C were 1.7-6.7-fold faster than at 10 °C. The Km of cellobiohydrolase and phosphatase significantly increased at 30 and 40 °C, indicating high enzyme conformational flexibility, or isoenzyme production at warm temperatures. We conclude that soil warming (at least up to 30°C) increases hotspot areas of enzyme activity and the maximum reaction rate (Vmax) in the detritusphere. This, in turn, leads to faster substrate exhaustion and shortens the duration of hot moments.

Aktivitas Antioksidan Danangiotensin-I Converting Enzyme Inhibitor oleh Yogurt dengan Ekstrak Daun Ficus glomerata Roxb

Directory of Open Access Journals (Sweden)

Baiq Rani Dewi Wulandani

2018-01-01

ABSTRAK  Ficus glomerata Roxb. telah diketahui memiliki senyawa flavonoid. Senyawa flavonoid yang terdapat didalam tanaman diketahui memiliki kemampuan sebagai antioksidan dan mampu sebagai Angiotensin Converting Enzyme I- inhibitor. Penelitian ini diawali dengan melakukan ekstraksi dengan menggunakan air pada daun Ficus glomerata Roxb untuk mendapatkan profil senyawa phenolik pada ekstrak daun Ficus glomerata Roxb [gallic acid, flavonol (quercetin dan rutin, flavanol (catechin, dan flavanone]. Tahap berikutnya adalah inokulasi dan perbanyakan starter yogurt untuk selanjutnya melakukan proses pembuatan yogurt dengan penambahan ekstrak daun Ficus glomerata Roxb. Pengujian aktivitas antioksidan, pengujian terhadap nilai o-phthalaldehyde (OPA dan pengujan terhadap angiotensin converting enzyme I- inhibitor dilakukan terhadap yogurt dengan ekstrak daun Ficus glomerata Roxb selama proses penyimpanan. Tujuan dari penelitian ini adalah untuk mempelajari kemampuan aktivitas antioksidan dan Angiotensin Converting Enzyme I- inhibitor pada yogurt dengan penambahan ekstrak daun Ficus glomerata Roxb selama penyimpanan (1, 7, 14, 21, dan 28 hari pada suhu 4 °C. Hasil yang diperoleh menunjukkan bahwa aktivitas antioksidan, nilai o-phthalaldehyde (OPA dan Angiotensin Converting Enzyme I- inhibitor pada yogurt dengan ekstrak daun Ficus glomerata Roxb selama proses penyimpanan di dalam refrigerator (4 °C adalah lebih tinggi dan menunjukkan perbedaan yang nyata (p < 0,05 dibandingkan dengan plain yogurt yang mencapai optimalnya pada hari ke-7 penyimpanan. Kata kunci: Angiotensin converting enzyme; Ficus glomerata Roxb; yogurt

Extracellular Enzymes Produced by the Cultivated Mushroom Lentinus edodes during Degradation of a Lignocellulosic Medium

Science.gov (United States)

Leatham, Gary F.

1985-01-01

Although the commercially important mushroom Lentinus (= Lentinula) edodes (Berk.) Sing. can be rapidly cultivated on supplemented wood particles, fruiting is not reliable. This study addressed the problem by developing more information about growth and development on a practical oakwood-oatmeal medium. The study determined (i) the components degraded during a 150-day incubation at 22°C, (ii) the apparent vegetative growth pattern, (iii) the likely growth-limiting nutrient, and (iv) assays that can be used to study key extracellular enzymes. All major components of the medium were degraded, lignin selectively so. The vegetative growth rate was most rapid during the initial 90 days, during which weight loss correlated with glucosamine accumulation (assayed after acid hydrolysis). The rate then slowed; in apparent preparation for fruiting, the cultures rapidly accumulated glucosamine (or its oligomer or polymer). Nitrogen was growth limiting. Certain enzyme activities were associated with the pattern of medium degradation, with growth, or with development. They included cellulolytic system enzymes, hemicellulases, the ligninolytic system, (gluco-)amylase, pectinase, acid protease, cell wall lytic enzymes (laminarinase, 1,4-β-d-glucosidase, β-N-acetyl-d-glucosaminidase, α-d-galactosidase, β-d-mannosidase), acid phosphatase, and laccase. Enzyme activities over the 150-day incubation period with and without a fruiting stimulus are reported. These results provide a basis for future investigations into the physiology and biochemistry of growth and fruiting. PMID:16346918

Three phase partitioning of zingibain, a milk-clotting enzyme from Zingiber officinale Roscoe rhizomes.

Science.gov (United States)

Gagaoua, Mohammed; Hoggas, Naouel; Hafid, Kahina

2015-02-01

The present work describes for the first time an elegant non-chromatographic method, the three phase partitioning for the purification and recovery of zingibain, a milk-clotting enzyme, from Zingiber officinale rhizomes. Factors affecting partitioning efficiency such as (NH4)2SO4 saturation, crude extract to t-butanol ratio and pH on zingibain partitioning were investigated. Optimal purification parameters were 50% (NH4)2SO4 saturation with 1.0:1.0 ratio of crude extract:t-butanol at pH 7.0, which gave 14.91 purification fold with 215% recovery of zingibain. The enzyme was found to be exclusively partitioned in the aqueous phase. The enzyme showed a prominent single band on SDS-PAGE. It is a monomeric protein of 33.8 kDa and its isoelectric point is 4.38. The enzyme exhibited maximal proteolytic activity at a temperature of 60 °C and pH 7.0. It was found to be stable at 40-65 °C during 2 h. The enzyme was found to be highly stable against numerous metal ions and its activity was enhanced by Ca(2+), K(+) and Na(+). It was completely inhibited by heavy metal ions such as Cu(2+) and Hg(2+) and partially by Cd(+). Zingibain milk-clotting activity (MCA) was found to be highly stable when stored under freezing (-20 °C) for 30 days compared at 4 °C. Copyright © 2014 Elsevier B.V. All rights reserved.

Toward mechanistic classification of enzyme functions.

Science.gov (United States)

Almonacid, Daniel E; Babbitt, Patricia C

2011-06-01

Classification of enzyme function should be quantitative, computationally accessible, and informed by sequences and structures to enable use of genomic information for functional inference and other applications. Large-scale studies have established that divergently evolved enzymes share conserved elements of structure and common mechanistic steps and that convergently evolved enzymes often converge to similar mechanisms too, suggesting that reaction mechanisms could be used to develop finer-grained functional descriptions than provided by the Enzyme Commission (EC) system currently in use. Here we describe how evolution informs these structure-function mappings and review the databases that store mechanisms of enzyme reactions along with recent developments to measure ligand and mechanistic similarities. Together, these provide a foundation for new classifications of enzyme function. Copyright © 2011 Elsevier Ltd. All rights reserved.

Production of Enzymes from Marine Actinobacteria.

Science.gov (United States)

Zhao, X Q; Xu, X N; Chen, L Y

Marine actinobacteria are well recognized for their capabilities to produce valuable natural products, which have great potential for applications in medical, agricultural, and fine chemical industries. In addition to producing unique enzymes responsible for biosynthesis of natural products, many marine actinobacteria also produce hydrolytic enzymes which are able to degrade various biopolymers, such as cellulose, xylan, and chitin. These enzymes are important to produce biofuels and biochemicals of interest from renewable biomass. In this chapter, the recent reports of novel enzymes produced by marine actinobacteria are reviewed, and advanced technologies that can be applied to search for novel marine enzymes as well as for improved enzyme production by marine actinobacteria are summarized, which include ribosome engineering, genome mining, as well as synthetic biology studies. © 2016 Elsevier Inc. All rights reserved.

Isotope partitioning for NAD-malic enzyme from Ascaris suum confirms a steady-state random kinetic mechanism

International Nuclear Information System (INIS)

Chen, C.Y.; Harris, B.G.; Cook, P.F.

1988-01-01

Isotope partitioning studies beginning with E-[ 14 C]NAD, E-[ 14 C] malate, E-[ 14 C] NAD-Mg 2+ , and E-Mg-[ 14 C]malate suggest a steady-state random mechanism for the NAD-malic enzyme. Isotope trapping beginning with E-[ 14 C]NAD and with varying concentrations of Mg 2+ and malate in the chase solution indicates that Mg 2+ is added in rapid equilibrium and must be added prior to malate for productive ternary complex formation. Equal percentage trapping from E-[ 14 C]NAD-Mg and E-Mg-[ 14 C] malate indicates the mechanism is steady-state random with equal off-rates for NAD and malate from E-NAD-Mg-malate. The off-rates for both do not change significantly in the ternary E-Mg-malate and E-NAD-Mg complexes, nor does the off-rate change for NAD from E-NAD. No trapping of malate was obtained from E-[ 14 C] malate, suggesting that this complex is nonproductive. A quantitative analysis of the data allows an estimation of values for a number of the rate constants along the reaction pathway

Zymography methods for visualizing hydrolytic enzymes

OpenAIRE

Vandooren, Jennifer; Geurts, Nathalie; Martens, Erik; Van den Steen, Philippe E.; Opdenakker, Ghislain

2013-01-01

Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful., but often misinterpreted, tool. yielding information on potential. hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tis...

A simple assay method for omega-oxidation of lauric acid by hepatic enzymes

International Nuclear Information System (INIS)

Giera, D.D.; van Lier, R.B.L.

1990-01-01

Routine assessment of hepatic ω-oxidation of fatty acids in toxicology studies requires a simpler method of enzymatic analysis than HPLC or TLC. A method depending upon selective solvent separation of 14 C-lauric acid and 14 C-11/12-hydroxy lauric acid was developed. Following enzymatic incubation and addition of 15% methanol to the acidified incubation mixtures, partitioning with an alkane solvent such as iso-octane, cyclohexane, or n-hexane separated the lauric acid substrate and ω-hydroxylated products into two immiscible phases. Approximately 98% of the substrate partitioned into the organic phase, and approximately 83% of the hydroxylated products partitioned into the aqueous phase. Subsequent quantitation of the enzymatic activity required only liquid scintillation counting of the aqueous phase. Hepatic homogenates from male rats treated with 0.01, 0.05, 0.125, and 0.25% clofibrate in the diet for 7 days had enzyme levels 1.3, 6.1, 11.1, and 15.9 times control values, respectively, when assayed by conventional TLC methods, and 1.3, 5.3, 12.3, and 15.3 times control values when assayed by the solvent extraction method. The data indicate that the selective solvent partitioning yields comparable precision and sensitivity to the more conventional TLC method when studying induction of hepatic microsomal enzymes

Studies on the enzymes produced by Basidiomycetes. Part 1. The production of crude enzymes