Exercise addiction risk and health in male and female amateur endurance cyclists.

PubMed

Mayolas-Pi, Carmen; Simón-Grima, Javier; Peñarrubia-Lozano, Carlos; Munguía-Izquierdo, Diego; Moliner-Urdiales, Diego; Legaz-Arrese, Alejandro

2017-03-01

Background and aims To determine the relationship between the risk of exercise addiction (REA) and health status in amateur endurance cyclists. Methods In 859 (751 men and 108 women) cyclists and 718 inactive subjects (307 men and 411 women), we examined the REA (Exercise Addiction Inventory), training status (volume, frequency, experience, and performance), socioeconomic status, quality of life (QoL) (SF-12), quality of sleep (Pittsburgh Sleep Quality Index), anxiety and depression (Hospital Anxiety and Depression Scale), and cardiometabolic risk: body mass index, physical activity (International Physical Activity Questionnaire), physical condition (International Fitness Scale), adherence to the Mediterranean diet (Mediterranean Diet Adherence Screener), alcohol and tobacco consumption. Results In total, 17% of the cyclists showed evidence of REA and 83% showed low REA. REA occurred independent of age, sex, training, and socioeconomic status (all ps > .05). Regardless of REA, the cyclists displayed a better physical QoL and a lower cardiometabolic risk than the inactive subjects (all ps < .05). The cyclists with REA displayed worse values of mental QoL, quality of sleep, and anxiety than cyclists with low REA (all ps < .05). The REA group had better values of mental QoL and anxiety and similar values of quality of sleep than the inactive subjects. The differences in mental QoL between the REA and low REA groups were significantly greater in women (p = .013). There was no Addiction × Sex interaction in the other analyzed variables. Conclusion Our results suggest that an increased prevalence of REA limits the benefits that amateur endurance cycling has on mental health and quality of sleep.

Feasibility of rapid ethical assessment for the Ethiopian health research ethics review system.

PubMed

Addissie, Adamu; Davey, Gail; Newport, Melanie; Farsides, Bobbie; Feleke, Yeweyenhareg

2015-01-01

One of the challenges in the process of ethical medical research in developing countries, including Ethiopia, is translating universal principles of medical ethics into appropriate informed consent documents and their implementation. Rapid Ethical Assessment (REA) has been suggested as a feasible approach to meet this application gap. In the past few years REA has been employed in few research project in Ethiopia and have been found to be a useful and practical approach. Feasibility assessment of REA for the Ethiopian research setting was conducted between 2012-2013 in order to inform the subsequent introduction of REA into research ethics review and governance system in the country. REA was found to be an appropriate, relevant and feasible venture. We argue that REA can be integrated as part of the ethics review and governance system in Ethiopia. REA tools and techniques are considered relevant and acceptable to the Ethiopian research community, with few practical challenges anticipated in their implementation. REA are considered feasible for integration in the Ethiopian ethics review system.

Numerical analysis of the angular motion of a neutrally buoyant spheroid in shear flow at small Reynolds numbers.

PubMed

Rosén, T; Einarsson, J; Nordmark, A; Aidun, C K; Lundell, F; Mehlig, B

2015-12-01

We numerically analyze the rotation of a neutrally buoyant spheroid in a shear flow at small shear Reynolds number. Using direct numerical stability analysis of the coupled nonlinear particle-flow problem, we compute the linear stability of the log-rolling orbit at small shear Reynolds number Re(a). As Re(a)→0 and as the box size of the system tends to infinity, we find good agreement between the numerical results and earlier analytical predictions valid to linear order in Re(a) for the case of an unbounded shear. The numerical stability analysis indicates that there are substantial finite-size corrections to the analytical results obtained for the unbounded system. We also compare the analytical results to results of lattice Boltzmann simulations to analyze the stability of the tumbling orbit at shear Reynolds numbers of order unity. Theory for an unbounded system at infinitesimal shear Reynolds number predicts a bifurcation of the tumbling orbit at aspect ratio λ(c)≈0.137 below which tumbling is stable (as well as log rolling). The simulation results show a bifurcation line in the λ-Re(a) plane that reaches λ≈0.1275 at the smallest shear Reynolds number (Re(a)=1) at which we could simulate with the lattice Boltzmann code, in qualitative agreement with the analytical results.

Remote identification of research and educational activities using spectral properties of nighttime light

NASA Astrophysics Data System (ADS)

Rybnikova, Nataliya A.; Portnov, Boris A.

2017-06-01

Research and educational activities (R&EAs) are major forces behind modern economic growth. However, data on geographic location of such activities are often poorly reported. According to our research hypothesis, intensities and spectral properties of artificial light-at-night (ALAN) can be used for remote identification of R&EAs, due to their unique ALAN signatures. In order to develop activity identification models, we carried out a series of in situ measurements of ALAN intensities and spectral properties in a major metropolitan area in Israel. For this task, we used an illuminance CL-500A spectrophotometer that measures the total intensity and spectral irradiance of ALAN, incremented by a 1-nm pitch, from 360 to 780 nm. As our analysis shows, logistic regressions, incorporating ALAN intensities at the peak or near-peak wavelengths, and geographical attributes of the measurement sites as controls, succeeded to predict correctly up to 98.6% of the actual locations of R&EAs. A digital camera satellite image, obtained from the Astronaut Photography Database, was used for the model's validation. According to the validation results, the actual locations of R&EAs coincided well with the estimated high probability areas, as confirmed by the values of Cohen's Kappa index of up to 64%, which indicate a reasonable level of agreement.

Comparative muscle transcriptome associated with carcass traits of Nellore cattle.

PubMed

Silva-Vignato, Bárbara; Coutinho, Luiz L; Cesar, Aline S M; Poleti, Mirele D; Regitano, Luciana C A; Balieiro, Júlio C C

2017-07-03

Commercial cuts yield is an important trait for beef production, which affects the final value of the products, but its direct determination is a challenging procedure to be implemented in practice. The measurement of ribeye area (REA) and backfat thickness (BFT) can be used as indirect measures of meat yield. REA and BFT are important traits studied in beef cattle due to their strong implication in technological (carcass yield) and nutritional characteristics of meat products, like the degree of muscularity and total body fat. Thus, the aim of this work was to study the Longissimus dorsi muscle transcriptome of Nellore cattle, associated with REA and BFT, to find differentially expressed (DE) genes, metabolic pathways, and biological processes that may regulate these traits. By comparing the gene expression level between groups with extreme genomic estimated breeding values (GEBV), 101 DE genes for REA and 18 for BFT (false discovery rate, FDR 10%) were identified. Functional enrichment analysis for REA identified two KEGG pathways, MAPK (Mitogen-Activated Protein Kinase) signaling pathway and endocytosis pathway, and three biological processes, response to endoplasmic reticulum stress, cellular protein modification process, and macromolecule modification. The MAPK pathway is responsible for fundamental cellular processes, such as growth, differentiation, and hypertrophy. For BFT, 18 biological processes were found to be altered and grouped into 8 clusters of semantically similar terms. The DE genes identified in the biological processes for BFT were ACHE, SRD5A1, RSAD2 and RSPO3. RSAD2 has been previously shown to be associated with lipid droplet content and lipid biosynthesis. In this study, we identified genes, metabolic pathways, and biological processes, involved in differentiation, proliferation, protein turnover, hypertrophy, as well as adipogenesis and lipid biosynthesis related to REA and BFT. These results enlighten some of the molecular processes involved in muscle and fat deposition, which are economically important carcass traits for beef production.

Repressor of Estrogen Receptor Activity (REA) Is Essential for Mammary Gland Morphogenesis and Functional Activities: Studies in Conditional Knockout Mice

PubMed Central

Park, Sunghee; Zhao, Yuechao; Yoon, Sangyeon; Xu, Jianming; Liao, Lan; Lydon, John; DeMayo, Franco; O'Malley, Bert W.

2011-01-01

Estrogen receptor (ER) is a key regulator of mammary gland development and is also implicated in breast tumorigenesis. Because ER-mediated activities depend critically on coregulator partner proteins, we have investigated the consequences of reduction or loss of function of the coregulator repressor of ER activity (REA) by conditionally deleting one allele or both alleles of the REA gene at different stages of mammary gland development. Notably, we find that heterozygosity and nullizygosity for REA result in very different mammary phenotypes and that REA has essential roles in the distinct morphogenesis and functions of the mammary gland at different stages of development, pregnancy, and lactation. During puberty, mice homozygous null for REA in the mammary gland (REAf/f PRcre/+) showed severely impaired mammary ductal elongation and morphogenesis, whereas mice heterozygous for REA (REAf/+ PRcre/+) displayed accelerated mammary ductal elongation, increased numbers of terminal end buds, and up-regulation of amphiregulin, the major paracrine mediator of estrogen-induced ductal morphogenesis. During pregnancy and lactation, mice with homozygous REA gene deletion in mammary epithelium (REAf/f whey acidic protein-Cre) showed a loss of lobuloalveolar structures and increased apoptosis of mammary alveolar epithelium, leading to impaired milk production and significant reduction in growth of their offspring, whereas body weights of the offspring nursed by females heterozygous for REA were slightly greater than those of control mice. Our findings reveal that REA is essential for mammary gland development and has a gene dosage-dependent role in the regulation of stage-specific physiological functions of the mammary gland. PMID:21862609

Demonstration that Australian Pasteurella multocida isolates from sporadic outbreaks of porcine pneumonia are non-toxigenic (toxA-) and display heterogeneous DNA restriction endonuclease profiles compared with toxigenic isolates from herds with progressive atrophic rhinitis.

PubMed

Djordjevic, S P; Eamens, G J; Ha, H; Walker, M J; Chin, J C

1998-08-01

Capsular types A and D of Pasteurella multocida cause economic losses in swine because of their association with progressive atrophic rhinitis (PAR) and enzootic pneumonia. There have been no studies comparing whole-cell DNA profiles of isolates associated with these two porcine respiratory diseases. Twenty-two isolates of P. multocida from diseased pigs in different geographic localities within Australia were characterised genotypically by restriction endonuclease analysis (REA) with the enzyme CfoI. Seven of 12 P. multocida isolates from nasal swabs from pigs in herds where PAR was either present or suspected displayed a capsular type D phenotype. These were shown to possess the toxA gene by polymerase chain reaction (PCR) and Southern hybridisation, and further substantiated by production of cytotoxin in vitro. The CfoI profile of one of these seven isolates, which was from the initial outbreak of PAR in Australia (in Western Australia, WA), was identical with profiles of all six other toxigenic isolates from sporadic episodes in New South Wales (NSW). The evidence suggests that the strain involved in the initial outbreak was responsible for the spread of PAR to the eastern states of Australia. Another 10 isolates, representing both capsular types A and D, were isolated exclusively from porcine lung lesions after sporadic outbreaks of enzootic pneumonia in NSW and WA. CfoI restriction endonuclease profiles of these isolates revealed considerable genomic heterogeneity. Furthermore, none of these possessed the toxA gene. This suggests that P. multocida strains with the toxA gene do not have a competitive survival advantage in the lower respiratory tract or that toxin production does not play a role in the pathology of pneumonic lesions, or both. REA with polyacrylamide gel electrophoresis and silver staining was found to be a practical and discriminatory tool for epidemiological tracing of P. multocida outbreaks associated with PAR or pneumonia in pigs.

Reflecting on explanatory ability: A mechanism for detecting gaps in causal knowledge.

PubMed

Johnson, Dan R; Murphy, Meredith P; Messer, Riley M

2016-05-01

People frequently overestimate their understanding-with a particularly large blind-spot for gaps in their causal knowledge. We introduce a metacognitive approach to reducing overestimation, termed reflecting on explanatory ability (REA), which is briefly thinking about how well one could explain something in a mechanistic, step-by-step, causally connected manner. Nine experiments demonstrated that engaging in REA just before estimating one's understanding substantially reduced overestimation. Moreover, REA reduced overestimation with nearly the same potency as generating full explanations, but did so 20 times faster (although only for high complexity objects). REA substantially reduced overestimation by inducing participants to quickly evaluate an object's inherent causal complexity (Experiments 4-7). REA reduced overestimation by also fostering step-by-step, causally connected processing (Experiments 2 and 3). Alternative explanations for REA's effects were ruled out including a general conservatism account (Experiments 4 and 5) and a covert explanation account (Experiment 8). REA's overestimation-reduction effect generalized beyond objects (Experiments 1-8) to sociopolitical policies (Experiment 9). REA efficiently detects gaps in our causal knowledge with implications for improving self-directed learning, enhancing self-insight into vocational and academic abilities, and even reducing extremist attitudes. (c) 2016 APA, all rights reserved).

Numerical investigation on the performance of fin and tube heat exchangers using rectangular vortex generators

NASA Astrophysics Data System (ADS)

Zeeshan, Mohd; Hazarika, Saheera Azmi; Nath, Sujit; Bhanja, Dipankar

2017-07-01

In the present work, a 3-D numerical investigation has been performed to explore the effect of attack angles on the thermal-hydraulic performance of fin and tube heat exchanger (FTHE) using rectangular winglet pairs (RWPs). RWPs are placed adjacent to the tubes and three attack angels are considered for the study i.e. 5°, 15° and 25°. The effect of attack angles are examined on the heat transfer characteristics as well as in pressure drop penalty with airside Reynolds number Rea ranges from 500 to 900. Two performance evaluation criteria namely PEC1 i.e. area goodness factor (j/f) and PEC2 i.e. heat transfer rate per unit fan power consumption (Q/Pf) are considered for the performance evaluation. Furthermore, MOORA method is applied to obtain the performance order of FTHE configurations by taking PEC1 and PEC2 as beneficial attributes and fan power Pf as a non-beneficial attribute, keeping equal importance to each attribute. The results show that 5° attack angle provides the better performance in terms of PEC1 as heat transfer coefficient is increased by 27.70% at Rea=500 and 32.73% at Rea=900 respectively with 13.01% increased pressure drop penalty at Rea=500 and 14.26% at Rea=900 respectively. In terms of PEC2, though the 5° attack angle provides the high values of Q/Pf factor among the 15° and 25° attack angles, but it is found insignificant to replace the baseline configuration i.e. plain fin and tube heat exchanger configuration without vortex generators. Moreover, in MOORA optimization analysis also, it is found that 5° attack angle provides the better thermal-hydraulic performance.

Bromination of Aromatic Compounds by Residual Bromide in Sodium Chloride Matrix Modifier Salt During Heated Headspace GC/MS Analysis

EPA Science Inventory

Analytical artifacts attributed to the bromination of toluene, xylenes, and trimethylbenzenes were found during the heated headspace gas chromatography/mass spectrometry (GC/MS) analysis of aqueous samples. The aqueous samples were produced from Fenton-like chemical oxidation rea...

Field comparison of an eddy accumulation and an aerodynamic-gradient system for measuring pesticide volatilization fluxes

USGS Publications Warehouse

Majewski, M.; Desjardina, R.; Rochette, P.; Pattey, E.; Selber, J.; Glotfelty, D.

1993-01-01

The field experiment reported here applied the relaxed eddy accumulation (REA) technique to the measurement of triallate (TA) and trifluralin (TF) volatilization from fallow soil. A critical analysis of the REA system used in this experiment is done, and the fluxes are compared to those obtained by the aerodynamic-gradient (AG) technique. The measured cumulative volatilization losses, corrected for the effective upwind source area (footprint), for the AG system were higher than with the REA system. The differences between the methods over the first 5 days of the experiment were 27 and 13% for TA and TF, respectively. A mass balance based on the amount of parent compounds volatilized from soil during the first 5 days of the experiment showed a 110 and 70% and a 79 and 61% accountability for triallate and trifluralin by the AG and REA methods, respectively. These results also show that the non-footprint-corrected AG flux values underestimated the volatilization flux by approximately 16%. The footprint correction model used in this experiment does not presently have the capability of accounting for changes in atmospheric stability. However, these values still provide an indication of the most likely upwind area affecting the evaporative flux estimations. The soil half-lives for triallate and trifluralin were 9.8 and 7.0 days, respectively. ?? 1992 American Chemical Society.

Ontology-Driven Business Modelling: Improving the Conceptual Representation of the REA Ontology

NASA Astrophysics Data System (ADS)

Gailly, Frederik; Poels, Geert

Business modelling research is increasingly interested in exploring how domain ontologies can be used as reference models for business models. The Resource Event Agent (REA) ontology is a primary candidate for ontology-driven modelling of business processes because the REA point of view on business reality is close to the conceptual modelling perspective on business models. In this paper Ontology Engineering principles are employed to reengineer REA in order to make it more suitable for ontology-driven business modelling. The new conceptual representation of REA that we propose uses a single representation formalism, includes a more complete domain axiomatizat-ion (containing definitions of concepts, concept relations and ontological axioms), and is proposed as a generic model that can be instantiated to create valid business models. The effects of these proposed improvements on REA-driven business modelling are demonstrated using a business modelling example.

QUALIS EVALUATION OF MEDICINE III: ANALYSIS OF ANESTHESIOLOGY AND GYNECOLOGY AND OBSTETRICS JOURNALS.

PubMed

Calderon, Iracema de Mattos Paranhos

2015-01-01

To know the current publication of Anesthesiology and Obstetrics and Gynecology subareas, to support the updating of Qualis Journals criteria in these specific subareas. Cross-sectional, descriptive study in which was evaluated in quantitatively and qualitatively way the bibliographic production of Anesthesiology and Obstetrics and Gynecology subareas, from January 2010 to December 2012. Were investigated the values ​​of the impact factor; calculated (i) the number (n) and the percentage of journals in each stratum Qualis A1, A2, B1, B2, B3, B4 and B5, and (ii) the median values ​​and their extreme limits (minimum values ​​and maximum) and quartiles (p25; p50; p75; p90) of the impact factors in the different strata. The bibliographic production of the three-year period 2010-2012 was published in 69 journals in Anesthesiology subarea and in 345 in Gynecology and Obstetrics. In Anesthesiology, 44% were within the limits of impact factor of superior A1, A2 and B1; in Obstetrics and Gynecology, 42.4% were in those limits and strata. Despite lagging behind by international standards, publications of Anesthesiology and Obstetrics and Gynecology showed tendency to improve the quality. In these sub-areas, the median of journals impact factor is beyond the limits defined by the area in the last assessment. Therefore, it must be reconsidered new indicators to assess this aspect. Conhecer a publicação atual das subáreas Anestesiologia e Ginecologia e Obstetrícia, para subsidiar a atualização dos critérios Qualis-periódicos, específicos dessas subáreas. Estudo de corte transversal, descritivo, onde avaliou-se, de modo quantitativo e qualitativo, a produção bibliográfica das subáreas Anestesiologia e Ginecologia e Obstetrícia, no período de janeiro de 2010 a dezembro de 2012. Foram definidos os valores do fator de impacto das revistas; foram calculados (i) o número (n) e percentual de periódicos em cada um dos estratos Qualis - A1, A2, B1, B2, B3, B4 e B5 e (ii) os valores de mediana e respectivos limites extremos (valores mínimo e máximo) e quartis (p25; p50; p75; p90) dos fatores de impacto nos diferentes estratos. A produção bibliográfica do triênio 2010-2012 foi divulgada em 69 periódicos na subárea Anestesiologia e em 345 na subárea Ginecologia e Obstetrícia. Na subárea Anestesiologia, 44% dessa produção estava nos limites de fator de impacto dos estratos superiores A1, A2 e B1; na subárea Ginecologia e Obstetrícia, 42,4% estavam nesses limites e estratos. Apesar de aquém do desejado para os padrões internacionais, as publicações das subáreas Anestesiologia e Ginecologia e Obstetrícia mostram tendência para melhorar a qualidade. Nessas subáreas, a mediana do fator de impacto dos periódicos está além dos limites definidos pela área na última avaliação. Impõe-se, portanto, novos indicadores para a avaliação desse quesito.

Uterine Development and Fertility Are Dependent on Gene Dosage of the Nuclear Receptor Coregulator REA

PubMed Central

Park, Sunghee; Yoon, Sangyeon; Zhao, Yuechao; Park, Seong-Eun; Liao, Lan; Xu, Jianming; Lydon, John P.; DeMayo, Francesco J.; O'Malley, Bert W.; Bagchi, Milan K.

2012-01-01

Although the effectiveness of nuclear hormone-receptor complexes is known to depend on coregulator partner proteins, relatively little is known about the roles of coregulators in uterine development and early stages of pregnancy and implantation. Because conventional genetic deletion of the coregulator, repressor of estrogen receptor activity (REA), was embryonic lethal, we here study REA conditional knockout mice generated by cre-loxP recombination, in which REA function was abrogated only in progesterone receptor-expressing tissues, to define the roles of REA in postembryonic stages and in a tissue-specific manner. We find that REA has gene dose-dependent activity impacting uterine development and fertility. Conditional homozygous mutant (REAd/d) mice developed to adulthood and showed normal ovarian function, but females were infertile with severely compromised uterine development and function characterized by cell cycle arrest, apoptosis, and altered adenogenesis (endometrial gland morphogenesis), resulting in failure of implantation and decidualization. By contrast, mice heterozygous for REA (REAf/d) had a very different phenotype, with estradiol treatment resulting in hyperstimulated, large uteri showing increased proliferation of luminal epithelial cells, and enhanced fluid imbibition associated with altered regulation of aquaporins. These REAf/d female mice showed a subfertility phenotype with reduced numbers and sizes of litters. These findings highlight that uterine development and regulation of estrogen receptor activities show a bimodal dependence on the gene dosage of REA. Optimal uterine development and functional activities require the normal gene dosage of REA, with partial or complete deletion resulting in hyperresponsiveness or underresponsiveness to hormone and subfertility or infertility, respectively. PMID:22585830

Clostridium difficile-associated reactive arthritis in two children.

PubMed

Löffler, Helga A; Pron, Benedicte; Mouy, Richard; Wulffraat, Nico M; Prieur, Anne-Marie

2004-01-01

In adults, reactive arthritis (ReA) following Clostridium difficile-enterocolitis has been documented. In children, only one case of C. difficile-associated ReA has been reported. We now describe two other cases of ReA associated with C. difficile in children. The characteristics of ReA due to C. difficile appear to be similar in adults and children. Both children show polyarthritis after an episode of diarrhoea with positive stool cultures for C. difficile. Arthritis is asymmetrical with a self-limiting course. Nonsteroidal antiinflammatory drug (NSAID) therapy is sufficient. One case is remarkable because of its prolonged course of ReA despite NSAID therapy, and its association with the presence of HLA-B27 antigen.

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

PubMed Central

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

2016-01-01

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

Genetic parameters for carcass traits and body weight using a Bayesian approach in the Canchim cattle.

PubMed

Meirelles, S L C; Mokry, F B; Espasandín, A C; Dias, M A D; Baena, M M; de A Regitano, L C

2016-06-10

Correlation between genetic parameters and factors such as backfat thickness (BFT), rib eye area (REA), and body weight (BW) were estimated for Canchim beef cattle raised in natural pastures of Brazil. Data from 1648 animals were analyzed using multi-trait (BFT, REA, and BW) animal models by the Bayesian approach. This model included the effects of contemporary group, age, and individual heterozygosity as covariates. In addition, direct additive genetic and random residual effects were also analyzed. Heritability estimated for BFT (0.16), REA (0.50), and BW (0.44) indicated their potential for genetic improvements and response to selection processes. Furthermore, genetic correlations between BW and the remaining traits were high (P > 0.50), suggesting that selection for BW could improve REA and BFT. On the other hand, genetic correlation between BFT and REA was low (P = 0.39 ± 0.17), and included considerable variations, suggesting that these traits can be jointly included as selection criteria without influencing each other. We found that REA and BFT responded to the selection processes, as measured by ultrasound. Therefore, selection for yearling weight results in changes in REA and BFT.

Factors associated to Caesarean delivery in public and private health care systems.

PubMed

Oliveira, Rosana Rosseto de; Melo, Emiliana Cristina; Novaes, Elisiane Soares; Ferracioli, Patrícia Louise Rodrigues Varela; Mathias, Thais Aidar de Freitas

2016-01-01

Identifying factors associated to Caesarean sections among the residents of Maringá-PR, according to the financing source for delivery. A cross-sectional study with data from 920 postpartum women interviewed between October 2013 and February 2014. Association analysis was performed by logistic regression. Caesarean section rates were 55.5% in the Unified Healthcare System (SUS) and 93.8% in the private system. Factors associated with Caesarean section in the SUS were: previous Caesarean section (OR=8.9; CI=4.6-16.9), desire for Caesarean section early in pregnancy (OR=2.0; CI=1.1-3.6), pregestational overweight/obesity (OR=1.8; CI=1.1-2.8), and per capita family income higher than one minimum wage (OR=2.1; CI=1.3-3.4). In the private system, desire for Caesarean section early in pregnancy (OR=25.3) and a previous Caesarean section (OR=11.3) were strongly associated to its performance. It is necessary to properly orientate all pregnant women who desire a Caesarean delivery, from both the SUS and the private system, about the inherent risks of the surgical procedure without indication. In the public health sector, guidelines should be focused on pregnant women with previous Caesarean delivery, with a per capita income higher than one minimum wage and those who are overweight or obese, as these women are more likely to have a Caesarean section. Identificar fatores associados à cesárea entre residentes de Maringá-PR, segundo a fonte de financiamento do parto. Estudo transversal com dados de 920 puérperas entrevistadas entre outubro de 2013 e fevereiro de 2014. A análise de associação foi feita por regressão logística. A taxa de cesariana foi de 55,5% e 93,8% no Sistema Único de Saúde (SUS) e no sistema privado, respectivamente. Associou-se à cesárea no SUS: realização de cesárea anterior (OR=8,9; IC=4,6-16,9), desejo pela cesárea no início da gestação (OR=2,0; IC=1,1-3,6), sobrepeso/obesidade pré-gestacional (OR=1,8; IC=1,1-2,8), e renda familiar per capita maior que um salário mínimo (OR=2,1; IC=1,3-3,4). No sistema privado, o desejo pela cesárea no início da gestação (OR=25,3) e uma cesárea anterior (OR=11,3) estiveram fortemente associados à sua realização. É necessário orientar adequadamente todas as gestantes que desejam o parto cesárea, no SUS e no sistema privado, sobre os riscos inerentes ao procedimento cirúrgico sem indicação. No setor público de saúde, devem ser foco das orientações as gestantes com parto cesárea anterior, as com renda familiar per capita maior que um salário mínimo e com sobrepeso ou obesidade, as quais têm mais chances de realizar cesárea.

Comparing LOPES measurements of air-shower radio emission with REAS 3.11 and CoREAS simulations

NASA Astrophysics Data System (ADS)

Apel, W. D.; Arteaga-Velázquez, J. C.; Bähren, L.; Bekk, K.; Bertaina, M.; Biermann, P. L.; Blümer, J.; Bozdog, H.; Brancus, I. M.; Cantoni, E.; Chiavassa, A.; Daumiller, K.; de Souza, V.; di Pierro, F.; Doll, P.; Engel, R.; Falcke, H.; Fuchs, B.; Fuhrmann, D.; Gemmeke, H.; Grupen, C.; Haungs, A.; Heck, D.; Hörandel, J. R.; Horneffer, A.; Huber, D.; Huege, T.; Isar, P. G.; Kampert, K.-H.; Kang, D.; Krömer, O.; Kuijpers, J.; Link, K.; Łuczak, P.; Ludwig, M.; Mathes, H. J.; Melissas, M.; Morello, C.; Oehlschläger, J.; Palmieri, N.; Pierog, T.; Rautenberg, J.; Rebel, H.; Roth, M.; Rühle, C.; Saftoiu, A.; Schieler, H.; Schmidt, A.; Schröder, F. G.; Sima, O.; Toma, G.; Trinchero, G. C.; Weindl, A.; Wochele, J.; Zabierowski, J.; Zensus, J. A.

2013-12-01

Cosmic ray air showers emit radio pulses at MHz frequencies, which can be measured with radio antenna arrays - like LOPES at the Karlsruhe Institute of Technology in Germany. To improve the understanding of the radio emission, we test theoretical descriptions with measured data. The observables used for these tests are the absolute amplitude of the radio signal, and the shape of the radio lateral distribution. We compare lateral distributions of more than 500 LOPES events with two recent and public Monte Carlo simulation codes, REAS 3.11 and CoREAS (v 1.0). The absolute radio amplitudes predicted by REAS 3.11 are in good agreement with the LOPES measurements. The amplitudes predicted by CoREAS are lower by a factor of two, and marginally compatible with the LOPES measurements within the systematic scale uncertainties. In contrast to any previous versions of REAS, REAS 3.11 and CoREAS now reproduce the shape of the measured lateral distributions correctly. This reflects a remarkable progress compared to the situation a few years ago, and it seems that the main processes for the radio emission of air showers are now understood: The emission is mainly due to the geomagnetic deflection of the electrons and positrons in the shower. Less important but not negligible is the Askaryan effect (net charge variation). Moreover, we confirm that the refractive index of the air plays an important role, since it changes the coherence conditions for the emission: Only the new simulations including the refractive index can reproduce rising lateral distributions which we observe in a few LOPES events. Finally, we show that the lateral distribution is sensitive to the energy and the mass of the primary cosmic ray particles.

Narrowing the gap: from semiconductor to semimetal in the homologous series of rare-earth zinc arsenides RE(2-y)Zn4As4·n(REAs) and Mn-substituted derivatives RE(2-y)Mn(x)Zn(4-x)As4·n(REAs) (RE = La-Nd, Sm, Gd).

PubMed

Lin, Xinsong; Tabassum, Danisa; Mar, Arthur

2015-12-14

A homologous series of ternary rare-earth zinc arsenides, prepared by reactions of the elements at 750 °C, has been identified with the formula RE(2-y)Zn4As4·n(REAs) (n = 2, 3, 4) for various RE members. They adopt trigonal structures: RE(4-y)Zn4As6 (RE = La-Nd), space group R3̄m1, Z = 3; RE(5-y)Zn4As7 (RE = Pr, Nd, Sm, Gd), space group P3̄m1, Z = 1; RE(6-y)Zn4As8 (RE = La-Nd, Sm, Gd), space group R3̄m1, Z = 3. The Zn atoms can be partially substituted by Mn atoms, resulting in quaternary derivatives RE(2-y)Mn(x)Zn(4-x)As4·n(REAs). Single-crystal structures were determined for nine ternary and quaternary arsenides RE(2-y)M4As4·n(REAs) (M = Mn, Zn) as representative examples of these series. The structures are built by stacking close-packed nets of As atoms, sometimes in very long sequences, with RE atoms occupying octahedral sites and M atoms occupying tetrahedral sites, resulting in an intergrowth of [REAs] and [M2As2] slabs. The recurring feature of all members of the homologous series is a sandwich of [M2As2]-[REAs]-[M2As2] slabs, while rocksalt-type blocks of [REAs] increase in thickness between these sandwiches with higher n. Similar to the previously known related homologous series REM(2-x)As2·n(REAs) which is deficient in M, this new series RE(2-y)M4As4·n(REAs) exhibits deficiencies in RE to reduce the electron excess that would be present in the fully stoichiometric formulas. Enthalpic and entropic factors are considered to account for the differences in site deficiencies in these two homologous series. Band structure calculations indicate that the semiconducting behaviour of the parent n = 0 member (with CaAl2Si2-type structure) gradually evolves, through a narrowing of the gap between valence and conduction bands, to semimetallic behaviour as the number of [REAs] blocks increases, to the limit of n = ∞ for rocksalt-type REAs.

Improved absolute calibration of LOPES measurements and its impact on the comparison with REAS 3.11 and CoREAS simulations

NASA Astrophysics Data System (ADS)

Apel, W. D.; Arteaga-Velázquez, J. C.; Bähren, L.; Bekk, K.; Bertaina, M.; Biermann, P. L.; Blümer, J.; Bozdog, H.; Brancus, I. M.; Cantoni, E.; Chiavassa, A.; Daumiller, K.; de Souza, V.; Di Pierro, F.; Doll, P.; Engel, R.; Falcke, H.; Fuchs, B.; Gemmeke, H.; Grupen, C.; Haungs, A.; Heck, D.; Hiller, R.; Hörandel, J. R.; Horneffer, A.; Huber, D.; Huege, T.; Isar, P. G.; Kampert, K.-H.; Kang, D.; Krömer, O.; Kuijpers, J.; Link, K.; Łuczak, P.; Ludwig, M.; Mathes, H. J.; Melissas, M.; Morello, C.; Nehls, S.; Oehlschläger, J.; Palmieri, N.; Pierog, T.; Rautenberg, J.; Rebel, H.; Roth, M.; Rühle, C.; Saftoiu, A.; Schieler, H.; Schmidt, A.; Schoo, S.; Schröder, F. G.; Sima, O.; Toma, G.; Trinchero, G. C.; Weindl, A.; Wochele, J.; Zabierowski, J.; Zensus, J. A.

2016-02-01

LOPES was a digital antenna array detecting the radio emission of cosmic-ray air showers. The calibration of the absolute amplitude scale of the measurements was done using an external, commercial reference source, which emits a frequency comb with defined amplitudes. Recently, we obtained improved reference values by the manufacturer of the reference source, which significantly changed the absolute calibration of LOPES. We reanalyzed previously published LOPES measurements, studying the impact of the changed calibration. The main effect is an overall decrease of the LOPES amplitude scale by a factor of 2.6 ± 0.2, affecting all previously published values for measurements of the electric-field strength. This results in a major change in the conclusion of the paper 'Comparing LOPES measurements of air-shower radio emission with REAS 3.11 and CoREAS simulations' published by Apel et al. (2013) : With the revised calibration, LOPES measurements now are compatible with CoREAS simulations, but in tension with REAS 3.11 simulations. Since CoREAS is the latest version of the simulation code incorporating the current state of knowledge on the radio emission of air showers, this new result indicates that the absolute amplitude prediction of current simulations now is in agreement with experimental data.

ReaDDy - A Software for Particle-Based Reaction-Diffusion Dynamics in Crowded Cellular Environments

PubMed Central

Schöneberg, Johannes; Noé, Frank

2013-01-01

We introduce the software package ReaDDy for simulation of detailed spatiotemporal mechanisms of dynamical processes in the cell, based on reaction-diffusion dynamics with particle resolution. In contrast to other particle-based reaction kinetics programs, ReaDDy supports particle interaction potentials. This permits effects such as space exclusion, molecular crowding and aggregation to be modeled. The biomolecules simulated can be represented as a sphere, or as a more complex geometry such as a domain structure or polymer chain. ReaDDy bridges the gap between small-scale but highly detailed molecular dynamics or Brownian dynamics simulations and large-scale but little-detailed reaction kinetics simulations. ReaDDy has a modular design that enables the exchange of the computing core by efficient platform-specific implementations or dynamical models that are different from Brownian dynamics. PMID:24040218

A Time-Harmonic Electromagnetic Analysis of Shielded Microstrip Circuits

DTIC Science & Technology

1986-09-01

surface current, J., we f i ndl Etiz.h = Viei Substituting in the Viand evaluating Et due to the TE field first E Te = J(-hENG,(Ax)G2’(Ay)gJx + jTEG(xG(A...he .. analysis..... is...... still......... pr vi in rea..n.. le results........ ..... .. onewoeetahrmec tpsiesaleog react.ais a t...e. de..........ail

QUALIS PERIODIC EVALUATION: ANALYSIS OF QUALIS UPGRADE IN MEDICINE III.

PubMed

Jukemura, José; Diniz, Márcio Augusto

2015-01-01

To evaluate the preliminary results related to journals up-grade that was used by Medicine III, through opportunity offered by Capes to all agency areas programs. Were used area document of Medicine I, II and III, besides other relevant topics available online at Capes site, between 2009 and 2013. The research was focused to answer two questions: 1) the stratification of Qualis is similar in the three areas of medicine? and 2) the evolution of Qualis in Medicine III was higher? Medicine III showed an increase in its Qualis classification and is publishing in journals with higher impact factors, virtually the same as the Medicine I and II. The area showed the strongest growth in recent three-year periods. Avaliar os resultados preliminares sobre a Medicina III do up-grade oportunizado pela Capes para todas as áreas. Foram utilizados os documentos de áreas e os relevantes ao tema disponíveis online no site da Capes entre 2009 e 2013. Procurou-se focar a pesquisa em dois aspectos para responder duas perguntas: 1) a estratificação do Qualis é semelhante nas três áreas da medicina? e 2) a evolução do Qualis da Medicina III foi maior? A Medicina III apresentou evolução em sua classificação Qualis e está publicando em revistas com maior fator de impacto e é praticamente igual ao da Medicina I e II. A área foi a que apresentou maior evolução nestes últimos triênios.

Southern Great Plains Rapid Ecoregional assessment—Volume I. Ecological communities

USGS Publications Warehouse

Reese, Gordon C.; Burris, Lucy; Carr, Natasha B.; Leinwand, Ian I.F.; Melcher, Cynthia P.

2017-10-19

The Southern Great Plains Rapid Ecoregional Assessment was conducted in partnership with the Bureau of Land Management (BLM) and the Great Plains Landscape Conservation Cooperative. The overall goal of the Rapid Ecoregional Assessments (REAs) is to compile and synthesize regional datasets to facilitate evaluation of the cumulative effects of change agents on priority ecological communities and species. In particular, the REAs identify and map the distribution of communities and wildlife habitats at broad spatial extents and provide assessments of ecological conditions. The REAs also identify where and to what degree ecological resources are currently at risk from change agents, such as development, fire, invasive species, and climate change. The REAs can help managers identify and prioritize potential areas for conservation or restoration, assess cumulative effects as required by the National Environmental Policy Act, and inform landscape-level planning and management decisions for multiple uses of public lands.Management questions form the basis for the REA framework and were developed in conjunction with the BLM and other stakeholders. Conservation elements are communities and species that are of regional management concern. Core management questions relate to the key ecological attributes and change agents associated with each conservation element. Integrated management questions synthesize the results of the primary core management questions into overall landscape-level ranks for each conservation element.The ecological communities evaluated as conservation elements are shortgrass, mixed-grass, and sand prairies; all grasslands; riparian and nonplaya wetlands; playa wetlands and saline lakes; and prairie streams and rivers. Species and species assemblages evaluated are the freshwater mussel assemblage, Arkansas River shiner (Notropis girardi), ferruginous hawk (Buteo regalis), lesser prairie chicken (Tympanuchus pallidicinctus), snowy plover (Charadrius nivosus), mountain plover (Charadrius montanus), long-billed curlew (Numenius americanus), interior least tern (Sternula antillarum athalassos), burrowing owl (Athene cunicularia hypugaea), black-tailed prairie dog (Cynomys ludovicianus), bat assemblage, swift fox (Vulpes velox), and mule deer (Odocoileus hemionus).The Southern Great Plains REA is summarized in a series of three reports and associated datasets. The pre-assessment report (available online at https://pubs.usgs.gov/of/2015/1003/) summarizes the process used by the REA stakeholders to select management questions, conservation elements, and change agents. It also provides background information for each conservation element. Volume I of the Southern Great Plains REA report (this volume) addresses the ecological communities. Volume II will address the species and species assemblages. All source and derived datasets used to produce the maps and graphs for REAs are available online at the BLM Landscape Approach Data Portal (https://landscape.blm.gov/geoportal/catalog/REAs/REAs.page).

Using Real-World Data in Health Technology Assessment (HTA) Practice: A Comparative Study of Five HTA Agencies.

PubMed

Makady, Amr; van Veelen, Ard; Jonsson, Páll; Moseley, Owen; D'Andon, Anne; de Boer, Anthonius; Hillege, Hans; Klungel, Olaf; Goettsch, Wim

2018-03-01

Reimbursement decisions are conventionally based on evidence from randomised controlled trials (RCTs), which often have high internal validity but low external validity. Real-world data (RWD) may provide complimentary evidence for relative effectiveness assessments (REAs) and cost-effectiveness assessments (CEAs). This study examines whether RWD is incorporated in health technology assessment (HTA) of melanoma drugs by European HTA agencies, as well as differences in RWD use between agencies and across time. HTA reports published between 1 January 2011 and 31 December 2016 were retrieved from websites of agencies representing five jurisdictions: England [National Institute for Health and Care Excellence (NICE)], Scotland [Scottish Medicines Consortium (SMC)], France [Haute Autorité de santé (HAS)], Germany [Institute for Quality and Efficacy in Healthcare (IQWiG)] and The Netherlands [Zorginstituut Nederland (ZIN)]. A standardized data extraction form was used to extract information on RWD inclusion for both REAs and CEAs. Overall, 52 reports were retrieved, all of which contained REAs; CEAs were present in 25 of the reports. RWD was included in 28 of the 52 REAs (54%), mainly to estimate melanoma prevalence, and in 22 of the 25 (88%) CEAs, mainly to extrapolate long-term effectiveness and/or identify drug-related costs. Differences emerged between agencies regarding RWD use in REAs; the ZIN and IQWiG cited RWD for evidence on prevalence, whereas the NICE, SMC and HAS additionally cited RWD use for drug effectiveness. No visible trend for RWD use in REAs and CEAs over time was observed. In general, RWD inclusion was higher in CEAs than REAs, and was mostly used to estimate melanoma prevalence in REAs or to predict long-term effectiveness in CEAs. Differences emerged between agencies' use of RWD; however, no visible trends for RWD use over time were observed.

Determination of representative elementary areas for soil redoximorphic features by digital image processing

USDA-ARS?s Scientific Manuscript database

Photography has been a welcome tool in documenting and conveying qualitative soil information. When coupled with image analysis software, the usefulness of digital cameras can be increased to advance the field of micropedology. The determination of a Representative Elementary Area (REA) still rema...

Continuous flow hygroscopicity-resolved relaxed eddy accumulation (Hy-Res REA) method of measuring size-resolved sodium chloride particle fluxes

EPA Science Inventory

The accurate representation of aerosols in climate models requires direct ambient measurement of the size- and composition-dependent particle production fluxes. Here, we present the design, testing, and analysis of data collected through the first instrument capable of measuring ...

Toward relaxed eddy accumulation measurements of sediment-water exchange in aquatic ecosystems

NASA Astrophysics Data System (ADS)

Lemaire, Bruno J.; Noss, Christian; Lorke, Andreas

2017-09-01

Solute transport across the sediment-water interface has major implications for water quality and biogeochemical cycling in aquatic ecosystems. Existing measurement techniques, however, are not capable of resolving sediment-water fluxes of most constituents under in situ flow conditions. We investigated whether relaxed eddy accumulation (REA), a micrometeorological technique with conditional sampling of turbulent updrafts and downdrafts, can be adapted to the aquatic environment. We simulated REA fluxes by reanalyzing eddy covariance measurements from a riverine lake. We found that the empirical coefficient that relates mass fluxes to the concentration difference between both REA samples is invariant with scalar and flow and responds as predicted by a joint Gaussian distribution of linearly correlated variables. Simulated REA fluxes differed on average by around 30% from eddy covariance fluxes (mean absolute error). Assessment of the lower quantification limit suggests that REA can potentially be applied for measuring benthic fluxes of a new range of constituents that cannot be assessed by standard eddy covariance methods.

Two-Photon Holographic Stimulation of ReaChR

PubMed Central

Chaigneau, Emmanuelle; Ronzitti, Emiliano; Gajowa, Marta A.; Soler-Llavina, Gilberto J.; Tanese, Dimitrii; Brureau, Anthony Y. B.; Papagiakoumou, Eirini; Zeng, Hongkui; Emiliani, Valentina

2016-01-01

Optogenetics provides a unique approach to remotely manipulate brain activity with light. Reaching the degree of spatiotemporal control necessary to dissect the role of individual cells in neuronal networks, some of which reside deep in the brain, requires joint progress in opsin engineering and light sculpting methods. Here we investigate for the first time two-photon stimulation of the red-shifted opsin ReaChR. We use two-photon (2P) holographic illumination to control the activation of individually chosen neurons expressing ReaChR in acute brain slices. We demonstrated reliable action potential generation in ReaChR-expressing neurons and studied holographic 2P-evoked spiking performances depending on illumination power and pulse width using an amplified laser and a standard femtosecond Ti:Sapphire oscillator laser. These findings provide detailed knowledge of ReaChR's behavior under 2P illumination paving the way for achieving in depth remote control of multiple cells with high spatiotemporal resolution deep within scattering tissue. PMID:27803649

How well the Reliable Ensemble Averaging Method (REA) for 15 CMIP5 GCMs simulations works for Mexico?

NASA Astrophysics Data System (ADS)

Colorado, G.; Salinas, J. A.; Cavazos, T.; de Grau, P.

2013-05-01

15 CMIP5 GCMs precipitation simulations were combined in a weighted ensemble using the Reliable Ensemble Averaging (REA) method, obtaining the weight of each model. This was done for a historical period (1961-2000) and for the future emissions based on low (RCP4.5) and high (RCP8.5) radiating forcing for the period 2075-2099. The annual cycle of simple ensemble of the historical GCMs simulations, the historical REA average and the Climate Research Unit (CRU TS3.1) database was compared in four zones of México. In the case of precipitation we can see the improvements by using the REA method, especially in the two northern zones of México where the REA average is more close to the observations (CRU) that the simple average. However in the southern zones although there is an improvement it is not as good as it is in the north, particularly in the southeast where instead of the REA average is able to reproduce qualitatively good the annual cycle with the mid-summer drought it was greatly underestimated. The main reason is because the precipitation is underestimated for all the models and the mid-summer drought do not even exists in some models. In the REA average of the future scenarios, as we can expected, the most drastic decrease in precipitation was simulated using the RCP8.5 especially in the monsoon area and in the south of Mexico in summer and in winter. In the center and southern of Mexico however, the same scenario in autumn simulates an increase of precipitation.

Rapid evidence assessment: increasing the transparency of an emerging methodology.

PubMed

Varker, Tracey; Forbes, David; Dell, Lisa; Weston, Adele; Merlin, Tracy; Hodson, Stephanie; O'Donnell, Meaghan

2015-12-01

Within the field of evidence-based practice, policy makers, health care professionals and consumers require timely reviews to inform decisions on efficacious health care and treatments. Rapid evidence assessment (REA), also known as rapid review, has emerged in recent years as a literature review methodology that fulfils this need. It highlights what is known in a clinical area to the target audience in a relatively short time frame. This article discusses the lack of transparency and limited critical appraisal that can occur in REA, and goes on to propose general principles for conducting a REA. The approach that we describe is consistent with the principles underlying systematic review methodology, but also makes allowances for the rapid delivery of information as required while utilizing explicit and reproducible methods at each stage. Our method for conducting REA includes: developing an explicit research question in consultation with the end-users; clear definition of the components of the research question; development of a thorough and reproducible search strategy; development of explicit evidence selection criteria; and quality assessments and transparent decisions about the level of information to be obtained from each study. In addition, the REA may also include an assessment of the quality of the total body of evidence. Transparent reporting of REA methodologies will provide greater clarity to end-users about how the information is obtained and about the trade-offs that are made between speed and rigour. © 2015 John Wiley & Sons, Ltd.

Canopy Level Emissions of 2-methyl-3-buten-2-ol ...

EPA Pesticide Factsheets

Emissions of biogenic volatile organic compounds (BVOC) observed during 2007 from a Pinus taeda experimental plantation in Central North Carolina are compared with model estimates from MEGAN 2.1. Relaxed Eddy Accumulation (REA) estimates of 2-methyl-3-buten-2-ol (MBO) fluxes are a factor of 3-4 higher than model estimates. MEGAN2.1 monoterpene emission estimates were approximately a factor of two higher than REA flux measurements. MEGAN2.1 β-caryophyllene emission estimates were within 60% of growing season REA flux estimates, but were several times higher than REA fluxes during cooler, dormant season periods. The sum of other sesquiterpene emissions estimated by MEGAN2.1 was several times higher than REA estimates throughout the year. Model components are examined to understand these discrepancies. Summertime LAI (and therefore foliar biomass) is a factor of two higher than assumed in MEGAN for the Pinus taeda default. Increasing the canopy mean MBO emission factor from 0.35 to 1.0 mg m-2 hr-1 also reduces MEGAN2.1 vs flux differences. This increase is within current emission factor uncertainties. The algorithm within MEGAN which adjusts isoprene emission estimates as a function of the previous 24 hour’s temperatures and light seems to also improve seasonal MEGAN MBO correlation with REA fluxes. Including the effects of the previous 240 hours, however, seems to degrade temporal model correlation with fluxes. This paper describes an emission inventory and mod

Trend analysis of tropospheric NO2 column density over East Asia during 2000-2010: multi-satellite observations and model simulations with the updated REAS emission inventory

NASA Astrophysics Data System (ADS)

Itahashi, S.; Uno, I.; Irie, H.; Kurokawa, J.; Ohara, T.

2013-04-01

Satellite observations of the tropospheric NO2 vertical column density (VCD) are closely correlated to surface NOx emissions and can thus be used to estimate the latter. In this study, the NO2 VCDs simulated by a regional chemical transport model with data from the updated Regional Emission inventory in ASia (REAS) version 2.1 were validated by comparison with multi-satellite observations (GOME, SCIAMACHY, GOME-2, and OMI) between 2000 and 2010. Rapid growth in NO2 VCD driven by expansion of anthropogenic NOx emissions was revealed above the central eastern China region, except during the economic downturn. In contrast, slightly decreasing trends were captured above Japan. The modeled NO2 VCDs using the updated REAS emissions reasonably reproduced the annual trends observed by multi-satellites, suggesting that the NOx emissions growth rate estimated by the updated inventory is robust. On the basis of the close linear relationship of modeled NO2 VCD, observed NO2 VCD, and anthropogenic NOx emissions, the NOx emissions in 2009 and 2010 were estimated. It was estimated that the NOx emissions from anthropogenic sources in China beyond doubled between 2000 and 2010, reflecting the strong growth of anthropogenic emissions in China with the rapid recovery from the economic downturn during late 2008 and mid-2009.

Finding One's Place: A Case Study of a Music Atelierista

ERIC Educational Resources Information Center

Bond, Vanessa L.

2018-01-01

The purpose of this exploratory case study was to document one teacher's journey as he negotiated the role of music atelierista (music studio teacher) in a school inspired by the Reggio Emilia approach (REA). Through the collection of qualitative data over a one-year time period and subsequent analysis, the author identified and described the…

Modelling of radio emission from cosmic ray air showers

NASA Astrophysics Data System (ADS)

Ludwig, Marianne

2011-06-01

Cosmic rays entering the Earth's atmosphere induce extensive air showers consisting of up to billions of secondary particles. Among them, a multitude of electrons and positrons are generated. These get deflected in the Earth's magnetic field, creating time-varying transverse currents. Thereby, the air shower emits coherent radiation in the MHz frequency range measured by radio antenna arrays on the ground such as LOPES at the KIT. This detection method provides a possibility to study cosmic rays with energies above 1017 eV. At this time, the radio technique undergoes the change from prototype experiments to large scale application. Thus, a detailed understanding of the radio emission process is needed more than ever. Before starting this work, different models made conflicting predictions on the pulse shape and the amplitude of the radio signal. It turned out that a radiation component caused by the variation of the number of charged particles within the air shower was missed in several models. The Monte Carlo code REAS2 superposing the radiation of the individual air shower electrons and positrons was one of those. At this time, it was not known how to take the missing component into account. For REAS3, we developed and implemented the endpoint formalism, a universal approach, to calculate the radiation from each single particle. For the first time, we achieve a good agreement between REAS3 and MGMR, an independent and completely different simulation approach. In contrast to REAS3, MGMR is based on a macroscopic approach and on parametrisations of the air shower. We studied the differences in the underlying air shower models to explain the remaining deviations. For comparisons with LOPES data, we developed a new method which allows "top-down" simulations of air showers. From this, we developed an air shower selection criterion based on the number of muons measured with KASCADE to take shower-to-shower fluctuations for a single event analysis into account. With this method, we simulate for the first time the radio emission from an air shower directly comparable with the measured one. We validated with a comparison between REAS3 simulations and LOPES data that the understanding of the radio emission from air showers increased significantly. REAS3 is the first simulation predicting lateral slope parameters and electric fields as measured with LOPES without using any free parameters. Last but not least, we show the didactic quality of the endpoint formalism. This formalism can be used to calculate any radiation from accelerated charged particles, which allows an increased insight in the radiation phenomena of classical electrodynamics. Thus, the endpoint formalism provides a universal tool which is relevant for scientists in many different fields.

Design of a compact all-permanent magnet ECR ion source injector for ReA at the MSU NSCL

NASA Astrophysics Data System (ADS)

Pham, Alfonse N.; Leitner, Daniela; Glennon, Patrick; Ottarson, Jack; Lawton, Don; Portillo, Mauricio; Machicoane, Guillaume; Wenstrom, John; Lajoie, Andrew

2016-06-01

The design of a compact all-permanent magnet electron cyclotron resonance (ECR) ion source injector for the ReAccelerator Facility (ReA) at the Michigan State University (MSU) National Superconducting Cyclotron Laboratory (NSCL) is currently being carried out. The ECR ion source injector will complement the electron beam ion trap (EBIT) charge breeder as an off-line stable ion beam injector for the ReA linac. The objective of the ECR ion source injector is to provide continuous-wave beams of heavy ions from hydrogen to masses up to 136Xe within the ReA charge-to-mass ratio (Q / A) operational range from 0.2 to 0.5. The ECR ion source will be mounted on a high-voltage platform that can be adjusted to obtain the required 12 keV/u injection energy into a room temperature radio-frequency quadrupole (RFQ) for further acceleration. The beam line consists of a 30 kV tetrode extraction system, mass analyzing section, and optical matching section for injection into the existing ReA low energy beam transport (LEBT) line. The design of the ECR ion source and the associated beam line are discussed.

Scientific and technological capabilities in health-related areas: opportunities, challenges, and interactions with the industrial sector.

PubMed

Vargas, Marco Antonio; Britto, Jorge

2016-11-03

Characterization of the scientific and technological infrastructure in health and its interactions with the industrial sector provides key elements for understanding the dynamics of innovation in health. This study conducts an exploratory analysis of the potentialities and limitations associated with scientific and technological capabilities in the health area in Brazil and the different links between the scientific and industrial sectors in health. The analysis points to important growth in internationally indexed research output, especially in certain areas such as pharmaceutics, public health, genetics, morphology, physiology, and microbiology. There has also been important growth in research groups that interact with the industrial sector in selected areas of health. The study highlights the importance of building more solid and permanent bridges between companies, research institutions, and the health system, linking the knowledge developed in research institutions to the dynamics of the industrial sector in health. Resumo: A caracterização da infraestrutura científica e tecnológica na área da saúde e das suas formas de articulação com a base produtiva representam elementos centrais na compreensão da dinâmica de inovação em saúde. Este estudo faz uma análise exploratória sobre as potencialidades e limitações associadas às capacitações científicas e tecnológicas na área da saúde no Brasil e as formas de articulação entre a base científica e a base produtiva em saúde. A análise aponta para o crescimento expressivo da produção bibliográfica com circulação internacional no campo da saúde, particularmente em determinadas áreas como farmácia, saúde coletiva, genética, morfologia, fisiologia e microbiologia. Além disso, observa-se um crescimento expressivo dos grupos de pesquisa com relacionamentos com o setor produtivo em áreas selecionadas da saúde. Destaca-se a importância da construção de pontes mais sólidas e permanentes entre empresas, instituições de pesquisa e sistema de saúde, articulando-se o conhecimento desenvolvido em instituições de pesquisa à dinamização da base produtiva em saúde.

Influence of the Relative Enamel Abrasivity (REA) of Toothpastes on the Uptake of KOH-soluble and Structurally Bound Fluoride.

PubMed

Elmazi, Valbona; Sener, Beatrice; Attin, Thomas; Imfeld, Thomas; Wegehaupt, Florian J

2015-01-01

To determine the influence of the relative enamel abrasivity (REA) of fluoridated toothpaste on the uptake of KOH-soluble and structurally bound fluoride into enamel. Bovine enamel samples were randomly allocated to 6 groups (n=36 per group). Groups A to C were treated with sodium fluoride (NaF) toothpastes and groups D to F with amine fluoride (AmF) toothpastes (1500 ppm F each). The REA in groups A and D was 2, in groups B and E it was 6 and in groups C and F it was 9. Twice a day, 18 samples of each group were immersed for 2 min in a slurry (toothpaste:artificial saliva=1:3), while the remaining samples were brushed with the respective slurry (2.5 N force; 60 strokes/min; 2 min). All samples were stored at 37°C and 100% humidity. After five days, the amount of KOH-soluble and structurally bound fluoride was determined and statistically compared by Scheffe's post-hoc tests. REA value and mode of application (immersion or brushing) had no significant influence on the amount of either kind of fluoride from NaF toothpastes. Only for the NaF toothpaste with REA 6 was the amount of KOH-soluble fluoride significantly higher after brushing. With AmF toothpastes, KOH-soluble and structurally bound fluoride concentrations were significantly higher when the samples were brushed. Furthermore, in the REA-2 group, the amounts of KOH-soluble fluoride (brushed or immersed) and structurally bound fluoride (brushed) were significantly higher than in the other groups. The REA dependency of KOH-soluble and structurally bound fluoride was found only for the AmF toothpastes. Using AmF toothpaste, the mode of application influenced the uptake of KOH-soluble and structurally bound fluoride into enamel.

A mixed-methods study on perceptions towards use of Rapid Ethical Assessment to improve informed consent processes for health research in a low-income setting.

PubMed

Addissie, Adamu; Davey, Gail; Newport, Melanie J; Addissie, Thomas; MacGregor, Hayley; Feleke, Yeweyenhareg; Farsides, Bobbie

2014-05-02

Rapid Ethical Assessment (REA) is a form of rapid ethnographic assessment conducted at the beginning of research project to guide the consent process with the objective of reconciling universal ethical guidance with specific research contexts. The current study is conducted to assess the perceived relevance of introducing REA as a mainstream tool in Ethiopia. Mixed methods research using a sequential explanatory approach was conducted from July to September 2012, including 241 cross-sectional, self-administered and 19 qualitative, in-depth interviews among health researchers and regulators including ethics committee members in Ethiopian health research institutions and universities. In their evaluation of the consent process, only 40.2% thought that the consent process and information given were adequately understood by study participants; 84.6% claimed they were not satisfied with the current consent process and 85.5% thought the best interests of study participants were not adequately considered. Commonly mentioned consent-related problems included lack of clarity (48.1%), inadequate information (34%), language barriers (28.2%), cultural differences (27.4%), undue expectations (26.6%) and power imbalances (20.7%). About 95.4% believed that consent should be contextualized to the study setting and 39.4% thought REA would be an appropriate approach to improve the perceived problems. Qualitative findings helped to further explore the gaps identified in the quantitative findings and to map-out concerns related to the current research consent process in Ethiopia. Suggestions included, conducting REA during the pre-test (pilot) phase of studies when applicable. The need for clear guidance for researchers on issues such as when and how to apply the REA tools was stressed. The study findings clearly indicated that there are perceived to be correctable gaps in the consent process of medical research in Ethiopia. REA is considered relevant by researchers and stakeholders to address these gaps. Exploring further the feasibility and applicability of REA is recommended.

A mixed-methods study on perceptions towards use of Rapid Ethical Assessment to improve informed consent processes for health research in a low-income setting

PubMed Central

2014-01-01

Background Rapid Ethical Assessment (REA) is a form of rapid ethnographic assessment conducted at the beginning of research project to guide the consent process with the objective of reconciling universal ethical guidance with specific research contexts. The current study is conducted to assess the perceived relevance of introducing REA as a mainstream tool in Ethiopia. Methods Mixed methods research using a sequential explanatory approach was conducted from July to September 2012, including 241 cross-sectional, self-administered and 19 qualitative, in-depth interviews among health researchers and regulators including ethics committee members in Ethiopian health research institutions and universities. Results In their evaluation of the consent process, only 40.2% thought that the consent process and information given were adequately understood by study participants; 84.6% claimed they were not satisfied with the current consent process and 85.5% thought the best interests of study participants were not adequately considered. Commonly mentioned consent-related problems included lack of clarity (48.1%), inadequate information (34%), language barriers (28.2%), cultural differences (27.4%), undue expectations (26.6%) and power imbalances (20.7%). About 95.4% believed that consent should be contextualized to the study setting and 39.4% thought REA would be an appropriate approach to improve the perceived problems. Qualitative findings helped to further explore the gaps identified in the quantitative findings and to map-out concerns related to the current research consent process in Ethiopia. Suggestions included, conducting REA during the pre-test (pilot) phase of studies when applicable. The need for clear guidance for researchers on issues such as when and how to apply the REA tools was stressed. Conclusion The study findings clearly indicated that there are perceived to be correctable gaps in the consent process of medical research in Ethiopia. REA is considered relevant by researchers and stakeholders to address these gaps. Exploring further the feasibility and applicability of REA is recommended. PMID:24885049

First charge breeding of a rare-isotope beam with the electron-beam ion trap of the ReA post-accelerator at the National Superconducting Cyclotron Laboratory.

PubMed

Lapierre, A; Schwarz, S; Baumann, T M; Cooper, K; Kittimanapun, K; Rodriguez, A J; Sumithrarachchi, C; Williams, S J; Wittmer, W; Leitner, D; Bollen, G

2014-02-01

An electron-beam ion trap (EBIT) charge breeder is being brought into operation at the National Superconducting Cyclotron Laboratory at Michigan State University. The EBIT is part of the ReA post-accelerator for reacceleration of rare isotopes, which are thermalized in a gas "stopping" cell after being produced at high energy by projectile fragmentation. The ReA EBIT has a distinctive design; it is characterized by a high-current electron gun and a two-field superconducting magnet to optimize the capture and charge-breeding efficiency of continuously injected singly charged ion beams. Following a brief overview of the reaccelerator system and the ReA EBIT, this paper presents the latest commissioning results, particularly, charge breeding and reacceleration of the highly charged rare isotopes, (76)Ga(24 +, 25 +).

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

PubMed

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.

Upgrades to the REA method for producing probabilistic climate change projections

NASA Astrophysics Data System (ADS)

Xu, Ying; Gao, Xuejie; Giorgi, Filippo

2010-05-01

We present an augmented version of the Reliability Ensemble Averaging (REA) method designed to generate probabilistic climate change information from ensembles of climate model simulations. Compared to the original version, the augmented one includes consideration of multiple variables and statistics in the calculation of the performance-based weights. In addition, the model convergence criterion previously employed is removed. The method is applied to the calculation of changes in mean and variability for temperature and precipitation over different sub-regions of East Asia based on the recently completed CMIP3 multi-model ensemble. Comparison of the new and old REA methods, along with the simple averaging procedure, and the use of different combinations of performance metrics shows that at fine sub-regional scales the choice of weighting is relevant. This is mostly because the models show a substantial spread in performance for the simulation of precipitation statistics, a result that supports the use of model weighting as a useful option to account for wide ranges of quality of models. The REA method, and in particular the upgraded one, provides a simple and flexible framework for assessing the uncertainty related to the aggregation of results from ensembles of models in order to produce climate change information at the regional scale. KEY WORDS: REA method, Climate change, CMIP3

K→(ππ)(I=2) decay amplitude from lattice QCD.

PubMed

Blum, T; Boyle, P A; Christ, N H; Garron, N; Goode, E; Izubuchi, T; Jung, C; Kelly, C; Lehner, C; Lightman, M; Liu, Q; Lytle, A T; Mawhinney, R D; Sachrajda, C T; Soni, A; Sturm, C

2012-04-06

We report on the first realistic ab initio calculation of a hadronic weak decay, that of the amplitude A(2) for a kaon to decay into two π mesons with isospin 2. We find ReA(2)=(1.436±0.063(stat)±0.258(syst))10(-8) GeV in good agreement with the experimental result and for the hitherto unknown imaginary part we find ImA(2)=-(6.83±0.51(stat)±1.30(syst))10(-13) GeV. Moreover combining our result for ImA(2) with experimental values of ReA(2), ReA(0), and ε'/ε, we obtain the following value for the unknown ratio ImA(0)/ReA(0) within the standard model: ImA(0)/ReA(0)=-1.63(19)(stat)(20(syst)×10(-4). One consequence of these results is that the contribution from ImA(2) to the direct CP violation parameter ε' (the so-called Electroweak Penguin contribution) is Re(ε'/ε)(EWP)=-(6.52±0.49(stat)±1.24(syst))×10(-4). We explain why this calculation of A(2) represents a major milestone for lattice QCD and discuss the exciting prospects for a full quantitative understanding of CP violation in kaon decays. © 2012 American Physical Society

K → ( π π ) I = 2 Decay Amplitude from Lattice QCD

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

Blum, T.; Boyle, P. A.; Christ, N. H.

2012-04-04

We report on the first realistic ab initio calculation of a hadronic weak decay, that of the amplitude A 2 for a kaon to decay into two π mesons with isospin 2. We find ReA 2=(1.436±0.063 stat±0.258 syst)10 -8 GeV in good agreement with the experimental result and for the hitherto unknown imaginary part we find ImA 2=-(6.83±0.51 stat±1.30 syst)10 -13 GeV. Moreover combining our result for ImA 2 with experimental values of ReA 2, ReA0, and ϵ'/ϵ, we obtain the following value for the unknown ratio ImA 0/ReA 0 within the standard model: ImA 0/ReA 0=-1.63(19) stat(20) syst×10 -4.more » One consequence of these results is that the contribution from ImA 2 to the direct CP violation parameter ϵ' (the so-called Electroweak Penguin contribution) is Re(ϵ'/ϵ)EWP=-(6.52±0.49 stat±1.24 syst)×10 -4. We explain why this calculation of A 2 represents a major milestone for lattice QCD and discuss the exciting prospects for a full quantitative understanding of CP violation in kaon decays.« less

Estimativas de possiveis recursos de petroleo e gas na America Central e na America do Sul [Estimates of possible petroleum and gas resources in Central American and South America

USGS Publications Warehouse

Schenk, C.S.

2001-01-01

O U.S. Geological Survey recentemente completou estimativas de possíveis recursos de petróleo e gás em 130 áreas petrolíferas pré-determinadas no mundo (USGS, 2000). Vinte e três destas áreas ficam na América do Sul, na América Central, e no Caribe (fig. 1). Os resultados estão apresentados na tabela 1. Nas 23 áreas, estimamos um total de 105 BBO e um total de 487 TCFG. A região composta de América Central mais América do Sul ficou em terceiro lugar no mundo em termos de possíveis recursos de petróleo e gás. No primeiro lugar ficou o Oriente Médio e no segundo lugar ficou a antiga União Soviética (USGS, 2000). As áreas com maiores probabilidades de encontrar depósitos gigantes de petróleo e gás se localizam nas áreas do Oceano Atlântico começando com a Bacia de Santos no sul até a Bacia Guyana-Suriname no norte. As possibilidades de existirem depósitos gigantes são maiores nas áreas submersas do mar até profundidades de 3,600 m. Diversos depósitos gigantes de petróleo foram descobertos no mar na Bacia de Campos e ainda podem serem encontrados depósitos similares na Bacia de Campos e suas imediações.

SURGERY AND CARDIOVASCULAR SURGERY JOURNALS ANALYSIS.

PubMed

Schanaider, Alberto

2015-01-01

To analyze critically the effectiveness and value of bibliometric indicators in journals of Surgery or Cardiovacular Surgery in the context of the postgraduate programs of CAPES Medicine III. A sampling with 16 academic programs and one professional master of Medicine III, encompassing the General and Digestive System Surgery, Cardiovascular Surgery and Multidisciplinary courses with such contents, was evaluated. Thomson Reuters/ISI (JCR), Elsevier/Scopus (SJR), and also Scielo databases were used. Only in seven programs, the teachers had an average of Qualis A1 articles greater than the others strata. Eleven journals in the surgical area are in stratum A1 (5%) and it reaches 25% in Cardiovascular Surgery. Among the six journals with the largest number of publications Qualis A1 in area Medicine III, five are from non-specific areas. The Acta Cirúrgica Brasileira represented 58% of the publications in the stratum A2. There are some obstacles in the Qualis classification with little uniformity among the Medicine areas I, II and III. A permanent committee should be set to update the Qualis, composed by the three medical areas. It should be considered using other index databases and the unification of the Qualis criteria for journals in medicine. Rating criteria of multi and transdisciplinary journals need to be reviewed. It is essential an institutional financial support for national journals chosen by peers aiming to provide a full computerization process and a professional reviewer of the English language, in order to increase the impact factor. Analisar criticamente a eficácia e valor de indicadores bibliométricos dos periódicos da Cirurgia e Cirurgia Cardiovascular no contexto dos Programas de Pós-Graduação da área Medicina III da CAPES. Foi avaliada uma amostragem com 16 programas acadêmicos e um mestrado profissional da área de Medicina III, compreendendo a Cirurgia Geral e do Aparelho Digestivo, a Cirurgia Cardiovascular e Cursos Multidisciplinares com estes conteúdos. Utilizaram-se as bases de dados Thomson Reuters/ISI (JCR), Elsevier/Scopus (SJR), além do Scielo. Somente nos programas 7, os docentes têm média de artigos Qualis A1 maior ou igual que os demais estratos. Apenas 11 periódicos da área de Cirurgia estão no estrato A1 (5%) e na Cirurgia Cardiovascular são 25%. Dos seis periódicos com o maior número de publicações na área Medicina III, Qualis A1, cinco não são específicos da área. A Acta Cirúrgica Brasileira representou 58% das publicações no estrato A2. Há alguns óbices na classificação Qualis com pouca uniformidade entre as Medicinas I, II e III. Se impõe a criação de um comitê permanente para atualização do Qualis, composto pelas três áreas da Medicina. Deve-se ponderar a utilização de outras bases de indexação e a unificação dos critérios Qualis para as revistas das áreas de medicina. Critérios de classificação dos periódicos multi e transdisciplinares precisam ser revistos. É imprescindível suporte financeiro institucional aos periódicos nacionais escolhidos pelos pares visando informatização completa e prover revisão profissional da língua inglesa, tendo como meta a elevação do fator de impacto.

Targeted proteome analysis of single-gene deletion strains of Saccharomyces cerevisiae lacking enzymes in the central carbon metabolism.

PubMed

Matsuda, Fumio; Kinoshita, Syohei; Nishino, Shunsuke; Tomita, Atsumi; Shimizu, Hiroshi

2017-01-01

Central carbon metabolism is controlled by modulating the protein abundance profiles of enzymes that maintain the essential systems in living organisms. In this study, metabolic adaptation mechanisms in the model organism Saccharomyces cerevisiae were investigated by direct determination of enzyme abundance levels in 30 wild type and mutant strains. We performed a targeted proteome analysis using S. cerevisiae strains that lack genes encoding the enzymes responsible for central carbon metabolism. Our analysis revealed that at least 30% of the observed variations in enzyme abundance levels could be explained by global regulatory mechanisms. A enzyme-enzyme co-abundance analysis revealed that the abundances of enzyme proteins involved in the trehalose metabolism and glycolysis changed in a coordinated manner under the control of the transcription factors for global regulation. The remaining variations were derived from local mechanisms such as a mutant-specific increase in the abundances of remote enzymes. The proteome data also suggested that, although the functional compensation of the deficient enzyme was attained by using more resources for protein biosynthesis, available resources for the biosynthesis of the enzymes responsible for central metabolism were not abundant in S. cerevisiae cells. These results showed that global and local regulation of enzyme abundance levels shape central carbon metabolism in S. cerevisiae by using a limited resource for protein biosynthesis.

Relative effectiveness assessments of oncology medicines for pricing and reimbursement decisions in European countries.

PubMed

Kleijnen, S; Lipska, I; Leonardo Alves, T; Meijboom, K; Elsada, A; Vervölgyi, V; d'Andon, A; Timoney, A; Leufkens, H G; De Boer, A; Goettsch, W G

2016-09-01

There is a debate on the added clinical value of new, expensive, anticancer treatments. Among European decision makers, the relevance of commonly used end points in trials, especially overall survival (OS), progression-free survival (PFS) and quality of life (QoL), varies, leading to the available evidence being valued differently. This research studies the extent to which the value of end points for cancer medicines differs among European decision makers. We compared guidelines and relative effectiveness assessments (REAs) of medicines for pricing or reimbursement decisions in England, France, Germany, The Netherlands, Poland, and Scotland. Anticancer medicines that received marketing authorization in Europe between 2011 and 2013 with at least four available national REAs were evaluated. A total of 79 REAs were included. Health technology assessment (HTA) guidelines indicate a preference for clinically and patient relevant end points such as OS and QoL above surrogate end points. Most guidelines do not specify whether PFS is considered a surrogate or patient-relevant end point. The number of REAs included per jurisdiction varied between 7 (The Netherlands) and 18 (Germany). OS data were included in all REAs and were the preferred end point by HTA agencies, but these data were not always mature or robust. QoL data are included in only 54% of the REAs, with a limited impact on the recommendations. PFS data are included in 70% of the REAs, but the extent to which HTA agencies find PFS relevant varies. European decision-making on relative effectiveness of anticancer medicines is affected by a gap in requested versus available clinical evidence, mainly because the regulator is willing to accept some degree of clinical uncertainty. A multi-stakeholder debate would be essential to align concrete robust evidence requirements in oncology and a collectively shared definition for relevant clinical benefit, which will benefit patients and society in general. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Enzyme Analysis to Determine Glucose Content

NASA Astrophysics Data System (ADS)

Carpenter, Charles; Ward, Robert E.

Enzyme analysis is used for many purposes in food science and technology. Enzyme activity is used to indicate adequate processing, to assess enzyme preparations, and to measure constituents of foods that are enzyme substrates. In this experiment, the glucose content of corn syrup solids is determined using the enzymes, glucose oxidase and peroxidase. Glucose oxidase catalyzes the oxidation of glucose to form hydrogen peroxide (H2O2), which then reacts with a dye in the presence of peroxidase to give a stable colored product.

Advanced Aircraft Electrical System Control Technology Demonstrator. Phase I. Requirements Analysis and Conceptual Design.

DTIC Science & Technology

1981-07-01

System 13 (7) Flight Critical Power 15 (8) Power Bus Configuration 16 b. System Control and Protection 20...includes the main buses, external power receptacles and distribution feeders. The function of the distribution protection system * is mainly to provide...TechnicaI rea Manager Power Systems Branch Power Systems B nch Aerospace Power Division Aerospace Power Division FOR .AKE D . REAMS Chief,

Cross-validation of two liquid water path retrieval algorithms applied to ground-based microwave radiation measurements by RPG-HATPRO instrument

NASA Astrophysics Data System (ADS)

Kostsov, Vladimir; Ionov, Dmitry; Biryukov, Egor; Zaitsev, Nikita

2017-04-01

A built-in operational regression algorithm (REA) of liquid water path (LWP) retrieval supplied by the manufacturer of the RPG-HATPRO microwave radiometer has been compared to a so-called physical algorithm (PHA) based on the inversion of the radiative transfer equation. The comparison has been performed for different scenarios of microwave observations by the RPG-HATPRO instrument that has been operating at St.Petersburg University since June 2012. The data for the scenarios have been collected within the time period December 2012 - December 2014. The estimations of bias and random error for both REA and PHA have been obtained. Special attention has been paid to the analysis of the quality of the LWP retrievals during and after rain events that have been detected by the built-in rain sensor. The estimation has been done of the time period after a rain event when the retrieval quality has to be considered as insufficient.

Radio Measurements of Air Showers with LOPES

NASA Astrophysics Data System (ADS)

Schröder, F. G.; Apel, W. D.; Arteaga-Velazquez, J. C.; Bähren, L.; Bekk, K.; Bertaina, M.; Biermann, P. L.; Blümer, J.; Bozdog, H.; Brancus, I. M.; Cantoni, E.; Chiavassa, A.; Daumiller, K.; de Souza, V.; Di Pierro, F.; Doll, P.; Engel, R.; Falcke, H.; Fuchs, B.; Fuhrmann, D.; Gemmeke, H.; Grupen, C.; Haungs, A.; Heck, D.; Hörandel, J. R.; Horneffer, A.; Huber, D.; Huege, T.; Isar, P. G.; Kampert, K.-H.; Kang, D.; Krömer, O.; Kuijpers, J.; Link, K.; Łuczak, P.; Ludwig, M.; Mathes, H. J.; Melissas, M.; Morello, C.; Oehlschläger, J.; Palmieri, N.; Pierog, T.; Rautenberg, J.; Rebel, H.; Roth, M.; Rühle, C.; Saftoiu, A.; Schieler, H.; Schmidt, A.; Sima, O.; Toma, G.; Trinchero, G. C.; Weindl, A.; Wochele, J.; Zabierowski, J.; Zensus, J. A.

2013-02-01

LOPES is a digital antenna array for the radio measurement of cosmic-ray air showers at energies around 1017 eV. It is triggered by the KASCADE-Grande air-shower array at the Karlsruhe Institute of Technology (KIT), Germany. Because of an absolute amplitude calibration and a sophisticated data analysis, LOPES can test models for the radio emission to an up-to-now unachieved level, thus improving our understanding of the radio emission mechanisms. Recent REAS simulations of the air-shower radio emission come closer to the measurements than any previously tested simulations. We have determined the radio-reconstruction precision of interesting air-shower parameters by comparing LOPES reconstructions to both REAS simulations and KASCADE-Grande measurements, and present our latest results for the angular resolution, the energy and the Xmax reconstruction based on the radio measurement of about 500 air showers. Although the precision of LOPES is limited by the high level of anthropogenic noise at KIT, it opens a promising perspective for next-generation radio arrays in regions with a lower ambient noise level.

Structural feasibility of parallel-laminated veneer crossarms

Treesearch

John Youngquist; Frank Brey; Joseph Jung

1977-01-01

Experimentally and commercially produced laminated M-19 crossarms were tested by standard Rural Electrification Administration (REA) crossarm tests. The laminated crossarms, produced by laminating veneer and by laminating solid-sawn dimension stock, generally performed satisfactorily according to REA specified standards. Materials tested are described and results on...

Queda dos homicídios em São Paulo, Brasil: uma análise descritiva

PubMed Central

Peres, Maria Fernanda Tourinho; Vicentin, Diego; Nery, Marcelo Batista; de Lima, Renato Sérgio; de Souza, Edinilsa Ramos; Cerda, Magdalena; Cardia, Nancy; Adorno, e Sérgio

2012-01-01

Objetivo Descrever a evolução da mortalidade por homicídios no Município de São Paulo segundo tipo de arma, sexo, raça ou cor, idade e áreas de exclusão/inclusão social entre 1996 e 2008. Métodos Estudo ecológico de série temporal. Os dados sobre óbitos ocorridos no Município foram coletados da base de dados do Programa de Aprimoramento das Informações sobre Mortalidade, seguindo a Classificação Internacional de Doenças, Décima Revisão (CID-10). Foram calculadas as taxas de mortalidade por homicídio (TMH) para a população total, por sexo, raça ou cor, faixa etária, tipo de arma e área de exclusão/inclusão social. As TMH foram padronizadas por idade pelo método direto. Foram calculados os percentuais de variação no período estudado. Para as áreas de exclusão/inclusão social foram calculados os riscos relativos de morte por homicídio. Resultados As TMH apresentaram queda de 73,7% entre 2001 e 2008. Foi observada redução da TMH em todos os grupos analisados, mais pronunciada em homens (−74,5%), jovens de 15 a 24 anos (−78,0%) e moradores de áreas de exclusão social extrema (−79,3%). A redução ocorreu, sobretudo, nos homicídios cometidos com armas de fogo (−74,1%). O risco relativo de morte por homicídio nas áreas de exclusão extrema (tendo como referência áreas com algum grau de exclusão social) foi de 2,77 em 1996, 3,9 em 2001 e 2,13 em 2008. Nas áreas de alta exclusão social, o risco relativo foi de 2,07 em 1996 e 1,96 em 2008. Conclusões Para compreender a redução dos homicídios no Município, é importante considerar macrodeterminantes que atingem todo o Município e todos os subgrupos populacionais e microdeterminantes que atuam localmente, influenciando de forma diferenciada os homicídios com armas de fogo e os homicídios na população jovem, no sexo masculino e em residentes em áreas de alta exclusão social. PMID:21390415

Retroperitoneal endoscopic adrenalectomy is safe and effective.

PubMed

Schreinemakers, J M J; Kiela, G J; Valk, G D; Vriens, M R; Rinkes, I H M Borel

2010-11-01

The aim of this study was to review an experience with retroperitoneal endoscopic adrenalectomy (REA). This is the procedure of choice for adrenal tumours at this institution. Between 1997 and 2008, 112 REAs were performed in a single university centre. Data were retrieved retrospectively from a prospectively collected database, including information on patient demographics, surgical procedure, complications and hospital stay. One hundred and twelve REAs were carried out successfully in 105 patients, including seven bilateral adrenalectomies. Thirty-nine patients with unilateral adrenal disease had a phaeochromocytoma, of whom 16 had multiple endocrine neoplasia syndrome type 2, 21 patients had Cushing's disease and 20 had Conn's disease. Median body mass index was 27 (interquartile range 23-29) kg/m(2). The median duration of unilateral operations was 100 (90-130) min with a median blood loss of 5 ml. Median tumour size was 3.1 (2.0-4.4) cm. Conversion from REA to open surgery was needed in two patients. Seven patients experienced postoperative complications (2 major, 5 minor). One patient needed a reoperation. The median postoperative hospital stay was 3 days. A learning curve with a significant decrease in operating time was observed over the years. REA appears to be a safe and effective surgical technique for adrenal gland tumours up to 6 cm in diameter, with a minimal complication rate. Copyright © 2010 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

77 FR 48172 - Comment Request for Information Collection for the Reemployment and Eligibility Assessments (REA...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-13

... of Labor, Employment and Training Administration, Office of Workforce Security, 200 Constitution... with reemployment and training services through the workforce investment system by linking them to... understand program dynamics, and to gather data to report on REAs, including the number of scheduled in...

POLICY ASSESSMENT FOR THE CARBON MONOXIDE NAAQS REVIEW

EPA Science Inventory

As part of the NAAQS review process, a Risk/Exposure Assessment (REA) has been developed by OAQPS and has receieved inital review by the Clean Air Scientific Advisory Committee (CASAC). A second review of the REA will be conducted in March 2010. The Policy Assessment for the Car...

Mariner Venus/Mercury 1973 rocket engine assembly

NASA Technical Reports Server (NTRS)

Snoke, D. R.; Williams, R. S.

1972-01-01

The fabrication and test of rocket engine assemblies (REA) for Mariner Venus/Mercury 1973 are reported. The fabrication, assembly and flight acceptance test of seven REA's including the type approval test of one engine and fabrication of one additional kit consisting of detail parts for an engine ready for catalyst loading are presented. The MV/M '73 REA which is a nominal 51 lbs thrust monopropellant engine is described. Under steady state operation the specific impulse is not less than 228 lb-sec at 55 lb and 218.5 lb-sec at 10 lb thrust varying linearly between these limits. The characteristic velocity is not less than 4100 ft/sec at any thrust level.

Description of FORTRAN Program DAWNA for Analysis of Muzzle Blast Field

DTIC Science & Technology

1976-04-01

meter feet 2/D ! P pressure N/m 2 lb/ft2 00 ’! u velocity m/sec ft/sec u/a» i OO { s entropy m2/sec2 0K ft2/sec2 0R (S...0,KK,i.L/ IPUNCH, IRSTRT, IMOVE , lUNl T, lUNQUT, 18« *** ** HÜUMBHl IFdMOVE. REA0(S,2) READ(5,2

Hemispheric Differences in Processing Dichotic Meaningful and Non-Meaningful Words

ERIC Educational Resources Information Center

Yasin, Ifat

2007-01-01

Classic dichotic-listening paradigms reveal a right-ear advantage (REA) for speech sounds as compared to non-speech sounds. This REA is assumed to be associated with a left-hemisphere dominance for meaningful speech processing. This study objectively probed the relationship between ear advantage and hemispheric dominance in a dichotic-listening…

The Retirement Equity Act, Survivor Benefits Protections, and Public Employee Retirement Systems.

ERIC Educational Resources Information Center

Liebig, Phoebe S.

The Retirement Equity Act (REA) sets out requirements for joint and survivor annuity coverage for married individuals who participate in federally regulated retirement plans. REA-mandated provisions do not apply to state and local government retirement systems. Because state and local government employees constitute a significant part of the work…

An alert system for triggering different levels of coastal management urgency: Tunisia case study using rapid environmental assessment data.

PubMed

Price, A R G; Jaoui, K; Pearson, M P; Jeudy de Grissac, A

2014-03-15

Rapid environmental assessment (REA) involves scoring abundances of ecosystems/species groups and magnitude of pressures, concurrently, using the same logarithmic (0-6) assessment scale. We demonstrate the utility of REA data for an alert system identifying different levels of coastal management concern. Thresholds set for abundances/magnitudes, when crossed, trigger proposed responses. Kerkennah, Tunisia, our case study, has significant natural assets (e.g. exceptional seagrass and invertebrate abundances), subjected to varying levels of disturbance and management concern. Using REA thresholds set, fishing, green algae/eutrophication and oil occurred at 'low' levels (scores 0-1): management not (currently) necessary. Construction and wood litter prevailed at 'moderate' levels (scores 2-4): management alerted for (further) monitoring. Solid waste densities were 'high' (scores 5-6): management alerted for action; quantities of rubbish were substantial (20-200 items m⁻¹ beach) but not unprecedented. REA is considered a robust methodology and complementary to other rapid assessment techniques, environmental frameworks and indicators of ecosystem condition. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative Bioinformatic Analysis of Active Site Structures in Evolutionarily Remote Homologues of α,β-Hydrolase Superfamily Enzymes.

PubMed

Suplatov, D A; Arzhanik, V K; Svedas, V K

2011-01-01

Comparative bioinformatic analysis is the cornerstone of the study of enzymes' structure-function relationship. However, numerous enzymes that derive from a common ancestor and have undergone substantial functional alterations during natural selection appear not to have a sequence similarity acceptable for a statistically reliable comparative analysis. At the same time, their active site structures, in general, can be conserved, while other parts may largely differ. Therefore, it sounds both plausible and appealing to implement a comparative analysis of the most functionally important structural elements - the active site structures; that is, the amino acid residues involved in substrate binding and the catalytic mechanism. A computer algorithm has been developed to create a library of enzyme active site structures based on the use of the PDB database, together with programs of structural analysis and identification of functionally important amino acid residues and cavities in the enzyme structure. The proposed methodology has been used to compare some α,β-hydrolase superfamily enzymes. The insight has revealed a high structural similarity of catalytic site areas, including the conservative organization of a catalytic triad and oxyanion hole residues, despite the wide functional diversity among the remote homologues compared. The methodology can be used to compare the structural organization of the catalytic and substrate binding sites of various classes of enzymes, as well as study enzymes' evolution and to create of a databank of enzyme active site structures.

PubMed

Fernández Alba, Juan Jesús; Paublete Herrera, María Del Carmen; González Macías, María Del Carmen; Carral San Laureano, Florentino; Carnicer Fuentes, Concepción; Vilar Sánchez, Ángel; Torrejón Cardoso, Rafael; Moreno Corral, Luis Javier

2016-11-29

Introducción: el sobrepeso y la obesidad se asocian a una mayor probabilidad de que el parto finalice en cesárea. Dado que dicho incremento del riesgo podría estar sesgado por variables de confusión como la diabetes o la hipertensión, en el presente trabajo pretendemos determinar si este riesgo persiste tras ser ajustado por numerosas variables de control.Objetivo: determinar si el sobrepeso y/o la obesidad son factores de riesgo independientes para que el parto finalice en cesárea.Métodos: estudio de cohortes retrospectivo. Se han incluido gestantes adscritas al Hospital Universitario de Puerto Real. Periodo de estudio: 2002-2011. Se incluyeron dos grupos de estudio: sobrepeso al inicio de la gestación (IMC entre 25 y 29,9) y obesidad al inicio de la gestación (IMC ≥ 30). Grupo ontrol: IMC al inicio de la gestación normal (entre 18,5 y 24,9). El riesgo de cesárea fue analizado mediante un estudio de regresión logística múltiple incluyendo como covariables: edad materna, parto inducido, diabetes gestacional, diabetes pregestacional, macrosomía, hipertensión arterial, nuliparidad, cesárea anterior, parto pretérmino y parto postérmino.Resultados: de los 18.243 partos registrados, el IMC al inicio de la gestación constaba en 4.711 casos (25,8%). El 26,1% presentaban sobrepeso, el 12,4% obesidad y el 58,9% IMC normal. Sin ajustar por variables control, se asociaron a un incremento del riesgo de cesárea: sobrepeso (OR 1,48; IC95% 1,27-1,73); obesidad grado 1 (OR 2,09; IC95% 1,66-2,64); obesidad grado 2 (OR 3,23; IC95% 2,31-4,53); obesidad grado 3 (OR 2,57; IC95% 1,56-4,22). El riesgo aumentado se mantuvo significativo en el análisis multivariante: sobrepeso (OR 1,51; IC95% 1,24-1,84); obesidad (OR 2,15; IC95% 1,67-2,76).Conclusiones: encontramos una asociación significativa e independiente entre el sobrepeso/obesidad maternos y la finalización del parto mediante cesárea incluso ajustando por numerosas variables de control como: edad materna, nuliparidad, cesárea anterior, hipertensión, diabetes, peso al nacer y edad gestacional al parto.

75 FR 28649 - State of Arizona Resource Advisory Council Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2010-05-21

... Policy and Management Act of 1976 and the Federal Advisory Committee Act of 1972, the U.S. Department of... Managers' Reports; and reports by RAC working groups. A public comment period will be provided at 11:30 a.m... brief review of the Recreation Enhancement Act (REA) Working Group Report, REA Working Group meeting...

The Effects of Background Noise on Dichotic Listening to Consonant-Vowel Syllables

ERIC Educational Resources Information Center

Sequeira, Sarah Dos Santos; Specht, Karsten; Hamalainen, Heikki; Hugdahl, Kenneth

2008-01-01

Lateralization of verbal processing is frequently studied with the dichotic listening technique, yielding a so called right ear advantage (REA) to consonant-vowel (CV) syllables. However, little is known about how background noise affects the REA. To address this issue, we presented CV-syllables either in silence or with traffic background noise…

Development of Activity-based Cost Functions for Cellulase, Invertase, and Other Enzymes

NASA Astrophysics Data System (ADS)

Stowers, Chris C.; Ferguson, Elizabeth M.; Tanner, Robert D.

As enzyme chemistry plays an increasingly important role in the chemical industry, cost analysis of these enzymes becomes a necessity. In this paper, we examine the aspects that affect the cost of enzymes based upon enzyme activity. The basis for this study stems from a previously developed objective function that quantifies the tradeoffs in enzyme purification via the foam fractionation process (Cherry et al., Braz J Chem Eng 17:233-238, 2000). A generalized cost function is developed from our results that could be used to aid in both industrial and lab scale chemical processing. The generalized cost function shows several nonobvious results that could lead to significant savings. Additionally, the parameters involved in the operation and scaling up of enzyme processing could be optimized to minimize costs. We show that there are typically three regimes in the enzyme cost analysis function: the low activity prelinear region, the moderate activity linear region, and high activity power-law region. The overall form of the cost analysis function appears to robustly fit the power law form.

Characterising Complex Enzyme Reaction Data

PubMed Central

Rahman, Syed Asad; Thornton, Janet M.

2016-01-01

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. PMID:26840640

AN ENZYME-IMMOBILIZATION PROCEDURE FOR THE ANALYSIS OF ENZYME-INHIBITING CHEMICALS IN WATER

EPA Science Inventory

The enzymes cholinesterase and urease were mixed individually with gelatin and immobilized onto the inside surface of glass capillary tubes. After the gelatin-enzyme mixture had dried, water samples containing various enzyme inhibiting test chemicals were pumped through the tubes...

Trends Influencing Researcher Education and Careers: What Do We Know, Need to Know and Do in Looking Forward

ERIC Educational Resources Information Center

Castelló, Montserrat; McAlpine, Lynn; Pyhältö, Kirsi

2015-01-01

EARLI SIG 24, Researcher Education and Careers (SIG-REaC), was founded because increasing interest has emerged within the EARLI community into understanding different aspects of doctoral and post-PhD researcher educational and career development. This special issue brings together the outcome of our first scholarly discussion at the SIG-REaC…

Análise da aplicação e dos resultados do modelo OPM3® para a área da saúde

PubMed Central

Augusto dos Santos, Luis; de Fátima Marin, Heimar

2015-01-01

Esta pesquisa procurou analisar se um modelo de questionário criado por uma comunidade internacional de gerenciamento de projetos e se é aplicavél a organizações voltadas a área de saúde. O modelo OPM3® (Organizational Project Management Maturity Model) foi criado para que organizações de qualquer área ou porte pudessem identificar a presença, ou ausência, de boas práticas de gerenciamento. O objetivo da aplicação desse modelo é avaliar sempre a organização e não o entrevistado. No presente artigo, são apresentados os resultados da aplicação desse modelo em uma organização que possuía produtos e serviços de tecnologia da informação aplicados à área de saúde. Este estudo verificou que o modelo é aplicável de forma rápida e que a organização analisada possuía um número expressivo de boas práticas. PMID:26924862

Biosensor for metal analysis and speciation

DOEpatents

Aiken, Abigail M.; Peyton, Brent M.; Apel, William A.; Petersen, James N.

2007-01-30

A biosensor for metal analysis and speciation is disclosed. The biosensor comprises an electron carrier immobilized to a surface of an electrode and a layer of an immobilized enzyme adjacent to the electrode. The immobilized enzyme comprises an enzyme having biological activity inhibited by a metal to be detected by the biosensor.

Functional Evolution of PLP-dependent Enzymes based on Active-Site Structural Similarities

PubMed Central

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-01-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5’-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the Comparison of Protein Active Site Structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. PMID:24920327

Functional evolution of PLP-dependent enzymes based on active-site structural similarities.

PubMed

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-10-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5'-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the comparison of protein active site structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional-fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. © 2014 Wiley Periodicals, Inc.

Kinetic analysis of a Michaelis-Menten mechanism in which the enzyme is unstable.

PubMed Central

Garrido-del Solo, C; García-Cánovas, F; Havsteen, B H; Varón-Castellanos, R

1993-01-01

A kinetic analysis of the Michaelis-Menten mechanism is made for the cases in which the free enzyme, or the enzyme-substrate complex, or both, are unstable, either spontaneously or as a result of the addition of a reagent. The explicit time-course equations of all of the species involved has been derived under conditions of limiting enzyme concentration. The validity of these equations has been checked by using numerical simulations. An experimental design and a kinetic data analysis allowing the evaluation of the parameters and kinetic constants are recommended. PMID:8373361

Production, optimization and characterization of fibrinolytic enzyme by Bacillus subtilis RJAS19.

PubMed

Kumar, D J Mukesh; Rakshitha, R; Vidhya, M Annu; Jennifer, P Sharon; Prasad, Sandip; Kumar, M Ravi; Kalaichelvan, P T

2014-04-01

The present study aimed at the production, purification and characterization of fibrinolytic nattokinase enzyme from the bacteria isolated from natto food. For the purpose, a fibrinolytic bacterium was isolated and identified as Bacillus subtilis based on 16S rDNA sequence analysis. The strain was employed for the production and optimization of fibrinolytic enzyme. The strain showed better enzyme production during 72nd h of incubation time with 50 degrees C at the pH 9. The lactose and peptone were found to be increasing the enzyme production rate. The enzyme produced was purified and also characterized with the help of SDS-PAGE analysis. The activity and stability profile of the purified enzyme was tested against different temperature and pH. The observations suggesting that the potential of fibrinolytic enzyme produced by Bacillus subtilis RJAS 19 for its applications in preventive medicines.

[Biosensor development in clinical analysis].

PubMed

Boitieux, J L; Desmet, G; Thomas, D

1985-01-01

The use of enzymes immobilized or as markers formed the subject of more than thousand publications in the field of industry or biomedical applications, during the last five years. Recently, some authors published works concerning immobilization of total microorganisms for catalytic purposes, others use the enzymatic activity for marking molecules involved in immunological analysis processes. Together industrial biotechnology and medical analysis laboratory are interested with the evolution of these procedures involving the activity of immobilized enzymes. Enzyme immobilization allowed the lowering of analysis costs for, in this case, the enzyme can be used several times. We take account of the two main cases which are encountered during utilization of immobilized enzymes of analytical purposes. The enzyme is used directly for the catalysed reaction or it is used as enzymatic marker. These both aspects are developed mainly for the elaboration of enzymatic and immunoenzymatic electrodes and the realization of automatic computerized devices allowing continuous estimation of numerous biological blood parameters. From these two precise examples, glucose and antigen determination, the authors show the evolution of these technologies in the field of immobilized enzymes or captors and the analysis of signals given by these electrodes requiring a computerized treatment. This new technology opens to important potentialities in the analytical field. The automatization of these devices allowing the control in real time, will probably make easier the optimization steps of procedures actually used in the biomedical sphere.

Mutations associated with reduced surotomycin susceptibility in Clostridium difficile and Enterococcus species.

PubMed

Adams, Hannah M; Li, Xiang; Mascio, Carmela; Chesnel, Laurent; Palmer, Kelli L

2015-07-01

Clostridium difficile infection (CDI) is an urgent public health concern causing considerable clinical and economic burdens. CDI can be treated with antibiotics, but recurrence of the disease following successful treatment of the initial episode often occurs. Surotomycin is a rapidly bactericidal cyclic lipopeptide antibiotic that is in clinical trials for CDI treatment and that has demonstrated superiority over vancomycin in preventing CDI relapse. Surotomycin is a structural analogue of the membrane-active antibiotic daptomycin. Previously, we utilized in vitro serial passage experiments to derive C. difficile strains with reduced surotomycin susceptibilities. The parent strains used included ATCC 700057 and clinical isolates from the restriction endonuclease analysis (REA) groups BI and K. Serial passage experiments were also performed with vancomycin-resistant and vancomycin-susceptible Enterococcus faecium and Enterococcus faecalis. The goal of this study is to identify mutations associated with reduced surotomycin susceptibility in C. difficile and enterococci. Illumina sequence data generated for the parent strains and serial passage isolates were compared. We identified nonsynonymous mutations in genes coding for cardiolipin synthase in C. difficile ATCC 700057, enoyl-(acyl carrier protein) reductase II (FabK) and cell division protein FtsH2 in C. difficile REA type BI, and a PadR family transcriptional regulator in C. difficile REA type K. Among the 4 enterococcal strain pairs, 20 mutations were identified, and those mutations overlap those associated with daptomycin resistance. These data give insight into the mechanism of action of surotomycin against C. difficile, possible mechanisms for resistance emergence during clinical use, and the potential impacts of surotomycin therapy on intestinal enterococci. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DEVELOPMENT OF METRICS FOR TECHNICAL PRODUCTION: QUALIS BOOKS AND BOOK CHAPTERS.

PubMed

Ribas-Filho, Jurandir Marcondes; Malafaia, Osvaldo; Czeczko, Nicolau Gregori; Ribas, Carmen A P Marcondes; Nassif, Paulo Afonso Nunes

2015-01-01

To propose metrics to qualify the publication in books and chapters, and from there, establish guidance for the evaluation of the Medicine III programs. Analysis of some of the 2013 area documents focusing this issue. Were analyzed the following areas: Computer Science; Biotechnology; Biological Sciences I; Public Health; Medicine I. Except for the Medicine I, which has not adopted the metric for books and chapters, all other programs established metrics within the intellectual production, although with unequal percentages. It´s desirable to include metrics for books and book chapters in the intellectual production of post-graduate programs in Area Document with percentage-value of 5% in publications of Medicine III programs. Propor a métrica para qualificar a produção veiculada através de livros e capítulos e, a partir daí, estabelecer orientação para a avaliação dos programas de pós-graduação da Medicina III. Análise dos documentos de área de 2013 dos programas de pós-graduação senso estrito das áreas: Ciência da Computação; Biotecnologia; Ciências Biológicas I; Saúde Coletiva; Medicina I. Excetuando-se o programa da Medicina I, que não adotou a métrica para classificação de livros e capítulos, todos os demais estabeleceram-na dentro da sua produção intelectual, embora com percentuais desiguais. É desejável inserir a métrica de livros e capitulos de livros na produção intelectual do Documento de Área dos programas, ortorgando a ela percentual de 5% das publicações qualificadas dos programas da Medicina III.

Harnessing Data to Assess Equity of Care by Race, Ethnicity and Language

PubMed Central

Gracia, Amber; Cheirif, Jorge; Veliz, Juana; Reyna, Melissa; Vecchio, Mara; Aryal, Subhash

2015-01-01

Objective: Determine any disparities in care based on race, ethnicity and language (REaL) by utilizing inpatient (IP) core measures at Texas Health Resources, a large, faith-based, non-profit health care delivery system located in a large, ethnically diverse metropolitan area in Texas. These measures, which were established by the U.S. Centers for Medicare and Medicaid Services (CMS) and The Joint Commission (TJC), help to ensure better accountability for patient outcomes throughout the U.S. health care system. Methods: Sample analysis to understand the architecture of race, ethnicity and language (REaL) variables within the Texas Health clinical database, followed by development of the logic, method and framework for isolating populations and evaluating disparities by race (non-Hispanic White, non-Hispanic Black, Native American/Native Hawaiian/Pacific Islander, Asian and Other); ethnicity (Hispanic and non-Hispanic); and preferred language (English and Spanish). The study is based on use of existing clinical data for four inpatient (IP) core measures: Acute Myocardial Infarction (AMI), Congestive Heart Failure (CHF), Pneumonia (PN) and Surgical Care (SCIP), representing 100% of the sample population. These comprise a high number of cases presenting in our acute care facilities. Findings are based on a sample of clinical data (N = 19,873 cases) for the four inpatient (IP) core measures derived from 13 of Texas Health’s wholly-owned facilities, formulating a set of baseline data. Results: Based on applied method, Texas Health facilities consistently scored high with no discernable race, ethnicity and language (REaL) disparities as evidenced by a low percentage difference to the reference point (non-Hispanic White) on IP core measures, including: AMI (0.3%–1.2%), CHF (0.7%–3.0%), PN (0.5%–3.7%), and SCIP (0–0.7%). PMID:26703665

Effect of nursing-calf implant timing on growth performance and carcass characteristics.

PubMed

Webb, M J; Harty, A A; Salverson, R R; Kincheloe, J J; Zuelly, S M S; Underwood, K R; Luebbe, M K; Olson, K C; Blair, A D

2017-12-01

The objective of this study was to compare pre- and postweaning growth performance, carcass characteristics, and meat quality attributes of calves that did not receive an implant or were implanted early or late in the nursing period. Crossbred steer calves ( = 135) were stratified by birth date and birth weight and randomly assigned to the following implant treatments: control (CON; no preweaning implant), 58 d (EARLY; 36 mg zeranol, administered at an average of 58 ± 13 d of age), and 121 d (LATE; 36 mg zeranol, administered at an average 121 ± 13 d of age). After weaning, steers were blocked by initial feed yard BW to 15 pens (5 pens/treatment and 9 steers/pen). All steers were implanted on d 21 after arrival at the feed yard and again on d 108 of finishing. Steer BW and ultrasound assessment of rib eye area (uREA), rib fat thickness (uRFT), and percent intramuscular fat (uIMF) were collected when implants were administered, at weaning, and on harvest day. Carcass measurements included HCW, rib eye area (REA), 12th-rib fat thickness (FT), and marbling score. Objective color (L*, a*, and b*) was recorded, and a 3.8-cm strip loin section was removed from both sides of each carcass and portioned into 2.54-cm steaks that were aged for 3 or 14 d for analysis of cook loss and Warner-Bratzler shear force (WBSF). The remaining portion of each sample was used for analysis of moisture and crude fat. Steer BW, ADG, and G:F did not differ among treatments ( > 0.05). Steers implanted in the EARLY treatment had a greater ( < 0.05) cumulative DMI than CON but were not different from steers implanted in the LATE treatment. Ultrasound REA and uRFT (averaged across all collection days) did not differ ( > 0.05); however, steers on the CON treatment had a greater ( ≤ 0.05) percent uIMF than EARLY implanted steers, whereas steers receiving the LATE implant were intermediate and not different from the other treatments. Hot carcass weight, REA, FT, USDA yield grade, marbling score, and objective color did not differ ( > 0.05) among treatments. The proportion of steers in each USDA yield and quality grade was similar ( > 0.05) among treatments, and no differences were detected for total carcass value or price per 45.4 kg (hundredweight; > 0.05). Treatment did not influence ( > 0.05) percent cook loss, crude fat, moisture, or WBSF. In conclusion, administering a nursing implant, regardless of timing, did not influence live performance, carcass characteristics, or meat quality of steers fed in this study.

Hemispheric Asymmetries Depend on the Phonetic Feature: A Dichotic Study of Place of Articulation and Voicing in French Stops

ERIC Educational Resources Information Center

Bedoin, Nathalie; Ferragne, Emmanuel; Marsico, Egidio

2010-01-01

Dichotic listening experiments show a right-ear advantage (REA), reflecting a left-hemisphere (LH) dominance. However, we found a decrease in REA when the initial stop consonants of two simultaneous French CVC words differed in voicing rather than place of articulation (Experiment 1). This result suggests that the right hemisphere (RH) is more…

Men of Good Will: The Religious Education Association, J. Elliot Ross, and the National Conference of Jews and Christians

ERIC Educational Resources Information Center

Nolan, Lucinda A.

2009-01-01

An impetus of the Religious Education Association (REA) toward becoming an actively intercultural and interreligious agency emerged in the third decade of its existence. This article explores this period through an examination of the involvement of the REA members, Father John Elliot Ross and others (1884-1946) in a series of seminars conducted by…

Large $N$ approach to kaon decays and mixing 28 years later: $$\\Delta I = 1/2$$ rule, $$\\hat B_K$$ and $$\\Delta M_K$$

DOE PAGES

Buras, Andrzej J.; Gérard, Jean -Marc; Bardeen, William A.

2014-05-20

We review and update our results for K → π π decays and K⁰- K¯⁰ mixing obtained by us in the 1980s within an approach based on the dual representation of QCD as a theory of weakly interacting mesons for large N colours. In our analytic approach the dynamics behind the enhancement of ReA 0 and suppression of ReA 2, the so-called ΔI = 1/2 rule for K → π π decays, has a simple structure: the usual octet enhancement through quark-gluon renormalization group evolution down to the scales O(1 GeV) is continued as a meson evolution down to zeromore » momentum scales at which the factorization of hadronic matrix elements is at work. The inclusion of lowest-lying vector meson contributions in addition to the pseudoscalar ones and of Wilson coefficients in a momentum scheme improves significantly the matching between quark-gluon and meson evolutions. In particular, the anomalous dimension matrix governing the meson evolution exhibits the structure of the known anomalous dimension matrix in the quark-gluon evolution. The recent results on ReA 2 and ReA 0 from the RBC-UKQC collaboration give support for our approach. In particular, the signs of the two main contractions found numerically by these authors follow uniquely from our analytic approach. At NLO in 1/N we obtain R = ReA 0/ReA 2= 16.0±1.5 which amounts to an order of magnitude enhancement over the strict large N limit value √2. QCD penguins contribute at 15% level to this result. We also find B^ K = 0.73± 0.02, with the smallness of 1/N corrections to the large N value B^ K = 3/4 resulting within our approach from an approximate cancellation between pseudoscalar and vector meson one-loop contributions. We summarize the status of ΔM K in this approach.« less

Source apportionment of secondary organic aerosol in China using a regional source-oriented chemical transport model and two emission inventories.

PubMed

Wang, Peng; Ying, Qi; Zhang, Hongliang; Hu, Jianlin; Lin, Yingchao; Mao, Hongjun

2018-06-01

A Community Multiscale Air Quality (CMAQ) model with source-oriented lumped SAPRC-11 (S11L) photochemical mechanism and secondary organic aerosol (SOA) module was applied to determine the contributions of anthropogenic and biogenic sources to SOA concentrations in China. A one-year simulation of 2013 using the Multi-resolution Emission Inventory for China (MEIC) shows that summer SOA are generally higher (10-15 μg m -3 ) due to large contributions of biogenic (country average 60%) and industrial sources (17%). In winter, SOA formation was mostly due to anthropogenic emissions from industries (40%) and residential sources (38%). Emissions from other countries in southeast China account for approximately 14% of the SOA in both summer and winter, and 46% in spring due to elevated open biomass burning in southeast Asia. The Regional Emission inventory in ASia v2.1 (REAS2) was applied in this study for January and August 2013. Two sets of simulations with the REAS2 inventory were conducted using two different methods to speciate total non-methane carbon into model species. One approach uses total non-methane hydrocarbon (NMHC) emissions and representative speciation profiles from the SPECIATE database. The other approach retains the REAS2 speciated species that can be directly mapped to S11L model species and uses source specific splitting factors to map other REAS2 lumped NMHC species. Biogenic emissions are still the most significant contributor in summer based on these two sets of simulations. However, contributions from the transportation sector to SOA in January are predicted to be much more important based on the two REAS2 emission inventories (∼30-40% vs. ∼5% by MEIC), and contributions from residential sources according to REAS2 was much lower (∼21-24% vs. ∼42%). These discrepancies in source contributions to SOA need to be further investigated as the country seeks for optimal emission control strategies to fight severe air pollution. Copyright © 2017 Elsevier Ltd. All rights reserved.

Estimated historical distribution of grassland communities of the Southern Great Plains

USGS Publications Warehouse

Reese, Gordon C.; Manier, Daniel J.; Carr, Natasha B.; Callan, Ramana; Leinwand, Ian I.F.; Assal, Timothy J.; Burris, Lucy; Ignizio, Drew A.

2016-12-07

The purpose of this project was to map the estimated distribution of grassland communities of the Southern Great Plains prior to Euro-American settlement. The Southern Great Plains Rapid Ecoregional Assessment (REA), under the direction of the Bureau of Land Management and the Great Plains Landscape Conservation Cooperative, includes four ecoregions: the High Plains, Central Great Plains, Southwestern Tablelands, and the Nebraska Sand Hills. The REA advisors and stakeholders determined that the mapping accuracy of available national land-cover maps was insufficient in many areas to adequately address management questions for the REA. Based on the recommendation of the REA stakeholders, we estimated the potential historical distribution of 10 grassland communities within the Southern Great Plains project area using data on soils, climate, and vegetation from the Natural Resources Conservation Service (NRCS) including the Soil Survey Geographic Database (SSURGO) and Ecological Site Information System (ESIS). The dominant grassland communities of the Southern Great Plains addressed as conservation elements for the REA area are shortgrass, mixed-grass, and sand prairies. We also mapped tall-grass, mid-grass, northwest mixed-grass, and cool season bunchgrass prairies, saline and foothill grasslands, and semi-desert grassland and steppe. Grassland communities were primarily defined using the annual productivity of dominant species in the ESIS data. The historical grassland community classification was linked to the SSURGO data using vegetation types associated with the predominant component of mapped soil units as defined in the ESIS data. We augmented NRCS data with Landscape Fire and Resource Management Planning Tools (LANDFIRE) Biophysical Settings classifications 1) where NRCS data were unavailable and 2) where fifth-level watersheds intersected the boundary of the High Plains ecoregion in Wyoming. Spatial data representing the estimated historical distribution of grassland communities of the Southern Great Plains are provided as a 30 x 30-meter gridded surface (raster dataset). This information will help to address the priority management questions for grassland communities for the Southern Great Plains REA and can be used to inform other regional-level land management decisions.

Word Frequency Analysis. MOS: 75C. Skill Levels 1 & 2.

DTIC Science & Technology

1981-05-01

A4Fi j, A REAS . I --- - ., ARISS 12 ARMY I A~K1h ’.-L ARRANGED 64 AS I ASCERTAIN 2 .L~u Ip ASSEMBLE 12 ASSEMBLED 1 ASSEMPLY I ASSI----------------2...GOVERNING .16 GRADE ’G.; S GRAA!TET I GRfATEST I GREEN I GFEP.I .4 ( AriSS I GROUPS s GUIDANCE I G lIn (;UItES V 1 E I GUIVIS/ 3 GWPM 1-’ 1 I.AO 1 HAE

Reactive arthritis and serum levels of mannose binding lectin – lack of association

PubMed Central

LOCHT, H; CHRISTIANSEN, M; LAURSEN, I

2003-01-01

The purpose was to evaluate the possible association of serum mannose binding lectin (s-MBL) levels on type of triggering microbe, duration of diarrhoea, incidence and course of reactive arthritis (ReA) caused by Salmonella, Yersinia and Campylobacter. Sixty patients with ReA of 1–228 months duration, 173 patients with ReA or uncomplicated enterocolitis caused by Campylobacter, 226 sera from patients with elevated antibody levels against Salmonella, Yersinia or Campylobacter, and 114 blood donors were tested for s-MBL using ELISA technique, both direct mannan binding assay and sandwich ELISA. s-MBL was compared with C-reactive protein (CRP) levels and with the ability of activating complement C4. Among the 114 donors 9% had s-MBL <50 µg/l, 16% had from 50–500 µg/l and 75% had >500 µg/l. The distribution of s-MBL levels in the three-patient groups did not differ significantly from the controls. There were no indications that low s-MBL was associated with prolonged duration of arthritis, diarrhoea or individual bacterial infections. The two MBL assays were comparable with respect to serum concentrations, indicating that the actual circulating MBL was also functionally active. s-MBL exhibited acute phase reactant behaviour and correlated to CRP level, but only in patients with s-MBL concentrations exceeding 1000 µg/l. MBL in 10 randomly selected ReA sera were tested for the ability to activate complement C4. The results did not differ from those of donor controls. This study demonstrates that the distributions of s-MBL levels in serum among patients with ReA are not different from donor controls. The course, outcome or triggering bacteria are not associated with a particular level of s-MBL. PMID:12519401

Rubiaceae in Brazilian Atlantic Forest remnants: floristic similarity and implications for conservation.

PubMed

de Paiva, Alessandra Marques; Barberena, Felipe Fajardo Villela Antolin; Lopes, Rosana Conrado

2016-06-01

Brazil holds most of the Atlantic Forest Domain and is also one of the Rubiaceae diversity centers in the Neotropics. Despite the urban expansion in the state of Rio de Janeiro, large areas of continuous vegetation with high connectivity degree can still be found. Recently, new Rubiaceae species have been described in the Rio de Janeiro flora, which present small populations and very particular distribution. The current paper analyzed the similarity in the floristic composition of the Rubiaceae in eight Atlantic Forest remnants of Rio de Janeiro state protected by Conservation Units. We also surveyed and set guidelines for conservation of microendemic species. The similarity analysis were based on previously published studies in Área de Proteção Ambiental de Grumari, Área de Proteção Ambiental Palmares, Parque Estadual da Serra da Tiririca, Parque Nacional do Itatiaia, Parque Nacional de Jurubatiba, Reserva Biológica de Poço das Antas, Reserva Biológica do Tinguá and Reserva Ecológica de Macaé de Cima - using the PAST software (“Paleontological Statistics”) with Sørensen coefficient. The floristic similarity analysis revealed two groups with distinct physiographic characteristics and different vegetation types. Group A consisted in two Restinga areas, Área de Proteção Ambiental de Grumari and Parque Nacional de Jurubatiba, which showed strong bootstrap support (98 %). Group B included forest remnants with distinct phytophisiognomies or altitudes, but with moderate bootstrap support. Low similarity levels among the eight areas were found due to the habitats’ heterogeneity. The current study pointed out 19 microendemic species from the Atlantic Forest, they present a single-site distribution or a distribution restricted to Mountain and Metropolitan regions of Rio de Janeiro state. Concerning the conservation status of microendemic species, discrepancies between the Catalogue of Flora of Rio de Janeiro and the Red Book of Brazilian Flora (two of the main reference catalogs of Brazilian flora) have been identified. We have also highlighted the need for recollecting microendemic species from the Atlantic Forest, and for properly assessing the degree of threat faced by these taxons early.

Urban versus rural lifestyle in adolescents: associations between environment, physical activity levels and sedentary behavior.

PubMed

Regis, Manuela Ferreira; Oliveira, Luciano Machado Ferreira Tenório de; Santos, Ana Raquel Mendes Dos; Leonidio, Ameliane da Conceição Reubens; Diniz, Paula Rejane Beserra; Freitas, Clara Maria Silvestre Monteiro de

2016-01-01

To analyze the levels of physical activity and sedentary behavior in adolescents living in urban and rural areas. An epidemiological, cross-section study with quantitative design, carried out at the regional level. The sample comprised 6,234 students aged 14 to 19 years, selected using random cluster sampling. The χ2 test and binary logistic regression were used in the analysis. A total of 74.5% of adolescents lived in urban areas. After adjustment, rural residents spent less time watching television (odds ratio - OR: 0.45; 95% confidence interval - 95%CI: 0.39-0.52), using a computer and/or playing video games (OR: 0.30; 95%CI: 0.22-0.42), or sitting down (OR: 0.66; 95%CI: 0.54-0.80); chose passive leisure less often (OR: 0.83; 95%IC: 0.72-0.95) and were less likely to be classified as insufficiently active (OR: 0.88; 95%IC: 0.78-0.99) when compared to urban residents, regardless of sex or age. The fact that adolescents living in rural areas who did not work were more likely to be classified as insufficiently active (OR: 2.59; 95%CI: 2.07-3.24) emphasized the significant role of occupation in physical activity levels in this group. Adolescents living in rural areas were less exposed to the sedentary behaviors, chose more active leisure, and had higher levels of physical activity. Place of residence and occupation may play a major role in youth lifestyle. Analisar os níveis de atividade física e o comportamento sedentário em adolescentes das áreas urbanas e rurais. Estudo epidemiológico, transversal, com abordagem quantitativa e abrangência estadual, cuja amostra foi constituída por 6.234 estudantes (14 a 19 anos), selecionados por meio de uma estratégia de amostragem aleatória de conglomerados. As análises foram realizadas por meio do teste χ2 e da regressão logística binária. Na amostra, 74,5% dos adolescentes eram residentes em área urbana. Após o ajuste, constatou-se que os adolescentes oriundos da área rural usavam menos televisão (odds ratio - OR: 0,45; intervalo de confiança de 95% - IC95%: 0,39-0,52), computador e/ou videogame (OR: 0,30; IC95%: 0,22-0,42), passavam menos tempo sentados (OR: 0,66; IC95%: 0,54-0,80), optaram menos pelo lazer passivo (OR: 0,83; IC95%: 0,72-0,95) e tinham menos chances de serem classificados como insuficientes ativos (OR: 0,88; IC95%: 0,78-0,99), quando comparados àqueles que residiam na área urbana, independentemente do sexo e da idade. Os adolescentes da área rural que não trabalhavam apresentaram mais chances de serem classificados como insuficientemente ativos (OR: 2,59; IC95%: 2,07-3,24), mostrando que a ocupação tinha um papel importante no nível de atividade física deste grupo. Os adolescentes residentes na área rural estiveram menos expostos aos comportamentos sedentários, optaram mais por um lazer ativo e apresentaram um melhor nível de atividade física, podendo a zona de domicílio e a ocupação influenciar no estilo de vida deles.

Chronic Sequelae of E. coli O157: Systematic Review and Meta-analysis of the Proportion of E. coli O157 Cases That Develop Chronic Sequelae

PubMed Central

Sargeant, Jan; Thomas, M. Kate; Fazil, Aamir

2014-01-01

Abstract Objective: This was a systematic review and meta-analysis to determine the proportion of Escherichia coli O157 cases that develop chronic sequelae. Data Sources: We conducted a systematic review of articles published prior to July 2011 in Pubmed, Agricola, CabDirect, or Food Safety and Technology Abstracts. Study Selection: Studies were selected that reported the number of E. coli O157 cases that developed reactive arthritis (ReA), hemolytic uremic syndrome (HUS), irritable bowel syndrome, inflammatory bowel disease, or Guillain Barré syndrome. Methods: Three levels of screening and data extraction of articles were conducted using predefined data fields. Meta-analysis was performed on unique outcome measures using a random-effects model, and heterogeneity was assessed using the I2 value. Meta-regression was used to explore the influence of nine study-level variables on heterogeneity. Results: A total of 82 studies were identified reporting 141 different outcome measures; 81 reported on HUS and one reported on ReA. Depending on the number of cases of E. coli O157, the estimate for the proportion of E. coli O157 cases that develop HUS ranged from 17.2% in extra-small studies (<50 cases) to 4.2% in extra-large studies (>1000 cases). Heterogeneity was significantly associated with group size (p<0.0001); however, the majority of the heterogeneity was unexplained. Conclusions: High unexplained heterogeneity indicated that the study-level factors examined had a minimal influence on the variation of estimates reported. PMID:24404780

The right ear advantage revisited: speech lateralisation in dichotic listening using consonant-vowel and vowel-consonant syllables.

PubMed

Sætrevik, Bjørn

2012-01-01

The dichotic listening task is typically administered by presenting a consonant-vowel (CV) syllable to each ear and asking the participant to report the syllable heard most clearly. The results tend to show more reports of the right ear syllable than of the left ear syllable, an effect called the right ear advantage (REA). The REA is assumed to be due to the crossing over of auditory fibres and the processing of language stimuli being lateralised to left temporal areas. However, the tendency for most dichotic listening experiments to use only CV syllable stimuli limits the extent to which the conclusions can be generalised to also apply to other speech phonemes. The current study re-examines the REA in dichotic listening by using both CV and vowel-consonant (VC) syllables and combinations thereof. Results showed a replication of the REA response pattern for both CV and VC syllables, thus indicating that the general assumption of left-side localisation of processing can be applied for both types of stimuli. Further, on trials where a CV is presented in one ear and a VC is presented in the other ear, the CV is selected more often than the VC, indicating that these phonemes have an acoustic or processing advantage.

Outcome of reactive arthritis after an extensive Finnish waterborne gastroenteritis outbreak: a 1-year prospective follow-up study.

PubMed

Uotila, Terhi M; Antonen, Jaakko A; Paakkala, Antti S; Mustonen, Jukka T; Korpela, Markku M

2013-08-01

The purpose of the study was to assess the 1-year outcome of definitive reactive arthritis (ReA) after a waterborne outbreak. A cohort of 21 patients (15 females and 6 males, median age 54 years) with ReA related to an extensive waterborne outbreak in Finland was clinically followed-up by rheumatologists with visits at baseline, at 1 month and 3, 6 and 12 months. Although the outcome was in general favourable, 1/3 of the patients had chronic course; 7 (33 %) of the 21 patients needed disease-modifying anti-rheumatic drugs (DMARDs) and even 8 (38 %) of them used glucocorticoids at 12 months. Four (19 %) were using non-steroidal anti-inflammatory drugs and nine (43 %) other analgesics. Many patients had articular pain and impaired physical function still at 12 months, even though inflammatory parameters and the number of swollen joints were low. Only one patient (5 %) was human leucocyte antigen-B27-positive. She had the most severe ReA and also additional infectious arthritis caused by Salmonella serotype enteritidis leading to osteonecrosis of her hip joint with subsequent need for arthroplasty. ReA as observed in our study was overall fairly mild, but in many individuals, postinfectious arthralgia and DMARD use continued at least up to 1 year.

Transcriptome mining and in silico structural and functional analysis of ascorbic acid and tartaric acid biosynthesis pathway enzymes in rose-scanted geranium.

PubMed

Narnoliya, Lokesh K; Sangwan, Rajender S; Singh, Sudhir P

2018-06-01

Rose-scented geranium (Pelargonium sp.) is widely known as aromatic and medicinal herb, accumulating specialized metabolites of high economic importance, such as essential oils, ascorbic acid, and tartaric acid. Ascorbic acid and tartaric acid are multifunctional metabolites of human value to be used as vital antioxidants and flavor enhancing agents in food products. No information is available related to the structural and functional properties of the enzymes involved in ascorbic acid and tartaric acid biosynthesis in rose-scented geranium. In the present study, transcriptome mining was done to identify full-length genes, followed by their bioinformatic and molecular modeling investigations and understanding of in silico structural and functional properties of these enzymes. Evolutionary conserved domains were identified in the pathway enzymes. In silico physicochemical characterization of the catalytic enzymes revealed isoelectric point (pI), instability index, aliphatic index, and grand average hydropathy (GRAVY) values of the enzymes. Secondary structural prediction revealed abundant proportion of alpha helix and random coil confirmations in the pathway enzymes. Three-dimensional homology models were developed for these enzymes. The predicted structures showed significant structural similarity with their respective templates in root mean square deviation analysis. Ramachandran plot analysis of the modeled enzymes revealed that more than 84% of the amino acid residues were within the favored regions. Further, functionally important residues were identified corresponding to catalytic sites located in the enzymes. To, our best knowledge, this is the first report which provides a foundation on functional annotation and structural determination of ascorbic acid and tartaric acid pathway enzymes in rose-scanted geranium.

Thrombin-like enzymes from snake venom: Structural characterization and mechanism of action.

PubMed

Ullah, Anwar; Masood, Rehana; Ali, Ijaz; Ullah, Kifayat; Ali, Hamid; Akbar, Haji; Betzel, Christian

2018-07-15

Snake venom thrombin-like enzymes (SVTLEs) constitute the major portion (10-24%) of snake venom and these are the second most abundant enzymes present in the crude venom. During envenomation, these enzymes had shown prominently the various pathological effects, such as disturbance in hemostatic system, fibrinogenolysis, fibrinolysis, platelet aggregation, thrombosis, neurologic disorders, activation of coagulation factors, coagulant, procoagulant etc. These enzymes also been used as a therapeutic agent for the treatment of various diseases such as congestive heart failure, ischemic stroke, thrombotic disorders etc. Although the crystal structures of five SVTLEs are available in the Protein Data Bank (PDB), there is no single article present in the literature that has described all of them. The current work describes the structural aspects, structure-based mechanism of action, processing and inhibition of these enzymes. The sequence analysis indicates that these enzymes show a high sequence identity (57-85%) with each other and low sequence identity with trypsin (36-43%), human alpha-thrombin (29-36%) and other snake venom serine proteinases (57-85%). Three-dimensional structural analysis indicates that the loops surrounding the active site are variable both in amino acids composition and length that may convey variable substrate specificity to these enzymes. The surface charge distributions also vary in these enzymes. Docking analysis with suramin shows that this inhibitor preferably binds to the C-terminal region of these enzymes and causes the destabilization of their three-dimensional structure. Copyright © 2018 Elsevier B.V. All rights reserved.

Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

PubMed

Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

2009-12-11

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

Secretome analysis of the thermophilic xylanase hyper-producer Thermomyces lanuginosus SSBP cultivated on corn cobs.

PubMed

Winger, A M; Heazlewood, J L; Chan, L J G; Petzold, C J; Permaul, K; Singh, S

2014-11-01

Thermomyces lanuginosus is a thermophilic fungus known for its ability to produce industrially important enzymes including large amounts of xylanase, the key enzyme in hemicellulose hydrolysis. The secretome of T. lanuginosus SSBP was profiled by shotgun proteomics to elucidate important enzymes involved in hemicellulose saccharification and to characterise the presence of other industrially interesting enzymes. This study reproducibly identified a total of 74 proteins in the supernatant following growth on corn cobs. An analysis of proteins revealed nine glycoside hydrolase (GH) enzymes including xylanase GH11, β-xylosidase GH43, β-glucosidase GH3, α-galactosidase GH36 and trehalose hydrolase GH65. Two commercially produced Thermomyces enzymes, lipase and amylase, were also identified. In addition, other industrially relevant enzymes not currently explored in Thermomyces were identified including glutaminase, fructose-bisphosphate aldolase and cyanate hydratase. Overall, these data provide insight into the novel ability of a cellulase-free fungus to utilise lignocellulosic material, ultimately producing a number of enzymes important to various industrial processes.

Thermophilic growth and enzymatic thermostability are polyphyletic traits within Chaetomiaceae.

PubMed

van den Brink, Joost; Facun, Kryss; de Vries, Michel; Stielow, J Benjamin

2015-12-01

Thermophilic fungi have the potential to produce industrial-relevant thermostable enzymes, in particular for the degradation of plant biomass. Sordariales is one of the few fungal orders containing several thermophilic taxa, of which many have been associated with the production of thermostable enzymes. The evolutionary affiliation of Sordariales fungi, especially between thermophiles and non-thermophilic relatives, is however poorly understood. Phylogenetic analysis within the current study was based on sequence data, derived from a traditional Sanger and highly multiplexed targeted next generation sequencing approach of 45 isolates. The inferred phylogeny and detailed growth analysis rendered the trait 'thermophily' as polyphyletic within Chaetomiaceae (Sordariales, Sordariomycetes), and characteristic to: Myceliophthora spp., Thielavia terrestris, Chaetomium thermophilum, and Mycothermus thermophilus. Compared to mesophiles, the isolates within thermophilic taxa produced enzyme mixtures with the highest thermostability of known cellulase activities. Temperature profiles of the enzyme activities correlated strongly with the optimal growth temperatures of the isolates but not with their phylogenetic relationships. This strong correlation between growth and enzyme characteristics indicated that detailed analysis of growth does give predictive information on enzyme physiology. The variation in growth and enzyme characteristics reveals these fungi as an excellent platform to better understand fungal thermophily and enzyme thermostability. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Vegetation Change Analysis User’s Manual

DTIC Science & Technology

2002-10-01

so slow and at so low of an altitude. At faster speeds motion blur is a problem. At lower altitudes safety may be an issue, especially in mountainous ...Heterogenool)l • Alea 0.13781 137809.9 Hole Alea Per ;&.rea 0 n013781 Hole RatiO Hclea ~ ~ol-d~~ M IIQI_oi..,.OI Pelin (corwe~<) p...best approximates the maximum plant cover value found from ground samplings. Values above that can be incorporated into a mountain mask that

Activity loss by H46A mutation in Mycobacterium tuberculosis isocitrate lyase is due to decrease in structural plasticity and collective motions of the active site.

PubMed

Shukla, Rohit; Shukla, Harish; Tripathi, Timir

2018-01-01

Mycobacterium tuberculosis isocitrate lyase (MtbICL) is a crucial enzyme of the glyoxylate cycle and is a validated anti-tuberculosis drug target. Structurally distant, non-active site mutation (H46A) in MtbICL has been found to cause loss of enzyme activity. The aim of the present work was to explore the structural alterations induced by H46A mutation that caused the loss of enzyme activity. The structural and dynamic consequences of H46A mutation were studied using multiple computational methods such as docking, molecular dynamics simulation and residue interaction network analysis (RIN). Principal component analysis and cross correlation analysis revealed the difference in conformational flexibility and collective modes of motions between the wild-type and mutant enzyme, particularly in the active site region. RIN analysis revealed that the active site geometry was disturbed in the mutant enzyme. Thus, the dynamic perturbation of the active site led to enzyme transition from its active form to inactive form upon mutation. The computational analyses elucidated the mutant-specific conformational alterations, differential dominant motions, and anomalous residue level interactions that contributed to the abrogated function of mutant MtbICL. An understanding of interactions of mutant enzymes may help in modifying the existing drugs and designing improved drugs for successful control of tuberculosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges.

PubMed

Qing, Taiping; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Wen, Li; Shangguan, Jingfang; Mao, Zhengui; Lei, Yanli

2016-04-01

Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.

Mission Connect Mild TBI Translational Research Consortium

DTIC Science & Technology

2010-08-01

Grill RJ, Parsley MA, Unabia G, Hulsebosch CE. Anti- inflammatory treatment of traumatic brain injury (TBI). Society for Neuroscience, 2009...Hulsebosch, CD, DeWitt DS, Dash PK, Grill RJ, Parsley MA, Unabia, BC, Rea HC, & Perez-Polo, JR Role of IL-1 and TNF receptor activation in neurological...deficits at TBI. Military health Research Forum, 2009. Hulsebosch, CD, DeWitt DS, Dash PK, Grill RJ, Parsley MA, Unabia, BC, Rea HC, & Perez-Polo, JR

Transaction based approach

NASA Astrophysics Data System (ADS)

Hunka, Frantisek; Matula, Jiri

2017-07-01

Transaction based approach is utilized in some methodologies in business process modeling. Essential parts of these transactions are human beings. The notion of agent or actor role is usually used for them. The paper on a particular example describes possibilities of Design Engineering Methodology for Organizations (DEMO) and Resource-Event-Agent (REA) methodology. Whereas the DEMO methodology can be regarded as a generic methodology having its foundation in the theory of Enterprise Ontology the REA methodology is regarded as the domain specific methodology and has its origin in accountancy systems. The results of these approaches is that the DEMO methodology captures everything that happens in the reality with a good empirical evidence whereas the REA methodology captures only changes connected with economic events. Economic events represent either change of the property rights to economic resource or consumption or production of economic resources. This results from the essence of economic events and their connection to economic resources.

A complete thermodynamic analysis of enzyme turnover links the free energy landscape to enzyme catalysis.

PubMed

Jones, Hannah B L; Wells, Stephen A; Prentice, Erica J; Kwok, Anthony; Liang, Liyin L; Arcus, Vickery L; Pudney, Christopher R

2017-09-01

Our understanding of how enzymes work is coloured by static structure depictions where the enzyme scaffold is presented as either immobile, or in equilibrium between well-defined static conformations. Proteins, however, exhibit a large degree of motion over a broad range of timescales and magnitudes and this is defined thermodynamically by the enzyme free energy landscape (FEL). The role and importance of enzyme motion is extremely contentious. Much of the challenge is in the experimental detection of so called 'conformational sampling' involved in enzyme turnover. Herein we apply combined pressure and temperature kinetics studies to elucidate the full suite of thermodynamic parameters defining an enzyme FEL as it relates to enzyme turnover. We find that the key thermodynamic parameters governing vibrational modes related to enzyme turnover are the isobaric expansivity term and the change in heat capacity for enzyme catalysis. Variation in the enzyme FEL affects these terms. Our analysis is supported by a range of biophysical and computational approaches that specifically capture information on protein vibrational modes and the FEL (all atom flexibility calculations, red edge excitation shift spectroscopy and viscosity studies) that provide independent evidence for our findings. Our data suggest that restricting the enzyme FEL may be a powerful strategy when attempting to rationally engineer enzymes, particularly to alter thermal activity. Moreover, we demonstrate how rational predictions can be made with a rapid computational approach. © 2017 Federation of European Biochemical Societies.

METRICS DEVELOPMENT FOR PATENTS.

PubMed

Veiga, Daniela Francescato; Ferreira, Lydia Masako

2015-01-01

To develop a proposal for metrics for patents to be applied in assessing the postgraduate programs of Medicine III - Capes. From the reading and analysis of the 2013 area documents of all the 48 areas of Capes, a proposal for metrics for patents was developed to be applied in Medicine III programs. Except for the areas Biotechnology, Food Science, Biological Sciences III, Physical Education, Engineering I, III and IV and Interdisciplinary, most areas do not adopt a scoring system for patents. The proposal developed was based on the criteria of Biotechnology, with adaptations. In general, it will be valued, in ascending order, the deposit, the granting and licensing/production. It will also be assigned higher scores to patents registered abroad and whenever there is a participation of students. This proposal can be applied to the item Intellectual Production of the evaluation form, in subsection Technical Production/Patents. The percentage of 10% for academic programs and 40% for Masters Professionals should be maintained. The program will be scored as Very Good when it reaches 400 points or over; Good, between 200 and 399 points; Regular, between 71 and 199 points; Weak up to 70 points; Insufficient, no punctuation. Desenvolver uma proposta de métricas para patentes a serem aplicadas na avaliação dos Programas de Pós-Graduação da Área Medicina III - Capes. A partir da leitura e análise dos documentos de área de 2013 de todas as 48 Áreas da Capes, desenvolveu-se uma proposta de métricas para patentes, a ser aplicada na avaliação dos programas da área. Constatou-se que, com exceção das áreas Biotecnologia, Ciência de Alimentos, Ciências Biológicas III, Educação Física, Engenharias I, III e IV e Interdisciplinar, a maioria não adota sistema de pontuação para patentes. A proposta desenvolvida baseou-se nos critérios da Biotecnologia, com adaptações. De uma forma geral, foi valorizado, em ordem crescente, o depósito, a concessão e o licenciamento/produção. Também foi atribuída maior pontuação a patentes registradas no exterior e com participação de discentes. Esta proposta poderá ser aplicada ao item Produção Intelectual da ficha de avaliação, no subitem Produções Técnicas, patentes e outras produções consideradas relevantes. Deverá ser mantida, neste subitem, a porcentagem de 10% para os programas acadêmicos e 40% para os mestrados profissionais. Será considerado Muito Bom o programa que obtiver 400 pontos/triênio ou mais, Bom entre 200 e 399 pontos; Regular entre 71 e 199 pontos; Fraco até 70 pontos; e Deficiente sem pontuação.

Comparison of Genomic Methods for Differentiating Strains of Enterococcus faecium: Assessment Using Clinical Epidemiologic Data

PubMed Central

Savor, Connie; Pfaller, Michael A.; Kruszynski, Julie A.; Hollis, Richard J.; Noskin, Gary A.; Peterson, Lance R.

1998-01-01

Genomic DNA extracted from 45 vancomycin-resistant Enterococcus faecium (VRE) isolates was cleaved with HindIII and HaeIII and subjected to agarose gel electrophoresis. The ability of this method (restriction endonuclease analysis [REA]) to distinguish strains at the subspecies level was compared with results previously determined by pulsed-field gel electrophoresis (PFGE). Chart reviews were performed to provide a clinical correlation of possible epidemiologic relatedness. A likely clinical association was found for 29 patients as part of two outbreaks. REA found 21 of 21 isolates were the same type in the first outbreak, with PFGE calling 19 strains the same type. In the second outbreak with eight patient isolates, HindIII found six were the same type and two were unique types. HaeIII found three strains were the same type, two strains were a separate type, and three more strains were unique types, while PFGE found three were the same type and five were unique types. No single “ideal” method can be used without clinical epidemiologic investigation, but any of these techniques is helpful in providing focus to infection control practitioners assessing possible outbreaks of nosocomial infection. PMID:9774587

Analysis of enzyme production by submerged culture of Aspergillus oryzae using whole barley.

PubMed

Masuda, Susumu; Kikuchi, Kaori; Matsumoto, Yuko; Sugimoto, Toshikazu; Shoji, Hiroshi; Tanabe, Masayuki

2009-10-01

We have reported on high enzyme production by submerged culture of Aspergillus kawachii using barley with the husk (whole barley). To elucidate the mechanism underlying this high enzyme production, we performed a detailed analysis. Aspergillus oryzae RIB40 was submerged-cultured using whole barley and milled whole barley. Enzyme production was analyzed in terms of changes in medium components and gene expression levels. When whole barley was used, high production of glucoamylase and alpha-amylase and high gene expression levels of these enzymes were observed. Low ammonium concentrations were maintained with nitrate ion uptake continuing into the late stage using whole barley. These findings suggest that the sustainability of nitrogen metabolism is related to high enzyme production, and that a mechanism other than that associated with the conventional amylase expression system is involved in this relationship.

Mexico and Trilateral Air Defense, Is NORAD the Answer?

DTIC Science & Technology

2009-02-20

accordance with a reciprocal agreement at Sistema Integrado de Vigilancia Aérea (the Mexican Air Operations Center [SIVA]) in Mexico City for the...government perceives the Secretaría de La Defensa Nacional or Mexican Army (SEDENA)/ Fuerza Aérea Mexicanos or Mexican Air Force (FAM) and the United...to internally focused missions like SEDENA and FAM. The “Ley Orgánica de la Armada de México” or the constitutional law of the Mexican Navy

Mission Connect Mild TBI Translational Research Consortium

DTIC Science & Technology

2009-08-01

modalities. We recruited to the project Margaret A. Parsley to carry out the Mild Traumatic Brain Injuries (MTBIs) under the supervision of Doug DeWitt...abstracts: Hulsebosch CE, Johnson KM, Dewitt DS, Dash PK, Grill R, Parsley MA, Unabia G, Rea H, Perez-Polo JR. Role of IL-1 and TNF receptor activation in...neurological deficits after TBI National Neurotrauma Society, 2010 Johnson KM, Dewitt DS, Dash PK, Grill R, Parsley MA, Unabia G, Rea H, Perez-Polo

Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion.

PubMed

Bibo-Verdugo, Betsaida; O'Donoghue, Anthony J; Rojo-Arreola, Liliana; Craik, Charles S; García-Carreño, Fernando

2016-04-01

Crustaceans are a diverse group, distributed in widely variable environmental conditions for which they show an equally extensive range of biochemical adaptations. Some digestive enzymes have been studied by purification/characterization approaches. However, global analysis is crucial to understand how digestive enzymes interplay. Here, we present the first proteomic analysis of the digestive fluid from a crustacean (Homarus americanus) and identify glycosidases and peptidases as the most abundant classes of hydrolytic enzymes. The digestion pathway of complex carbohydrates was predicted by comparing the lobster enzymes to similar enzymes from other crustaceans. A novel and unbiased substrate profiling approach was used to uncover the global proteolytic specificity of gastric juice and determine the contribution of cysteine and aspartic acid peptidases. These enzymes were separated by gel electrophoresis and their individual substrate specificities uncovered from the resulting gel bands. This new technique is called zymoMSP. Each cysteine peptidase cleaves a set of unique peptide bonds and the S2 pocket determines their substrate specificity. Finally, affinity chromatography was used to enrich for a digestive cathepsin D1 to compare its substrate specificity and cold-adapted enzymatic properties to mammalian enzymes. We conclude that the H. americanus digestive peptidases may have useful therapeutic applications, due to their cold-adaptation properties and ability to hydrolyze collagen.

Spatial localization of the first and last enzymes effectively connects active metabolic pathways in bacteria.

PubMed

Meyer, Pablo; Cecchi, Guillermo; Stolovitzky, Gustavo

2014-12-14

Although much is understood about the enzymatic cascades that underlie cellular biosynthesis, comparatively little is known about the rules that determine their cellular organization. We performed a detailed analysis of the localization of E.coli GFP-tagged enzymes for cells growing exponentially. We found that out of 857 globular enzymes, at least 219 have a discrete punctuate localization in the cytoplasm and catalyze the first or the last reaction in 60% of biosynthetic pathways. A graph-theoretic analysis of E.coli's metabolic network shows that localized enzymes, in contrast to non-localized ones, form a tree-like hierarchical structure, have a higher within-group connectivity, and are traversed by a higher number of feed-forward and feedback loops than their non-localized counterparts. A Gene Ontology analysis of these enzymes reveals an enrichment of terms related to essential metabolic functions in growing cells. Given that these findings suggest a distinct metabolic role for localization, we studied the dynamics of cellular localization of the cell wall synthesizing enzymes in B. subtilis and found that enzymes localize during exponential growth but not during stationary growth. We conclude that active biochemical pathways inside the cytoplasm are organized spatially following a rule where their first or their last enzymes localize to effectively connect the different active pathways and thus could reflect the activity state of the cell's metabolic network.

Multifamily Housing Rehabilitation Process Improvements

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

Sweet, Marshall L.; Francisco, Abby; Roberts, Sydney G.

Rea Ventures Group, LLC, (Rea Ventures) partnered with Southface Energy Institute (Southface) on the rehabilitation of 418 low-income rental multifamily apartments located at 14 different properties in Georgia (Climate Zones 2-4). These 22-year old, individually-metered units were arranged in rowhouse or townhouse style units. Rehabilitation plans were developed using a process prescribed by the US Department of Agriculture (USDA) Rural Development program, who partially funded the building upgrades. The USDA is responsible for building, upgrading, and subsidizing housing in rural areas nationwide. In 2012, over $100 million was allocated in grants and loans. Due to the unique financing mechanism asmore » well as long-term ownership requirements, property owners are especially motivated to invest in upgrades that will increase durability and tenant retention. These buildings represent a large stock of rural affordable housing that have the potential for significant energy and cost savings for property owners and tenants. Southface analyzed the energy upgrade potential of one stereotypical property in the Rea Ventures portfolio. This study will provide insight into the most cost-effective, implementable energy efficiency and durability upgrades for this age multifamily housing, having an enormous impact not only on the portfolio of Rea Ventures but on the vast USDA and larger Federal portfolio. Additionally, Southface will identify gaps in the current capital needs assessment process, examine available audit and simulation tools and protocols, and evaluate additional auditor training or certification needs.« less

The Conservation of Structure and Mechanism of Catalytic Action in a Family of Thiamin Pyrophosphate (TPP)-dependent Enzymes

NASA Technical Reports Server (NTRS)

Dominiak, P.; Ciszak, Ewa

2004-01-01

Thiamin pyrophosphate (TPP)-dependent enzymes are a divergent family of TPP and metal ion binding proteins that perform a wide range of functions with the common decarboxylation steps of a -(O=)C-C(OH)- fragment of alpha-ketoacids and alpha- hydroxyaldehydes. To determine how structure and catalytic action are conserved in the context of large sequence differences existing within this family of enzymes, we have carried out an analysis of TPP-dependent enzymes of known structures. The common structure of TPP-dependent enzymes is formed at the interface of four alpha/beta domains from at least two subunits, which provide for two metal and TPP-binding sites. Residues around these catalytic sites are conserved for functional purpose, while those further away from TPP are conserved for structural reasons. Together they provide a network of contacts required for flip-flop catalytic action within TPP-dependent enzymes. Thus our analysis defines a TPP-action motif that is proposed for annotating TPP-dependent enzymes for advancing functional proteomics.

County-scale spatial distribution of soil enzyme activities and enzyme activity indices in agricultural land: implications for soil quality assessment.

PubMed

Tan, Xiangping; Xie, Baoni; Wang, Junxing; He, Wenxiang; Wang, Xudong; Wei, Gehong

2014-01-01

Here the spatial distribution of soil enzymatic properties in agricultural land was evaluated on a county-wide (567 km(2)) scale in Changwu, Shaanxi Province, China. The spatial variations in activities of five hydrolytic enzymes were examined using geostatistical methods. The relationships between soil enzyme activities and other soil properties were evaluated using both an integrated total enzyme activity index (TEI) and the geometric mean of enzyme activities (GME). At the county scale, soil invertase, phosphatase, and catalase activities were moderately spatially correlated, whereas urease and dehydrogenase activities were weakly spatially correlated. Correlation analysis showed that both TEI and GME were better correlated with selected soil physicochemical properties than single enzyme activities. Multivariate regression analysis showed that soil OM content had the strongest positive effect while soil pH had a negative effect on the two enzyme activity indices. In addition, total phosphorous content had a positive effect on TEI and GME in orchard soils, whereas alkali-hydrolyzable nitrogen and available potassium contents, respectively, had negative and positive effects on these two enzyme indices in cropland soils. The results indicate that land use changes strongly affect soil enzyme activities in agricultural land, where TEI provides a sensitive biological indicator for soil quality.

A Century of Enzyme Kinetic Analysis, 1913 to 2013

PubMed Central

Johnson, Kenneth A.

2013-01-01

This review traces the history and logical progression of methods for quantitative analysis of enzyme kinetics from the 1913 Michaelis and Menten paper to the application of modern computational methods today. Following a brief review of methods for fitting steady state kinetic data, modern methods are highlighted for fitting full progress curve kinetics based upon numerical integration of rate equations, including a re-analysis of the original Michaelis-Menten full time course kinetic data. Finally, several illustrations of modern transient state kinetic methods of analysis are shown which enable the elucidation of reactions occurring at the active sites of enzymes in order to relate structure and function. PMID:23850893

Continuous determination of land-atmosphere Hg0 exchange using a novel Relaxed Eddy Accumulation design

NASA Astrophysics Data System (ADS)

Osterwalder, Stefan; Fritsche, Johannes; Nilsson, Mats B.; Alewell, Christine; Bishop, Kevin

2015-04-01

The fate of anthropogenic emissions to the atmosphere is influenced by the exchange of elemental mercury (Hg0) with the earth surface. However, it remains challenging to quantify these exchanges which hold the key to a better understanding of mercury cycling at different scales, from the entire earth to specific environments. To better test hypotheses about land-atmosphere Hg interactions, we applied dynamic flux chambers (DFCs) for short term measurements and developed a novel Relaxed Eddy Accumulation (REA) design for continuous flux monitoring. Accurate determination of Hg0 fluxes has proven difficult due to the technical challenges presented by the small concentration differences (< 1 ng m-3) between updrafts and downdrafts. To address this we present a dual-intake, single analyzer REA system including a calibration module for periodic quality-control measurements with reference gases. To demonstrate the system performance, we present results from two contrasting environments: In February 2012 REA monitored a heterogeneous urban surface in the center of Basel, Switzerland where an average flux of 14 ng m-2 h-1 was detected with a distinct diurnal pattern. In May 2012, the REA monitored a boreal mire in northern Sweden with different turbulence regimes and Hg0 sink/source characteristics. During the snowmelt period in May 2012 the Hg0 flux averaged at 2 ng m-2 h-1. In order to better quantify inputs and outputs of Hg from boreal landscapes, we subsequently monitored the land-atmosphere exchange of Hg0 during a course of a year and compared the fluxes occasionally with DFC measurements. The amount of Hg0 volatilized from boreal mires was at a similar level as the annual export of Hg in stream water, identifying the mire as net source of Hg to neighboring environments. We believe that this dual-inlet, single detector approach is a significant innovation which can help realize the potential of REA for continuous, long-term determination of land-atmosphere Hg0 exchange.

Proteomic analysis of temperature dependent extracellular proteins from Aspergillus fumigatus grown under solid-state culture condition.

PubMed

Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

2013-06-07

Fungal species of the genus Aspergillus are filamentous ubiquitous saprophytes that play a major role in lignocellulosic biomass recycling and also are considered as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. Analysis of extracellular secreted biomass degrading enzymes using complex lignocellulosic biomass as a substrate by solid-state fermentation could be a more practical approach to evaluate application of the enzymes for lignocellulosic biorefinery. This study isolated a fungal strain from compost, identified as Aspergillus fumigatus, and further analyzed it for lignocellulolytic enzymes at different temperatures using label free quantitative proteomics. The profile of secretome composition discovered cellulases, hemicellulases, lignin degrading proteins, peptidases and proteases, and transport and hypothetical proteins; while protein abundances and further their hierarchical clustering analysis revealed temperature dependent expression of these enzymes during solid-state fermentation of sawdust. The enzyme activities and protein abundances as determined by exponentially modified protein abundance index (emPAI) indicated the maximum activities at the range of 40-50 °C, demonstrating the thermophilic nature of the isolate A. fumigatus LF9. Characterization of the thermostability of secretome suggested the potential of the isolated fungal strain in the production of thermophilic biomass degrading enzymes for industrial application.

Kinetic analysis of enzyme systems with suicide substrate in the presence of a reversible competitive inhibitor, tested by simulated progress curves.

PubMed

Moruno-Dávila, M A; Garrido-del Solo, C; García-Moreno, M; Havsteen, B H; Garcia-Sevilla, F; Garcia-Cánovas, F; Varón, R

2001-02-01

The use of suicide substrates remains a very important and useful method in enzymology for studying enzyme mechanisms and designing potential drugs. Suicide substrates act as modified substrates for the target enzymes and bind to the active site. Therefore the presence of a competitive reversible inhibitor decreases the rate of substrate-induced inactivation and protects the enzyme from this inactivation. This lowering on the inactivation rate has evident physiological advantages, since it allows the easy acquisition of experimental data and facilitates kinetic data analysis by providing another variable (inhibitor concentration). However despite the importance of the simultaneous action of a suicide substrate and a competitive reversible inhibition, to date no corresponding kinetic analysis has been carried out. Therefore we present a general kinetic analysis of a Michaelis-Menten reaction mechanism with double inhibition caused by both, a suicide substrate and a competitive reversible inhibitor. We assume rapid equilibrium of the reversible reaction steps involved, while the time course equations for the reaction product have been derived with the assumption of a limiting enzyme. The goodness of the analytical solutions has been tested by comparison with the simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters from the time progress curve of the product is suggested. In conclusion, we present a complete kinetic analysis of an enzyme reaction mechanism as described above in an attempt to fill a gap in the theoretical treatment of this type of system.

Understanding Regulation of Metabolism through Feasibility Analysis

PubMed Central

Nikerel, Emrah; Berkhout, Jan; Hu, Fengyuan; Teusink, Bas; Reinders, Marcel J. T.; de Ridder, Dick

2012-01-01

Understanding cellular regulation of metabolism is a major challenge in systems biology. Thus far, the main assumption was that enzyme levels are key regulators in metabolic networks. However, regulation analysis recently showed that metabolism is rarely controlled via enzyme levels only, but through non-obvious combinations of hierarchical (gene and enzyme levels) and metabolic regulation (mass action and allosteric interaction). Quantitative analyses relating changes in metabolic fluxes to changes in transcript or protein levels have revealed a remarkable lack of understanding of the regulation of these networks. We study metabolic regulation via feasibility analysis (FA). Inspired by the constraint-based approach of Flux Balance Analysis, FA incorporates a model describing kinetic interactions between molecules. We enlarge the portfolio of objectives for the cell by defining three main physiologically relevant objectives for the cell: function, robustness and temporal responsiveness. We postulate that the cell assumes one or a combination of these objectives and search for enzyme levels necessary to achieve this. We call the subspace of feasible enzyme levels the feasible enzyme space. Once this space is constructed, we can study how different objectives may (if possible) be combined, or evaluate the conditions at which the cells are faced with a trade-off among those. We apply FA to the experimental scenario of long-term carbon limited chemostat cultivation of yeast cells, studying how metabolism evolves optimally. Cells employ a mixed strategy composed of increasing enzyme levels for glucose uptake and hexokinase and decreasing levels of the remaining enzymes. This trade-off renders the cells specialized in this low-carbon flux state to compete for the available glucose and get rid of over-overcapacity. Overall, we show that FA is a powerful tool for systems biologists to study regulation of metabolism, interpret experimental data and evaluate hypotheses. PMID:22808034

The probiotic Lactobacillus johnsonii NCC 533 produces high-molecular-mass inulin from sucrose by using an inulosucrase enzyme.

PubMed

Anwar, Munir A; Kralj, Slavko; van der Maarel, Marc J E C; Dijkhuizen, Lubbert

2008-06-01

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (open reading frame AAS08734) annotated as a putative levansucrase precursor. However, (13)C nuclear magnetic resonance (NMR) analysis of the fructan product synthesized in situ revealed that this is of the inulin type. The ftf gene of L. johnsonii was cloned and expressed to elucidate its exact identity. The purified L. johnsonii protein was characterized as an inulosucrase enzyme, producing inulin from sucrose, as identified by (13)C NMR analysis. Thin-layer chromatographic analysis of the reaction products showed that InuJ synthesized, besides the inulin polymer, a broad range of fructose oligosaccharides. Maximum InuJ enzyme activity was observed in a pH range of 4.5 to 7.0, decreasing sharply at pH 7.5. InuJ exhibited the highest enzyme activity at 55 degrees C, with a drastic decrease at 60 degrees C. Calcium ions were found to have an important effect on enzyme activity and stability. Kinetic analysis showed that the transfructosylation reaction of the InuJ enzyme does not obey Michaelis-Menten kinetics. The non-Michaelian behavior of InuJ may be attributed to the oligosaccharides that were initially formed in the reaction and which may act as better acceptors than the growing polymer chain. This is only the second example of the isolation and characterization of an inulosucrase enzyme and its inulin (oligosaccharide) product from a Lactobacillus strain. Furthermore, this is the first Lactobacillus strain shown to produce inulin polymer in situ.

U.S. Border Patrol OCONUS: Possible Contributions to the Whole of Government Approach to Stability Operations

DTIC Science & Technology

2010-06-11

Force–Rural Area Police Patrol Unit (Unidad Móvil Policial Para Reas Rurales) also known as Los Leopardos (the Leopards ). USBP U.S. Border Patrol...99Ibid. 38 Antinarcotics Force--Rural Area Police Patrol Unit (Unidad Móvil Policial Para Reas Rurales) also known as Los Leopardos (the Leopards ). The...training or preparation time for this mission. Upon arrival he found that the inclement cold weather included snow , sleet, ice, and frigid cold

Population Dynamics of Streptococcus mitis in Its Natural Habitat

PubMed Central

Hohwy, Jesper; Reinholdt, Jesper; Kilian, Mogens

2001-01-01

The purpose of this study was to examine the genetic structure of the typical commensal Streptococcus mitis biovar 1 in its natural habitat in the human oral cavity and pharynx and to investigate the role that selected microbial properties and host, spatial, and temporal factors play in determining the structure of the bacterial population. Consecutive samples were collected from buccal and pharyngeal mucosal surfaces of two infants, their four parents, and two elderly individuals over a period of approximately 1 year. A total of 751 isolates identified as S. mitis biovar 1 were typed by restriction endonuclease analysis (REA) and representative clones were typed by multilocus enzyme electrophoresis (MLEE). The genetic diversity of the S. mitis biovar 1 isolates collected from single infant hosts over a period of 9 to 10 months was found to be between 0.69 and 0.76, which is considerably higher than that previously observed for intestinal populations of Escherichia coli. The study provides evidence of the existence of both transient and persistent clones in adult individuals. In the two infants, however, none of 42 demonstrated clones were detected on more than a single occasion. Statistical calculations showed that the ability to persist was not distributed at random in the S. mitis biovar 1 population. However, neither immunoglobulin A1 protease activity nor the ability to bind α-amylase from saliva was a preferential characteristic of persistent genotypes. In contrast to current concepts of climax ecosystems, the species niche in the habitat appears to be maintained predominantly by a succession of clones rather than by stable strains. Several lines of evidence suggest that the major origin of “new” clones is the many other habitats in the respiratory tract that are occupied by this species. PMID:11553543

Identification of the sequence motif of glycoside hydrolase 13 family members

PubMed Central

Kumar, Vikash

2011-01-01

A bioinformatics analysis of sequences of enzymes of the glycoside hydrolase (GH) 13 family members such as α-amylase, cyclodextrin glycosyltransferase (CGTase), branching enzyme and cyclomaltodextrinase has been carried out in order to find out the sequence motifs that govern the reactions specificities of these enzymes by using hidden Markov model (HMM) profile. This analysis suggests the existence of such sequence motifs and residues of these motifs constituting the −1 to +3 catalytic subsites of the enzyme. Hence, by introducing mutations in the residues of these four subsites, one can change the reaction specificities of the enzymes. In general it has been observed that α -amylase sequence motif have low sequence conservation than rest of the motifs of the GH13 family members. PMID:21544166

Use of immobilized exopeptidases and volatile buffers for analysis of peptides by fast atom bombardment mass spectrometry.

PubMed

Wagner, R M; Fraser, B A

1987-05-01

beta-Lipotrophin (62-77) or Ac-gastrin releasing peptide was incubated with immobilized carboxypeptidase Y or aminopeptidase M. Subsequent aliquots of each incubation mixture were analysed by fast atom bombardment mass spectrometry using a dithiothreitol/dithioerythritol liquid matrix. The use of immobilized enzymes and volatile buffers for exopeptidase digestions enabled rapid and facile separation of enzyme from digestion products. This approach to mass spectral peptide analysis reduced spectral background arising from a glycerol matrix, buffer salts, or enzyme proteins and contaminants, enabling analysis of as little as 200 picomoles of a suitable peptide.

Impulsive versus premeditated aggression: implications for mens rea decisions.

PubMed

Barratt, Ernest S; Felthous, Alan R

2003-01-01

Science can provide more information about the nature of aggressive acts, and therefore the mens rea of criminal offenses, than is commonly assumed. For example, progress has been made in classifying aggression as impulsive or premeditated within the context of the role of conscious experience in controlling behavior. This review of the status of the scientific ability to distinguish conscious from unconscious acts and more specifically impulsive from premeditated aggressive acts is organized around four themes: (i) How is aggression defined and measured in general? (ii) How does the distinction between impulsive and premeditated aggression relate to the legal concept of mens rea? (iii) How do various scientific disciplines contribute to the mind/body discourse? (iv) What risk factors are associated with impulsive and premeditated aggression respectively? The authors conclude that the most promising approach to researching the nature of behavioral intention and motivation is to apply a discipline neutral model that integrates the data from multiple disciplines, collectively designated the cognitive neurosciences. Copyright 2003 John Wiley & Sons, Ltd.

Transformation of cyclodextrin glucanotransferase (CGTase) from aqueous suspension to fine solid particles via electrospraying.

PubMed

Saallah, Suryani; Naim, M Nazli; Mokhtar, Mohd Noriznan; Abu Bakar, Noor Fitrah; Gen, Masao; Lenggoro, I Wuled

2014-10-01

In this study, the potential of electrohydrodynamic atomization or electrospraying to produce nanometer-order CGTase particles from aqueous suspension was demonstrated. CGTase enzyme was prepared in acetate buffer solution (1% v/v), followed by electrospraying in stable Taylor cone-jet mode. The deposits were collected on aluminium foil (collector) at variable distances from the tip of spraying needle, ranging from 10 to 25 cm. The Coulomb fission that occurs during electrospraying process successfully transformed the enzyme to the solid state without any functional group deterioration. The functional group verification was conducted by FTIR analysis. Comparison between the deposit and the as-received enzyme in dry state indicates almost identical spectra. By increasing the distance of the collector from the needle tip, the average particle size of the solidified enzyme was reduced from 200±117 nm to 75±34 nm. The average particle sizes produced from the droplet fission were in agreement with the scaling law models. Enzyme activity analysis showed that the enzyme retained its initial activity after the electrospraying process. The enzyme particles collected at the longest distance (25 cm) demonstrated the highest enzyme activity, which indicates that the activity was controlled by the enzyme particle size. Copyright © 2014 Elsevier Inc. All rights reserved.

Caenorhabditis elegans glutamylating enzymes function redundantly in male mating.

PubMed

Chawla, Daniel G; Shah, Ruchi V; Barth, Zachary K; Lee, Jessica D; Badecker, Katherine E; Naik, Anar; Brewster, Megan M; Salmon, Timothy P; Peel, Nina

2016-09-15

Microtubule glutamylation is an important modulator of microtubule function and has been implicated in the regulation of centriole stability, neuronal outgrowth and cilia motility. Glutamylation of the microtubules is catalyzed by a family of tubulin tyrosine ligase-like (TTLL) enzymes. Analysis of individual TTLL enzymes has led to an understanding of their specific functions, but how activities of the TTLL enzymes are coordinated to spatially and temporally regulate glutamylation remains relatively unexplored. We have undertaken an analysis of the glutamylating TTLL enzymes in C. elegans We find that although all five TTLL enzymes are expressed in the embryo and adult worm, loss of individual enzymes does not perturb microtubule function in embryonic cell divisions. Moreover, normal dye-filling, osmotic avoidance and male mating behavior indicate the presence of functional amphid cilia and male-specific neurons. A ttll-4(tm3310); ttll-11(tm4059); ttll-5(tm3360) triple mutant, however, shows reduced male mating efficiency due to a defect in the response step, suggesting that these three enzymes function redundantly, and that glutamylation is required for proper function of the male-specific neurons. © 2016. Published by The Company of Biologists Ltd.

Coupling between Catalytic Loop Motions and Enzyme Global Dynamics

PubMed Central

Kurkcuoglu, Zeynep; Bakan, Ahmet; Kocaman, Duygu; Bahar, Ivet; Doruker, Pemra

2012-01-01

Catalytic loop motions facilitate substrate recognition and binding in many enzymes. While these motions appear to be highly flexible, their functional significance suggests that structure-encoded preferences may play a role in selecting particular mechanisms of motions. We performed an extensive study on a set of enzymes to assess whether the collective/global dynamics, as predicted by elastic network models (ENMs), facilitates or even defines the local motions undergone by functional loops. Our dataset includes a total of 117 crystal structures for ten enzymes of different sizes and oligomerization states. Each enzyme contains a specific functional/catalytic loop (10–21 residues long) that closes over the active site during catalysis. Principal component analysis (PCA) of the available crystal structures (including apo and ligand-bound forms) for each enzyme revealed the dominant conformational changes taking place in these loops upon substrate binding. These experimentally observed loop reconfigurations are shown to be predominantly driven by energetically favored modes of motion intrinsically accessible to the enzyme in the absence of its substrate. The analysis suggests that robust global modes cooperatively defined by the overall enzyme architecture also entail local components that assist in suitable opening/closure of the catalytic loop over the active site. PMID:23028297

Enhanced efficiency of biological excess sludge hydrolysis under anaerobic digestion by additional enzymes.

PubMed

Yang, Qi; Luo, Kun; Li, Xiao-ming; Wang, Dong-bo; Zheng, Wei; Zeng, Guang-ming; Liu, Jing-jin

2010-05-01

In this investigation, the effects of commercial enzyme preparation containing alpha amylase and neutral protease on hydrolysis of excess sludge and the kinetic analysis of hydrolysis process were evaluated. The results indicated that amylase treatment displayed higher hydrolysis efficiency than that of protease. VSS reduction greatly increased to 39.70% for protease and 54.24% for amylase at the enzyme dosage of 6% (w/w), respectively. The hydrolysis rate of sludge improved with temperature increasing from 40 to 50 degrees Celsius, which could be well described by the amended Arrhenius equation. Mixed-enzyme had great impact on sludge solubilisation than single enzyme. The mixture of two enzymes (protease:amylase=1:3) resulted in optimum hydrolysis efficiency, the efficiency of solids hydrolysis increased from 10% (control test) to 68.43% at the temperature of 50 degrees Celsius. Correspondingly, the concentration of reducing sugar and NH(4)(+)-N improved about 377% and 201%, respectively. According to the kinetic analysis of enzymatic hydrolysis process, VSS solubilisation process within prior 4 h followed first-order kinetics. Compared with control test, the hydrolysis rate improved significantly at 50 degrees Celsius when either single enzyme or mixed-enzyme was added. Copyright 2009. Published by Elsevier Ltd.

Rapid Ethical Assessment on Informed Consent Content and Procedure in Hintalo-Wajirat, Northern Ethiopia: A Qualitative Study.

PubMed

Abay, Serebe; Addissie, Adamu; Davey, Gail; Farsides, Bobbie; Addissie, Thomas

2016-01-01

Informed consent is a key component of bio-medical research involving human participants. However, obtaining informed consent is challenging in low literacy and resource limited settings. Rapid Ethical Assessment (REA) can be used to contextualize and simplify consent information within a given study community. The current study aimed to explore the effects of social, cultural, and religious factors during informed consent process on a proposed HPV-serotype prevalence study. A qualitative community-based REA was conducted in Adigudom and Mynebri Kebeles, Northern Ethiopia, from July to August 2013. Data were collected by a multi-disciplinary team using open ended questions concerning informed consent components in relation to the parent study. The team conducted one-to-one In-Depth Interviews (IDI) and Focus Group Discussions (FGDs) with key informants and community members to collect data based on the themes of the study. Tape recorded data were transcribed in Tigrigna and then translated into English. Data were categorized and thematically analyzed using open coding and content analysis based on pre-defined themes. The REA study revealed a number of socio-cultural issues relevant to the proposed study. Low community awareness about health research, participant rights and cervical cancer were documented. Giving a vaginal sample for testing was considered to be highly embarrassing, whereas giving a blood sample made participants worry that they might be given a result without the possibility of treatment. Verbal consent was preferred to written consent for the proposed study. This rapid ethical assessment disclosed important socio-cultural issues which might act as barriers to informed decision making. The findings were important for contextual modification of the Information Sheet, and to guide the best consent process for the proposed study. Both are likely to have enabled participants to understand the informed consent better and consequently to comply with the study.

Speech processing asymmetry revealed by dichotic listening and functional brain imaging.

PubMed

Hugdahl, Kenneth; Westerhausen, René

2016-12-01

In this article, we review research in our laboratory from the last 25 to 30 years on the neuronal basis for laterality of speech perception focusing on the upper, posterior parts of the temporal lobes, and its functional and structural connections to other brain regions. We review both behavioral and brain imaging data, with a focus on dichotic listening experiments, and using a variety of imaging modalities. The data have come in most parts from healthy individuals and from studies on normally functioning brain, although we also review a few selected clinical examples. We first review and discuss the structural model for the explanation of the right-ear advantage (REA) and left hemisphere asymmetry for auditory language processing. A common theme across many studies have been our interest in the interaction between bottom-up, stimulus-driven, and top-down, instruction-driven, aspects of hemispheric asymmetry, and how perceptual factors interact with cognitive factors to shape asymmetry of auditory language information processing. In summary, our research have shown laterality for the initial processing of consonant-vowel syllables, first observed as a behavioral REA when subjects are required to report which syllable of a dichotic syllable-pair they perceive. In subsequent work we have corroborated the REA with brain imaging, and have shown that the REA is modulated through both bottom-up manipulations of stimulus properties, like sound intensity, and top-down manipulations of cognitive properties, like attention focus. Copyright © 2015 Elsevier Ltd. All rights reserved.

Building America Case Study: Rehabilitation of USDA Multifamily Homes, Georgia (Climate Zones 2-4)

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

Rea Ventures Group, LLC, (Rea Ventures) partnered with Southface Energy Institute (Southface) on the rehabilitation of 418 low-income rental multifamily apartments located at 14 different properties in Georgia (Climate Zones 2-4). These 22-year old, individually-metered units were arranged in rowhouse or townhouse style units. Rehabilitation plans were developed using a process prescribed by the US Department of Agriculture (USDA) Rural Development program, who partially funded the building upgrades. The USDA is responsible for building, upgrading, and subsidizing housing in rural areas nationwide. In 2012, over $100 million was allocated in grants and loans. Due to the unique financing mechanism asmore » well as long-term ownership requirements, property owners are especially motivated to invest in upgrades that will increase durability and tenant retention. These buildings represent a large stock of rural affordable housing that have the potential for significant energy and cost savings for property owners and tenants. Southface analyzed the energy upgrade potential of one stereotypical property in the Rea Ventures portfolio. This study will provide insight into the most cost-effective, implementable energy efficiency and durability upgrades for this age multifamily housing, having an enormous impact not only on the portfolio of Rea Ventures but on the vast USDA and larger Federal portfolio. Additionally, Southface will identify gaps in the current capital needs assessment process, examine available audit and simulation tools and protocols, and evaluate additional auditor training or certification needs.« less

The Thiamine-Pyrophosphate-Motif

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Dominiak, Paulina

2004-01-01

Thiamin pyrophosphate (TPP), a derivative of vitamin B1, is a cofactor for enzymes performing catalysis in pathways of energy production including the well known decarboxylation of a-keto acid dehydrogenases followed by transketolation. TPP-dependent enzymes constitute a structurally and functionally diverse group exhibiting multimeric subunit organization, multiple domains and two chemically equivalent catalytic centers. Annotation of functional TPP-dependcnt enzymes, therefore, has not been trivial due to low sequence similarity related to this complex organization. Our approach to analysis of structures of known TPP-dependent enzymes reveals for the first time features common to this group, which we have termed the TPP-motif. The TPP-motif consists of specific spatial arrangements of structural elements and their specific contacts to provide for a flip-flop, or alternate site, enzymatic mechanism of action. Analysis of structural elements entrained in the flip-flop action displayed by TPP-dependent enzymes reveals a novel definition of the common amino acid sequences. These sequences allow for annotation of TPP-dependent enzymes, thus advancing functional proteomics. Further details of three-dimensional structures of TPP-dependent enzymes will be discussed.

Enzyme-based logic gates and circuits-analytical applications and interfacing with electronics.

PubMed

Katz, Evgeny; Poghossian, Arshak; Schöning, Michael J

2017-01-01

The paper is an overview of enzyme-based logic gates and their short circuits, with specific examples of Boolean AND and OR gates, and concatenated logic gates composed of multi-step enzyme-biocatalyzed reactions. Noise formation in the biocatalytic reactions and its decrease by adding a "filter" system, converting convex to sigmoid response function, are discussed. Despite the fact that the enzyme-based logic gates are primarily considered as components of future biomolecular computing systems, their biosensing applications are promising for immediate practical use. Analytical use of the enzyme logic systems in biomedical and forensic applications is discussed and exemplified with the logic analysis of biomarkers of various injuries, e.g., liver injury, and with analysis of biomarkers characteristic of different ethnicity found in blood samples on a crime scene. Interfacing of enzyme logic systems with modified electrodes and semiconductor devices is discussed, giving particular attention to the interfaces functionalized with signal-responsive materials. Future perspectives in the design of the biomolecular logic systems and their applications are discussed in the conclusion. Graphical Abstract Various applications and signal-transduction methods are reviewed for enzyme-based logic systems.

The CoFactor database: organic cofactors in enzyme catalysis.

PubMed

Fischer, Julia D; Holliday, Gemma L; Thornton, Janet M

2010-10-01

Organic enzyme cofactors are involved in many enzyme reactions. Therefore, the analysis of cofactors is crucial to gain a better understanding of enzyme catalysis. To aid this, we have created the CoFactor database. CoFactor provides a web interface to access hand-curated data extracted from the literature on organic enzyme cofactors in biocatalysis, as well as automatically collected information. CoFactor includes information on the conformational and solvent accessibility variation of the enzyme-bound cofactors, as well as mechanistic and structural information about the hosting enzymes. The database is publicly available and can be accessed at http://www.ebi.ac.uk/thornton-srv/databases/CoFactor.

Development of Ultrasonically Levitated Drops as Microreactors for Study of Enzyme Kinetics and Potential as a Universal Portable Analysis System

DTIC Science & Technology

2008-12-01

1 DEVELOPMENT OF ULTRASONICALLY LEVITATED DROPS AS MICROREAC- TORS FOR STUDY OF ENZYME KINETICS AND POTENTIAL AS A UNIVERSAL PORTABLE ANALYSIS...microfluidic systems are incompatible with the chemistry one wishes to study. We have devel- oped an alternative approach. We use ultrasonically levitated ...since at least the 1940’s, we are the second group to carry out enzyme reactions in levitated drops, (Weis; Nardozzi 2005) and have fab- ricated the

Discriminative structural approaches for enzyme active-site prediction.

PubMed

Kato, Tsuyoshi; Nagano, Nozomi

2011-02-15

Predicting enzyme active-sites in proteins is an important issue not only for protein sciences but also for a variety of practical applications such as drug design. Because enzyme reaction mechanisms are based on the local structures of enzyme active-sites, various template-based methods that compare local structures in proteins have been developed to date. In comparing such local sites, a simple measurement, RMSD, has been used so far. This paper introduces new machine learning algorithms that refine the similarity/deviation for comparison of local structures. The similarity/deviation is applied to two types of applications, single template analysis and multiple template analysis. In the single template analysis, a single template is used as a query to search proteins for active sites, whereas a protein structure is examined as a query to discover the possible active-sites using a set of templates in the multiple template analysis. This paper experimentally illustrates that the machine learning algorithms effectively improve the similarity/deviation measurements for both the analyses.

Multi-enzyme logic network architectures for assessing injuries: digital processing of biomarkers.

PubMed

Halámek, Jan; Bocharova, Vera; Chinnapareddy, Soujanya; Windmiller, Joshua Ray; Strack, Guinevere; Chuang, Min-Chieh; Zhou, Jian; Santhosh, Padmanabhan; Ramirez, Gabriela V; Arugula, Mary A; Wang, Joseph; Katz, Evgeny

2010-12-01

A multi-enzyme biocatalytic cascade processing simultaneously five biomarkers characteristic of traumatic brain injury (TBI) and soft tissue injury (STI) was developed. The system operates as a digital biosensor based on concerted function of 8 Boolean AND logic gates, resulting in the decision about the physiological conditions based on the logic analysis of complex patterns of the biomarkers. The system represents the first example of a multi-step/multi-enzyme biosensor with the built-in logic for the analysis of complex combinations of biochemical inputs. The approach is based on recent advances in enzyme-based biocomputing systems and the present paper demonstrates the potential applicability of biocomputing for developing novel digital biosensor networks.

Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

PubMed

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.

Measurement of the area of venous ulcers using two software programs.

PubMed

Eberhardt, Thaís Dresch; Lima, Suzinara Beatriz Soares de; Lopes, Luis Felipe Dias; Borges, Eline de Lima; Weiller, Teresinha Heck; Fonseca, Graziele Gorete Portella da

2016-12-19

to compare the measurement area of venous ulcers using AutoCAD(r) and Image Tool software. this was an assessment of reproducibility tests conducted in a angiology clinic of a university hospital. Data were collected from 21 patients with venous ulcers, in the period from March to July of 2015, using a collection form and photograph of wounds. Five nurses (evaluators) of the hospital skin wound study group participated. The wounds were measured using both software programs. Data were analyzed using intraclass correlation coefficient, concordance correlation coefficient and Bland-Altman analysis. The study met the ethical aspects in accordance with current legislation. the size of ulcers varied widely, however, without significant difference between the measurements; an excellent intraclass and concordance correlation was found between both software programs, which seem to be more accurate when measuring a wound area >10 cm². the use of both software programs is appropriate for measurement of venous ulcers, appearing to be more accurate when used to measure a wound area > 10 cm². comparar a mensuração de área de úlceras venosas por meio dos softwares AutoCAD(r) e Image Tool. trata-se de um estudo de avaliação de reprodutibilidade de testes, realizado em um ambulatório de angiologia de um hospital universitário. Os dados foram coletados de 21 pacientes com úlceras venosas, no período de março a julho de 2015, por meio de formulário de coleta e fotografia das feridas. Cinco enfermeiros (avaliadores) do Grupo de Estudos de Lesões de Pele do hospital participaram da pesquisa. As feridas foram mensuradas em ambos os softwares. Os dados foram analisados por meio do Coeficiente de correlação intraclasse, Coeficiente de correlação de concordância e procedimento de Bland e Altman. A pesquisa respeitou os aspectos éticos de acordo com a legislação vigente. os tamanhos das úlceras apresentaram grande amplitude, porém, sem diferença significativa entre as mensurações, existe excelente correlação intraclasse e de concordância entre os softwares, os quais parecem ser mais precisos na mensuração de feridas com área > 10 cm². o uso de ambos os softwares é indicado para a mensuração de úlceras venosas, parecendo ser mais precisos quando utilizados para mensurar feridas com área > 10 cm². comparar la medida del área de úlceras venosas por medio de los softwares AutoCAD(r) e Image Tool. se trata de un estudio de evaluación de reproducibilidad de pruebas, realizado en un ambulatorio de angiología de un hospital universitario. Los datos fueron recolectados de 21 pacientes con úlceras venosas, en el período de marzo a julio de 2015, por medio de formulario de recolección y fotografías de las heridas. Cinco enfermeros (evaluadores) del Grupo de Estudios de Lesiones de Piel del hospital participaron de la investigación. Las heridas fueron medidas en ambos softwares. Los datos fueron analizados por medio de: Coeficiente de correlación intraclase, Coeficiente de correlación de concordancia y Procedimiento de Bland y Altman. La investigación respetó los aspectos éticos de acuerdo con la legislación vigente. los tamaños de las úlceras presentaron gran amplitud, sin embargo, sin diferencia significativa entre las medidas; existe excelente correlación intraclase y de concordancia entre los softwares, los que parecen ser más precisos en medidas de heridas con área > 10 cm². el uso de ambos softwares es indicado para medir úlceras venosas, pareciendo ser más precisos cuando utilizados para medir heridas con área > 10 cm².

A century of enzyme kinetic analysis, 1913 to 2013.

PubMed

Johnson, Kenneth A

2013-09-02

This review traces the history and logical progression of methods for quantitative analysis of enzyme kinetics from the 1913 Michaelis and Menten paper to the application of modern computational methods today. Following a brief review of methods for fitting steady state kinetic data, modern methods are highlighted for fitting full progress curve kinetics based upon numerical integration of rate equations, including a re-analysis of the original Michaelis-Menten full time course kinetic data. Finally, several illustrations of modern transient state kinetic methods of analysis are shown which enable the elucidation of reactions occurring at the active sites of enzymes in order to relate structure and function. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Preliminary evaluation of infrared spectroscopy for the differentiation of Brettanomyces bruxellensis strains isolated from red wines.

PubMed

Oelofse, A; Malherbe, S; Pretorius, I S; Du Toit, M

2010-10-15

The objective of this study was to evaluate different infrared spectroscopy methods in combination with chemometrics for the differentiation between Brettanomyces bruxellensis strains. These methods of discrimination were applied to intact yeast cells of B. bruxellensis strains and on wines spoiled by the same strains. Eleven wine isolates of B. bruxellensis were evaluated for volatile phenol production in red wine and their genetic diversity was determined by Restriction Endonuclease Analysis-Pulsed Field Gel Electrophoresis (REA-PFGE). Fourier transform mid-infrared (FTMIR) spectroscopy was used to obtain spectral fingerprints of the spoiled wines. Attenuated total reflectance (ATR) was used to obtain spectral fingerprints from the intact cells of the 11 B. bruxellensis strains. The groupings from the genetic fingerprints obtained with REA-PFGE were used as reference firstly for comparison with the groupings observed with the FTMIR spectral fingerprint of the wines and secondly for the FTIR-ATR spectral fingerprints from the whole cells. Results indicated that ATR-IR spectra obtained by scanning whole cells of B. bruxellensis could be useful for rapid strain typing in comparison or complementary to molecular techniques and FTMIR spectra from wines provide a useful resource for the discrimination between B. bruxellensis contaminated wines. Copyright © 2010 Elsevier B.V. All rights reserved.

Growth curves of carcass traits obtained by ultrasonography in three lines of Nellore cattle selected for body weight.

PubMed

Coutinho, C C; Mercadante, M E Z; Jorge, A M; Paz, C C P; El Faro, L; Monteiro, F M

2015-10-30

The effect of selection for postweaning weight was evaluated within the growth curve parameters for both growth and carcass traits. Records of 2404 Nellore animals from three selection lines were analyzed: two selection lines for high postweaning weight, selection (NeS) and traditional (NeT); and a control line (NeC) in which animals were selected for postweaning weight close to the average. Body weight (BW), hip height (HH), rib eye area (REA), back fat thickness (BFT), and rump fat thickness (RFT) were measured and records collected from animals 8 to 20 (males) and 11 to 26 (females) months of age. The parameters A (asymptotic value) and k (growth rate) were estimated using the nonlinear model procedure of the Statistical Analysis System program, which included fixed effect of line (NeS, NeT, and NeC) in the model, with the objective to evaluate differences in the estimated parameters between lines. Selected animals (NeS and NeT) showed higher growth rates than control line animals (NeC) for all traits. Line effect on curves parameters was significant (P < 0.001) for BW, HH, and REA in males, and for BFT and RFT in females. Selection for postweaning weight was effective in altering growth curves, resulting in animals with higher growth potential.

novPTMenzy: a database for enzymes involved in novel post-translational modifications

PubMed Central

Khater, Shradha; Mohanty, Debasisa

2015-01-01

With the recent discoveries of novel post-translational modifications (PTMs) which play important roles in signaling and biosynthetic pathways, identification of such PTM catalyzing enzymes by genome mining has been an area of major interest. Unlike well-known PTMs like phosphorylation, glycosylation, SUMOylation, no bioinformatics resources are available for enzymes associated with novel and unusual PTMs. Therefore, we have developed the novPTMenzy database which catalogs information on the sequence, structure, active site and genomic neighborhood of experimentally characterized enzymes involved in five novel PTMs, namely AMPylation, Eliminylation, Sulfation, Hydroxylation and Deamidation. Based on a comprehensive analysis of the sequence and structural features of these known PTM catalyzing enzymes, we have created Hidden Markov Model profiles for the identification of similar PTM catalyzing enzymatic domains in genomic sequences. We have also created predictive rules for grouping them into functional subfamilies and deciphering their mechanistic details by structure-based analysis of their active site pockets. These analytical modules have been made available as user friendly search interfaces of novPTMenzy database. It also has a specialized analysis interface for some PTMs like AMPylation and Eliminylation. The novPTMenzy database is a unique resource that can aid in discovery of unusual PTM catalyzing enzymes in newly sequenced genomes. Database URL: http://www.nii.ac.in/novptmenzy.html PMID:25931459

Different digestion enzymes used for human pancreatic islet isolation: A mixed treatment comparison (MTC) meta-analysis

PubMed Central

Rheinheimer, Jakeline; Ziegelmann, Patrícia Klarmann; Carlessi, Rodrigo; Reck, Luciana Ross; Bauer, Andrea Carla; Leitão, Cristiane Bauermann; Crispim, Daisy

2014-01-01

Collagenases are critical reagents determining yield and quality of isolated human pancreatic islets and may affect islet transplantation outcome. Some islet transplantation centers have compared 2 or more collagenase blends; however, the results regarding differences in quantity and quality of islets are conflicting. Thus, for the first time, a mixed treatment comparison (MTC) meta-analysis was carried out to compile data about the effect of different collagenases used for human pancreas digestion on islet yield, purity, viability and stimulation index (SI). Pubmed, Embase and Cochrane libraries were searched. Of 755 articles retrieved, a total of 15 articles fulfilled the eligibility criteria and were included in the MTC meta-analysis. Our results revealed that Vitacyte and Liberase MTF were associated with a small increase in islet yield (islet equivalent number/g pancreas) when compared with Sevac enzyme [standardized mean difference (95% credible interval – CrI) = −2.19 (−4.25 to −0.21) and −2.28 (−4.49 to −0.23), respectively]. However, all other enzyme comparisons did not show any significant difference regarding islet yield. Purity and viability percentages were not significantly different among any of the analyzed digestion enzymes. Interestingly, Vitacyte and Serva NB1 were associated with increased SI when compared with Liberase MTF enzyme [unstandardized weighted mean difference (95% CrI) = −1.69 (−2.87 to −0.51) and −1.07 (−1.79 to −0.39), respectively]. In conclusion, our MTC meta-analysis suggests that the digestion enzymes currently being used for islet isolation works with similar efficiency regarding islet yield, purity and viability; however, Vitacyte and Serva NB1 enzymes seem to be associated with an improved SI as compared with Liberase MTF. PMID:25437379

Distinct Kynureninase and Hydroxykynureninase Activities in Microorganisms: Occurrence and Properties of a Single Physiologically Discrete Enzyme in Yeast

PubMed Central

Shetty, A. S.; Gaertner, F. H.

1973-01-01

(i) Saccharomyces cerevisiae grown in the presence of 1.0 mM l-tryptophan slowly excreted fluorescent material that was chromatographically identifiable as 3-hydroxyanthranilate but did not excrete detectable amounts of anthranilate nor rapidly deplete the medium of l-tryptophan. Under similar growth conditions, Neurospora crassa rapidly excretes anthranilate and rapidly depletes the medium of l-tryptophan. (ii) Chromatographic analysis of crude extracts from yeast revealed a single kynureninase-type enzyme whose synthesis was not measurably affected by the presence of tryptophan in the medium. Previous studies have provided evidence for two kynureninase-type enzymes in N. crassa, an inducible kynureninase and a constitutive hydroxykynureninase. (iii) Kinetic analysis of the partially purified yeast enzyme provided Michaelis constants for l-3-hydroxykynurenine and l-kynurenine of 6.7 × 10−6 and 5.4 × 10−4 M, respectively. This and other kinetic properties of the yeast enzyme are comparable to those reported for the constitutive enzyme from N. crassa. (iv) These findings suggest that S. cerevisiae has in common with N. crassa the biosynthetic enzyme hydroxykynureninase but lacks the catabolic enzyme kynureninase. Therefore, it can be predicted that, unlike N. crassa, S. cerevisiae does not carry out the tryptophan-anthranilate cycle. Distinct kynureninase-type enzymes may exist in other microorganisms and in mammals. PMID:4266242

Dynamic Perturbation of the Active Site Determines Reversible Thermal Inactivation in Glycoside Hydrolase Family 12.

PubMed

Jiang, Xukai; Li, Wen; Chen, Guanjun; Wang, Lushan

2017-02-27

The temperature dependence of enzyme catalysis is highly debated. Specifically, how high temperatures induce enzyme inactivation has broad implications for both fundamental and applied science. Here, we explored the mechanism of the reversible thermal inactivation in glycoside hydrolase family 12 (GH12) using comparative molecular dynamics simulations. First, we investigated the distribution of structural flexibility over the enzyme and found that the active site was the general thermal-sensitive region in GH12 cellulases. The dynamic perturbation of the active site before enzyme denaturation was explored through principal-component analysis, which indicated that variations in the collective motion and conformational ensemble of the active site may precisely correspond to enzyme transition from its active form to the inactive form. Furthermore, the degree of dynamic perturbation of the active site was found to be negatively correlated with the melting temperatures of GH12 enzymes, further proving the importance of the dynamic stability of the active site. Additionally, analysis of the residue-interaction network revealed that the active site in thermophilic enzyme was capable of forming additional contacts with other amino acids than those observed in the mesophilic enzyme. These interactions are likely the key mechanisms underlying the differences in rigidity of the active site. These findings provide further biophysical insights into the reversible thermal inactivation of enzymes and potential applications in future protein engineering.

Purification and characterization of a milk-clotting aspartic protease from Withania coagulans fruit.

PubMed

Salehi, Mahmoud; Aghamaali, Mahmoud Reza; Sajedi, Reza H; Asghari, S Mohsen; Jorjani, Eisa

2017-05-01

Withania coagulans fruit has traditionally been used as milk coagulant. The present study reports the purification and characterization of an aspartic protease from W. coagulans fruit. The enzyme was purified via fractional ammonium sulfate precipitation and cation exchange chromatography. SDS-PAGE analysis revealed the presence of a monomeric protein with molecular weight of 31kDa. Proteolytic activity (PA) of the protease was evaluated using casein, and the milk-clotting activity (MCA) was analyzed by skim milk. The K m and V max values of the enzyme for casein were obtained to be 1.29mg/ml and 0.035μmol Tyr/min, respectively. Optimal temperature and pH were 65°C and 5.5, respectively. After incubation of enzyme at 65°C for 1h, 73% of PA was remained which demonstrated high thermal stability of the enzyme. Mass spectrometry analysis of the purified protease and enzyme assays in the presence of protease inhibitors indicated that aspartic protease was the only responsible enzyme in milk coagulation. Furthermore, by investigating the effect of salts on enzyme activity, it was observed that both NaCl and CaCl 2 reduced enzyme activity. These characteristics of the protease suggest that the enzyme may be suitable for producing low salt content cheeses. Copyright © 2017 Elsevier B.V. All rights reserved.

An Approach for Identification of Novel Drug Targets in Streptococcus pyogenes SF370 Through Pathway Analysis.

PubMed

Singh, Satendra; Singh, Dev Bukhsh; Singh, Anamika; Gautam, Budhayash; Ram, Gurudayal; Dwivedi, Seema; Ramteke, Pramod W

2016-12-01

Streptococcus pyogenes is one of the most important pathogens as it is involved in various infections affecting upper respiratory tract and skin. Due to the emergence of multidrug resistance and cross-resistance, S. Pyogenes is becoming more pathogenic and dangerous. In the present study, an in silico comparative analysis of total 65 metabolic pathways of the host (Homo sapiens) and the pathogen was performed. Initially, 486 paralogous enzymes were identified so that they can be removed from possible drug target list. The 105 enzymes of the biochemical pathways of S. pyogenes from the KEGG metabolic pathway database were compared with the proteins from the Homo sapiens by performing a BLASTP search against the non-redundant database restricted to the Homo sapiens subset. Out of these, 83 enzymes were identified as non-human homologous while 30 enzymes of inadequate amino acid length were removed for further processing. Essential enzymes were finally mined from remaining 53 enzymes. Finally, 28 essential enzymes were identified in S. pyogenes SF370 (serotype M1). In subcellular localization study, 18 enzymes were predicted with cytoplasmic localization and ten enzymes with the membrane localization. These ten enzymes with putative membrane localization should be of particular interest. Acyl-carrier-protein S-malonyltransferase, DNA polymerase III subunit beta and dihydropteroate synthase are novel drug targets and thus can be used to design potential inhibitors against S. pyogenes infection. 3D structure of dihydropteroate synthase was modeled and validated that can be used for virtual screening and interaction study of potential inhibitors with the target enzyme.

Technology Prospecting on Enzymes: Application, Marketing and Engineering

PubMed Central

Li, Shuang; Yang, Xiaofeng; Yang, Shuai; Zhu, Muzi; Wang, Xiaoning

2012-01-01

Enzymes are protein molecules functioning as specialized catalysts for chemical reactions. They have contributed greatly to the traditional and modern chemical industry by improving existing processes. In this article, we first give a survey of representative industrial applications of enzymes, focusing on the technical applications, feed industry, food processing and cosmetic products. The recent important developments and applications of enzymes in industry are reviewed. Then large efforts are dedicated to the worldwide enzyme market from the demand and production perspectives. Special attention is laid on the Chinese enzyme market. Although enzyme applications are being developed in full swing, breakthroughs are needed to overcome their weaknesses in maintaining activities during the catalytic processes. Strategies of metagomic analysis, cell surface display technology and cell-free system might give valuable solutions in novel enzyme exploiting and enzyme engineering. PMID:24688658

dEMBF: A Comprehensive Database of Enzymes of Microalgal Biofuel Feedstock.

PubMed

Misra, Namrata; Panda, Prasanna Kumar; Parida, Bikram Kumar; Mishra, Barada Kanta

2016-01-01

Microalgae have attracted wide attention as one of the most versatile renewable feedstocks for production of biofuel. To develop genetically engineered high lipid yielding algal strains, a thorough understanding of the lipid biosynthetic pathway and the underpinning enzymes is essential. In this work, we have systematically mined the genomes of fifteen diverse algal species belonging to Chlorophyta, Heterokontophyta, Rhodophyta, and Haptophyta, to identify and annotate the putative enzymes of lipid metabolic pathway. Consequently, we have also developed a database, dEMBF (Database of Enzymes of Microalgal Biofuel Feedstock), which catalogues the complete list of identified enzymes along with their computed annotation details including length, hydrophobicity, amino acid composition, subcellular location, gene ontology, KEGG pathway, orthologous group, Pfam domain, intron-exon organization, transmembrane topology, and secondary/tertiary structural data. Furthermore, to facilitate functional and evolutionary study of these enzymes, a collection of built-in applications for BLAST search, motif identification, sequence and phylogenetic analysis have been seamlessly integrated into the database. dEMBF is the first database that brings together all enzymes responsible for lipid synthesis from available algal genomes, and provides an integrative platform for enzyme inquiry and analysis. This database will be extremely useful for algal biofuel research. It can be accessed at http://bbprof.immt.res.in/embf.

Metagenomic insights into the rumen microbial fibrolytic enzymes in Indian crossbred cattle fed finger millet straw.

PubMed

Jose, V Lyju; Appoothy, Thulasi; More, Ravi P; Arun, A Sha

2017-12-01

The rumen is a unique natural habitat, exhibiting an unparalleled genetic resource of fibrolytic enzymes of microbial origin that degrade plant polysaccharides. The objectives of this study were to identify the principal plant cell wall-degrading enzymes and the taxonomic profile of rumen microbial communities that are associated with it. The cattle rumen microflora and the carbohydrate-active enzymes were functionally classified through a whole metagenomic sequencing approach. Analysis of the assembled sequences by the Carbohydrate-active enzyme analysis Toolkit identified the candidate genes encoding fibrolytic enzymes belonging to different classes of glycoside hydrolases(11,010 contigs), glycosyltransferases (6366 contigs), carbohydrate esterases (4945 contigs), carbohydrate-binding modules (1975 contigs), polysaccharide lyases (480 contigs), and auxiliary activities (115 contigs). Phylogenetic analysis of CAZyme encoding contigs revealed that a significant proportion of CAZymes were contributed by bacteria belonging to genera Prevotella, Bacteroides, Fibrobacter, Clostridium, and Ruminococcus. The results indicated that the cattle rumen microbiome and the CAZymes are highly complex, structurally similar but compositionally distinct from other ruminants. The unique characteristics of rumen microbiota and the enzymes produced by resident microbes provide opportunities to improve the feed conversion efficiency in ruminants and serve as a reservoir of industrially important enzymes for cellulosic biofuel production.

Model-Assisted Analysis of Sugar Metabolism throughout Tomato Fruit Development Reveals Enzyme and Carrier Properties in Relation to Vacuole Expansion[W

PubMed Central

Beauvoit, Bertrand P.; Colombié, Sophie; Monier, Antoine; Andrieu, Marie-Hélène; Biais, Benoit; Bénard, Camille; Chéniclet, Catherine; Dieuaide-Noubhani, Martine; Nazaret, Christine; Mazat, Jean-Pierre; Gibon, Yves

2014-01-01

A kinetic model combining enzyme activity measurements and subcellular compartmentation was parameterized to fit the sucrose, hexose, and glucose-6-P contents of pericarp throughout tomato (Solanum lycopersicum) fruit development. The model was further validated using independent data obtained from domesticated and wild tomato species and on transgenic lines. A hierarchical clustering analysis of the calculated fluxes and enzyme capacities together revealed stage-dependent features. Cell division was characterized by a high sucrolytic activity of the vacuole, whereas sucrose cleavage during expansion was sustained by both sucrose synthase and neutral invertase, associated with minimal futile cycling. Most importantly, a tight correlation between flux rate and enzyme capacity was found for fructokinase and PPi-dependent phosphofructokinase during cell division and for sucrose synthase, UDP-glucopyrophosphorylase, and phosphoglucomutase during expansion, thus suggesting an adaptation of enzyme abundance to metabolic needs. In contrast, for most enzymes, flux rates varied irrespectively of enzyme capacities, and most enzymes functioned at <5% of their maximal catalytic capacity. One of the major findings with the model was the high accumulation of soluble sugars within the vacuole together with organic acids, thus enabling the osmotic-driven vacuole expansion that was found during cell division. PMID:25139005

dEMBF: A Comprehensive Database of Enzymes of Microalgal Biofuel Feedstock

PubMed Central

Misra, Namrata; Panda, Prasanna Kumar; Parida, Bikram Kumar; Mishra, Barada Kanta

2016-01-01

Microalgae have attracted wide attention as one of the most versatile renewable feedstocks for production of biofuel. To develop genetically engineered high lipid yielding algal strains, a thorough understanding of the lipid biosynthetic pathway and the underpinning enzymes is essential. In this work, we have systematically mined the genomes of fifteen diverse algal species belonging to Chlorophyta, Heterokontophyta, Rhodophyta, and Haptophyta, to identify and annotate the putative enzymes of lipid metabolic pathway. Consequently, we have also developed a database, dEMBF (Database of Enzymes of Microalgal Biofuel Feedstock), which catalogues the complete list of identified enzymes along with their computed annotation details including length, hydrophobicity, amino acid composition, subcellular location, gene ontology, KEGG pathway, orthologous group, Pfam domain, intron-exon organization, transmembrane topology, and secondary/tertiary structural data. Furthermore, to facilitate functional and evolutionary study of these enzymes, a collection of built-in applications for BLAST search, motif identification, sequence and phylogenetic analysis have been seamlessly integrated into the database. dEMBF is the first database that brings together all enzymes responsible for lipid synthesis from available algal genomes, and provides an integrative platform for enzyme inquiry and analysis. This database will be extremely useful for algal biofuel research. It can be accessed at http://bbprof.immt.res.in/embf. PMID:26727469

Microbiological characterization and probiotic potential of koko and koko sour water, African spontaneously fermented millet porridge and drink.

PubMed

Lei, V; Jakobsen, M

2004-01-01

To identify and examine the diversity of predominant lactic acid bacteria (LAB) in koko and koko sour water (KSW) from different Ghanaian production sites with regard to pattern of fermentation (API 50 CHL), genotype, antimicrobial activity, and resistance to low pH and bile salts. In total 215 LAB were isolated from koko and KSW. The isolates were identified using intergenic transcribed spacers (ITS)-PCR restriction fragment length polymorphism (RFLP), API 50 CHL, restriction enzyme analysis with pulsed-field gel electrophoresis (REA-PFGE) and sequencing of the 16S rRNA gene. The dominating micro-organisms in koko was found to be Weisella confusa and Lactobacillus fermentum, followed by Lact. salivarius and Pediococcus spp. Chemometric data analysis were used to link the LAB species to the different production stages and production sites. At intra-species level the isolates were found to have a great diversity. The isolates were investigated for antimicrobial activity using agar diffusion assays, and acid and bile tolerance. Most isolates showed low levels of antimicrobial activity towards the indicator strain Listeria innocua, but not towards the bacteriocin-sensitive Lact. sakei. Growth of all LAB isolates was unaffected by the presence of 0.3% (v/v) oxgall bile. The isolates were able to survive, but were not able to grow in growth medium adjusted to pH 2.5. The dominating LAB of koko and KSW were W. confusa and Lact. fermentum showing a pronounced taxonomic biodiversity at sub-species level between stages within the production as well as between production sites. Other species observed in KSW were Lact. salivarius, Ped. pentosaceus, Ped. acidilactici and Lact. paraplantarum. They occurred in levels of 108 CFU ml-1 in fresh KSW and showed uniform antimicrobial activity, and acid and bile tolerance. The present study gives a detailed picture of the taxonomy and diversity of LAB in an African-fermented millet product that may have potential as a probiotic product for the local population. The chemometric tools Principal Component Analysis and anova Partial Least Squares Regression were proven to be useful in the analysis of microbial groupings and associations with specific sites and stages in the production of koko and KSW.

Biochemical characterization of Aspergillus awamori exoinulinase: substrate binding characteristics and regioselectivity of hydrolysis.

PubMed

Kulminskaya, Anna A; Arand, Michael; Eneyskaya, Elena V; Ivanen, Dina R; Shabalin, Konstantin A; Shishlyannikov, Sergei M; Saveliev, Andrew N; Korneeva, Olga S; Neustroev, Kirill N

2003-08-21

1H-NMR analysis was applied to investigate the hydrolytic activity of Aspergillus awamori inulinase. The obtained NMR signals and deduced metabolite pattern revealed that the enzyme cleaves off only fructose from inulin and does not possess transglycosylating activity. Kinetics for the enzyme hydrolysis of inulooligosaccharides with different degree of polymerization (d.p.) were recorded. The enzyme hydrolyzed both beta2,1- as well as beta2,6-fructosyl linkages in fructooligosaccharides. From the k(cat)/K(m) ratios obtained with inulooligosaccharides with d.p. from 2 to 7, we deduce that the catalytic site of the inulinase contains at least five fructosyl-binding sites and can be classified as exo-acting enzyme. Product analysis of inulopentaose and inulohexaose hydrolysis by the Aspergillus inulinase provided no evidence for a possible multiple-attack mode of action, suggesting that the enzyme acts exclusively as an exoinulinase.

Topological analysis of metabolic control.

PubMed

Sen, A K

1990-12-01

A topological approach is presented for the analysis of control and regulation in metabolic pathways. In this approach, the control structure of a metabolic pathway is represented by a weighted directed graph. From an inspection of the topology of the graph, the control coefficients of the enzymes are evaluated in a heuristic manner in terms of the enzyme elasticities. The major advantage of the topological approach is that it provides a visual framework for (1) calculating the control coefficients of the enzymes, (2) analyzing the cause-effect relationships of the individual enzymes, (3) assessing the relative importance of the enzymes in metabolic regulation, and (4) simplifying the structure of a given pathway, from a regulatory viewpoint. Results are obtained for (a) an unbranched pathway in the absence of feedback the feedforward regulation and (b) an unbranched pathway with feedback inhibition. Our formulation is based on the metabolic control theory of Kacser and Burns (1973) and Heinrich and Rapoport (1974).

An evaluation of the lamb vision system as a predictor of lamb carcass red meat yield percentage.

PubMed

Brady, A S; Belk, K E; LeValley, S B; Dalsted, N L; Scanga, J A; Tatum, J D; Smith, G C

2003-06-01

An objective method for predicting red meat yield in lamb carcasses is needed to accurately assess true carcass value. This study was performed to evaluate the ability of the lamb vision system (LVS; Research Management Systems USA, Fort Collins, CO) to predict fabrication yields of lamb carcasses. Lamb carcasses (n = 246) were evaluated using LVS and hot carcass weight (HCW), as well as by USDA expert and on-line graders, before fabrication of carcass sides to either bone-in or boneless cuts. On-line whole number, expert whole-number, and expert nearest-tenth USDA yield grades and LVS + HCW estimates accounted for 53, 52, 58, and 60%, respectively, of the observed variability in boneless, saleable meat yields, and accounted for 56, 57, 62, and 62%, respectively, of the variation in bone-in, saleable meat yields. The LVS + HCW system predicted 77, 65, 70, and 87% of the variation in weights of boneless shoulders, racks, loins, and legs, respectively, and 85, 72, 75, and 86% of the variation in weights of bone-in shoulders, racks, loins, and legs, respectively. Addition of longissimus muscle area (REA), adjusted fat thickness (AFT), or both REA and AFT to LVS + HCW models resulted in improved prediction of boneless saleable meat yields by 5, 3, and 5 percentage points, respectively. Bone-in, saleable meat yield estimations were improved in predictive accuracy by 7.7, 6.6, and 10.1 percentage points, and in precision, when REA alone, AFT alone, or both REA and AFT, respectively, were added to the LVS + HCW output models. Use of LVS + HCW to predict boneless red meat yields of lamb carcasses was more accurate than use of current on-line whole-number, expert whole-number, or expert nearest-tenth USDA yield grades. Thus, LVS + HCW output, when used alone or in combination with AFT and/or REA, improved on-line estimation of boneless cut yields from lamb carcasses. The ability of LVS + HCW to predict yields of wholesale cuts suggests that LVS could be used as an objective means for pricing carcasses in a value-based marketing system.

CURRENT SITUATION OF MEDICINE III AND CHALLENGES.

PubMed

Ferreira, Lydia Masako

2015-01-01

Describe the current situation of the area Medicine III of CAPES and detect challenges for the next four years of evaluation. The area's documents and reports of meetings were read from 2004 to 2013 Medicine III Capes as well as reports and evaluation form of each Postgraduate Program (PPG) of the area and the sub-page of the area from the Capes website. The data relating to the evaluation process, the assessment form and faculty, student and scientific production data of all of Post-Graduate Programs of Medicine III were computed and analyzed. From these data were detected the challenges of the area for the next four years (2013-2016). Among the 3,806 PPG, Medicine III had 41 PPG during last triennial evaluation and progressed from 18% to 43% of PPG very good or more concept (triennium 2001-2003 and 2010-2012). Most PPG were located in the South-East region (32), three in the South and two in the North-East. There was no PPG in North or Central-West regions. In 2013 and 2014 there were four approved Professional Master Degree Programs and one Master (M) and Doctorate (PhD). The average of permanent professors was 558 teachers with about three students/professor. The number of PhD graduates has increased as well as the reason PhD/MD. The proportion of in high impact periodicals (A1, A2, B1 and B2) jumped from 30% to 50% demonstrating positive community response to the policy area. The challenges identified were: decrease regional asymmetry, increase the number of masters and doctors of excellence, reassessment of Brazilian journals, stimulate and set internationalization indicators, including post-doctors and definition of its indicators, the PPG nucleation analysis, PPG 3x3, include primary and secondary education, professional master and indicators of technological scientific production and solidarity. Medicine III has been scientifically consolidated and their scientific researchers demonstrated maturity reaching a high level and matched to areas of greatest tradition and history. For the maintenance and advancement of the area some challenges and goals were established to be developed in the period from 2013 to 2016. Descrever a situação atual da área Medicina III da Capes e detectar os desafios para o próximo quadriênio de avaliação. Os documentos e relatórios de reuniões de área a partir de 2004 até 2013 da Medicina III da Capes foram lidos assim como os relatórios e as fichas de avaliação de cada Programa de Pós Graduação (PPG) da área e a subpágina da área no site da Capes. Os dados referentes ao Processo de Avaliação, à Ficha de Avaliação e aos dados do Corpo Docente, Discente e Produção científica de todos os PPG da Medicina III foram computados e analisados. A partir desses dados foram detectados os desafios da área para o próximo quadriênio (2013-2016). Dentre os 3.806 PPG, a Medicina III apresentava 41 PPG durante última avaliação trienal e evoluiu de 18% para 43% de PPG > conceito muito bom (Triênios 2001-2003 e 2010-2012). A maioria dos PPG se localizavam na Região Sudeste (32), três no Sul e dois no Nordeste. Não havia nenhum PPG no Norte ou Centro-Oeste. Em 2013 e 2014 foram aprovadas quatro mestrados profissionais e um mestrado e doutorado acadêmico. A média de docente permanente no triênio foi de 558 docentes com cerca de três alunos titulados/docente. O número de doutores titulados tem aumentado assim como a razão Doutorado/Mestrado. A proporção de publicações em periódicos de maior impacto (A1, A2, B1 e B2) saltou de 30% para 50% demonstrando resposta positiva da comunidade à política da área. Os desafios detectados foram: diminuir assimetria regional, elevar o número de mestres e doutores de excelência, reavaliação das revistas nacionais, estimular e definir indicadores de internacionalização, inclusão de pós-doutores e definição de seus indicadores, análise da nucleação do PPG, PPG 3x3, inclusão ensino médio e fundamental, mestrado profissional e indicadores da produção científico-tecnológica e solidariedade. A Medicina III tem se consolidado cientificamente e seus pesquisadores demonstraram maturidade científica chegando a um patamar elevado e igualado às áreas de maior tradição e história. Para a manutenção e avanço da área alguns desafios e metas foram traçadas para serem desenvolvidas no quadriênio 2013-2016.

Policies for Use of Real-World Data in Health Technology Assessment (HTA): A Comparative Study of Six HTA Agencies.

PubMed

Makady, Amr; Ham, Renske Ten; de Boer, Anthonius; Hillege, Hans; Klungel, Olaf; Goettsch, Wim

2017-04-01

Randomized controlled trials provide robust data on the efficacy of interventions rather than on effectiveness. Health technology assessment (HTA) agencies worldwide are thus exploring whether real-world data (RWD) may provide alternative sources of data on effectiveness of interventions. Presently, an overview of HTA agencies' policies for RWD use in relative effectiveness assessments (REA) is lacking. To review policies of six European HTA agencies on RWD use in REA of drugs. A literature review and stakeholder interviews were conducted to collect information on RWD policies for six agencies: the Dental and Pharmaceutical Benefits Agency (Sweden), the National Institute for Health and Care Excellence (United Kingdom), the Institute for Quality and Efficiency in Healthcare (Germany), the High Authority for Health (France), the Italian Medicines Agency (Italy), and the National Healthcare Institute (The Netherlands). The following contexts for RWD use in REA of drugs were reviewed: initial reimbursement discussions, pharmacoeconomic analyses, and conditional reimbursement schemes. We identified 13 policy documents and 9 academic publications, and conducted 6 interviews. Policies for RWD use in REA of drugs notably differed across contexts. Moreover, policies differed between HTA agencies. Such variations might discourage the use of RWD for HTA. To facilitate the use of RWD for HTA across Europe, more alignment of policies seems necessary. Recent articles and project proposals of the European network of HTA may provide a starting point to achieve this. Copyright © 2017 International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc. All rights reserved.

Herd-of-origin effect on the post-weaning performance of centrally tested Nellore beef cattle.

PubMed

de Rezende Neves, Haroldo Henrique; Polin dos Reis, Felipe; Motta Paterno, Flávia; Rocha Guarini, Aline; Carvalheiro, Roberto; da Silva, Lilian Regina; de Oliveira, João Ademir; Aidar de Queiroz, Sandra

2014-10-01

The objective of a performance test station is to evaluate the performance of potential breeding bulls earlier in order to decrease the generation interval and increase genetic gain as well. This study evaluates the herd-of-origin influence on end-of-test weight (ETW), average daily weight gain during testing (ADG), average daily weight gain during the adjustment period (ADGadj), rib eye area (REA), marbling (MARB), subcutaneous fat thickness (SFT), conformation (C), early finishing (EF), muscling (M), navel (N) and temperament (T) scores, and scrotal circumference (SC) of Nellore cattle that underwent a performance test. We evaluated 664 animals that participated in the performance tests conducted at the Center for Performance CRV Lagoa between 2007 and 2012. Components of variance for each trait were estimated by an animal model (model 1), using the restricted maximum likelihood method. An alternative animal model (model 2) included, in addition to the fixed effects present in S1, the non-correlated random effect of herd-year (HY). A significant HY effect was observed on ETW, REA, SFT, ADGadj, C, and Cw (p < 0.05). The estimated heritability of all traits decreased when the HY effect was included in the model; also, the bull rank, in deciles, changed significantly for traits ETW, REA, SFT, and C. The adjustment period did not completely remove the environmental effect of herd of origin on ETW, REA, SFT, and C. It is recommended that the herd-of-origin effect should be included in the statistical models used to predict the breeding values of the participants of these performance tests.

Regulation of yeast central metabolism by enzyme phosphorylation

PubMed Central

Oliveira, Ana Paula; Ludwig, Christina; Picotti, Paola; Kogadeeva, Maria; Aebersold, Ruedi; Sauer, Uwe

2012-01-01

As a frequent post-translational modification, protein phosphorylation regulates many cellular processes. Although several hundred phosphorylation sites have been mapped to metabolic enzymes in Saccharomyces cerevisiae, functionality was demonstrated for few of them. Here, we describe a novel approach to identify in vivo functionality of enzyme phosphorylation by combining flux analysis with proteomics and phosphoproteomics. Focusing on the network of 204 enzymes that constitute the yeast central carbon and amino-acid metabolism, we combined protein and phosphoprotein levels to identify 35 enzymes that change their degree of phosphorylation during growth under five conditions. Correlations between previously determined intracellular fluxes and phosphoprotein abundances provided first functional evidence for five novel phosphoregulated enzymes in this network, adding to nine known phosphoenzymes. For the pyruvate dehydrogenase complex E1 α subunit Pda1 and the newly identified phosphoregulated glycerol-3-phosphate dehydrogenase Gpd1 and phosphofructose-1-kinase complex β subunit Pfk2, we then validated functionality of specific phosphosites through absolute peptide quantification by targeted mass spectrometry, metabolomics and physiological flux analysis in mutants with genetically removed phosphosites. These results demonstrate the role of phosphorylation in controlling the metabolic flux realised by these three enzymes. PMID:23149688

Applying functional metagenomics to search for novel lignocellulosic enzymes in a microbial consortium derived from a thermophilic composting phase of sugarcane bagasse and cow manure.

PubMed

Colombo, Lívia Tavares; de Oliveira, Marcelo Nagem Valério; Carneiro, Deisy Guimarães; de Souza, Robson Assis; Alvim, Mariana Caroline Tocantins; Dos Santos, Josenilda Carlos; da Silva, Cynthia Canêdo; Vidigal, Pedro Marcus Pereira; da Silveira, Wendel Batista; Passos, Flávia Maria Lopes

2016-09-01

Environments where lignocellulosic biomass is naturally decomposed are sources for discovery of new hydrolytic enzymes that can reduce the high cost of enzymatic cocktails for second-generation ethanol production. Metagenomic analysis was applied to discover genes coding carbohydrate-depleting enzymes from a microbial laboratory subculture using a mix of sugarcane bagasse and cow manure in the thermophilic composting phase. From a fosmid library, 182 clones had the ability to hydrolyse carbohydrate. Sequencing of 30 fosmids resulted in 12 contigs encoding 34 putative carbohydrate-active enzymes belonging to 17 glycosyl hydrolase (GH) families. One third of the putative proteins belong to the GH3 family, which includes β-glucosidase enzymes known to be important in the cellulose-deconstruction process but present with low activity in commercial enzyme preparations. Phylogenetic analysis of the amino acid sequences of seven selected proteins, including three β-glucosidases, showed low relatedness with protein sequences deposited in databases. These findings highlight microbial consortia obtained from a mixture of decomposing biomass residues, such as sugar cane bagasse and cow manure, as a rich resource of novel enzymes potentially useful in biotechnology for saccharification of lignocellulosic substrate.

The kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats: calibration to the indirect immunofluorescence assay and computerized standardization of results through normalization to control values.

PubMed Central

Barlough, J E; Jacobson, R H; Downing, D R; Lynch, T J; Scott, F W

1987-01-01

The computer-assisted, kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats was calibrated to the conventional indirect immunofluorescence assay by linear regression analysis and computerized interpolation (generation of "immunofluorescence assay-equivalent" titers). Procedures were developed for normalization and standardization of kinetics-based enzyme-linked immunosorbent assay results through incorporation of five different control sera of predetermined ("expected") titer in daily runs. When used with such sera and with computer assistance, the kinetics-based enzyme-linked immunosorbent assay minimized both within-run and between-run variability while allowing also for efficient data reduction and statistical analysis and reporting of results. PMID:3032390

The kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats: calibration to the indirect immunofluorescence assay and computerized standardization of results through normalization to control values.

PubMed

Barlough, J E; Jacobson, R H; Downing, D R; Lynch, T J; Scott, F W

1987-01-01

The computer-assisted, kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats was calibrated to the conventional indirect immunofluorescence assay by linear regression analysis and computerized interpolation (generation of "immunofluorescence assay-equivalent" titers). Procedures were developed for normalization and standardization of kinetics-based enzyme-linked immunosorbent assay results through incorporation of five different control sera of predetermined ("expected") titer in daily runs. When used with such sera and with computer assistance, the kinetics-based enzyme-linked immunosorbent assay minimized both within-run and between-run variability while allowing also for efficient data reduction and statistical analysis and reporting of results.

Analysis of genes involved in glycogen degradation in Escherichia coli.

PubMed

Strydom, Lindi; Jewell, Jonathan; Meier, Michael A; George, Gavin M; Pfister, Barbara; Zeeman, Samuel; Kossmann, Jens; Lloyd, James R

2017-02-01

Escherichia coli accumulate or degrade glycogen depending on environmental carbon supply. Glycogen phosphorylase (GlgP) and glycogen debranching enzyme (GlgX) are known to act on the glycogen polymer, while maltodextrin phosphorylase (MalP) is thought to remove maltodextrins released by GlgX. To examine the roles of these enzymes in more detail, single, double and triple mutants lacking all their activities were produced. GlgX and GlgP were shown to act directly on the glycogen polymer, while MalP most likely catabolised soluble malto-oligosaccharides. Interestingly, analysis of a triple mutant lacking all three enzymes indicates the presence of another enzyme that can release maltodextrins from glycogen. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Conceptual design study for the use of COBE rocket engines on the Tropical Rainfall Measuring Mission

NASA Technical Reports Server (NTRS)

1992-01-01

The objective of this conceptual design study is to verify that the Cosmic Background Explorer (COBE) Hydrazine Propulsion Subsystem (HPS) Rocket Engine Assembly (REA) will satisfy the Tropical Rainfall Measuring Mission (TRMM) requirements and to develop a preliminary thruster module design using the existing REAs. The performance of the COBE HPS 5 lbf thrusters meet the TRMM mission requirements. The preliminary design consists of a single 5 lbf REA REM which is isolation mounted to a spacecraft interface angle bracket (5 or 10 deg angle). The REM incorporates a catalyst bed heater and sensor assembly, and propellant thermal control is achieved by thermostatically controlled heaters on the thruster valves. A ROM cost of approx. $950 K has been estimated for the phase 2 program to finalize the design, fabricate, and test the hardware using mechanical thermostats for thermal control. In the event that solid state thermostats are used, the cost is estimated to be $160 K higher. A ROM cost is approx. $145 K is estimated to study the effects of using Japanese manufactured hydrazine for the TRMM mission.

The influence of memory and attention on the ear advantage in dichotic listening.

PubMed

D'Anselmo, Anita; Marzoli, Daniele; Brancucci, Alfredo

2016-12-01

The role of memory retention and attentional control on hemispheric asymmetry was investigated using a verbal dichotic listening paradigm, with the consonant-vowel syllables (/ba/,/da/,/ga/,/ka/,/pa/and/ta/), while manipulating the focus of attention and the time interval between stimulus and response. Attention was manipulated using three conditions: non-forced (NF), forced left (FL) and forced right (FR) attention. Memory involvement was varied using four delays (0, 1, 3 and 4 s) between stimulus presentation and response. Results showed a significant right ear advantage (REA) in the NF condition and an increased REA in the FR condition. A left ear advantage (LEA) was found in FL condition. The REA increased significantly in the NF attention condition at the 3-s compared to the 0-s delay and in the FR condition at the 1-s compared to the 0-s delay. No modulation of the left ear advantage was observed in the FL condition. These results are discussed in terms of an interaction between attentional processes and memory retention. Copyright © 2016 Elsevier B.V. All rights reserved.

Lignocellulose-converting enzyme activity profiles correlate with molecular systematics and phylogeny grouping in the incoherent genus Phlebia (Polyporales, Basidiomycota).

PubMed

Kuuskeri, Jaana; Mäkelä, Miia R; Isotalo, Jarkko; Oksanen, Ilona; Lundell, Taina

2015-10-19

The fungal genus Phlebia consists of a number of species that are significant in wood decay. Biotechnological potential of a few species for enzyme production and degradation of lignin and pollutants has been previously studied, when most of the species of this genus are unknown. Therefore, we carried out a wider study on biochemistry and systematics of Phlebia species. Isolates belonging to the genus Phlebia were subjected to four-gene sequence analysis in order to clarify their phylogenetic placement at species level and evolutionary relationships of the genus among phlebioid Polyporales. rRNA-encoding (5.8S, partial LSU) and two protein-encoding gene (gapdh, rpb2) sequences were adopted for the evolutionary analysis, and ITS sequences (ITS1+5.8S+ITS2) were aligned for in-depth species-level phylogeny. The 49 fungal isolates were cultivated on semi-solid milled spruce wood medium for 21 days in order to follow their production of extracellular lignocellulose-converting oxidoreductases and carbohydrate active enzymes. Four-gene phylogenetic analysis confirmed the polyphyletic nature of the genus Phlebia. Ten species-level subgroups were formed, and their lignocellulose-converting enzyme activity profiles coincided with the phylogenetic grouping. The highest enzyme activities for lignin modification (manganese peroxidase activity) were obtained for Phlebia radiata group, which supports our previous studies on the enzymology and gene expression of this species on lignocellulosic substrates. Our study implies that there is a species-level connection of molecular systematics (genotype) to the efficiency in production of both lignocellulose-converting carbohydrate active enzymes and oxidoreductases (enzyme phenotype) on spruce wood. Thus, we may propose a similar phylogrouping approach for prediction of lignocellulose-converting enzyme phenotypes in new fungal species or genetically and biochemically less-studied isolates of the wood-decay Polyporales.

Proteomic analysis of scallop hepatopancreatic extract provides insights into marine polysaccharide digestion.

PubMed

Lyu, Qianqian; Jiao, Wenqian; Zhang, Keke; Bao, Zhenmin; Wang, Shi; Liu, Weizhi

2016-12-16

Marine polysaccharides are used in a variety of applications, and the enzymes that degrade these polysaccharides are of increasing interest. The main food source of herbivorous marine mollusks is seaweed, and several polysaccharide-degrading enzymes have been extracted from mollusk digestive glands (hepatopancreases). Here, we used a comprehensive proteomic approach to examine the hepatopancreatic proteins of the Zhikong scallop (Chlamys farreri). We identified 435 proteins, the majority of which were lysosomal enzymes and carbohydrate and protein metabolism enzymes. However, several new enzymes related to polysaccharide metabolism were also identified. Phylogenetic and structural analyses of these enzymes suggest that these polysaccharide-degrading enzymes may have a variety of potential substrate specificities. Taken together, our study characterizes several novel polysaccharide-degrading enzymes in the scallop hepatopancreas and provides an enhanced view of these enzymes and a greater understanding of marine polysaccharide digestion.

MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

EPA Science Inventory

Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

The Kinetics and Inhibition of Gamma-Glutamyl Transpeptidase: A Biochemistry Laboratory Experiment.

ERIC Educational Resources Information Center

Splittgerber, A. G.; Sohl, Julie

1988-01-01

Discusses an enzyme kinetics laboratory experiment involving a two substrate system for undergraduate biochemistry. Uses the enzyme gamma-glutamyl transpeptidase as this enzyme in blood serum is of clinical significance. Notes elevated levels are seen in liver disease, alcoholism, and epilepsy. Uses a spectrophotometer for the analysis. (MVL)

Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

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

Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

Enzyme class identification in cleaning products by hydrolysis followed by derivatization with o-phthaldialdehyde, HPLC and linear discriminant analysis.

PubMed

Beneito-Cambra, M; Bernabé-Zafón, V; Herrero-Martínez, J M; Simó-Alfonso, E F; Ramis-Ramos, G

2009-07-15

The enzymes present in raw materials of the cleaning industry (enzyme industrial concentrates) and in household cleaners were isolated by precipitation with acetone and hydrolyzed with HCl. The resulting amino acids were derivatized with o-phthaldialdehyde, and the derivatives were separated by HPLC. The peaks of 14 amino acids were observed using a C18 column and a multi-segmented gradient of acetonitrile-water in the presence of a 5 mM citric/citrate buffer of pH 6.5. Using either normalized peak areas (divided by the sum of the peak areas of the chromatogram) or ratios of pairs of peak areas as predictor variables, linear discriminant analysis models, capable of predicting the enzyme class, including proteases, lipases, amylases and cellulases, were constructed. For this purpose, both enzyme industrial concentrates and detergent bases spiked with them were included in the training set. In all cases, the enzymes of the evaluation set, including industrial concentrates, spiked detergent bases and commercial cleaners were correctly classified with assignment probabilities higher than 99%.

Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

DOE PAGES

Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.; ...

2017-07-07

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

Network Analysis of Enzyme Activities and Metabolite Levels and Their Relationship to Biomass in a Large Panel of Arabidopsis Accessions[C][W][OA

PubMed Central

Sulpice, Ronan; Trenkamp, Sandra; Steinfath, Matthias; Usadel, Bjorn; Gibon, Yves; Witucka-Wall, Hanna; Pyl, Eva-Theresa; Tschoep, Hendrik; Steinhauser, Marie Caroline; Guenther, Manuela; Hoehne, Melanie; Rohwer, Johann M.; Altmann, Thomas; Fernie, Alisdair R.; Stitt, Mark

2010-01-01

Natural genetic diversity provides a powerful resource to investigate how networks respond to multiple simultaneous changes. In this work, we profile maximum catalytic activities of 37 enzymes from central metabolism and generate a matrix to investigate species-wide connectivity between metabolites, enzymes, and biomass. Most enzyme activities change in a highly coordinated manner, especially those in the Calvin-Benson cycle. Metabolites show coordinated changes in defined sectors of metabolism. Little connectivity was observed between maximum enzyme activities and metabolites, even after applying multivariate analysis methods. Measurements of posttranscriptional regulation will be required to relate these two functional levels. Individual enzyme activities correlate only weakly with biomass. However, when they are used to estimate protein abundances, and the latter are summed and expressed as a fraction of total protein, a significant positive correlation to biomass is observed. The correlation is additive to that obtained between starch and biomass. Thus, biomass is predicted by two independent integrative metabolic biomarkers: preferential investment in photosynthetic machinery and optimization of carbon use. PMID:20699391

Declarative Business Process Modelling and the Generation of ERP Systems

NASA Astrophysics Data System (ADS)

Schultz-Møller, Nicholas Poul; Hølmer, Christian; Hansen, Michael R.

We present an approach to the construction of Enterprise Resource Planning (ERP) Systems, which is based on the Resources, Events and Agents (REA) ontology. This framework deals with processes involving exchange and flow of resources in a declarative, graphically-based manner describing what the major entities are rather than how they engage in computations. We show how to develop a domain-specific language on the basis of REA, and a tool which automatically can generate running web-applications. A main contribution is a proof-of-concept showing that business-domain experts can generate their own applications without worrying about implementation details.

NOAA Weather Radio

Science.gov Websites

Search For Go NWS All NOAA NWR Recepción El área de la recepción nominal para un receptor de Radio cosas afectan la recepción de señas de la radio. Por ejemplo, las extensiones grandes de agua de sal de NWR. Siga leyendo si está dentro del área pero tiene dificultad con recepción. Su seña de la

Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

PubMed Central

Janssen, George; Christis, Chantal; Kooy-Winkelaar, Yvonne; Edens, Luppo; Smith, Drew

2015-01-01

Background Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). Methods Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. Findings The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. Conclusion Currently available digestive enzyme supplements are ineffective in degrading immunogenic gluten epitopes. PMID:26030273

Secretome analysis of Pleurotus eryngii reveals enzymatic composition for ramie stalk degradation.

PubMed

Xie, Chunliang; Luo, Wei; Li, Zhimin; Yan, Li; Zhu, Zuohua; Wang, Jing; Hu, Zhenxiu; Peng, Yuande

2016-01-01

Pleurotus eryngii (P. eryngii) can secrete large amount of hydrolytic and oxidative enzymes to degrade lignocellulosic biomass. In spite of several researches on the individual lignolytic enzymes, a direct deconstruction of lignocellulose by enzyme mixture is not yet possible. Identifying more high-performance enzymes or enzyme complexes will lead to efficient in vitro lignocelluloses degradation. In this report, secretomic analysis was used to search for the new or interesting enzymes for lignocellulose degradation. Besides, the utilization ability of P. eryngii to ramie stalk substrate was evaluated from the degradation of cellulose, hemicellulose, and lignin in medium and six extracellular enzymes activities during different growth stages were discussed. The results showed that a high biological efficiency of 71% was obtained; cellulose, hemicelluloses, and lignin decomposition rates of P. eryngii were 29.2, 26.0, and 51.2%, respectively. Enzyme activity showed that carboxymethyl cellulase, xylanase, laccase, and peroxidase activity peaks appeared at the primordial initiation stage. In addition, we profiled a global view of the secretome of P. eryngii cultivated in ramie stalk media to understand the mechanism behind lignocellulosic biomass hydrolysis. Eighty-seven nonredundant proteins were identified and a diverse group of enzymes, including cellulases, hemicellulases, pectinase, ligninase, protease, peptidases, and phosphatase implicated in lignocellulose degradation were found. In conclusion, the information in this report will be helpful to better understand the lignocelluloses degradation mechanisms of P. eryngii. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Monooxygenase, a Novel Beta-Cypermethrin Degrading Enzyme from Streptomyces sp

PubMed Central

Xiao, Ying; Deng, Yinyue; Chang, Changqing; Zhong, Guohua; Hu, Meiying; Zhang, Lian-Hui

2013-01-01

The widely used insecticide beta-cypermethrin has become a public concern because of its environmental contamination and toxic effects on mammals. In this study, a novel beta-cypermethrin degrading enzyme designated as CMO was purified to apparent homogeneity from a Streptomyces sp. isolate capable of utilizing beta-cypermethrin as a growth substrate. The native enzyme showed a monomeric structure with a molecular mass of 41 kDa and pI of 5.4. The enzyme exhibited the maximal activity at pH 7.5 and 30°C. It was fairly stable in the pH range from 6.5–8.5 and at temperatures below 10°C. The enzyme activity was significantly stimulated by Fe2+, but strongly inhibited by Ag+, Al3+, and Cu2+. The enzyme catalyzed the degradation of beta-cypermethrin to form five products via hydroxylation and diaryl cleavage. A novel beta-cypermethrin detoxification pathway was proposed based on analysis of these products. The purified enzyme was identified as a monooxygenase by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis (MALDI-TOF-MS) and N-terminal protein sequencing. Given that all the characterized pyrethroid-degrading enzymes are the members of hydrolase family, CMO represents the first pyrethroid-degrading monooxygenase identified from environmental microorganisms. Taken together, our findings depict a novel pyrethroid degradation mechanism and indicate that the purified enzyme may be a promising candidate for detoxification of beta-cypermethrin and environmental protection. PMID:24098697

DNA Knots: Theory and Experiments

NASA Astrophysics Data System (ADS)

Sumners, D. W.

Cellular DNA is a long, thread-like molecule with remarkably complex topology. Enzymes that manipulate the geometry and topology of cellular DNA perform many vital cellular processes (including segregation of daughter chromosomes, gene regulation, DNA repair, and generation of antibody diversity). Some enzymes pass DNA through itself via enzyme-bridged transient breaks in the DNA; other enzymes break the DNA apart and reconnect it to different ends. In the topological approach to enzymology, circular DNA is incubated with an enzyme, producing an enzyme signature in the form of DNA knots and links. By observing the changes in DNA geometry (supercoiling) and topology (knotting and linking) due to enzyme action, the enzyme binding and mechanism can often be characterized. This paper will discuss some personal research history, and the tangle model for the analysis of site-specific recombination experiments on circular DNA.

A phylogenetic analysis of normal modes evolution in enzymes and its relationship to enzyme function

PubMed Central

Lai, Jason; Jin, Jing; Kubelka, Jan; Liberles, David A.

2012-01-01

Since the dynamic nature of protein structures is essential for enzymatic function, it is expected that the functional evolution can be inferred from the changes in the protein dynamics. However, dynamics can also diverge neutrally with sequence substitution between enzymes without changes of function. In this study, a phylogenetic approach is implemented to explore the relationship between enzyme dynamics and function through evolutionary history. Protein dynamics are described by normal mode analysis based on a simplified harmonic potential force field applied to the reduced Cα representation of the protein structure while enzymatic function is described by Enzyme Commission (EC) numbers. Similarity of the binding pocket dynamics at each branch of the protein family’s phylogeny was analyzed in two ways: 1) explicitly by quantifying the normal mode overlap calculated for the reconstructed ancestral proteins at each end and 2) implicitly using a diffusion model to obtain the reconstructed lineage-specific changes in the normal modes. Both explicit and implicit ancestral reconstruction identified generally faster rates of change in dynamics compared with the expected change from neutral evolution at the branches of potential functional divergences for the alpha-amylase, D-isomer specific 2-hydroxyacid dehydrogenase, and copper-containing amine oxidase protein families. Normal modes analysis added additional information over just comparing the RMSD of static structures. However, the branch-specific changes were not statistically significant compared to background function-independent neutral rates of change of dynamic properties and blind application of the analysis would not enable prediction of changes in enzyme specificity. PMID:22651983

A phylogenetic analysis of normal modes evolution in enzymes and its relationship to enzyme function.

PubMed

Lai, Jason; Jin, Jing; Kubelka, Jan; Liberles, David A

2012-09-21

Since the dynamic nature of protein structures is essential for enzymatic function, it is expected that functional evolution can be inferred from the changes in protein dynamics. However, dynamics can also diverge neutrally with sequence substitution between enzymes without changes of function. In this study, a phylogenetic approach is implemented to explore the relationship between enzyme dynamics and function through evolutionary history. Protein dynamics are described by normal mode analysis based on a simplified harmonic potential force field applied to the reduced C(α) representation of the protein structure while enzymatic function is described by Enzyme Commission numbers. Similarity of the binding pocket dynamics at each branch of the protein family's phylogeny was analyzed in two ways: (1) explicitly by quantifying the normal mode overlap calculated for the reconstructed ancestral proteins at each end and (2) implicitly using a diffusion model to obtain the reconstructed lineage-specific changes in the normal modes. Both explicit and implicit ancestral reconstruction identified generally faster rates of change in dynamics compared with the expected change from neutral evolution at the branches of potential functional divergences for the α-amylase, D-isomer-specific 2-hydroxyacid dehydrogenase, and copper-containing amine oxidase protein families. Normal mode analysis added additional information over just comparing the RMSD of static structures. However, the branch-specific changes were not statistically significant compared to background function-independent neutral rates of change of dynamic properties and blind application of the analysis would not enable prediction of changes in enzyme specificity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Peri-Implant Crevicular Fluid Analysis, Enzymes and Biomarkers: a Systemetic Review

PubMed Central

Dursun, Erhan

2016-01-01

ABSTRACT Objectives To review the current understanding of the biomarkers and enzymes associated with different forms peri-implant diseases and how their level changes influence the pathogenesis of the inflammatory diseases around dental implants. Material and Methods An electronic search in two different databases was performed including MEDLINE (PubMed) and EMBASE between 1996 to 2016. Human studies analyse peri-implant crevicular fluid (PICF) biomarker and enzyme levels of implants having peri-implant mucositis and peri-implantitis published in English language, were evaluated. A systematic review was performed to assess which biomarkers and enzymes in PICF were used to identify the inflammatory conditions around dental implants. Results Fifty-one articles were identified of which 41 were further evaluated and included in the analysis. Due to significant heterogeneity between included studies, a meta-analysis could not be performed. Instead, a systematic descriptive review was performed. Conclusions Biomarkers and enzymes in peri-implant crevicular fluid have shown promising results in differentiating from peri-implant disease condition to health. However, due to inconsistent results and acquiring much evidence from cross-sectional studies, additional evidence supported by randomized-controlled trials is needed to validate the links reported. PMID:27833734

GEN1 from a thermophilic fungus is functionally closely similar to non-eukaryotic junction-resolving enzymes.

PubMed

Freeman, Alasdair D J; Liu, Yijin; Déclais, Anne-Cécile; Gartner, Anton; Lilley, David M J

2014-12-12

Processing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1nt 3' to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1. Copyright © 2014. Published by Elsevier Ltd.

Systems analysis in Cellvibrio japonicus resolves predicted redundancy of β-glucosidases and determines essential physiological functions: Functional analysis of C. japonicus β-glucosidases

DOE PAGES

Nelson, Cassandra E.; Rogowski, Artur; Morland, Carl; ...

2017-02-28

Degradation of polysaccharides forms an essential arc in the carbon cycle, provides a percentage of our daily caloric intake, and is a major driver in the renewable chemical industry. Microorganisms proficient at degrading insoluble polysaccharides possess large numbers of carbohydrate active enzymes, many of which have been categorized as functionally redundant. Here we present data that suggests that carbohydrate active enzymes that have overlapping enzymatic activities can have unique, non-overlapping biological functions in the cell. Our comprehensive study to understand cellodextrin utilization in the soil saprophyte Cellvibrio japonicus found that only one of four predicted β-glucosidases is required in amore » physiological context. Gene deletion analysis indicated that only the cel3B gene product is essential for efficient cellodextrin utilization in C. japonicus and is constitutively expressed at high levels. Interestingly, expression of individual β-glucosidases in Escherichia coli K-12 enabled this non-cellulolytic bacterium to be fully capable of using cellobiose as a sole carbon source. Furthermore, enzyme kinetic studies indicated that the Cel3A enzyme is significantly more active than the Cel3B enzyme on the oligosaccharides but not disaccharides. Finally, our approach for parsing related carbohydrate active enzymes to determine actual physiological roles in the cell can be applied to other polysaccharide-degradation systems.« less

Computational active site analysis of molecular pathways to improve functional classification of enzymes.

PubMed

Ozyurt, A Sinem; Selby, Thomas L

2008-07-01

This study describes a method to computationally assess the function of homologous enzymes through small molecule binding interaction energy. Three experimentally determined X-ray structures and four enzyme models from ornithine cyclo-deaminase, alanine dehydrogenase, and mu-crystallin were used in combination with nine small molecules to derive a function score (FS) for each enzyme-model combination. While energy values varied for a single molecule-enzyme combination due to differences in the active sites, we observe that the binding energies for the entire pathway were proportional for each set of small molecules investigated. This proportionality of energies for a reaction pathway appears to be dependent on the amino acids in the active site and their direct interactions with the small molecules, which allows a function score (FS) to be calculated to assess the specificity of each enzyme. Potential of mean force (PMF) calculations were used to obtain the energies, and the resulting FS values demonstrate that a measurement of function may be obtained using differences between these PMF values. Additionally, limitations of this method are discussed based on: (a) larger substrates with significant conformational flexibility; (b) low homology enzymes; and (c) open active sites. This method should be useful in accurately predicting specificity for single enzymes that have multiple steps in their reactions and in high throughput computational methods to accurately annotate uncharacterized proteins based on active site interaction analysis. 2008 Wiley-Liss, Inc.

Structural and Biochemical Characterization of Acinetobacter spp. Aminoglycoside Acetyltransferases Highlights Functional and Evolutionary Variation among Antibiotic Resistance Enzymes.

PubMed

Stogios, Peter J; Kuhn, Misty L; Evdokimova, Elena; Law, Melissa; Courvalin, Patrice; Savchenko, Alexei

2017-02-10

Modification of aminoglycosides by N-acetyltransferases (AACs) is one of the major mechanisms of resistance to these antibiotics in human bacterial pathogens. More than 50 enzymes belonging to the AAC(6') subfamily have been identified in Gram-negative and Gram-positive clinical isolates. Our understanding of the molecular function and evolutionary origin of these resistance enzymes remains incomplete. Here we report the structural and enzymatic characterization of AAC(6')-Ig and AAC(6')-Ih from Acinetobacter spp. The crystal structure of AAC(6')-Ig in complex with tobramycin revealed a large substrate-binding cleft remaining partially unoccupied by the substrate, which is in stark contrast with the previously characterized AAC(6')-Ib enzyme. Enzymatic analysis indicated that AAC(6')-Ig and -Ih possess a broad specificity against aminoglycosides but with significantly lower turnover rates as compared to other AAC(6') enzymes. Structure- and function-informed phylogenetic analysis of AAC(6') enzymes led to identification of at least three distinct subfamilies varying in oligomeric state, active site composition, and drug recognition mode. Our data support the concept of AAC(6') functionality originating through convergent evolution from diverse Gcn5-related-N-acetyltransferase (GNAT) ancestral enzymes, with AAC(6')-Ig and -Ih representing enzymes that may still retain ancestral nonresistance functions in the cell as provided by their particular active site properties.

A Novel Enzyme Portfolio for Red Algal Polysaccharide Degradation in the Marine Bacterium Paraglaciecola hydrolytica S66T Encoded in a Sizeable Polysaccharide Utilization Locus.

PubMed

Schultz-Johansen, Mikkel; Bech, Pernille K; Hennessy, Rosanna C; Glaring, Mikkel A; Barbeyron, Tristan; Czjzek, Mirjam; Stougaard, Peter

2018-01-01

Marine microbes are a rich source of enzymes for the degradation of diverse polysaccharides. Paraglaciecola hydrolytica S66 T is a marine bacterium capable of hydrolyzing polysaccharides found in the cell wall of red macroalgae. In this study, we applied an approach combining genomic mining with functional analysis to uncover the potential of this bacterium to produce enzymes for the hydrolysis of complex marine polysaccharides. A special feature of P. hydrolytica S66 T is the presence of a large genomic region harboring an array of carbohydrate-active enzymes (CAZymes) notably agarases and carrageenases. Based on a first functional characterization combined with a comparative sequence analysis, we confirmed the enzymatic activities of several enzymes required for red algal polysaccharide degradation by the bacterium. In particular, we report for the first time, the discovery of novel enzyme activities targeting furcellaran, a hybrid carrageenan containing both β-carrageenan and κ/β-carrageenan motifs. Some of these enzymes represent a new subfamily within the CAZy classification. From the combined analyses, we propose models for the complete degradation of agar and κ/β-type carrageenan by P. hydrolytica S66 T . The novel enzymes described here may find value in new bio-based industries and advance our understanding of the mechanisms responsible for recycling of red algal polysaccharides in marine ecosystems.

Methods for the Measurement of a Bacterial Enzyme Activity in Cell Lysates and Extracts

PubMed Central

Mendz, George; Hazell, Stuart

1998-01-01

The kinetic characteristics and regulation of aspartate carbamoyltransferase activity were studied in lysates and cell extracts of Helicobacter pylori by three diffirent methods. Nuclear magnetic resonance spectroscopy, radioactive tracer analysis, and spectrophotometry were employed in conjunction to identify the properties of the enzyme activity and to validate the results obtained with each assay. NMR spectroscopy was the most direct method to provide proof of ACTase activity; radioactive tracer analysis was the most sensitive technique and a microtitre-based colorimetric assay was the most cost-and time-efficient for large scale analyses. Freeze-thawing was adopted as the preferred method for cell lysis in studying enzyme activity in situ. This study showed the benefits of employing several different complementary methods to investigate bacterial enzyme activity. PMID:12734591

Cost evaluation of cellulase enzyme for industrial-scale cellulosic ethanol production based on rigorous Aspen Plus modeling.

PubMed

Liu, Gang; Zhang, Jian; Bao, Jie

2016-01-01

Cost reduction on cellulase enzyme usage has been the central effort in the commercialization of fuel ethanol production from lignocellulose biomass. Therefore, establishing an accurate evaluation method on cellulase enzyme cost is crucially important to support the health development of the future biorefinery industry. Currently, the cellulase cost evaluation methods were complicated and various controversial or even conflict results were presented. To give a reliable evaluation on this important topic, a rigorous analysis based on the Aspen Plus flowsheet simulation in the commercial scale ethanol plant was proposed in this study. The minimum ethanol selling price (MESP) was used as the indicator to show the impacts of varying enzyme supply modes, enzyme prices, process parameters, as well as enzyme loading on the enzyme cost. The results reveal that the enzyme cost drives the cellulosic ethanol price below the minimum profit point when the enzyme is purchased from the current industrial enzyme market. An innovative production of cellulase enzyme such as on-site enzyme production should be explored and tested in the industrial scale to yield an economically sound enzyme supply for the future cellulosic ethanol production.

DEVELOPMENT OF METRICS FOR ARTICLES PUBLISHED IN PROFESSIONAL MASTER'S COURSE.

PubMed

Brito, Marcus Vinicius Henriques; Carneiro, Francisca Regina Oliveira

2015-01-01

Propose metric to qualify the production conveyed through articles published in Professional Master's Programs and, from there, to establish guidance for the evaluation of postgraduate programs of Medicine III. Analysis of the documents of 2013 area graduate programs strict sense concerning the application and measurement of score the articles published, and creation of proposal for metric of the theme with the quadrennial review of Medicine III. Were evaluated the medicines area documents I, II and III; Biological Sciences (I) and Interdisciplinary, as well as the 2013 reports of CAPES. All programs establish metrics for "Classification of Published Articles" within its bibliographic production although with different percentages respecting its specificities. With these data collected and correlating their relevance with the surgical areas, was drafted proposal for quantification of the quality of the published articles to "Professional Postgraduate Programs" at surgical area that have specific characteristics according to their guidelines, directing their scientific production to technique journals preferably. The metric suggested for published articles, that should be included in intellectual production of the Area Document, should be considered for the extract A1 = 100 points; A2 = 85 points; B1 = 80 points; B2 = 70 points; B3 = 60 points; B4 = 40 points and B5 = 20 points. Propor métrica para qualificar a produção veiculada através de artigos publicados em programas de mestrado profissional e, a partir daí, estabelecer orientação para a avaliação dos programas de pós-graduação da Medicina III. Análise dos documentos de área de 2013 dos programas de pós-graduação senso estrito no que concerne à aplicação e mensuração de pontuação a artigos publicados, e criação de proposta para métrica do tema com vistas à avaliação quadrienal da Medicina III. Foram avaliados os documentos de área das Medicinas I, II e III; Ciências Biológicas I e Interdisciplinar, assim como os relatórios de 2013 da CAPES. Todos os programas estabelecem métrica para "Classificação de Artigos Publicados" dentro de sua produção bibliográfica embora com percentuais diferentes respeitando suas especificidades. Com esses dados levantados e correlacionando-se sua pertinência com as áreas cirúrgicas, foi elaborada proposta de quantificação da qualidade dos artigos publicados para "Programas de Pós-graduação Profissionais" na área cirúrgica que possuem características próprias de acordo com suas diretrízes, direcionando assim sua produção científica preferencialmente para revistas técnicas. A métrica sugerida para artigos publicados, que deve ser incluída na produção intelectual do Documento de Área, deverá ser considerada para o extrato A1 = 100 pontos; A2 = 85 pontos; B1 = 80 pontos; B2 = 70 pontos; B3 = 60 pontos; B4 = 40 pontos e B5 = 20 pontos.

DEVELOPMENT OF METRICS FOR PROTOCOLS AND OTHER TECHNICAL PRODUCTS.

PubMed

Veiga, Daniela Francescato; Ferreira, Lydia Masako

2015-01-01

To develop a proposal for metrics for protocols and other technical products to be applied in assessing the Postgraduate Programs of Medicine III - Capes. The 2013 area documents of all the 48 Capes areas were read. From the analysis of the criteria used by the areas at the 2013's Triennal Assessment, a proposal for metrics for protocols and other technical products was developed to be applied in assessing the Postgraduate Programs of Medicine III. This proposal was based on the criteria of Biological Sciences I and Interdisciplinary areas. Only seven areas have described a scoring system for technical products. The products considered and the scoring varied widely. Due to the wide range of different technical products which could be considered relevant, and that would not be punctuated if they were not previously specified, it was developed, for the Medicine III, a proposal for metrics in which five specific criteria to be analyzed: Demand, Relevance/Impact, Scope, Complexity and Adherence to the Program. Based on these criteria, each product can receive 10 to 100 points. This proposal can be applied to the item Intellectual Production of the evaluation form, in subsection "Technical production, patents and other relevant production". The program will be scored as Very Good when it reaches mean ≥150 points/permanent professor/quadrennium; Good, mean between 100 and 149 points; Regular, mean between 60 and 99 points; Weak mean between 30 and 59 points; Insufficient, up to 29 points/permanent professor/quadrennium. Desenvolver proposta de métricas para protocolos e outras produções técnicas a serem aplicadas na avaliação dos Programas de Pós-Graduação da Área Medicina III da Capes. Foram lidos os documentos de área de 2013 de todas as 48 Áreas da Capes. A partir da análise dos critérios utilizados por elas na avaliação trienal 2013, foi desenvolvida uma proposta de métricas para protocolos e outras produções técnicas. Esta proposta foi baseada nos critérios adotados pelas Áreas Ciências Biológicas I e Interdisciplinar. Apenas sete áreas descreveram sistema de pontuação para produtos técnicos, e as produções consideradas e a pontuação variaram amplamente. Dada à imensa gama de produções técnicas diferentes que podem ser consideradas relevantes, e que não seriam contempladas em sistema de pontuação caso fossem especificadas, foi desenvolvida para a Medicina III uma proposta de métricas em que são analisados cinco critérios específicos para cada produção: Demanda, Relevância/Impacto, Abrangência, Complexidade e Aderência ao Programa. Com base nestes critérios, cada produção pode receber de 10 a 100 pontos. Esta proposta poderá ser aplicada ao item Produção Intelectual da Ficha de Avaliação, subitem "Produção técnica, patentes e outras produções consideradas relevantes". Será considerado Muito Bom o Programa que obtiver média ≥150 pontos/docente permanente/quadriênio; Bom, média entre 100 e 149 pontos; Regular, média entre 60 e 99 pontos; Fraco, média entre 30 e 59 pontos; e Deficiente média ≤29 pontos/docente permanente/quadriênio.

Population data of five genetic markers in the Turkish population: comparison with four American population groups.

PubMed

Kurtuluş-Ulküer, M; Ulküer, U; Kesici, T; Menevşe, S

2002-09-01

In this study, the phenotype and allele frequencies of five enzyme systems were determined in a total of 611 unrelated Turkish individuals and analyzed by using the exact and the chi 2 test. The following five red cell enzymes were identified by cellulose acetate electrophoresis: phosphoglucomutase (PGM), adenosine deaminase (ADA), phosphoglucose isomerase (PGI), adenylate kinase (AK), and 6-phosphogluconate dehydrogenase (6-PGD). The ADA, PGM and AK enzymes were found to be polymorphic in the Turkish population. The results of the statistical analysis showed, that the phenotype frequencies of the five enzyme under study are in Hardy-Weinberg equilibrium. Statistical analysis was performed in order to examine whether there are significant differences in the phenotype frequencies between the Turkish population and four American population groups. This analysis showed, that there are some statistically significant differences between the Turkish and the other groups. Moreover, the observed phenotype and allele frequencies were compared with those obtained in other population groups of Turkey.

Genomewide analysis of polysaccharides degrading enzymes in 11 white- and brown-rot Polyporales provides insight into mechanisms of wood decay

Treesearch

Chiaki Hori; Jill Gaskell; Kiyohiko Igarashi; Masahiro Samejima; David Hibbett; Bernard Henrissat; Dan Cullen

2013-01-01

To degrade the polysaccharides, wood-decay fungi secrete a variety of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) classified into various sequence-based families of carbohydrate-active enzymes (CAZys) and their appended carbohydrate-binding modules (CBM). Oxidative enzymes, such as cellobiose dehydrogenase (CDH) and lytic polysaccharide monooxygenase (...

Development of a Multianalyte Enzyme-Linked Immunosorbent Assay for Permethrin and Aroclors and Its Implementation for Analysis of Soil/Sediment and House Dust ExtractsExtracts

EPA Science Inventory

Development of a multianalyte enzyme-linked immunosorbent assay (ELISA) for detection of permethrin and aroclors 1248 or 1254, and implementation of the assay for analysis of soil/sediment samples are described. The feasibility of using the multianalyte ELISA to monitor aroclors ...

Concentration measurement of lysosome enzymes in blood by fluorimetric analysis method

NASA Astrophysics Data System (ADS)

Strinadko, Marina M.; Strinadko, Elena M.

2002-02-01

The diagnostics of heritable disease series and sugar diabetes, myocardial infarction, collagenosis and kidney diseases widely uses the measurement of lysosomic enzymes in blood. In the present research work the definition procedure of concentration (beta) -glucuronidase with the help of fluorimetric analysis is offered, which allows using microamounts of biological fluids and samples with low enzyme activity which is especially important in paediatric practice. Due to the sharp sensibility of fluorimetric analysis and high speed of luminescent reactions the procedure gives an opportunity to obtain the result in the minimum terms as well as the use of small amounts of reaction mixture. The incubation in large dilution leads thereby to the elimination of influence of endogenic inhibitors and activators.

Marvels of enzyme catalysis at true atomic resolution: distortions, bond elongations, hidden flips, protonation states and atom identities.

PubMed

Neumann, Piotr; Tittmann, Kai

2014-12-01

Although general principles of enzyme catalysis are fairly well understood nowadays, many important details of how exactly the substrate is bound and processed in an enzyme remain often invisible and as such elusive. In fortunate cases, structural analysis of enzymes can be accomplished at true atomic resolution thus making possible to shed light on otherwise concealed fine-structural traits of bound substrates, intermediates, cofactors and protein groups. We highlight recent structural studies of enzymes using ultrahigh-resolution X-ray protein crystallography showcasing its enormous potential as a tool in the elucidation of enzymatic mechanisms and in unveiling fundamental principles of enzyme catalysis. We discuss the observation of seemingly hyper-reactive, physically distorted cofactors and intermediates with elongated scissile substrate bonds, the detection of 'hidden' conformational and chemical equilibria and the analysis of protonation states with surprising findings. In delicate cases, atomic resolution is required to unambiguously disclose the identity of atoms as demonstrated for the metal cluster in nitrogenase. In addition to the pivotal structural findings and the implications for our understanding of enzyme catalysis, we further provide a practical framework for resolution enhancement through optimized data acquisition and processing. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of Carboxylic Acid Reductases as Enzymes in the Toolbox for Synthetic Chemistry.

PubMed

Finnigan, William; Thomas, Adam; Cromar, Holly; Gough, Ben; Snajdrova, Radka; Adams, Joseph P; Littlechild, Jennifer A; Harmer, Nicholas J

2017-03-20

Carboxylic acid reductase enzymes (CARs) meet the demand in synthetic chemistry for a green and regiospecific route to aldehydes from their respective carboxylic acids. However, relatively few of these enzymes have been characterized. A sequence alignment with members of the ANL (Acyl-CoA synthetase/ NRPS adenylation domain/Luciferase) superfamily of enzymes shed light on CAR functional dynamics. Four unstudied enzymes were selected by using a phylogenetic analysis of known and hypothetical CARs, and for the first time, a thorough biochemical characterization was performed. Kinetic analysis of these enzymes with various substrates shows that they have a broad but similar substrate specificity. Electron-rich acids are favored, which suggests that the first step in the proposed reaction mechanism, attack by the carboxylate on the α-phosphate of adenosine triphosphate (ATP), is the step that determines the substrate specificity and reaction kinetics. The effects of pH and temperature provide a clear operational window for the use of these CARs, whereas an investigation of product inhibition by NADP + , adenosine monophosphate, and pyrophosphate indicates that the binding of substrates at the adenylation domain is ordered with ATP binding first. This study consolidates CARs as important and exciting enzymes in the toolbox for sustainable chemistry and provides specifications for their use as a biocatalyst.

A comparison of maximal bioenergetic enzyme activities obtained with commonly used homogenization techniques.

PubMed

Grace, M; Fletcher, L; Powers, S K; Hughes, M; Coombes, J

1996-12-01

Homogenization of tissue for analysis of bioenergetic enzyme activities is a common practice in studies examining metabolic properties of skeletal muscle adaptation to disease, aging, inactivity or exercise. While numerous homogenization techniques are in use today, limited information exists concerning the efficacy of specific homogenization protocols. Therefore, the purpose of this study was to compare the efficacy of four commonly used approaches to homogenizing skeletal muscle for analysis of bioenergetic enzyme activity. The maximal enzyme activity (Vmax) of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured from homogenous muscle samples (N = 48 per homogenization technique) and used as indicators to determine which protocol had the highest efficacy. The homogenization techniques were: (1) glass-on-glass pestle; (2) a combination of a mechanical blender and a teflon pestle (Potter-Elvehjem); (3) a combination of the mechanical blender and a biological detergent; and (4) the combined use of a mechanical blender and a sonicator. The glass-on-glass pestle homogenization protocol produced significantly higher (P < 0.05) enzyme activities compared to all other protocols for both enzymes. Of the four protocols examined, the data demonstrate that the glass-on-glass pestle homogenization protocol is the technique of choice for studying bioenergetic enzyme activity in skeletal muscle.

Enzyme inhibitory metabolites from endophytic Penicillium citrinum isolated from Boswellia sacra.

PubMed

Ali, Sajid; Khan, Abdul Latif; Ali, Liaqat; Rizvi, Tania Shamim; Khan, Sumera Afzal; Hussain, Javid; Hamayun, Muhammad; Al-Harrasi, Ahmed

2017-07-01

Fungal endophytes establish an important niche within the host plant through the secretion of chemical constituents. Isolation of bioactive metabolites could be a vital source for inhibiting the function of enzymes such as α-glucosidase and urease. The present study aimed to elucidate the potential of endophytes associated with Boswellia sacra through bioassay-guided isolation and identification of secondary metabolites with enzyme inhibitory ability. Endophytic fungal strains viz. Penicillium citrinum, P. spinulosum, Fusarium oxysporum, Alternaria alternata and Aspergillus caespitosus were identified through genomic DNA extraction, PCR amplification, sequencing and phylogenetic analysis. The enzymes inhibition analysis of the ethyl acetate extract from pure cultures suggested that P. citrinum possess significantly higher enzyme inhibitory activities compared to other strains. The active strain was subjected to chromatographic isolation and nuclear magnetic resonance methods to identify bioactive compounds. The bioactive extracts resulted in the isolation of 11-oxoursonic acid benzyl ester (1), n-nonane (2), 3-decene-1-ol (3), 2-Hydroxyphenyl acetic acid (4), and Glochidacuminosides A (5). Among pure compound, 11-oxoursonic acid benzyl ester (1) showed significantly higher enzyme inhibition activity compared to other metabolites. Our results suggest that the endophytic microorganism associated with the arid-land tree can offer a rich source of biologically active chemical constituents that could help discover lead drugs for enzyme inhibition.

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

Theisen, Matthew K.; Lafontaine Rivera, Jimmy G.; Liao, James C.

Stability in a metabolic system may not be obtained if incorrect amounts of enzymes are used. Without stability, some metabolites may accumulate or deplete leading to the irreversible loss of the desired operating point. Even if initial enzyme amounts achieve a stable steady state, changes in enzyme amount due to stochastic variations or environmental changes may move the system to the unstable region and lose the steady-state or quasi-steady-state flux. This situation is distinct from the phenomenon characterized by typical sensitivity analysis, which focuses on the smooth change before loss of stability. Here we show that metabolic networks differ significantlymore » in their intrinsic ability to attain stability due to the network structure and kinetic forms, and that after achieving stability, some enzymes are prone to cause instability upon changes in enzyme amounts. We use Ensemble Modelling for Robustness Analysis (EMRA) to analyze stability in four cell-free enzymatic systems when enzyme amounts are changed. Loss of stability in continuous systems can lead to lower production even when the system is tested experimentally in batch experiments. The predictions of instability by EMRA are supported by the lower productivity in batch experimental tests. Finally, the EMRA method incorporates properties of network structure, including stoichiometry and kinetic form, but does not require specific parameter values of the enzymes.« less

Genome-Scale Architecture of Small Molecule Regulatory Networks and the Fundamental Trade-Off between Regulation and Enzymatic Activity

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

Reznik, Ed; Christodoulou, Dimitris; Goldford, Joshua E.

Metabolic flux is in part regulated by endogenous small molecules that modulate the catalytic activity of an enzyme, e.g., allosteric inhibition. In contrast to transcriptional regulation of enzymes, technical limitations have hindered the production of a genome-scale atlas of small molecule-enzyme regulatory interactions. Here, we develop a framework leveraging the vast, but fragmented, biochemical literature to reconstruct and analyze the small molecule regulatory network (SMRN) of the model organism Escherichia coli, including the primary metabolite regulators and enzyme targets. Using metabolic control analysis, we prove a fundamental trade-off between regulation and enzymatic activity, and we combine it with metabolomic measurementsmore » and the SMRN to make inferences on the sensitivity of enzymes to their regulators. By generalizing the analysis to other organisms, we identify highly conserved regulatory interactions across evolutionarily divergent species, further emphasizing a critical role for small molecule interactions in the maintenance of metabolic homeostasis.« less

On origin and evolution of carbonic anhydrase isozymes: A phylogenetic analysis from whole-enzyme to active site.

PubMed

Banerjee, Srijoni; Deshpande, Parag A

2016-04-01

Genetic evolution of carbonic anhydrase enzyme provides an interesting instance of functional similarity in spite of structural diversity of the members of a given family of enzymes. Phylogenetic analysis of α-, β- and γ-carbonic anhydrase was carried out to determine the evolutionary relationships among various members of the family with the enzyme marking its presence in a wide range of cellular and chromosomal locations. The presence of more than one class of enzymes in a particular organism was revealed by phylogenetic time tree. The evolutionary relationships among the members of animal, plant and microbial kingdom were developed. The study revises a long-established notion of kingdom-specificity of the different classes of carbonic anhydrases and provides a new version of the presence of multiple classes of carbonic anhydrases in a single organism and the presence of a given class of carbonic anhydrase across different kingdoms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome-Scale Architecture of Small Molecule Regulatory Networks and the Fundamental Trade-Off between Regulation and Enzymatic Activity

DOE PAGES

Reznik, Ed; Christodoulou, Dimitris; Goldford, Joshua E.; ...

2017-09-12

Metabolic flux is in part regulated by endogenous small molecules that modulate the catalytic activity of an enzyme, e.g., allosteric inhibition. In contrast to transcriptional regulation of enzymes, technical limitations have hindered the production of a genome-scale atlas of small molecule-enzyme regulatory interactions. Here, we develop a framework leveraging the vast, but fragmented, biochemical literature to reconstruct and analyze the small molecule regulatory network (SMRN) of the model organism Escherichia coli, including the primary metabolite regulators and enzyme targets. Using metabolic control analysis, we prove a fundamental trade-off between regulation and enzymatic activity, and we combine it with metabolomic measurementsmore » and the SMRN to make inferences on the sensitivity of enzymes to their regulators. By generalizing the analysis to other organisms, we identify highly conserved regulatory interactions across evolutionarily divergent species, further emphasizing a critical role for small molecule interactions in the maintenance of metabolic homeostasis.« less

Application of activity-based protein profiling to study enzyme function in adipocytes.

PubMed

Galmozzi, Andrea; Dominguez, Eduardo; Cravatt, Benjamin F; Saez, Enrique

2014-01-01

Activity-based protein profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways. © 2014 Elsevier Inc. All rights reserved.

Identification and modification of dynamical regions in proteins for alteration of enzyme catalytic effect

DOEpatents

Agarwal, Pratul K.

2015-11-24

A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.

Identification and modification of dynamical regions in proteins for alteration of enzyme catalytic effect

DOEpatents

Agarwal, Pratul K.

2013-04-09

A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.

Integrating enzyme fermentation in lignocellulosic ethanol production: life-cycle assessment and techno-economic analysis.

PubMed

Olofsson, Johanna; Barta, Zsolt; Börjesson, Pål; Wallberg, Ola

2017-01-01

Cellulase enzymes have been reported to contribute with a significant share of the total costs and greenhouse gas emissions of lignocellulosic ethanol production today. A potential future alternative to purchasing enzymes from an off-site manufacturer is to integrate enzyme and ethanol production, using microorganisms and part of the lignocellulosic material as feedstock for enzymes. This study modelled two such integrated process designs for ethanol from logging residues from spruce production, and compared it to an off-site case based on existing data regarding purchased enzymes. Greenhouse gas emissions and primary energy balances were studied in a life-cycle assessment, and cost performance in a techno-economic analysis. The base case scenario suggests that greenhouse gas emissions per MJ of ethanol could be significantly lower in the integrated cases than in the off-site case. However, the difference between the integrated and off-site cases is reduced with alternative assumptions regarding enzyme dosage and the environmental impact of the purchased enzymes. The comparison of primary energy balances did not show any significant difference between the cases. The minimum ethanol selling price, to reach break-even costs, was from 0.568 to 0.622 EUR L -1 for the integrated cases, as compared to 0.581 EUR L -1 for the off-site case. An integrated process design could reduce greenhouse gas emissions from lignocellulose-based ethanol production, and the cost of an integrated process could be comparable to purchasing enzymes produced off-site. This study focused on the environmental and economic assessment of an integrated process, and in order to strengthen the comparison to the off-site case, more detailed and updated data regarding industrial off-site enzyme production are especially important.

Emissions of selected VOC from forests: First results on measurements needed for improvement and validation of emission models

NASA Astrophysics Data System (ADS)

Steigner, D.; Steinbrecher, R.; Rappenglück, B.; Gasche, R.; Hansel, A.; Graus, M.; Lindinger, Ch.

2003-04-01

Biogenic volatile organic compounds (BVOCs) play a crucial role in the formation of photo-oxidants and particles through the diverse BVOC degradation pathways. Yet, current estimations about temporal and spatial BVOC emissions, including the specific BVOC mix are rather vague. This project addresses this issue by: the determination of (a) BVOC net emission rates and (b) primary emissions of BVOCs from the trees and soils. Measurement campaigns were carried out at the Waldstein site in the Fichtelgebirge in 2001 and 2002. Primary emissions of isoprenoids from the soil and from twigs of Norway spruce (Picea abies [L.] Karst.) and stand fluxes of isoprenoids were quantified by means of REA-technique with in situ GC-FID analysis and GC-MS analysis in the laboratory. Moreover, REA-samples obtained by the system were analysed by a PTR-MS. A critical value when using the REA approach is the Businger-Oncley parameter b. For this canopy type a b value of 0.39 (threshold velocity w_o = 0.6) was determined. The PTR-MS data show clear diurnal variations of ambient air mixing ratios of VOC such as isoprene and monoterpenes, but also of oxygenated VOCs such as carbonyls and alcohols and methylvinylketone (MVK) and methacrolein (MAK), products from isoprene degradation. Four selected trees (Picea abies [L.] Karst.) were intensively screened for primary BVOC emission rates. Most abundant species are b-pinene/sabinene and camphene. They show typical diurnal patterns with high emissions during daytime. Soil emissions of NO reached 250 nmol N m-2 s-1 at soil temperatures (in 3 cm depth) of 13^oC and at a relative air humidity of 60%. Ambient air mixing ratios near the soil surface of NO reached values of up to 0.7 ppb. NO_2 and ozone mixing ratios varied between 0.1 to 1.5 ppb and 10 to 37 ppb, respectively. As expected nitrogen oxide emissions rates tend to increase with increasing surface temperature. Isoprenoid emission from the soil was low and in general near the detection limit of 0.05 nmol m-2 s-1. Canopy fluxes of isoprene reached up to 7 nmol m-2 s-1 during daytime. At the same time MVK+MAK fluxes were about 30% lower. The fluxes of the sum of monoterpenes were in the same range. Striking were the sometimes very high fluxes of methanol and acetaldehyde of up to 80 nmol m-2 s-1.

Tracing the Repertoire of Promiscuous Enzymes along the Metabolic Pathways in Archaeal Organisms.

PubMed

Martínez-Núñez, Mario Alberto; Rodríguez-Escamilla, Zuemy; Rodríguez-Vázquez, Katya; Pérez-Rueda, Ernesto

2017-07-13

The metabolic pathways that carry out the biochemical transformations sustaining life depend on the efficiency of their associated enzymes. In recent years, it has become clear that promiscuous enzymes have played an important role in the function and evolution of metabolism. In this work we analyze the repertoire of promiscuous enzymes in 89 non-redundant genomes of the Archaea cellular domain. Promiscuous enzymes are defined as those proteins with two or more different Enzyme Commission (E.C.) numbers, according the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. From this analysis, it was found that the fraction of promiscuous enzymes is lower in Archaea than in Bacteria. A greater diversity of superfamily domains is associated with promiscuous enzymes compared to specialized enzymes, both in Archaea and Bacteria, and there is an enrichment of substrate promiscuity rather than catalytic promiscuity in the archaeal enzymes. Finally, the presence of promiscuous enzymes in the metabolic pathways was found to be heterogeneously distributed at the domain level and in the phyla that make up the Archaea. These analyses increase our understanding of promiscuous enzymes and provide additional clues to the evolution of metabolism in Archaea.

Structure and function of α-glucan debranching enzymes.

PubMed

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

2016-07-01

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 (GH13_41) in two-domain amylase-pullulanases. GH57 harbours type II pullulanases. Specificity differences, domain organisation, carbohydrate binding modules, sequence motifs, three-dimensional structures and specificity determinants are discussed. The phylogenetic analysis indicated that GH13_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.

The maximum entropy production and maximum Shannon information entropy in enzyme kinetics

NASA Astrophysics Data System (ADS)

Dobovišek, Andrej; Markovič, Rene; Brumen, Milan; Fajmut, Aleš

2018-04-01

We demonstrate that the maximum entropy production principle (MEPP) serves as a physical selection principle for the description of the most probable non-equilibrium steady states in simple enzymatic reactions. A theoretical approach is developed, which enables maximization of the density of entropy production with respect to the enzyme rate constants for the enzyme reaction in a steady state. Mass and Gibbs free energy conservations are considered as optimization constraints. In such a way computed optimal enzyme rate constants in a steady state yield also the most uniform probability distribution of the enzyme states. This accounts for the maximal Shannon information entropy. By means of the stability analysis it is also demonstrated that maximal density of entropy production in that enzyme reaction requires flexible enzyme structure, which enables rapid transitions between different enzyme states. These results are supported by an example, in which density of entropy production and Shannon information entropy are numerically maximized for the enzyme Glucose Isomerase.

Determination of glutamine and glutamic acid in mammalian cell cultures using tetrathiafulvalene modified enzyme electrodes.

PubMed

Mulchandani, A; Bassi, A S

1996-01-01

Tetrathiafulvalene (TTF) mediated amperometric enzyme electrodes have been developed for the monitoring of L-glutamine and L-glutamic acid in growing mammalian cell cultures. The detection of glutamine was accomplished by a coupled enzyme system comprised of glutaminase plus glutamate oxidase, while the detection of glutamic acid was carried out by a single enzyme, glutamate oxidase. The appropriate enzyme(s) were immoblized on the Triton-X treated surface of tetrathiafulvalene modified carbon paste electrodes by adsorption, in conjunction with entrapment by an electrochemically deposited copolymer film of 1,3-phenylenediamine and resorcinol. Operating conditions for the glutamine enzyme electrode were optimized with respect to the amount of enzymes immoblized, pH, temperature and mobile phase flow rate for operation in a flow injection (FIA) system. When applied to glutamine and glutamic acid measurements in mammalian cell culture in FIA, the results obtained with enzyme electrodes were in excellent agreement with those determined by enzymatic analysis.

A meta-analysis of soil exoenzyme responses to simulated climate change

NASA Astrophysics Data System (ADS)

Gebhardt, M.; Espinosa, N. J.; Blankinship, J. C.; Gallery, R. E.

2017-12-01

Microorganisms produce extracellular enzymes to decompose plant matter and drive biogeochemical transformations in soils. Climate change factors, such as warming and altered precipitation patterns, can impact enzyme activity through both direct and indirect mechanisms. Although many individual studies have examined how soil exoenzyme activities respond to climate change manipulations, there is disagreement surrounding the direction of these responses. We performed a synthesis of published studies to examine the influence of warming and altered precipitation on microbial exoenzyme activity. We found that warming increased enzyme activity with a more pronounced effect for oxidative relative to hydrolytic enzymes. Reduced precipitation consistently decreased exoenzyme activity. These responses, however, varied by season, biome, and enzyme type. The majority of studies fitting our criteria (e.g., experiments lasting a minimum of one growing season, paired treatments and controls) were located in North America and Europe. Inferences from this analysis therefore exclude many important ecosystems such as hyper-arid, wetlands, and artic systems. Carbon degrading enzyme activities were less sensitive to climate change manipulations when compared to phosphorus and nitrogen degrading enzyme activities. Linking enzyme activity to biogeochemical processes requires concomitant measurements of organic and inorganic carbon pools, mineralogy, nutrients, microbial biomass and community structure, and heterotrophic respiration within individual studies. Furthermore, linking these parameters to climate and environmental factors will require a comprehensive and consistent inclusion of biotic and abiotic variables among researchers and experiments. Globally, soils contain the largest carbon pools. Understanding the impacts of large-scale perturbations on soil enzyme activity will help to constrain predictions on the fate of biogeochemical transformations and improve model projections.

Laterality of basic auditory perception.

PubMed

Sininger, Yvonne S; Bhatara, Anjali

2012-01-01

Laterality (left-right ear differences) of auditory processing was assessed using basic auditory skills: (1) gap detection, (2) frequency discrimination, and (3) intensity discrimination. Stimuli included tones (500, 1000, and 4000 Hz) and wide-band noise presented monaurally to each ear of typical adult listeners. The hypothesis tested was that processing of tonal stimuli would be enhanced by left ear (LE) stimulation and noise by right ear (RE) presentations. To investigate the limits of laterality by (1) spectral width, a narrow-band noise (NBN) of 450-Hz bandwidth was evaluated using intensity discrimination, and (2) stimulus duration, 200, 500, and 1000 ms duration tones were evaluated using frequency discrimination. A left ear advantage (LEA) was demonstrated with tonal stimuli in all experiments, but an expected REA for noise stimuli was not found. The NBN stimulus demonstrated no LEA and was characterised as a noise. No change in laterality was found with changes in stimulus durations. The LEA for tonal stimuli is felt to be due to more direct connections between the left ear and the right auditory cortex, which has been shown to be primary for spectral analysis and tonal processing. The lack of a REA for noise stimuli is unexplained. Sex differences in laterality for noise stimuli were noted but were not statistically significant. This study did establish a subtle but clear pattern of LEA for processing of tonal stimuli.

Laterality of Basic Auditory Perception

PubMed Central

Sininger, Yvonne S.; Bhatara, Anjali

2010-01-01

Laterality (left-right ear differences) of auditory processing was assessed using basic auditory skills: 1) gap detection 2) frequency discrimination and 3) intensity discrimination. Stimuli included tones (500, 1000 and 4000 Hz) and wide-band noise presented monaurally to each ear of typical adult listeners. The hypothesis tested was: processing of tonal stimuli would be enhanced by left ear (LE) stimulation and noise by right ear (RE) presentations. To investigate the limits of laterality by 1) spectral width, a narrow band noise (NBN) of 450 Hz bandwidth was evaluated using intensity discrimination and 2) stimulus duration, 200, 500 and 1000 ms duration tones were evaluated using frequency discrimination. Results A left ear advantage (LEA) was demonstrated with tonal stimuli in all experiments but an expected REA for noise stimuli was not found. The NBN stimulus demonstrated no LEA and was characterized as a noise. No change in laterality was found with changes in stimulus durations. The LEA for tonal stimuli is felt to be due to more direct connections between the left ear and the right auditory cortex which has been shown to be primary for spectral analysis and tonal processing. The lack of a REA for noise stimuli is unexplained. Sex differences in laterality for noise stimuli were noted but were not statistically significant. This study did establish a subtle but clear pattern of LEA for processing of tonal stimuli. PMID:22385138

Molecular epidemiology of Mycobacterium avium subspecies paratuberculosis in ruminants in different parts of India.

PubMed

Sonawane, Ganesh G; Narnaware, Shirish D; Tripathi, Bhupendra N

2016-03-01

Paratuberculosis is an economically important, chronic, and incurable disease in ruminants, caused by Mycobacterium avium subspecies paratuberculosis (MAP). Understanding the genetic variability of MAP strains is important in diagnosis, epidemiological investigation, and the formation of strategies for prevention and control of the disease. In the present study, a total of 61 MAP isolates obtained from different parts and species of India were typed using IS1311 polymerase chain reaction-restriction endonuclease analysis (PCR-REA) to analyze the genetic difference(s), if any, between them and the host adaptation. Based on PCR-REA results, bison B type was detected in 54 (87%) MAP isolates obtained from cattle, sheep, and goats. Of these, 19 were from sheep of the Rajasthan (n=17) and Bareilly (n=2), North India regions, 28 were from cattle of Chennai, South India (n=3), Bareilly, North India (n=3), and Nagpur, West India (n=22), and seven goat isolates from Bareilly, North India region. The 'C' type strain was detected in only seven cattle isolates obtained from the Bareilly region. The study revealed that in India, bison B-type MAP strains were prevalent in most of the ruminant species. These results have important epidemiological implications with regard to control and prevention of paratuberculosis in India. Copyright © 2015 Asian African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

CR extension from hypersurfaces of higher type

NASA Astrophysics Data System (ADS)

Baracco, Luca

2007-07-01

We prove extension of CR functions from a hypersurface M of in presence of the so-called sector property. If M has finite type in the Bloom-Graham sense, then our result is already contained in [C. Rea, Prolongement holomorphe des fonctions CR, conditions suffisantes, C. R. Acad. Sci. Paris 297 (1983) 163-166] by Rea. We think however, that the argument of our proof carries an expressive geometric meaning and deserves interest on its own right. Also, our method applies in some case to hypersurfaces of infinite type; note that for these, the classical methods fail. CR extension is treated by many authors mainly in two frames: extension in directions of iterated of commutators of CR vector fields (cf., for instance, [A. Boggess, J. Pitts, CR extension near a point of higher type, Duke Math. J. 52 (1) (1985) 67-102; A. Boggess, J.C. Polking, Holomorphic extension of CR functions, Duke Math. J. 49 (1982) 757-784. ; M.S. Baouendi, L. Rothschild, Normal forms for generic manifolds and holomorphic extension of CR functions, J. Differential Geom. 25 (1987) 431-467. ]); extension through minimality towards unprecised directions [A.E. Tumanov, Extension of CR-functions into a wedge, Mat. Sb. 181 (7) (1990) 951-964. ; A.E. Tumanov, Analytic discs and the extendibility of CR functions, in: Integral Geometry, Radon Transforms and Complex Analysis, Venice, 1996, in: Lecture Notes in Math., vol. 1684, Springer, Berlin, 1998, pp. 123-141].

Prescribed burning effects on soil enzyme activity in a southern Ohio hardwood forest: A landscape-scale analysis

Treesearch

Ralph E. J. Boerner; Kelly L. M. Decker; Elaine K. Sutherland

2000-01-01

We assessed the effect of a single, dormant season prescribed fire on soil enzyme activity in oak-hickory (Quercus-Carya) forests in southern Ohio, USA. Four enzymes specific for different C sources were chosen for monitoring: acid phosphatase, beta-glucosidase, chitinase and phenol oxidase. Postfire acid phosphatase activity was generally reduced by burning and...

Comparison of monolithic and microparticulate columns for reversed-phase liquid chromatography of tryptic digests of industrial enzymes in cleaning products.

PubMed

Beneito-Cambra, M; Herrero-Martínez, J M; Ramis-Ramos, G; Lindner, W; Lämmerhofer, M

2011-10-14

Enzymes of several classes used in the formulations of cleaning products were characterized by trypsin digestion followed by HPLC with UV detection. A polymeric monolithic column (ProSwift) was used to optimize the separation of both the intact enzymes and their tryptic digests. This column was adequate for the quality control of raw industrial enzyme concentrates. Then, monolithic and microparticulate columns were compared for peptide analysis. Under optimized conditions, the analysis of tryptic digests of enzymes of different classes commonly used in the formulation of cleaning products was carried out. Number of peaks, peak capacity and global resolution were obtained in order to evaluate the chromatographic performance of each column. Particulate shell-core C18 columns (Kinetex, 2.6 μm) showed the best performance, followed by a silica monolithic column (Chromolith RP-18e) and the conventional C18 packings (Gemini, 5 μm or 3 μm). A polymeric monolithic column (ProSwift) gave the worst performances. The proposed method was satisfactorily applied to the characterization of the enzymes present in spiked detergent bases and commercial cleaners. Copyright © 2011 Elsevier B.V. All rights reserved.

Analogs of diadenosine tetraphosphate (Ap4A).

PubMed

Guranowski, Andrzej

2003-01-01

This review summarizes our knowledge of analogs and derivatives of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), the most extensively studied member of the dinucleoside 5',5"'-P1,Pn-polyphosphate (NpnN) family. After a short discussion of enzymes that may be responsible for the accumulation and degradation of Np4)N's in the cell, this review focuses on chemically and/or enzymatically produced analogs and their practical applications. Particular attention is paid to compounds that have aided the study of enzymes involved in the metabolism of Ap4A (Np4N'). Certain Ap4A analogs were alternative substrates of Ap4A-degrading enzymes and/or acted as enzyme inhibitors, some other helped to establish enzyme mechanisms, increased the sensitivity of certain enzyme assays or produced stable enzyme:ligand complexes for structural analysis.

A thermal biosensor based on enzyme reaction.

PubMed

Zheng, Yi-Hua; Hua, Tse-Chao; Xu, Fei

2005-01-01

Application of the thermal biosensor as analytical tool is promising due to advantages as universal, simplicity and quick response. A novel thermal biosensor based on enzyme reaction has been developed. This biosensor is a flow injection analysis system and consists of two channels with enzyme reaction column and reference column. The reference column, which is set for eliminating the unspecific heat, is inactived on special enzyme reaction of the ingredient to be detected. The special enzyme reaction takes places in the enzyme reaction column at a constant temperature realizing by a thermoelectric thermostat. Thermal sensor based on the thermoelectric module containing 127 serial BiTe-thermocouples is used to monitor the temperature difference between two streams from the enzyme reaction column and the reference column. The analytical example for dichlorvos shows that this biosensor can be used as analytical tool in medicine and biology.

Sequential capillary electrophoresis analysis using optically gated sample injection and UV/vis detection.

PubMed

Liu, Xiaoxia; Tian, Miaomiao; Camara, Mohamed Amara; Guo, Liping; Yang, Li

2015-10-01

We present sequential CE analysis of amino acids and L-asparaginase-catalyzed enzyme reaction, by combing the on-line derivatization, optically gated (OG) injection and commercial-available UV-Vis detection. Various experimental conditions for sequential OG-UV/vis CE analysis were investigated and optimized by analyzing a standard mixture of amino acids. High reproducibility of the sequential CE analysis was demonstrated with RSD values (n = 20) of 2.23, 2.57, and 0.70% for peak heights, peak areas, and migration times, respectively, and the LOD of 5.0 μM (for asparagine) and 2.0 μM (for aspartic acid) were obtained. With the application of the OG-UV/vis CE analysis, sequential online CE enzyme assay of L-asparaginase-catalyzed enzyme reaction was carried out by automatically and continuously monitoring the substrate consumption and the product formation every 12 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays. The study demonstrated the feasibility and reliability of integrating the OG injection with UV/vis detection for sequential online CE analysis, which could be of potential value for online monitoring various chemical reaction and bioprocesses. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alkaline active cyanide dihydratase of Flavobacterium indicum MTCC 6936: Growth optimization, purification, characterization and in silico analysis.

PubMed

Kumar, Virender; Kumar, Vijay; Bhalla, Tek Chand

2018-05-15

The present work explores a rare cyanide dihydratase of Flavobacterium indicum MTCC 6936 for its potential of cyanide degradation. The enzyme is purified to 12 fold with a yield of 76%. SDS and native-PAGE analysis revealed that enzyme was monomer of 40 kDa size. The enzyme works well in mesophilic range at wide array of pH. The thermostability profile of cyanide dihydratase revealed that the enzyme is quite stable at 30 °C and 35 °C with half-life of 6 h 30 min and 5 h respectively. K m and V max for cyanide dihydratase of F. indicum was measured to be 4.76 mM and 45 U mg -1 with k cat calculated to be 27.3 s -1 and specificity constant (k cat /K m ) to be around 5.67 mM -1  s -1 . MALDI-TOF analysis of purified protein revealed that the amino acid sequence has 50% and 43% sequence identity with putative amino acid sequence of F. indicum and earlier reported cyanide dihydratase of Bacillus pumilus respectively. Homology modeling studies of cyanide dihydratase of F. indicum predicted the catalytic triad of the enzyme indicating Cys at 164, Glu at 46 and Lys at 130th position. The purified enzyme has potential applications in bioremediation and analytical sector. Copyright © 2018 Elsevier B.V. All rights reserved.

Mapping the polysaccharide degradation potential of Aspergillus niger

PubMed Central

2012-01-01

Background The degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation. Results Through the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from Aspergillus niger, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan. Conclusions The mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in A. niger. PMID:22799883

Mapping the polysaccharide degradation potential of Aspergillus niger.

PubMed

Andersen, Mikael R; Giese, Malene; de Vries, Ronald P; Nielsen, Jens

2012-07-16

The degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation. Through the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from Aspergillus niger, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan. The mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in A. niger.

Patient safety: knowledge between multiprofessional residents.

PubMed

Oliveira, João Lucas Campos de; Silva, Simone Viana da; Santos, Pamela Regina Dos; Matsuda, Laura Misue; Tonini, Nelsi Salete; Nicola, Anair Lazzari

2017-01-01

To assess the knowledge of multiprofesional residents in health about the security of the patient theme. Cross-sectional study, quantitative, developed with graduate courses/residence specialties of health in a public university of Paraná, Brazil. Participants (n=78) answered a questionnaire containing nine objective questions related to patient safety. Data were analyzed using descriptive statistics, in proportion measures. The minimum 75% of correct answers was considered the cutoff for positive evaluation. The sample was predominantly composed of young people from medical programs. Almost half of the items evaluated (n=5) achieved the established positive pattern, especially those who dealt with the hand hygiene moments (98.8%) and goal of the Patient Safety National Program (92.3%). The identification of the patient was the worst rated item (37.7%). In the analysis by professional areas, only the Nursing reached the standard of hits established. Knowledge of the residents was threshold. Verificar o conhecimento de residentes multiprofissionais na área da saúde sobre o tema segurança do paciente. Estudo transversal, quantitativo, desenvolvido com pós-graduandos dos cursos/especialidades de residência da área da saúde de uma universidade pública do Paraná. Os participantes (n=78) responderam um questionário contendo nove questões objetivas relacionadas com a segurança do paciente. Os dados foram analisados por estatística descritiva, em medidas de proporção. O mínimo de 75% de acertos foi considerado ponto de corte para avaliação positiva. A amostra foi composta por profissionais predominantemente jovens, oriundos de programas médicos. Quase metade dos itens avaliados (n=5) alcançou o padrão de positividade estabelecido, com destaque para os que trataram dos momentos de higienização das mãos (98,8%) e o objetivo do Programa Nacional de Segurança do Paciente (92,3%). A identificação do paciente foi o pior item avaliado (37,7%). Na análise por áreas profissionais, somente a Enfermagem alcançou o padrão de acertos estabelecido. O conhecimento dos residentes foi limiar.

Postinfectious bronchiolitis obliterans

PubMed

2018-06-01

La bronquiolitis obliterante es una enfermedad pulmonar crónica infrecuente y grave producto de una lesión del tracto respiratorio inferior. En nuestro país, es más frecuente observarla secundaria a una lesión viral grave, en especial, por adenovirus. La bronquiolitis obliterante se caracteriza por la oclusión parcial o total del lumen de los bronquiolos respiratorios y terminales por tejido inflamatorio y fibrosis, que produce la obstrucción crónica de la vía aérea. Este consenso discute el estado actual del conocimiento en las diferentes áreas de la bronquiolitis obliterante secundaria a una lesión infecciosa.

A right-ear bias of auditory selective attention is evident in alpha oscillations.

PubMed

Payne, Lisa; Rogers, Chad S; Wingfield, Arthur; Sekuler, Robert

2017-04-01

Auditory selective attention makes it possible to pick out one speech stream that is embedded in a multispeaker environment. We adapted a cued dichotic listening task to examine suppression of a speech stream lateralized to the nonattended ear, and to evaluate the effects of attention on the right ear's well-known advantage in the perception of linguistic stimuli. After being cued to attend to input from either their left or right ear, participants heard two different four-word streams presented simultaneously to the separate ears. Following each dichotic presentation, participants judged whether a spoken probe word had been in the attended ear's stream. We used EEG signals to track participants' spatial lateralization of auditory attention, which is marked by interhemispheric differences in EEG alpha (8-14 Hz) power. A right-ear advantage (REA) was evident in faster response times and greater sensitivity in distinguishing attended from unattended words. Consistent with the REA, we found strongest parietal and right frontotemporal alpha modulation during the attend-right condition. These findings provide evidence for a link between selective attention and the REA during directed dichotic listening. © 2016 Society for Psychophysiological Research.

Interaction of attention and acoustic factors in dichotic listening for fused words.

PubMed

McCulloch, Katie; Lachner Bass, Natascha; Dial, Heather; Hiscock, Merrill; Jansen, Ben

2017-07-01

Two dichotic listening experiments examined the degree to which the right-ear advantage (REA) for linguistic stimuli is altered by a "top-down" variable (i.e., directed attention) in conjunction with selected "bottom-up" (acoustic) variables. Halwes fused dichotic words were administered to 99 right-handed adults with instructions to attend to the left or right ear, or to divide attention equally. Stimuli in Experiment 1 were presented without noise or mixed with noise that was high-pass or low-pass filtered, or unfiltered. The stimuli themselves in Experiment 2 were high-pass or low-pass filtered, or unfiltered. The initial consonants of each dichotic pair were categorized according to voice onset time (VOT) and place of articulation (PoA). White noise extinguished both the REA and selective attention, and filtered noise nullified selective attention without extinguishing the REA. Frequency filtering of the words themselves did not alter performance. VOT effects were inconsistent across experiments but PoA analyses indicated that paired velar consonants (/k/ and /g/) yield a left-ear advantage and paradoxical selective-attention results. The findings show that ear asymmetry and the effectiveness of directed attention can be altered by bottom-up variables.

Binding Pattern Elucidation of NNK and NNAL Cigarette Smoke Carcinogens with NER Pathway Enzymes: an Onco- Informatics Study.

PubMed

Jamal, Qazi Mohammad Sajid; Dhasmana, Anupam; Lohani, Mohtashim; Firdaus, Sumbul; Ansari, Md Yousuf; Sahoo, Ganesh Chandra; Haque, Shafiul

2015-01-01

Cigarette smoke derivatives like NNK (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone) and NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol) are well-known carcinogens. We analyzed the interaction of enzymes involved in the NER (nucleotide excision repair) pathway with ligands (NNK and NNAL). Binding was characterized for the enzymes sharing equivalent or better interaction as compared to +Ve control. The highest obtained docking energy between NNK and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.13 kcal/mol, -7.27 kcal/mol, -8.05 kcal/mol and -7.58 kcal/mol respectively. Similarly the highest obtained docking energy between NNAL and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.46 kcal/mol, -7.94 kcal/mol, -7.83 kcal/mol and -7.67 kcal/mol respectively. In order to find out the effect of NNK and NNAL on enzymes involved in the NER pathway applying protein-protein interaction and protein-complex (i.e. enzymes docked with NNK/NNAL) interaction analysis. It was found that carcinogens are well capable to reduce the normal functioning of genes like RAD23A (HR23A), CCNH, CDK7 and CETN2. In silico analysis indicated loss of functions of these genes and their corresponding enzymes, which possibly might be a cause for alteration of DNA repair pathways leading to damage buildup and finally contributing to cancer formation.

Design and commissioning of a 16.1 MHz multiharmonic buncher for the reaccelerator at NSCL

NASA Astrophysics Data System (ADS)

Alt, Daniel Maloney

The ReAccelerator (ReA) linear accelerator facility at the National Superconducting Cyclotron Laboratory is a unique resource for the nuclear physics community. The particle fragmentation beam production technique, combined with the ability to stop and then reaccelerate the beam to energies of astrophysical interest, give experimenters an unprecedented range of rare isotopes at energies of nuclear and astrophysical interest. The ReAccelerator also functions as a testbed for technology to be incorporated in the upcoming Facility for Rare Isotope Beams linear accelerator, which will eventually in turn become the beam source for ReA. This prototype nature of the ReAccelerator, however, dictated some design choices which have resulted in a final beam with a time structure that is less than ideal for certain classes of experiments. The cavities and RFQ used in ReA have an operating frequency of 80.5 MHz, which corresponds to a separation between particle bunches at the detectors of 12.4 ns. While this separation is acceptable for many experiments, sensitive time of flight measurements require a greater separation between pulses. As nuclear physics experiments rely on statistics, a solution to increasing bunch separation without simply discarding a large fraction of the beam particles was desired. This document describes the design and construction of such a device, a 16.1 MHz multiharmonic buncher. The first chapter provides backgound information on the NSCL and ReA, and some basic concepts in accelerator physics to lay the groundwork for the project.Next, more specifics are provided on the time structure of accelerated beams, and the experimental motivation for greater separation. The third chapter outlines the basic principles of multiharmonic bunching. In order to evaluate the feasibility of any buncher design, the exact acceptance of the Radiofrequency Quadrupole (RFQ) of the ReAccelerator needed to be empirically measured. Chapter 4 describes the results of that measurement. Chapter 5 outlines the simulations and calculations that went into the design choices for this particular buncher, incorporating the results of the RFQ measurements. The next two chapters describe the construction, installation, and testing of the device, and give experimental results. Finally, Chapter 8 summarizes the project and the final steps which need to be undertaken to make the device a simple to use asset for future experimentalists at ReA.

Emissions of air pollutants and greenhouse gases over Asian regions during 2000-2008: Regional Emission inventory in ASia (REAS) version 2

NASA Astrophysics Data System (ADS)

Kurokawa, J.; Ohara, T.; Morikawa, T.; Hanayama, S.; Janssens-Maenhout, G.; Fukui, T.; Kawashima, K.; Akimoto, H.

2013-11-01

We have updated the Regional Emission inventory in ASia (REAS) as version 2.1. REAS 2.1 includes most major air pollutants and greenhouse gases from each year during 2000 and 2008 and following areas of Asia: East, Southeast, South, and Central Asia and the Asian part of Russia. Emissions are estimated for each country and region using updated activity data and parameters. Monthly gridded data with a 0.25° × 0.25° resolution are also provided. Asian emissions for each species in 2008 are as follows (with their growth rate from 2000 to 2008): 56.9 Tg (+34%) for SO2, 53.9 Tg (+54%) for NOx, 359.5 Tg (+34%) for CO, 68.5 Tg (+46%) for non-methane volatile organic compounds, 32.8 Tg (+17%) for NH3, 36.4 Tg (+45%) for PM10, 24.7 Tg (+42%) for PM2.5, 3.03 Tg (+35%) for black carbon, 7.72 Tg (+21%) for organic carbon, 182.2 Tg (+32%) for CH4, 5.80 Tg (+18%) for N2O, and 16.0 Pg (+57%) for CO2. By country, China and India were respectively the largest and second largest contributors to Asian emissions. Both countries also had higher growth rates in emissions than others because of their continuous increases in energy consumption, industrial activities, and infrastructure development. In China, emission mitigation measures have been implemented gradually. Emissions of SO2 in China increased from 2000 to 2006 and then began to decrease as flue-gas desulphurization was installed to large power plants. On the other hand, emissions of air pollutants in total East Asia except for China decreased from 2000 to 2008 owing to lower economic growth rates and more effective emission regulations in Japan, South Korea, and Taiwan. Emissions from other regions generally increased from 2000 to 2008, although their relative shares of total Asian emissions are smaller than those of China and India. Tables of annual emissions by country and region broken down by sub-sector and fuel type, and monthly gridded emission data with a resolution of 0.25° × 0.25° for the major sectors are available from the following URL: http://www.nies.go.jp/REAS/.

Emissions of air pollutants and greenhouse gases over Asian regions during 2000-2008: Regional Emission inventory in ASia (REAS) version 2

NASA Astrophysics Data System (ADS)

Kurokawa, J.; Ohara, T.; Morikawa, T.; Hanayama, S.; Greet, J.-M.; Fukui, T.; Kawashima, K.; Akimoto, H.

2013-04-01

We have updated the Regional Emission inventory in ASia (REAS) as version 2.1. REAS 2.1 includes most major air pollutants and greenhouse gases from each year during 2000 and 2008 and following areas of Asia: East, Southeast, South, and Central Asia and the Asian part of Russia. Emissions are estimated for each country and region using updated activity data and parameters. Monthly gridded data with a 0.25 × 0.25° resolution are also provided. Asian emissions for each species in 2008 are as follows (with their growth rate from 2000 to 2008): 56.9 Tg (+34%) for SO2, 53.9 Tg (+54%) for NOx, 359.5 Tg (+34%) for CO, 68.5 Tg (+46%) for non-methane volatile organic compounds, 32.8 Tg (+17%) for NH3, 36.4 Tg (+45%) for PM10, 24.7 Tg (+42%) for PM2.5, 3.03 Tg (+35%) for black carbon, 7.72 Tg (+21%) for organic carbon, 182.2 Tg (+32%) for CH4, 5.80 Tg (+18%) for N2O, and 16.7 Pg (+59%) for CO2. By country, China and India were respectively the largest and second largest contributors to Asian emissions. Both countries also had higher growth rates in emissions than others because of their continuous increases in energy consumption, industrial activities, and infrastructure development. In China, emission mitigation measures have been implemented gradually. Emissions of SO2 in China increased from 2000 to 2006 and then began to decrease as flue-gas desulfurization was installed to large power plants. On the other hand, emissions of air pollutants in total East Asia except for China decreased from 2000 to 2008 owing to lower economic growth rates and more effective emission regulations in Japan, South Korea, and Taiwan. Emissions from other regions generally increased from 2000 to 2008, although their relative shares of total Asian emissions are smaller than those of China and India. Tables of annual emissions by country and region broken down by sub-sector and fuel type, and monthly gridded emission data with a resolution of 0.25 × 0.25° for the major sectors are available from the following url: http://www.nies.go.jp/REAS/ .

The post-menopausal ovary displays a unique pattern of steroidogenic enzyme expression.

PubMed

Havelock, Jon C; Rainey, William E; Bradshaw, Karen D; Carr, Bruce R

2006-01-01

While menopause results in the loss of cyclic steroid production, evidence exists for persistent, albeit reduced, ovarian androgen production. In order to continue to synthesize ovarian androgens, the steroidogenic enzymes necessary for androgen biosynthesis must be present. Few studies have selectively analysed some of the steroidogenic enzymes present in the post-menopausal ovary (PMO), and a comprehensive study of this matter has never been undertaken. RNA and protein were obtained from PMO, pre-menopausal ovarian stroma, corpora lutea (CL), ovarian follicles, placenta, and myometrium. Oligonucleotide microarray analysis was performed to compare the gene expression profiles of PMO with pre-menopausal ovarian stroma. Real-time RT-PCR was performed for LH/HCG receptor (LHCGR), steroidogenic acute regulatory (StAR), cholesterol side-chain cleavage (CYP11A), 3beta-hydroxysteroid dehydrogenase type I (HSD3B1) and type II (HSD3B2, 3betaHSD), 17a-hydroxylase (CYP17), cytochrome b5 (CytB5), and aromatase (CYP19). Western blot analysis was performed for StAR, CYP11A, CYP17,and 3betaHSD. The PMO and pre-menopausal ovarian stroma had a similar pattern of steroidogenic enzyme expression. The PMO had persistent, but reduced, levels of LHCGR and most steroidogenic enzymes. CYP19 and HSD3B2 mRNA were greatly reduced in PMO in comparison with CL (50-fold and 2000-fold less respectively). HSD3B2 was not detectable in PMO by western analysis. This study supports the idea that the PMO retains some steroidogenic capacity. However, based on steroidogenic enzyme expression, the PMO has a unique pattern of steroidogenic enzyme expression that favors Delta5 steroid formation over Delta4 steroid formation.

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

PubMed Central

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

Proteomic analysis reveals large amounts of decomposition enzymes and major metabolic pathways involved in algicidal process of Trametes versicolor F21a.

PubMed

Gao, Xueyan; Wang, Congyan; Dai, Wei; Ren, Shenrong; Tao, Fang; He, Xingbing; Han, Guomin; Wang, Wei

2017-06-20

A recent algicidal mode indicates that fungal mycelia can wrap and eliminate almost all co-cultivated algal cells within a short time span. However, the underlying molecular mechanism is rarely understood. We applied proteomic analysis to investigate the algicidal process of Trametes versicolor F21a and identified 3,754 fungal proteins. Of these, 30 fungal enzymes with endo- or exoglycosidase activities such as β-1,3-glucanase, α-galactosidase, α-glucosidase, alginate lyase and chondroitin lyase were significantly up-regulated. These proteins belong to Glycoside Hydrolases, Auxiliary Activities, Carbohydrate Esterases and Polysaccharide Lyases, suggesting that these enzymes may degrade lipopolysaccharides, peptidoglycans and alginic acid of algal cells. Additionally, peptidase, exonuclease, manganese peroxidase and cytochrome c peroxidase, which decompose proteins and DNA or convert other small molecules of algal cells, could be other major decomposition enzymes. Gene Ontology and KEGG pathway enrichment analysis demonstrated that pyruvate metabolism and tricarboxylic acid cycle pathways play a critical role in response to adverse environment via increasing energy production to synthesize lytic enzymes or uptake molecules. Carbon metabolism, selenocompound metabolism, sulfur assimilation and metabolism, as well as several amino acid biosynthesis pathways could play vital roles in the synthesis of nutrients required by fungal mycelia.

Conformation Analysis of T1 Lipase on Alcohols Solvent using Molecular Dynamics Simulation

NASA Astrophysics Data System (ADS)

Putri, A. M.; Sumaryada, T.; Wahyudi, S. T.

2017-07-01

Biodiesel usually is produced commercially via a transesterification reaction of vegetable oil with alcohol and alkali catalyst. The alkali catalyst has some drawbacks, such as the soap formation during the reaction. T1 Lipase enzyme had been known as a thermostable biocatalyst which is able to produce biodiesel through a cleaner process. In this paper the performance of T1 lipase enzyme as catalyst for transesterification reaction in pure ethanol, methanol, and water solvents were studied using a Molecular Dynamics (MD) Simulation at temperature of 300 K for 10 nanoseconds. The results have shown that in general the conformation of T1 lipase enzyme in methanol is more dynamics as shown by the value of root mean square deviation (RMSD), root mean squared fluctuation (RMSF), and radius of gyration. The highest solvent accessible surface area (SASA) total was also found in methanol due to the contribution of non-polar amino acid in the interior of the protein. Analysis of MD simulation has also revealed that the enzyme structure tend to be more rigid in ethanol environment. The analysis of electrostatic interactions have shown that Glu359-Arg270 salt-bridge pair might hold the key of thermostability of T1 lipase enzyme as shown by its strong and stable binding in all three solvents.

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis.

PubMed

Florencio, Camila; Cunha, Fernanda M; Badino, Alberto C; Farinas, Cristiane S; Ximenes, Eduardo; Ladisch, Michael R

2016-08-01

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei. Copyright © 2016 Elsevier Inc. All rights reserved.

Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.

PubMed

Biner, Olivier; Trachsel, Christian; Moser, Aline; Kopp, Lukas; Langenegger, Nicolas; Kämpfer, Urs; von Ballmoos, Christoph; Nentwig, Wolfgang; Schürch, Stefan; Schaller, Johann; Kuhn-Nentwig, Lucia

2015-01-01

Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40-60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.

Stability of Ensemble Models Predicts Productivity of Enzymatic Systems

DOE PAGES

Theisen, Matthew K.; Lafontaine Rivera, Jimmy G.; Liao, James C.

2016-03-10

Stability in a metabolic system may not be obtained if incorrect amounts of enzymes are used. Without stability, some metabolites may accumulate or deplete leading to the irreversible loss of the desired operating point. Even if initial enzyme amounts achieve a stable steady state, changes in enzyme amount due to stochastic variations or environmental changes may move the system to the unstable region and lose the steady-state or quasi-steady-state flux. This situation is distinct from the phenomenon characterized by typical sensitivity analysis, which focuses on the smooth change before loss of stability. Here we show that metabolic networks differ significantlymore » in their intrinsic ability to attain stability due to the network structure and kinetic forms, and that after achieving stability, some enzymes are prone to cause instability upon changes in enzyme amounts. We use Ensemble Modelling for Robustness Analysis (EMRA) to analyze stability in four cell-free enzymatic systems when enzyme amounts are changed. Loss of stability in continuous systems can lead to lower production even when the system is tested experimentally in batch experiments. The predictions of instability by EMRA are supported by the lower productivity in batch experimental tests. Finally, the EMRA method incorporates properties of network structure, including stoichiometry and kinetic form, but does not require specific parameter values of the enzymes.« less

Evolutionary analysis of the TPP-dependent enzyme family.

PubMed

Costelloe, Seán J; Ward, John M; Dalby, Paul A

2008-01-01

The evolutionary relationships of the thiamine pyrophosphate (TPP)-dependent family of enzymes was investigated by generation of a neighbor joining phylogenetic tree using sequences from the conserved pyrophosphate (PP) and pyrimidine (Pyr) binding domains of 17 TPP-dependent enzymes. This represents the most comprehensive analysis of TPP-dependent enzyme evolution to date. The phylogeny was shown to be robust by comparison with maximum likelihood trees generated for each individual enzyme and also broadly confirms the evolutionary history proposed recently from structural comparisons alone (Duggleby 2006). The phylogeny is most parsimonious with the TPP enzymes having arisen from a homotetramer which subsequently diverged into an alpha(2)beta(2) heterotetramer. The relationship between the PP- and Pyr-domains and the recruitment of additional protein domains was examined using the transketolase C-terminal (TKC)-domain as an example. This domain has been recruited by several members of the family and yet forms no part of the active site and has unknown function. Removal of the TKC-domain was found to increase activity toward beta-hydroxypyruvate and glycolaldehyde. Further truncations of the Pyr-domain yielded several variants with retained activity. This suggests that the influence of TKC-domain recruitment on the evolution of the mechanism and specificity of transketolase (TK) has been minor, and that the smallest functioning unit of TK comprises the PP- and Pyr-domains, whose evolutionary histories extend to all TPP-dependent enzymes.

Portable Enzyme-Paper Biosensors Based on Redox-Active CeO2 Nanoparticles.

PubMed

Karimi, A; Othman, A; Andreescu, S

2016-01-01

Portable, nanoparticle (NP)-enhanced enzyme sensors have emerged as powerful devices for qualitative and quantitative analysis of a variety of analytes for biomedicine, environmental applications, and pharmaceutical fields. This chapter describes a method for the fabrication of a portable, paper-based, inexpensive, robust enzyme biosensor for the detection of substrates of oxidase enzymes. The method utilizes redox-active NPs of cerium oxide (CeO2) as a sensing platform which produces color in response to H2O2 generated by the action of oxidase enzymes on their corresponding substrates. This avoids the use of peroxidases which are routinely used in conjunction with glucose oxidase. The CeO2 particles serve dual roles, as high surface area supports to anchor high loadings of the enzyme as well as a color generation reagent, and the particles are recycled multiple times for the reuse of the biosensor. These sensors are small, light, disposable, inexpensive, and they can be mass produced by standard, low-cost printing methods. All reagents needed for the analysis are embedded within the paper matrix, and sensors stored over extended periods of time without performance loss. This novel sensor is a general platform for the in-field detection of analytes that are substrates for oxidase enzymes in clinical, food, and environmental samples. © 2016 Elsevier Inc. All rights reserved.

Analysis of the enzyme network involved in cattle milk production using graph theory.

PubMed

Ghorbani, Sholeh; Tahmoorespur, Mojtaba; Masoudi Nejad, Ali; Nasiri, Mohammad; Asgari, Yazdan

2015-06-01

Understanding cattle metabolism and its relationship with milk products is important in bovine breeding. A systemic view could lead to consequences that will result in a better understanding of existing concepts. Topological indices and quantitative characterizations mostly result from the application of graph theory on biological data. In the present work, the enzyme network involved in cattle milk production was reconstructed and analyzed based on available bovine genome information using several public datasets (NCBI, Uniprot, KEGG, and Brenda). The reconstructed network consisted of 3605 reactions named by KEGG compound numbers and 646 enzymes that catalyzed the corresponding reactions. The characteristics of the directed and undirected network were analyzed using Graph Theory. The mean path length was calculated to be4.39 and 5.41 for directed and undirected networks, respectively. The top 11 hub enzymes whose abnormality could harm bovine health and reduce milk production were determined. Therefore, the aim of constructing the enzyme centric network was twofold; first to find out whether such network followed the same properties of other biological networks, and second, to find the key enzymes. The results of the present study can improve our understanding of milk production in cattle. Also, analysis of the enzyme network can help improve the modeling and simulation of biological systems and help design desired phenotypes to increase milk production quality or quantity.

Analysis of 16S rRNA gene lactic acid bacteria (LAB) isolate from Markisa fruit (Passiflora sp.) as a producer of protease enzyme and probiotics

NASA Astrophysics Data System (ADS)

Hidayat, Habibi

2017-03-01

16S rRNA gene analysis of bacteria lactic acid (LAB) isolate from Markisa Kuning Fruit (Passiflora edulis var. flavicarpa) as a producer of protease enzyme and probiotics has been done. The aim of the study is to determine the protease enzyme activity and 16S rRNA gene amplification using PCR. The calculation procedure was done to M4 isolate bacteria lactic acid (LAB) Isolate which has been resistant to acids with pH 2.0 in the manner of screening protease enzyme activity test result 6.5 to clear zone is 13 mm againts colony diametre is 2 mm. The results of study enzyme activity used spectrophotometer UV-Vis obtainable the regression equation Y=0.02983+0.001312X, with levels of protein M4 isolate is 0.6594 mg/mL and enzyme activity of obtainable is 0.8626 unit/ml while the spesific enzyme activity produced is 1.308 unit/mg. Then, 16S rRNA gene amplificatiom and DNA sequencing has been done. The results of study showed that the bacteria species contained from M4 bacteria lactic acid (LAB) isolate is Weisella cibiria strain II-I-59. Weisella cibiria strain II-I-59 is one of bacteria could be utilized in the digestive tract.

A global characterization and identification of multifunctional enzymes.

PubMed

Cheng, Xian-Ying; Huang, Wei-Juan; Hu, Shi-Chang; Zhang, Hai-Lei; Wang, Hao; Zhang, Jing-Xian; Lin, Hong-Huang; Chen, Yu-Zong; Zou, Quan; Ji, Zhi-Liang

2012-01-01

Multi-functional enzymes are enzymes that perform multiple physiological functions. Characterization and identification of multi-functional enzymes are critical for communication and cooperation between different functions and pathways within a complex cellular system or between cells. In present study, we collected literature-reported 6,799 multi-functional enzymes and systematically characterized them in structural, functional, and evolutionary aspects. It was found that four physiochemical properties, that is, charge, polarizability, hydrophobicity, and solvent accessibility, are important for characterization of multi-functional enzymes. Accordingly, a combinational model of support vector machine and random forest model was constructed, based on which 6,956 potential novel multi-functional enzymes were successfully identified from the ENZYME database. Moreover, it was observed that multi-functional enzymes are non-evenly distributed in species, and that Bacteria have relatively more multi-functional enzymes than Archaebacteria and Eukaryota. Comparative analysis indicated that the multi-functional enzymes experienced a fluctuation of gene gain and loss during the evolution from S. cerevisiae to H. sapiens. Further pathway analyses indicated that a majority of multi-functional enzymes were well preserved in catalyzing several essential cellular processes, for example, metabolisms of carbohydrates, nucleotides, and amino acids. What's more, a database of known multi-functional enzymes and a server for novel multi-functional enzyme prediction were also constructed for free access at http://bioinf.xmu.edu.cn/databases/MFEs/index.htm.

Nonlinear Analysis of Experimental Measurements 7.6. Theoretical Chemistry

DTIC Science & Technology

2015-01-26

Jianshu Cao, Robert J. Silbey, Jaeyoung Sung. Quantitative Interpretation of the Randomness in Single Enzyme Turnover Times, Biophysical Journal...Universality of Poisson Indicator and Fano Factor of Transport Event Statistics in Ion Channels and Enzyme Kinetics., J. Phys. B: At. Mol. Opt. Phys...TOTAL: 4 01/26/2015 Received Book 4.00 Jianshu Cao, Jianlan Wu. GENERALIZED MICHAELIS–MENTENEQUATION FOR CONFORMATION- MODULATEDMONOMERIC ENZYMES , New

SGR 1822-1606 (Swift 1822.3-1606): Spin-down rate and inferred dipole field strength

NASA Astrophysics Data System (ADS)

Gogus, Ersin; Strohmayer, Tod; Kouveliotou, Chryssa

2011-07-01

We have been monitoring the new source Swift 1822.3-1606 (Cummings et al. GCN 12159) with RXTE. We acquired a total exposure of 20.6 ks in 5 pointings, spanning a time baseline of 5 days. We clearly detect the 8.44 s pulsations reported earlier (Pagani et al. ATel #3489, Gogus et al ATel #3491, Rea et al Atel #3501). We employed an epoch folding technique to determine the spin ephemeris. Our preliminary analysis reveal the spin period, P = 8.4377158(9) s and the spin-down rate, Pdot = 2.2(5) x 10-11 s/s (Epoch: 55758.5 MJD).

[Effect of low-intensity 900 MHz frequency electromagnetic radiation on rat liver and blood serum enzyme activities].

PubMed

Nersesova, L S; Petrosian, M S; Gazariants, M G; Mkrtchian, Z S; Meliksetian, G O; Pogosian, L G; Akopian, Zh I

2014-01-01

The comparative analysis of the rat liver and blood serum creatine kinase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and purine nucleoside phosphorylase post-radiation activity levels after a total two-hour long single and fractional exposure of the animals to low-intensity 900 MHz frequency electromagnetic field showed that the most sensitive enzymes to the both schedules of radiation are the liver creatine kinase, as well as the blood serum creatine kinase and alkaline phosphatase. According to the comparative analysis of the dynamics of changes in the activity level of the liver and blood serum creatine kinase, alanine aminotransferase, aspartate aminotransferase and purine nucleoside phosphorylase, both single and fractional radiation schedules do not affect the permeability of a hepatocyte cell membrane, but rather cause changes in their energetic metabolism. The correlation analysis of the post-radiation activity level changes of the investigated enzymes did not reveal a clear relationship between them. The dynamics of post-radiation changes in the activity of investigated enzyme levels following a single and short-term fractional schedules of radiation did not differ essentially.

Quantitative Structure-Activity Relationship Modeling Coupled with Molecular Docking Analysis in Screening of Angiotensin I-Converting Enzyme Inhibitory Peptides from Qula Casein Hydrolysates Obtained by Two-Enzyme Combination Hydrolysis.

PubMed

Lin, Kai; Zhang, Lanwei; Han, Xue; Meng, Zhaoxu; Zhang, Jianming; Wu, Yifan; Cheng, Dayou

2018-03-28

In this study, Qula casein derived from yak milk casein was hydrolyzed using a two-enzyme combination approach, and high angiotensin I-converting enzyme (ACE) inhibitory activity peptides were screened by quantitative structure-activity relationship (QSAR) modeling integrated with molecular docking analysis. Hydrolysates (<3 kDa) derived from combinations of thermolysin + alcalase and thermolysin + proteinase K demonstrated high ACE inhibitory activities. Peptide sequences in hydrolysates derived from these two combinations were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). On the basis of the QSAR modeling prediction, a total of 16 peptides were selected for molecular docking analysis. The docking study revealed that four of the peptides (KFPQY, MPFPKYP, MFPPQ, and QWQVL) bound the active site of ACE. These four novel peptides were chemically synthesized, and their IC 50 was determined. Among these peptides, KFPQY showed the highest ACE inhibitory activity (IC 50 = 12.37 ± 0.43 μM). Our study indicated that Qula casein presents an excellent source to produce ACE inhibitory peptides.

Principles for circadian orchestration of metabolic pathways.

PubMed

Thurley, Kevin; Herbst, Christopher; Wesener, Felix; Koller, Barbara; Wallach, Thomas; Maier, Bert; Kramer, Achim; Westermark, Pål O

2017-02-14

Circadian rhythms govern multiple aspects of animal metabolism. Transcriptome-, proteome- and metabolome-wide measurements have revealed widespread circadian rhythms in metabolism governed by a cellular genetic oscillator, the circadian core clock. However, it remains unclear if and under which conditions transcriptional rhythms cause rhythms in particular metabolites and metabolic fluxes. Here, we analyzed the circadian orchestration of metabolic pathways by direct measurement of enzyme activities, analysis of transcriptome data, and developing a theoretical method called circadian response analysis. Contrary to a common assumption, we found that pronounced rhythms in metabolic pathways are often favored by separation rather than alignment in the times of peak activity of key enzymes. This property holds true for a set of metabolic pathway motifs (e.g., linear chains and branching points) and also under the conditions of fast kinetics typical for metabolic reactions. By circadian response analysis of pathway motifs, we determined exact timing separation constraints on rhythmic enzyme activities that allow for substantial rhythms in pathway flux and metabolite concentrations. Direct measurements of circadian enzyme activities in mouse skeletal muscle confirmed that such timing separation occurs in vivo.

Principles for circadian orchestration of metabolic pathways

PubMed Central

Thurley, Kevin; Herbst, Christopher; Wesener, Felix; Koller, Barbara; Wallach, Thomas; Maier, Bert; Kramer, Achim

2017-01-01

Circadian rhythms govern multiple aspects of animal metabolism. Transcriptome-, proteome- and metabolome-wide measurements have revealed widespread circadian rhythms in metabolism governed by a cellular genetic oscillator, the circadian core clock. However, it remains unclear if and under which conditions transcriptional rhythms cause rhythms in particular metabolites and metabolic fluxes. Here, we analyzed the circadian orchestration of metabolic pathways by direct measurement of enzyme activities, analysis of transcriptome data, and developing a theoretical method called circadian response analysis. Contrary to a common assumption, we found that pronounced rhythms in metabolic pathways are often favored by separation rather than alignment in the times of peak activity of key enzymes. This property holds true for a set of metabolic pathway motifs (e.g., linear chains and branching points) and also under the conditions of fast kinetics typical for metabolic reactions. By circadian response analysis of pathway motifs, we determined exact timing separation constraints on rhythmic enzyme activities that allow for substantial rhythms in pathway flux and metabolite concentrations. Direct measurements of circadian enzyme activities in mouse skeletal muscle confirmed that such timing separation occurs in vivo. PMID:28159888

Expanding the Knowledge on Lignocellulolytic and Redox Enzymes of Worker and Soldier Castes from the Lower Termite Coptotermes gestroi.

PubMed

Franco Cairo, João P L; Carazzolle, Marcelo F; Leonardo, Flávia C; Mofatto, Luciana S; Brenelli, Lívia B; Gonçalves, Thiago A; Uchima, Cristiane A; Domingues, Romênia R; Alvarez, Thabata M; Tramontina, Robson; Vidal, Ramon O; Costa, Fernando F; Costa-Leonardo, Ana M; Paes Leme, Adriana F; Pereira, Gonçalo A G; Squina, Fabio M

2016-01-01

Termites are considered one of the most efficient decomposers of lignocelluloses on Earth due to their ability to produce, along with its microbial symbionts, a repertoire of carbohydrate-active enzymes (CAZymes). Recently, a set of Pro-oxidant, Antioxidant, and Detoxification enzymes (PAD) were also correlated with the metabolism of carbohydrates and lignin in termites. The lower termite Coptotermes gestroi is considered the main urban pest in Brazil, causing damage to wood constructions. Recently, analysis of the enzymatic repertoire of C. gestroi unveiled the presence of different CAZymes. Because the gene profile of CAZy/PAD enzymes endogenously synthesized by C. gestroi and also by their symbiotic protists remains unclear, the aim of this study was to explore the eukaryotic repertoire of these enzymes in worker and soldier castes of C. gestroi . Our findings showed that worker and soldier castes present similar repertoires of CAZy/PAD enzymes, and also confirmed that endo-glucanases (GH9) and beta-glucosidases (GH1) were the most important glycoside hydrolase families related to lignocellulose degradation in both castes. Classical cellulases such as exo-glucanases (GH7) and endo-glucanases (GH5 and GH45), as well as classical xylanases (GH10 and GH11), were found in both castes only taxonomically related to protists, highlighting the importance of symbiosis in C. gestroi . Moreover, our analysis revealed the presence of Auxiliary Activity enzyme families (AAs), which could be related to lignin modifications in termite digestomes. In conclusion, this report expanded the knowledge on genes and proteins related to CAZy/PAD enzymes from worker and soldier castes of lower termites, revealing new potential enzyme candidates for second-generation biofuel processes.

Sequence divergence and diversity suggests ongoing functional diversification of vertebrate NAD metabolism

PubMed Central

Gossmann, Toni I.; Ziegler, Mathias

2014-01-01

NAD is not only an important cofactor in redox reactions but has also received attention in recent years because of its physiological importance in metabolic regulation, DNA repair and signaling. In contrast to the redox reactions, these regulatory processes involve degradation of NAD and therefore necessitate a constant replenishment of its cellular pool. NAD biosynthetic enzymes are common to almost all species in all clades, but the number of NAD degrading enzymes varies substantially across taxa. In particular, vertebrates, including humans, have a manifold of NAD degrading enzymes which require a high turnover of NAD. As there is currently a lack of a systematic study of how natural selection has shaped enzymes involved in NAD metabolism we conducted a comprehensive evolutionary analysis based on intraspecific variation and interspecific divergence. We compare NAD biosynthetic and degrading enzymes in four eukaryotic model species and subsequently focus on human NAD metabolic enzymes and their orthologs in other vertebrates. We find that the majority of enzymes involved in NAD metabolism are subject to varying levels of purifying selection. While NAD biosynthetic enzymes appear to experience a rather high level of evolutionary constraint, there is evidence for positive selection among enzymes mediating NAD-dependent signaling. This is particularly evident for members of the PARP family, a diverse protein family involved in DNA damage repair and programmed cell death. Based on haplotype information and substitution rate analysis we pinpoint sites that are potential targets of positive selection. We also link our findings to a three dimensional structure, which suggests that positive selection occurs in domains responsible for DNA binding and polymerization rather than the NAD catalytic domain. Taken together, our results indicate that vertebrate NAD metabolism is still undergoing functional diversification. PMID:25084685

Sequence divergence and diversity suggests ongoing functional diversification of vertebrate NAD metabolism.

PubMed

Gossmann, Toni I; Ziegler, Mathias

2014-11-01

NAD is not only an important cofactor in redox reactions but has also received attention in recent years because of its physiological importance in metabolic regulation, DNA repair and signaling. In contrast to the redox reactions, these regulatory processes involve degradation of NAD and therefore necessitate a constant replenishment of its cellular pool. NAD biosynthetic enzymes are common to almost all species in all clades, but the number of NAD degrading enzymes varies substantially across taxa. In particular, vertebrates, including humans, have a manifold of NAD degrading enzymes which require a high turnover of NAD. As there is currently a lack of a systematic study of how natural selection has shaped enzymes involved in NAD metabolism we conducted a comprehensive evolutionary analysis based on intraspecific variation and interspecific divergence. We compare NAD biosynthetic and degrading enzymes in four eukaryotic model species and subsequently focus on human NAD metabolic enzymes and their orthologs in other vertebrates. We find that the majority of enzymes involved in NAD metabolism are subject to varying levels of purifying selection. While NAD biosynthetic enzymes appear to experience a rather high level of evolutionary constraint, there is evidence for positive selection among enzymes mediating NAD-dependent signaling. This is particularly evident for members of the PARP family, a diverse protein family involved in DNA damage repair and programmed cell death. Based on haplotype information and substitution rate analysis we pinpoint sites that are potential targets of positive selection. We also link our findings to a three dimensional structure, which suggests that positive selection occurs in domains responsible for DNA binding and polymerization rather than the NAD catalytic domain. Taken together, our results indicate that vertebrate NAD metabolism is still undergoing functional diversification. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Expanding the Knowledge on Lignocellulolytic and Redox Enzymes of Worker and Soldier Castes from the Lower Termite Coptotermes gestroi

PubMed Central

Franco Cairo, João P. L.; Carazzolle, Marcelo F.; Leonardo, Flávia C.; Mofatto, Luciana S.; Brenelli, Lívia B.; Gonçalves, Thiago A.; Uchima, Cristiane A.; Domingues, Romênia R.; Alvarez, Thabata M.; Tramontina, Robson; Vidal, Ramon O.; Costa, Fernando F.; Costa-Leonardo, Ana M.; Paes Leme, Adriana F.; Pereira, Gonçalo A. G.; Squina, Fabio M.

2016-01-01

Termites are considered one of the most efficient decomposers of lignocelluloses on Earth due to their ability to produce, along with its microbial symbionts, a repertoire of carbohydrate-active enzymes (CAZymes). Recently, a set of Pro-oxidant, Antioxidant, and Detoxification enzymes (PAD) were also correlated with the metabolism of carbohydrates and lignin in termites. The lower termite Coptotermes gestroi is considered the main urban pest in Brazil, causing damage to wood constructions. Recently, analysis of the enzymatic repertoire of C. gestroi unveiled the presence of different CAZymes. Because the gene profile of CAZy/PAD enzymes endogenously synthesized by C. gestroi and also by their symbiotic protists remains unclear, the aim of this study was to explore the eukaryotic repertoire of these enzymes in worker and soldier castes of C. gestroi. Our findings showed that worker and soldier castes present similar repertoires of CAZy/PAD enzymes, and also confirmed that endo-glucanases (GH9) and beta-glucosidases (GH1) were the most important glycoside hydrolase families related to lignocellulose degradation in both castes. Classical cellulases such as exo-glucanases (GH7) and endo-glucanases (GH5 and GH45), as well as classical xylanases (GH10 and GH11), were found in both castes only taxonomically related to protists, highlighting the importance of symbiosis in C. gestroi. Moreover, our analysis revealed the presence of Auxiliary Activity enzyme families (AAs), which could be related to lignin modifications in termite digestomes. In conclusion, this report expanded the knowledge on genes and proteins related to CAZy/PAD enzymes from worker and soldier castes of lower termites, revealing new potential enzyme candidates for second-generation biofuel processes. PMID:27790186

Characterization and identification of enzyme-producing microflora isolated from the gut of sea cucumber Apostichopus japonicus

NASA Astrophysics Data System (ADS)

Li, Fenghui; Gao, Fei; Tan, Jie; Fan, Chaojing; Sun, Huiling; Yan, Jingping; Chen, Siqing; Wang, Xiaojun

2016-01-01

Gut microorganisms play an important role in the digestion of their host animals. The purpose of this research was to isolate and assess the enzyme-producing microbes from the Apostichopus japonicus gut. Thirty-nine strains that can produce at least one of the three digestive enzymes (protease, amylase, and cellulase) were qualitatively screened based on their extracellular enzyme-producing abilities. The enzyme-producing strains clustered into eight groups at the genetic similarity level of 100% by analyzing the restriction patterns of 16S rDNA amplified with Mbo I. Phylogenetic analysis revealed that 37 strains belonged to the genus Bacillus and two were members of the genus Virgibacillus. Enzyme-producing capability results indicate that the main enzyme-producing microflora in the A. japonicus gut was Bacillus, which can produce protease, amylase, and cellulase. Virgibacillus, however, can only produce protease. The high enzyme-producing capability of the isolates suggests that the gut microbiota play an important role in the sea cucumber digestive process.

The biodiscovery potential of marine bacteria: an investigation of phylogeny and function

PubMed Central

Mühling, Martin; Joint, Ian; Willetts, Andrew J

2013-01-01

Summary A collection of marine bacteria isolated from a temperate coastal zone has been screened in a programme of biodiscovery. A total of 34 enzymes with biotechnological potential were screened in 374 isolates of marine bacteria. Only two enzymes were found in all isolates while the majority of enzyme activities were present in a smaller proportion of the isolates. A cluster analysis demonstrated no significant correlation between taxonomy and enzyme function. However, there was evidence of co-occurrence of some enzyme activity in the same isolate. In this study marine Proteobacteria had a higher complement of enzymes with biodiscovery potential than Actinobacteria; this contrasts with the terrestrial environment where the Actinobacteria phylum is a proven source of enzymes with important industrial applications. In addition, a number of novel enzyme functions were more abundant in this marine culture collection than would be expected on the basis of knowledge from terrestrial bacteria. There is a strong case for future investigation of marine bacteria as a source for biodiscovery. PMID:23557256

Production, partial purification and characterization of xylanase using Nicotiana tabacum leaf dust as substrate.

PubMed

Acharya, Komal P; Shilpkar, Prateek

2016-03-01

Isolated Bacillus sp. was used in the present study for production of xylanase from Nicotiana tabacum leaf dust. The strain was able to give a maximum of 1.77 Uml⁻¹ xylanase activity under optimized fermentation conditions which was further increased upto 2.77 Uml⁻¹ after extraction and partial purification of enzyme. After partial purification, the enzyme was characterized and it gave the highest xylanase activity at pH 7.0, when 0.2 ml enzyme was incubated with 2.0% substrate (Nicotiana tabacum leaf dust) for 60 min at 60°C. Saccharification study of Nicotiana tabacum leaf dust with partially purified enzyme revealed that 18.4% reducing sugar was released in 20 hrs incubation, and TLC and HPTLC analysis showed that xylose and glucose sugars were obtained after hydrolysis of substrate. FTIR analysis confirmed decomposition of substrate.

Chemical and protein structural basis for biological crosstalk between PPAR α and COX enzymes

NASA Astrophysics Data System (ADS)

Cleves, Ann E.; Jain, Ajay N.

2015-02-01

We have previously validated a probabilistic framework that combined computational approaches for predicting the biological activities of small molecule drugs. Molecule comparison methods included molecular structural similarity metrics and similarity computed from lexical analysis of text in drug package inserts. Here we present an analysis of novel drug/target predictions, focusing on those that were not obvious based on known pharmacological crosstalk. Considering those cases where the predicted target was an enzyme with known 3D structure allowed incorporation of information from molecular docking and protein binding pocket similarity in addition to ligand-based comparisons. Taken together, the combination of orthogonal information sources led to investigation of a surprising predicted relationship between a transcription factor and an enzyme, specifically, PPAR α and the cyclooxygenase enzymes. These predictions were confirmed by direct biochemical experiments which validate the approach and show for the first time that PPAR α agonists are cyclooxygenase inhibitors.

Techno-economic analysis of lipase enzyme production from agro-industry waste with solid state fermentation method

NASA Astrophysics Data System (ADS)

Hidayatullah, I. M.; Arbianti, R.; Utami, T. S.; Suci, M.; Sahlan, M.; Wijanarko, A.; Gozan, M.; Hermansyah, H.

2018-03-01

Needs for this kind of catalyst derived from biological raw materials (biocatalysts) has increased along with development of products based on eco-friendly. To achieve the needs of biocatalyst (enzyme), large production is necessary. This study aimed to get the best conditions and design equipment to produce lipase enzyme based on solid state fermentation using SuperPro Designer v9.0. Several equipment such as Tray Bioreactor, Mixing Tank 1, Filter Press, centrifuge, Mixing Tank 2, and a dryer have been improved during the simulation. Economic analysis in the form of NPV, IRR, Payback Period, and the Benefit Cost Ratio was evaluated respectively. The result showed that production of 10 kg enzyme with NPV Rp112.796.147.423,00; IRR 54.20%; Payback Period 1.95 years; and Benefit Cost Ratio of 3.36 was more advantageous.

Soil enzymes as biodiagnostics indicator of heavy metal pollution of urbanozem

NASA Astrophysics Data System (ADS)

Novosyolova, E. I.; Volkova, O. O.; Turyanova, R. R.

2018-01-01

The article presents a comparative analysis of the impact of the introduction of different doses of copper and cadmium on the activity of redox enzymes of urbanozem, collected from different territories of Ufa. The studies established the inverse relationship of the activity of catalase and polyphenol oxidase, and the direct one of the activity of peroxidase that depends on the doses of heavy metals, that allows to recommend their use as bioindicator of pollution of urbanozem with these metals. The reaction of the studied enzymes on the introduction of heavy metals is an indicator of their toxicity to living things at the molecular level. Comparative analysis of the impact of cadmium and copper in different doses on the activity of soil enzymes did not reveal a uniform regularity. Each of the metals showed their toxicity in different ways depending on the duration of their impact.

A B12-dependent radical SAM enzyme involved in Oxetanocin-A biosynthesis

PubMed Central

Bridwell-Rabb, Jennifer; Zhong, Aoshu; Sun, He G.; Drennan, Catherine L.; Liu, Hung-wen

2017-01-01

Summary Oxetanocin-A (OXT-A, 1) is a potent antitumor, antiviral, and antibacterial compound. Biosynthesis of OXT-A has been linked to a plasmid-borne, Bacillus megaterium gene cluster that contains four genes, oxsA, oxsB, oxrA, and oxrB. Here, we show that the oxsA and oxsB genes are both required for the production of OXT-A. Biochemical analysis of the encoded proteins, a cobalamin (Cbl)-dependent S-adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD-domain phosphohydrolase, OxsA, revealed that OXT-A is derived from 2′-deoxyadenosine phosphate in an OxsB-catalyzed ring contraction reaction initiated by H-atom abstraction from C2′. Hence, OxsB represents the first biochemically characterized non-methylating Cbl-dependent AdoMet radical enzyme. X-ray analysis of OxsB reveals the fold of a Cbl-dependent AdoMet radical enzyme for which there are an estimated 7000 members. Overall, this work provides a framework for understanding the interplay of AdoMet and Cbl cofactors and expands the catalytic repertoire of Cbl-dependent AdoMet radical enzymes. PMID:28346939

Intrinsic fluorescence spectroscopy of glutamate dehydrogenase: Integrated behavior and deconvolution analysis

NASA Astrophysics Data System (ADS)

Pompa, P. P.; Cingolani, R.; Rinaldi, R.

2003-07-01

In this paper, we present a deconvolution method aimed at spectrally resolving the broad fluorescence spectra of proteins, namely, of the enzyme bovine liver glutamate dehydrogenase (GDH). The analytical procedure is based on the deconvolution of the emission spectra into three distinct Gaussian fluorescing bands Gj. The relative changes of the Gj parameters are directly related to the conformational changes of the enzyme, and provide interesting information about the fluorescence dynamics of the individual emitting contributions. Our deconvolution method results in an excellent fitting of all the spectra obtained with GDH in a number of experimental conditions (various conformational states of the protein) and describes very well the dynamics of a variety of phenomena, such as the dependence of hexamers association on protein concentration, the dynamics of thermal denaturation, and the interaction process between the enzyme and external quenchers. The investigation was carried out by means of different optical experiments, i.e., native enzyme fluorescence, thermal-induced unfolding, and fluorescence quenching studies, utilizing both the analysis of the “average” behavior of the enzyme and the proposed deconvolution approach.

Two-dimensional cross correlation analysis of protein unfolding: Portrayal of the thermal denaturation of CMP kinases in the absence and presence of substrates

NASA Astrophysics Data System (ADS)

Schultz, Christian P.; Bârzu, Octavian; Mantsch, Henry H.

2000-03-01

The functional role of CMP kinases is to regenerate mono-phosphate nucleotides in cells by transferring phosphate residues from tri-phosphorylated nucleotides to monophosphorylated nucleotides. These enzymes possess two binding sites and maintain a highly conserved secondary structure. They are essential for cell survival. Herein we compare the infrared spectra of two similar, but not identical enzymes, the CMP kinases from Escherichia coli and Bacillus subtilis. A two-dimensional cross correlation analysis of the infrared spectra reveals differences in the denaturation behavior of the two proteins. Different secondary structure elements show different time-delayed or advanced unfolding events in the two enzymes. When bound to the active sites, the two nucleotide-substrates CMP and ATP exert a stabilizing effect on the structure of both proteins. The changes observed upon thermal denaturation are different for the two enzymes. Model 2D correlations are used to simulate the different denaturation of the two enzymes. Thermal denaturation and aggregation can be distinguished as two processes separated in time.

Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme.

PubMed

Torrens-Spence, Michael P; Gillaspy, Glenda; Zhao, Bingyu; Harich, Kim; White, Robert H; Li, Jianyong

2012-02-10

Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46mM) and dopa (km 1.40mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine. Copyright © 2012 Elsevier Inc. All rights reserved.

Rational design for the stability improvement of Armillariella tabescens β-mannanase MAN47 based on N-glycosylation modification.

PubMed

Hu, Weixiong; Liu, Xiaoyun; Li, Yufeng; Liu, Daling; Kuang, Zhihe; Qian, Chuiwen; Yao, Dongsheng

2017-02-01

β-Mannanase has been widely used in industries such as food and feed processing and thus has been a target enzyme for biotechnological development. In this study, we sought to improve the stability and protease resistance of a recombinant β-mannanase, MAN47 from Armillariella tabescens, through rationally designed N-glycosylation. Based on homology modeling, molecular docking, secondary structure analysis and glycosylation feasibility analysis, an enhanced aromatic sequon sequence was introduced into specific MAN47 loop regions to facilitate N-glycosylation. The mutant enzymes were expressed in Pichia pastoris SMD1168, and their thermal stability, pH stability, trypsin resistance and pepsin resistance were determined. Two mutant MAN47 enzymes, g-123 and g-347, were glycosylated as expected when expressed in yeast, and their thermal stability, pH stability, and protease resistance were significantly improved compared to the wild-type enzyme. An enzyme with multiple stability characterizations has broad prospects in practical applications, and the rational design N-glycosylation strategy may have applications in simultaneously improving several properties of other biotechnological targets. Copyright © 2016 Elsevier Inc. All rights reserved.

Tips on the analysis of phosphatidic acid by the fluorometric coupled enzyme assay.

PubMed

Hassaninasab, Azam; Han, Gil-Soo; Carman, George M

2017-06-01

The fluorometric coupled enzyme assay to measure phosphatidic acid (PA) involves the solubilization of extracted lipids in Triton X-100, deacylation, and the oxidation of PA-derived glycerol-3-phosphate to produce hydrogen peroxide for conversion of Amplex Red to resorufin. The enzyme assay is sensitive, but plagued by high background fluorescence from the peroxide-containing detergent and incomplete heat inactivation of lipoprotein lipase. These problems affecting the assay reproducibility were obviated by the use of highly pure Triton X-100 and by sufficient heat inactivation of the lipase enzyme. The enzyme assay could accurately measure the PA content from the subcellular fractions of yeast cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Halophiles and their enzymes: negativity put to good use.

PubMed

DasSarma, Shiladitya; DasSarma, Priya

2015-06-01

Halophilic microorganisms possess stable enzymes that function in very high salinity, an extreme condition that leads to denaturation, aggregation, and precipitation of most other proteins. Genomic and structural analyses have established that the enzymes of halophilic Archaea and many halophilic Bacteria are negatively charged due to an excess of acidic over basic residues, and altered hydrophobicity, which enhance solubility and promote function in low water activity conditions. Here, we provide an update on recent bioinformatic analysis of predicted halophilic proteomes as well as experimental molecular studies on individual halophilic enzymes. Recent efforts on discovery and utilization of halophiles and their enzymes for biotechnology, including biofuel applications are also considered. Copyright © 2015 Elsevier Ltd. All rights reserved.

Halophiles and their enzymes: Negativity put to good use

PubMed Central

DasSarma, Shiladitya; DasSarma, Priya

2015-01-01

Halophilic microorganisms possess stable enzymes that function in very high salinity, an extreme condition that leads to denaturation, aggregation, and precipitation of most other proteins. Genomic and structural analyses have established that the enzymes of halophilic Archaea and many halophilic Bacteria are negatively charged due to an excess of acidic over basic residues, and altered hydrophobicity, which enhance solubility and promote function in low water activity conditions. Here, we provide an update on recent bioinformatic analysis of predicted halophilic proteomes as well as experimental molecular studies on individual halophilic enzymes. On-going efforts on discovery and utilization of halophiles and their enzymes for biotechnology, including biofuel applications are also considered. PMID:26066288

Glycan analysis of recombinant Aspergillus niger endo-polygalacturonase A.

PubMed

Woosley, Bryan D; Kim, Young Hwan; Kumar Kolli, V S; Wells, Lance; King, Dan; Poe, Ryan; Orlando, Ron; Bergmann, Carl

2006-10-16

The enzyme endo-polygalacturonase A, or PGA, is produced by the fungus, Aspergillus niger, and appears to play a critical role during invasion of plant cell walls. The enzyme has been homologously overexpressed in order to provide sufficient quantities of purified enzyme for structural and biological studies. We have characterized this enzyme in terms of its post-translational modifications (PTMs) and found it to be both N- and O-glycosylated. Additionally, we have characterized the glycosyl moieties using MALDI-TOF and LC-ESI mass spectrometry. The characterization of all PTMs on PGA, along with molecular modeling, allows us to reveal potential roles played by the glycans in modulating the interaction of the enzyme with other macromolecules.

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

PubMed Central

Corcoran, Callan C.; Grady, Cameron R.; Pisitkun, Trairak; Parulekar, Jaya

2017-01-01

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. PMID:27974320

Coproduction of detergent compatible bacterial enzymes and stain removal evaluation.

PubMed

Niyonzima, Francois N; More, Sunil S

2015-10-01

Most of the detergents that are presently produced contain the detergent compatible enzymes to improve and accelerate the washing performance by removing tough stains. The process is environment friendly as the use of enzymes in the detergent formulation reduces the utilization of toxic detergent constituents. The current trend is to use the detergent compatible enzymes that are active at low and ambient temperature in order to save energy and maintain fabric quality. As the detergent compatible bacterial enzymes are used together in the detergent formulation, it is important to co-produce the detergent enzymes in a single fermentation medium as the enzyme stability is assured, and production cost gets reduced enormously. The review reports on the production, purification, characterization and application of detergent compatible amylases, lipases, and proteases are available. However, there is no specific review or minireview on the concomitant production of detergent compatible amylases, lipases, and proteases. In this minireview, the coproduction of detergent compatible enzymes by bacterial species, enzyme stability towards detergents and detergent components, and stain release analysis were discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional and structural characterization of the pentapeptide insertion of Theileria annulata lactate dehydrogenase by site-directed mutagenesis, comparative modeling and molecular dynamics simulations.

PubMed

Erdemir, Aysegul; Mutlu, Ozal

2017-06-01

Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Metagenomic mining pectinolytic microbes and enzymes from an apple pomace-adapted compost microbial community.

PubMed

Zhou, Man; Guo, Peng; Wang, Tao; Gao, Lina; Yin, Huijun; Cai, Cheng; Gu, Jie; Lü, Xin

2017-01-01

Degradation of pectin in lignocellulosic materials is one of the key steps for biofuel production. Biological hydrolysis of pectin, i.e., degradation by pectinolytic microbes and enzymes, is an attractive paradigm because of its obvious advantages, such as environmentally friendly procedures, low in energy demand for lignin removal, and the possibility to be integrated in consolidated process. In this study, a metagenomics sequence-guided strategy coupled with enrichment culture technique was used to facilitate targeted discovery of pectinolytic microbes and enzymes. An apple pomace-adapted compost (APAC) habitat was constructed to boost the enrichment of pectinolytic microorganisms. Analyses of 16S rDNA high-throughput sequencing revealed that microbial communities changed dramatically during composting with some bacterial populations being greatly enriched. Metagenomics data showed that apple pomace-adapted compost microbial community (APACMC) was dominated by Proteobacteria and Bacteroidetes . Functional analysis and carbohydrate-active enzyme profiles confirmed that APACMC had been successfully enriched for the targeted functions. Among the 1756 putative genes encoding pectinolytic enzymes, 129 were predicted as novel (with an identity <30% to any CAZy database entry) and only 1.92% were more than 75% identical with proteins in NCBI environmental database, demonstrating that they have not been observed in previous metagenome projects. Phylogenetic analysis showed that APACMC harbored a broad range of pectinolytic bacteria and many of them were previously unrecognized. The immensely diverse pectinolytic microbes and enzymes found in our study will expand the arsenal of proficient degraders and enzymes for lignocellulosic biofuel production. Our study provides a powerful approach for targeted mining microbes and enzymes in numerous industries.

Techniques for quantitative LC-MS/MS analysis of protein therapeutics: advances in enzyme digestion and immunocapture.

PubMed

Fung, Eliza N; Bryan, Peter; Kozhich, Alexander

2016-04-01

LC-MS/MS has been investigated to quantify protein therapeutics in biological matrices. The protein therapeutics is digested by an enzyme to generate surrogate peptide(s) before LC-MS/MS analysis. One challenge is isolating protein therapeutics in the presence of large number of endogenous proteins in biological matrices. Immunocapture, in which a capture agent is used to preferentially bind the protein therapeutics over other proteins, is gaining traction. The protein therapeutics is eluted for digestion and LC-MS/MS analysis. One area of tremendous potential for immunocapture-LC-MS/MS is to obtain quantitative data where ligand-binding assay alone is not sufficient, for example, quantitation of antidrug antibody complexes. Herein, we present an overview of recent advance in enzyme digestion and immunocapture applicable to protein quantitation.

Meta-analysis of expression and function of neprilysin in Alzheimer's disease.

PubMed

Zhang, Huifeng; Liu, Dan; Wang, Yixing; Huang, Huanhuan; Zhao, Yujia; Zhou, Hui

2017-09-14

Neprilysin (NEP) is one of the most important Aβ-degrading enzymes, and its expression and activity in Alzheimer's brain have been widely reported, but the results remain debatable. Thus, the meta-analysis was performed to elucidate the role of NEP in Alzheimer's disease (AD). The relevant case-control or cohort studies were retrieved according to our inclusion/exclusion criteria. Six studies with 123 controls and 141 AD cases, seven studies with 102 controls and 90 AD cases, and four studies with 93 controls and 132 AD cases were included in meta-analysis of NEP's protein, mRNA, and enzyme activity respectively. We conducted Meta regression to detect the sources of heterogeneity and further performed cumulative meta-analysis or subgroup analysis. Our meta-analysis revealed a significantly lower level of NEP mRNA (SMD=-0.44, 95%CI: -0.87, -0.00, p=0.049) in AD cases than in non-AD cases, and such pattern was not altered over time in the cumulative meta-analysis. However, the decrease of NEP protein (SMD=-0.18, 95%CI: -0.62, 0.25) and enzyme activity (SMD=-0.35, 95%CI: -1.03, 0.32) in AD cases did not pass the significance check, while the cumulative meta-analysis by average age showed the pooled effect became insignificant as adding the studies with younger subjects, which indicates that the protein expression and enzyme activity of NEP in the cortex are affected by age. Therefore, the present meta-analysis suggests the need of further investigation of roles of NEP in AD pathogenesis and treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Mechanisms of Enhanced Catalysis in Enzyme-DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis.

PubMed

Gao, Yingning; Roberts, Christopher C; Toop, Aaron; Chang, Chia-En A; Wheeldon, Ian

2016-08-03

Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme-DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9- and 2.4-fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4-aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural adaptation of cold-active RTX lipase from Pseudomonas sp. strain AMS8 revealed via homology and molecular dynamics simulation approaches.

PubMed

Mohamad Ali, Mohd Shukuri; Mohd Fuzi, Siti Farhanie; Ganasen, Menega; Abdul Rahman, Raja Noor Zaliha Raja; Basri, Mahiran; Salleh, Abu Bakar

2013-01-01

The psychrophilic enzyme is an interesting subject to study due to its special ability to adapt to extreme temperatures, unlike typical enzymes. Utilizing computer-aided software, the predicted structure and function of the enzyme lipase AMS8 (LipAMS8) (isolated from the psychrophilic Pseudomonas sp., obtained from the Antarctic soil) are studied. The enzyme shows significant sequence similarities with lipases from Pseudomonas sp. MIS38 and Serratia marcescens. These similarities aid in the prediction of the 3D molecular structure of the enzyme. In this study, 12 ns MD simulation is performed at different temperatures for structural flexibility and stability analysis. The results show that the enzyme is most stable at 0°C and 5°C. In terms of stability and flexibility, the catalytic domain (N-terminus) maintained its stability more than the noncatalytic domain (C-terminus), but the non-catalytic domain showed higher flexibility than the catalytic domain. The analysis of the structure and function of LipAMS8 provides new insights into the structural adaptation of this protein at low temperatures. The information obtained could be a useful tool for low temperature industrial applications and molecular engineering purposes, in the near future.

Molecular dynamics investigation of the ionic liquid/enzyme interface: application to engineering enzyme surface charge.

PubMed

Burney, Patrick R; Nordwald, Erik M; Hickman, Katie; Kaar, Joel L; Pfaendtner, Jim

2015-04-01

Molecular simulations of the enzymes Candida rugosa lipase and Bos taurus α-chymotrypsin in aqueous ionic liquids 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium ethyl sulfate were used to study the change in enzyme-solvent interactions induced by modification of the enzyme surface charge. The enzymes were altered by randomly mutating lysine surface residues to glutamate, effectively decreasing the net surface charge by two for each mutation. These mutations resemble succinylation of the enzyme by chemical modification, which has been shown to enhance the stability of both enzymes in ILs. After establishing that the enzymes were stable on the simulated time scales, we focused the analysis on the organization of the ionic liquid substituents about the enzyme surface. Calculated solvent charge densities show that for both enzymes and in both solvents that changing positively charged residues to negative charge does indeed increase the charge density of the solvent near the enzyme surface. The radial distribution of IL constituents with respect to the enzyme reveals decreased interactions with the anion are prevalent in the modified systems when compared to the wild type, which is largely accompanied by an increase in cation contact. Additionally, the radial dependence of the charge density and ion distribution indicates that the effect of altering enzyme charge is confined to short range (≤1 nm) ordering of the IL. Ultimately, these results, which are consistent with that from prior experiments, provide molecular insight into the effect of enzyme surface charge on enzyme stability in ILs. © 2015 Wiley Periodicals, Inc.

Application of liquid chromatography-electrospray ionization mass spectrometry for study of steroid-converting enzymes.

PubMed

Miksík, Ivan; Mikulíková, Katerina; Pácha, Jirí; Kucka, Marek; Deyl, Zdenek

2004-02-05

A high-performance liquid chromatography-atmospheric pressure ionization-electrospray ionization mass spectrometry (HPLC-API-ESI-MS) method was developed for the analysis of steroids in a study of steroid-converting enzymes. Separations ware done on a Zorbax Eclipse XDB-C18 column (eluted with a linear methanol-water-acetic acid gradient) and identification of the steroids involved was done by API-ESI-MS using positive ion mode and extracted ion analysis. The applicability of the present method for studying steroid metabolism was proven in assaying two steroid-converting enzymes (20beta-hydroxysteroid dehydrogenase and 11beta-hydroxysteroid dehydrogenase) in various biological samples (rat and chicken intestine, chicken oviduct).

Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation

PubMed Central

Yunus, Ali A.; Lima, Christopher D.

2009-01-01

SUMO conjugation to protein substrates requires the concerted action of a dedicated E2 ubiquitin conjugation enzyme (Ubc9) and associated E3 ligases. Although Ubc9 can directly recognize and modify substrate lysine residues that occur within a consensus site for SUMO modification, E3 ligases can redirect specificity and enhance conjugation rates during SUMO conjugation in vitro and in vivo. In this chapter, we will describe methods utilized to purify SUMO conjugating enzymes and model substrates which can be used for analysis of SUMO conjugation in vitro. We will also describe methods to extract kinetic parameters during E3-dependent or E3-independent substrate conjugation. PMID:19107417

qPIPSA: Relating enzymatic kinetic parameters and interaction fields

PubMed Central

Gabdoulline, Razif R; Stein, Matthias; Wade, Rebecca C

2007-01-01

Background The simulation of metabolic networks in quantitative systems biology requires the assignment of enzymatic kinetic parameters. Experimentally determined values are often not available and therefore computational methods to estimate these parameters are needed. It is possible to use the three-dimensional structure of an enzyme to perform simulations of a reaction and derive kinetic parameters. However, this is computationally demanding and requires detailed knowledge of the enzyme mechanism. We have therefore sought to develop a general, simple and computationally efficient procedure to relate protein structural information to enzymatic kinetic parameters that allows consistency between the kinetic and structural information to be checked and estimation of kinetic constants for structurally and mechanistically similar enzymes. Results We describe qPIPSA: quantitative Protein Interaction Property Similarity Analysis. In this analysis, molecular interaction fields, for example, electrostatic potentials, are computed from the enzyme structures. Differences in molecular interaction fields between enzymes are then related to the ratios of their kinetic parameters. This procedure can be used to estimate unknown kinetic parameters when enzyme structural information is available and kinetic parameters have been measured for related enzymes or were obtained under different conditions. The detailed interaction of the enzyme with substrate or cofactors is not modeled and is assumed to be similar for all the proteins compared. The protein structure modeling protocol employed ensures that differences between models reflect genuine differences between the protein sequences, rather than random fluctuations in protein structure. Conclusion Provided that the experimental conditions and the protein structural models refer to the same protein state or conformation, correlations between interaction fields and kinetic parameters can be established for sets of related enzymes. Outliers may arise due to variation in the importance of different contributions to the kinetic parameters, such as protein stability and conformational changes. The qPIPSA approach can assist in the validation as well as estimation of kinetic parameters, and provide insights into enzyme mechanism. PMID:17919319

Evaluation of partial 16S ribosomal DNA sequencing for identification of nocardia species by using the MicroSeq 500 system with an expanded database.

PubMed

Cloud, Joann L; Conville, Patricia S; Croft, Ann; Harmsen, Dag; Witebsky, Frank G; Carroll, Karen C

2004-02-01

Identification of clinically significant nocardiae to the species level is important in patient diagnosis and treatment. A study was performed to evaluate Nocardia species identification obtained by partial 16S ribosomal DNA (rDNA) sequencing by the MicroSeq 500 system with an expanded database. The expanded portion of the database was developed from partial 5' 16S rDNA sequences derived from 28 reference strains (from the American Type Culture Collection and the Japanese Collection of Microorganisms). The expanded MicroSeq 500 system was compared to (i). conventional identification obtained from a combination of growth characteristics with biochemical and drug susceptibility tests; (ii). molecular techniques involving restriction enzyme analysis (REA) of portions of the 16S rRNA and 65-kDa heat shock protein genes; and (iii). when necessary, sequencing of a 999-bp fragment of the 16S rRNA gene. An unknown isolate was identified as a particular species if the sequence obtained by partial 16S rDNA sequencing by the expanded MicroSeq 500 system was 99.0% similar to that of the reference strain. Ninety-four nocardiae representing 10 separate species were isolated from patient specimens and examined by using the three different methods. Sequencing of partial 16S rDNA by the expanded MicroSeq 500 system resulted in only 72% agreement with conventional methods for species identification and 90% agreement with the alternative molecular methods. Molecular methods for identification of Nocardia species provide more accurate and rapid results than the conventional methods using biochemical and susceptibility testing. With an expanded database, the MicroSeq 500 system for partial 16S rDNA was able to correctly identify the human pathogens N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. otitidiscaviarum, and N. veterana.

Cost analysis of DAWT innovative wind energy systems

NASA Astrophysics Data System (ADS)

Foreman, K. M.

The results of a diffuser augmented wind turbine (DAWT) preliminary design study of three constructional material approaches and cost analysis of DAWT electrical energy generation are presented. Costs are estimated assuming a limited production run (100 to 500 units) of factory-built subassemblies and on-site final assembly and erection within 200 miles of regional production centers. It is concluded that with the DAWT the (busbar) cost of electricity (COE) can range between 2.0 and 3.5 cents/kW-hr for farm and REA cooperative end users, for sites with annual average wind speeds of 16 and 12 mph respectively, and 150 kW rated units. No tax credit incentives are included in these figures. For commercial end users of the same units and site characteristics, the COE ranges between 4.0 and 6.5 cents/kW-hr.

Psychometric properties of the Body Checking Questionnaire in college women.

PubMed

White, Emily K; Claudat, Kim; Jones, Sarah C; Barchard, Kimberly A; Warren, Cortney S

2015-03-01

While the Body Checking Questionnaire (BCQ; Reas et al., 2002) is the most commonly-used measure of body checking behaviors, findings on the factor structure in nonclinical samples are mixed. This study investigated the factor structure and psychometric properties of the BCQ among nonclinical college women. In Study 1 (n=326), an exploratory factor analysis indicated factors corresponding to (a) behavioral and (b) visual checking. In Study 2 (n=1013), a confirmatory factor analysis revealed adequate fit for these factors, better than the original or total score solutions. Scales based upon the two factors demonstrated good internal consistency, convergent validity with measures of eating pathology, and sensitivity for detecting at-risk eating pathology. Results suggest an alternate two-factor solution that differs from the original three-factor solution. Copyright © 2015. Published by Elsevier Ltd.

Primary and secondary structural analyses of glutathione S-transferase pi from human placenta.

PubMed

Ahmad, H; Wilson, D E; Fritz, R R; Singh, S V; Medh, R D; Nagle, G T; Awasthi, Y C; Kurosky, A

1990-05-01

The primary structure of glutathione S-transferase (GST) pi from a single human placenta was determined. The structure was established by chemical characterization of tryptic and cyanogen bromide peptides as well as automated sequence analysis of the intact enzyme. The structural analysis indicated that the protein is comprised of 209 amino acid residues and gave no evidence of post-translational modifications. The amino acid sequence differed from that of the deduced amino acid sequence determined by nucleotide sequence analysis of a cDNA clone (Kano, T., Sakai, M., and Muramatsu, M., 1987, Cancer Res. 47, 5626-5630) at position 104 which contained both valine and isoleucine whereas the deduced sequence from nucleotide sequence analysis identified only isoleucine at this position. These results demonstrated that in the one individual placenta studied at least two GST pi genes are coexpressed, probably as a result of allelomorphism. Computer assisted consensus sequence evaluation identified a hydrophobic region in GST pi (residues 155-181) that was predicted to be either a buried transmembrane helical region or a signal sequence region. The significance of this hydrophobic region was interpreted in relation to the mode of action of the enzyme especially in regard to the potential involvement of a histidine in the active site mechanism. A comparison of the chemical similarity of five known human GST complete enzyme structures, one of pi, one of mu, two of alpha, and one microsomal, gave evidence that all five enzymes have evolved by a divergent evolutionary process after gene duplication, with the microsomal enzyme representing the most divergent form.

In silico studies on tryparedoxin peroxidase of Leishmania infantum: structural aspects.

PubMed

Singh, Bishal Kumar; Dubey, Vikash Kumar

2009-09-01

Tryparedoxin peroxidase (TryP) is a key enzyme of the trypanothione-dependent metabolism for removal of oxidative stress in leishmania. These enzymes function as antioxidants through their peroxidase and peroxynitrite reductase activities. Inhibitors of this enzyme are presumed to be antilesihmania drugs and structural studies are prerequisite of rational drug design. We have constructed three dimensional structure of TryP of Leishmania infantum using comparative modeling. Structural analysis reveals several interesting features. Moreover, it shows remarkable structural difference with human host glutathione peroxidase, an enzyme involved in similar function and TryP from Leishmania major.

Biosensors based on enzyme field-effect transistors for determination of some substrates and inhibitors.

PubMed

Dzyadevych, Sergei V; Soldatkin, Alexey P; Korpan, Yaroslav I; Arkhypova, Valentyna N; El'skaya, Anna V; Chovelon, Jean-Marc; Martelet, Claude; Jaffrezic-Renault, Nicole

2003-10-01

This paper is a review of the authors' publications concerning the development of biosensors based on enzyme field-effect transistors (ENFETs) for direct substrates or inhibitors analysis. Such biosensors were designed by using immobilised enzymes and ion-selective field-effect transistors (ISFETs). Highly specific, sensitive, simple, fast and cheap determination of different substances renders them as promising tools in medicine, biotechnology, environmental control, agriculture and the food industry. The biosensors based on ENFETs and direct enzyme analysis for determination of concentrations of different substrates (glucose, urea, penicillin, formaldehyde, creatinine, etc.) have been developed and their laboratory prototypes were fabricated. Improvement of the analytical characteristics of such biosensors may be achieved by using a differential mode of measurement, working solutions with different buffer concentrations and specific agents, negatively or positively charged additional membranes, or genetically modified enzymes. These approaches allow one to decrease the effect of the buffer capacity influence on the sensor response in an aim to increase the sensitivity of the biosensors and to extend their dynamic ranges. Biosensors for the determination of concentrations of different toxic substances (organophosphorous pesticides, heavy metal ions, hypochlorite, glycoalkaloids, etc.) were designed on the basis of reversible and/or irreversible enzyme inhibition effect(s). The conception of an enzymatic multibiosensor for the determination of different toxic substances based on the enzyme inhibition effect is also described. We will discuss the respective advantages and disadvantages of biosensors based on the ENFETs developed and also demonstrate their practical application.

Computational Functional Analysis of Lipid Metabolic Enzymes.

PubMed

Bagnato, Carolina; Have, Arjen Ten; Prados, María B; Beligni, María V

2017-01-01

The computational analysis of enzymes that participate in lipid metabolism has both common and unique challenges when compared to the whole protein universe. Some of the hurdles that interfere with the functional annotation of lipid metabolic enzymes that are common to other pathways include the definition of proper starting datasets, the construction of reliable multiple sequence alignments, the definition of appropriate evolutionary models, and the reconstruction of phylogenetic trees with high statistical support, particularly for large datasets. Most enzymes that take part in lipid metabolism belong to complex superfamilies with many members that are not involved in lipid metabolism. In addition, some enzymes that do not have sequence similarity catalyze similar or even identical reactions. Some of the challenges that, albeit not unique, are more specific to lipid metabolism refer to the high compartmentalization of the routes, the catalysis in hydrophobic environments and, related to this, the function near or in biological membranes.In this work, we provide guidelines intended to assist in the proper functional annotation of lipid metabolic enzymes, based on previous experiences related to the phospholipase D superfamily and the annotation of the triglyceride synthesis pathway in algae. We describe a pipeline that starts with the definition of an initial set of sequences to be used in similarity-based searches and ends in the reconstruction of phylogenies. We also mention the main issues that have to be taken into consideration when using tools to analyze subcellular localization, hydrophobicity patterns, or presence of transmembrane domains in lipid metabolic enzymes.

Rapid Identification of Sequences for Orphan Enzymes to Power Accurate Protein Annotation

PubMed Central

Ojha, Sunil; Watson, Douglas S.; Bomar, Martha G.; Galande, Amit K.; Shearer, Alexander G.

2013-01-01

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the “back catalog” of enzymology – “orphan enzymes,” those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme “back catalog” is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology’s “back catalog” another powerful tool to drive accurate genome annotation. PMID:24386392

Use of plasmid analysis and determination of aminoglycoside-modifying enzymes to characterize isolates from an outbreak of methicillin-resistant Staphylococcus aureus.

PubMed Central

Licitra, C M; Brooks, R G; Terry, P M; Shaw, K J; Hare, R S

1989-01-01

We compared disk susceptibility, plasmid analysis, aminoglycoside resistance patterns, and DNA hybridization for their usefulness in characterizing isolates from a hospital outbreak of methicillin-resistant Staphylococcus aureus. Fifteen isolates were susceptible (group 1) and 28 were resistant (group 2) to gentamicin. A total of 15 of 15 (100%) group 1 and 22 of 28 (79%) group 2 isolates carried a 21.5-megadalton plasmid. All group 2 isolates and none of the group 1 isolates possessed a 33-megadalton plasmid. Aminoglycoside resistance pattern determinations revealed the presence of the ANT(4')-I enzyme (aminoglycoside 4' adenyltransferase) in all group 1 isolates but was unable to demonstrate presence of this enzyme in group 2 organisms. The APH(2") + AAC(6')-II enzyme (aminoglycoside 2" phosphotransferase plus 6' acetyltransferase) was found in all of the group 2 isolates but in none of the group 1 isolates. Use of DNA hybridization revealed the presence of the ANT(4')-I enzyme in both groups (group 1, 14 of 15; group 2, 26 of 28). In this hospital outbreak, we found good correlation between disk susceptibility, plasmid profile, aminoglycoside resistance patterns, and DNA hybridization results. It was difficult to predict the presence of the ANT(4')-I enzyme in the presence of the bifunctional [APH(2") + AAC(6')-II] enzyme by the aminoglycoside resistance pattern method because of overlap of the substrate profile. Images PMID:2808676

Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

PubMed

Ramkissoon, Kevin R; Miller, Jennifer K; Ojha, Sunil; Watson, Douglas S; Bomar, Martha G; Galande, Amit K; Shearer, Alexander G

2013-01-01

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.

Superconducting Resonators Development for the FRIB and ReA Linacs at MSU: Recent Achievements and Future Goals

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

Facco, A; Binkowski, J; Compton, C

2012-07-01

The superconducting driver and post-accelerator linacs of the FRIB project, the large scale radioactive beam facility under construction at MSU, require the construction of about 400 low-{beta} Quarter-wave (QWR) and Half-wave resonators (HWR) with four different optimum velocities. 1st and 2nd generation prototypes of {beta}{sub 0} = 0.041 and 0.085 QWRs and {beta}{sub 0} = 0.53 HWRs have been built and tested, and have more than fulfilled the FRIB and ReA design goals. The present cavity surface preparation at MSU allowed production of low-{beta} cavities nearly free from field emission. The first two cryostats of {beta}{sub 0} = 0.041 QWRsmore » are now in operation in the ReA3 linac. A 3rd generation design of the FRIB resonators allowed to further improve the cavity parameters, reducing the peak magnetic field in operation and increasing the possible operation gradient, with consequent reduction of the number of required resonators. The construction of the cavities for FRIB, which includes three phases for each cavity type (development, pre-production and production runs) has started. Cavity design, construction, treatment and performance will be described and discussed.« less

Dichotic listening in patients with situs inversus: brain asymmetry and situs asymmetry.

PubMed

Tanaka, S; Kanzaki, R; Yoshibayashi, M; Kamiya, T; Sugishita, M

1999-06-01

In order to investigate the relation between situs asymmetry and functional asymmetry of the human brain, a consonant-vowel syllable dichotic listening test known as the Standard Dichotic Listening Test (SDLT) was administered to nine subjects with situs inversus (SI) that ranged in age from 6 to 46 years old (mean of 21.8 years old, S.D. = 15.6); the four males and five females all exhibited strong right-handedness. The SDLT was also used to study twenty four age-matched normal subjects that were from 6 to 48 years old (mean 21.7 years old, S.D. = 15.3); the twelve males and twelve females were all strongly right-handed and served as a control group. Eight out of the nine subjects (88.9%) with SI more often reproduced the sounds from the right ear than sounds from the left ear; this is called right ear advantage (REA). The ratio of REA in the control group was almost the same, i.e., nineteen out of the twenty-four subjects (79.1%) showed REA. Results of the present study suggest that the left-right reversal in situs inversus does not involve functional asymmetry of the brain. As such, the system that produces functional asymmetry in the human brain must independently recognize laterality from situs asymmetry.

PIERO ontology for analysis of biochemical transformations: effective implementation of reaction information in the IUBMB enzyme list.

PubMed

Kotera, Masaaki; Nishimura, Yosuke; Nakagawa, Zen-ichi; Muto, Ai; Moriya, Yuki; Okamoto, Shinobu; Kawashima, Shuichi; Katayama, Toshiaki; Tokimatsu, Toshiaki; Kanehisa, Minoru; Goto, Susumu

2014-12-01

Genomics is faced with the issue of many partially annotated putative enzyme-encoding genes for which activities have not yet been verified, while metabolomics is faced with the issue of many putative enzyme reactions for which full equations have not been verified. Knowledge of enzymes has been collected by IUBMB, and has been made public as the Enzyme List. To date, however, the terminology of the Enzyme List has not been assessed comprehensively by bioinformatics studies. Instead, most of the bioinformatics studies simply use the identifiers of the enzymes, i.e. the Enzyme Commission (EC) numbers. We investigated the actual usage of terminology throughout the Enzyme List, and demonstrated that the partial characteristics of reactions cannot be retrieved by simply using EC numbers. Thus, we developed a novel ontology, named PIERO, for annotating biochemical transformations as follows. First, the terminology describing enzymatic reactions was retrieved from the Enzyme List, and was grouped into those related to overall reactions and biochemical transformations. Consequently, these terms were mapped onto the actual transformations taken from enzymatic reaction equations. This ontology was linked to Gene Ontology (GO) and EC numbers, allowing the extraction of common partial reaction characteristics from given sets of orthologous genes and the elucidation of possible enzymes from the given transformations. Further future development of the PIERO ontology should enhance the Enzyme List to promote the integration of genomics and metabolomics.

Proteomic researches for lignocellulose-degrading enzymes: A mini-review.

PubMed

Guo, Hongliang; Wang, Xiao-Dong; Lee, Duu-Jong

2018-05-31

Protective action of lignin/hemicellulose networks and crystalline structures of embedded cellulose render lignocellulose material resistant to external enzymatic attack. To eliminate this bottleneck, research has been conducted in which advanced proteomic techniques are applied to identify effective commercial hydrolytic enzymes. This mini-review summarizes researches on lignocellulose-degrading enzymes, the mechanisms of the responses of various lignocellulose-degrading strains and microbial communities to various carbon sources and various biomass substrates, post-translational modifications of lignocellulose-degrading enzymes, new lignocellulose-degrading strains, new lignocellulose-degrading enzymes and a new method of secretome analysis. The challenges in the practical use of enzymatic hydrolysis process to realize lignocellulose biorefineries are discussed, along with the prospects for the same. Copyright © 2018 Elsevier Ltd. All rights reserved.

Novel HLA-B27-restricted epitopes from Chlamydia trachomatis generated upon endogenous processing of bacterial proteins suggest a role of molecular mimicry in reactive arthritis.

PubMed

Alvarez-Navarro, Carlos; Cragnolini, Juan J; Dos Santos, Helena G; Barnea, Eilon; Admon, Arie; Morreale, Antonio; López de Castro, José A

2013-09-06

Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na(+)-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27(+) cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330-338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211-223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211-223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.

Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis*

PubMed Central

Alvarez-Navarro, Carlos; Cragnolini, Juan J.; Dos Santos, Helena G.; Barnea, Eilon; Admon, Arie; Morreale, Antonio; López de Castro, José A.

2013-01-01

Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na+-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27+ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA. PMID:23867464

A Defined and Flexible Pocket Explains Aryl Substrate Promiscuity by the Cahuitamycin Starter Unit Activating Enzyme CahJ.

PubMed

Sherman, David H; Tripathi, Ashootosh; Park, Sung Ryeol; Sikkema, Andrew; Cho, Hyo Je; Wu, Jianfeng; Lee, Brian; Xi, Chuanwu; Smith, Janet L

2018-05-09

Cahuitamycins are biofilm inhibitors biosynthesized by a convergent NRPS pathway. Previous genetic analysis indicated that a discrete enzyme, CahJ, serves as a gatekeeper for cahuitamycin structural diversification. Herein, the CahJ protein was probed structurally, functionally and through mutasynthesis. This analysis enabled production of a new cahuitamycin congener through targeted precursor incorporation. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acute Exposure of Medaka to Carcinogens: An Ultrastructural, Cytochemical and Morphometric Analysis of Liver and Kidney

DTIC Science & Technology

1993-05-20

statements regarding acid phosphatase activity are not possible because of the inconsistency of labeling. Morphometric analysis were conducted on ratios of...may result from the combining of TCE and other toxic substances. The two enzyme systems selected for examination, acid phosphatase and peroxidase are...administration of DENA have demonstrated altered activity of several enzymes, including acid phosphatase (Fischer et al., 1983). Long term treatment of rodents

Enzyme-based electrochemical biosensors for determination of organophosphorus and carbamate pesticides

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

Everett, W.R.; Rechnitz, G.A.

1999-01-01

A mini review of enzyme-based electrochemical biosensors for inhibition analysis of organophosphorus and carbamate pesticides is presented. Discussion includes the most recent literature to present advances in detection limits, selectivity and real sample analysis. Recent reviews on the monitoring of pesticides and their residues suggest that the classical analytical techniques of gas and liquid chromatography are the most widely used methods of detection. These techniques, although very accurate in their determinations, can be quite time consuming and expensive and usually require extensive sample clean up and pro-concentration. For these and many other reasons, the classical techniques are very difficult tomore » adapt for field use. Numerous researchers, in the past decade, have developed and made improvements on biosensors for use in pesticide analysis. This mini review will focus on recent advances made in enzyme-based electrochemical biosensors for the determinations of organophosphorus and carbamate pesticides.« less

Enzyme/non-enzyme discrimination and prediction of enzyme active site location using charge-based methods.

PubMed

Bate, Paul; Warwicker, Jim

2004-07-02

Calculations of charge interactions complement analysis of a characterised active site, rationalising pH-dependence of activity and transition state stabilisation. Prediction of active site location through large DeltapK(a)s or electrostatic strain is relevant for structural genomics. We report a study of ionisable groups in a set of 20 enzymes, finding that false positives obscure predictive potential. In a larger set of 156 enzymes, peaks in solvent-space electrostatic properties are calculated. Both electric field and potential match well to active site location. The best correlation is found with electrostatic potential calculated from uniform charge density over enzyme volume, rather than from assignment of a standard atom-specific charge set. Studying a shell around each molecule, for 77% of enzymes the potential peak is within that 5% of the shell closest to the active site centre, and 86% within 10%. Active site identification by largest cleft, also with projection onto a shell, gives 58% of enzymes for which the centre of the largest cleft lies within 5% of the active site, and 70% within 10%. Dielectric boundary conditions emphasise clefts in the uniform charge density method, which is suited to recognition of binding pockets embedded within larger clefts. The variation of peak potential with distance from active site, and comparison between enzyme and non-enzyme sets, gives an optimal threshold distinguishing enzyme from non-enzyme. We find that 87% of the enzyme set exceeds the threshold as compared to 29% of the non-enzyme set. Enzyme/non-enzyme homologues, "structural genomics" annotated proteins and catalytic/non-catalytic RNAs are studied in this context.

Integrative computational approach for genome-based study of microbial lipid-degrading enzymes.

PubMed

Vorapreeda, Tayvich; Thammarongtham, Chinae; Laoteng, Kobkul

2016-07-01

Lipid-degrading or lipolytic enzymes have gained enormous attention in academic and industrial sectors. Several efforts are underway to discover new lipase enzymes from a variety of microorganisms with particular catalytic properties to be used for extensive applications. In addition, various tools and strategies have been implemented to unravel the functional relevance of the versatile lipid-degrading enzymes for special purposes. This review highlights the study of microbial lipid-degrading enzymes through an integrative computational approach. The identification of putative lipase genes from microbial genomes and metagenomic libraries using homology-based mining is discussed, with an emphasis on sequence analysis of conserved motifs and enzyme topology. Molecular modelling of three-dimensional structure on the basis of sequence similarity is shown to be a potential approach for exploring the structural and functional relationships of candidate lipase enzymes. The perspectives on a discriminative framework of cutting-edge tools and technologies, including bioinformatics, computational biology, functional genomics and functional proteomics, intended to facilitate rapid progress in understanding lipolysis mechanism and to discover novel lipid-degrading enzymes of microorganisms are discussed.

Nickel-impregnated silica nanoparticle synthesis and their evaluation for biocatalyst immobilization.

PubMed

Prakasham, Reddy Shetty; Devi, G Sarala; Rao, Chaganti Subba; Sivakumar, V S S; Sathish, T; Sarma, P N

2010-04-01

In the present investigation, impact of nickel-impregnated silica paramagnetic particles (NSP) as biocatalyst immobilization matrices was investigated. These nanoparticles were synthesized by sol-gel route using a nonionic surfactant block co polymer [poly (ethylene glycol)-block-poly-(propylene glycol)-block-poly (ethylene glycol)]. Diastase enzyme was immobilized on these particles (enzyme-impregnated NSP) as model enzyme and characterized using Fourier-transform infrared spectroscopy and X-ray crystallography. Analysis of enzyme-binding nature with these nanoparticles at different physiological conditions revealed that binding pattern and activity profile varied with the pH of the reaction mixture. The immobilized enzyme was further characterized for its biocatalytic activity with respect to kinetic properties such as Km and Vmax and compared with free enzyme. Paramagnetic nanoparticle-immobilized enzyme showed more affinity for substrate compared to free one. The nature of silica and nickel varied from amorphous to crystalline nature and vice versa upon immobilization of enzyme. To the best of our knowledge, this is the first report of its kind for change of nature from one form to other under normal temperatures upon diastase interaction with NSP.

Efficient elimination of nonstoichiometric enzyme inhibitors from HTS hit lists.

PubMed

Habig, Michael; Blechschmidt, Anke; Dressler, Sigmar; Hess, Barbara; Patel, Viral; Billich, Andreas; Ostermeier, Christian; Beer, David; Klumpp, Martin

2009-07-01

High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC(50) values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms.

Enzyme Sorption onto Soil and Biocarbon Amendments Alters Catalytic Capacity and Depends on the Specific Protein and pH

NASA Astrophysics Data System (ADS)

Foster, E.; Fogle, E. J.; Cotrufo, M. F.

2017-12-01

Enzymes catalyze biogeochemical reactions in soils and play a key role in nutrient cycling in agricultural systems. Often, to increase soil nutrients, agricultural managers add organic amendments and have recently experimented with charcoal-like biocarbon products. These amendments can enhance soil water and nutrient holding capacity through increasing porosity. However, the large surface area of the biocarbon has the potential to sorb nutrients and other organic molecules. Does the biocarbon decrease nutrient cycling through sorption of enzymes? In a laboratory setting, we compared the interaction of two purified enzymes β-glucosidase and acid phosphatase with a sandy clay loam and two biocarbons. We quantified the sorbed enzymes at three different pHs using a Bradford protein assay and then measured the activity of the sorbed enzyme via high-throughput fluorometric analysis. Both sorption and activity depended upon the solid phase, pH, and specific enzyme. Overall the high surface area biocarbon impacted the catalytic capacity of the enzymes more than the loam soil, which may have implications for soil nutrient management with these organic amendments.

Characterization of cellulolytic enzyme system of Schizophyllum commune mutant and evaluation of its efficiency on biomass hydrolysis.

PubMed

Sornlake, Warasirin; Rattanaphanjak, Phatcharamon; Champreda, Verawat; Eurwilaichitr, Lily; Kittisenachai, Suthathip; Roytrakul, Sittiruk; Fujii, Tatsuya; Inoue, Hiroyuki

2017-07-01

Schizophyllum commune is a basidiomycete equipped with an efficient cellulolytic enzyme system capable of growth on decaying woods. In this study, production of lignocellulose-degrading enzymes from S. commune mutant G-135 (SC-Cel) on various cellulosic substrates was examined. The highest cellulase activities including CMCase, FPase, and β-glucosidase were obtained on Avicel-PH101 while a wider range of enzymes attacking non-cellulosic polysaccharides and lignin were found when grown on alkaline-pretreated biomass. Proteomic analysis of SC-Cel also revealed a complex enzyme system comprising seven glycosyl hydrolase families with an accessory carbohydrate esterase, polysaccharide lyase, and auxiliary redox enzymes. SC-Cel obtained on Avicel-PH101 effectively hydrolyzed all agricultural residues with the maximum glucan conversion of 98.0% using corn cobs with an enzyme dosage of 5 FPU/g-biomass. The work showed potential of SC-Cel on hydrolysis of various herbaceous biomass with enhanced efficiency by addition external β-xylosidase.

Following the compositional changes of fresh grape skin cell walls during the fermentation process in the presence and absence of maceration enzymes.

PubMed

Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Trygg, Johan; Vivier, Melané A

2015-03-18

Cell wall profiling technologies were used to follow compositional changes that occurred in the skins of grape berries (from two different ripeness levels) during fermentation and enzyme maceration. Multivariate data analysis showed that the fermentation process yielded cell walls enriched in hemicellulose components because pectin was solubilized (and removed) with a reduction as well as exposure of cell wall proteins usually embedded within the cell wall structure. The addition of enzymes caused even more depectination, and the enzymes unravelled the cell walls enabling better access to, and extraction of, all cell wall polymers. Overripe grapes had cell walls that were extensively hydrolyzed and depolymerized, probably by natural grape-tissue-ripening enzymes, and this enhanced the impact that the maceration enzymes had on the cell wall monosaccharide profile. The combination of the techniques that were used is an effective direct measurement of the hydrolysis actions of maceration enzymes on the cell walls of grape berry skin.

Comparative genomic and phylogenetic investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family

USDA-ARS?s Scientific Manuscript database

Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes characterized in several bacteria and eukaryotic organisms. We report a comprehensive phylogenetic analysis employing an exhaustive dataset of NAT-homologous sequences recovered through inspection of 2445 genomes. We describe ...

Diversity and strain specificity of plant cell wall degrading enzymes revealed by the draft genome of Ruminococcus flavefaciens FD-1.

PubMed

Berg Miller, Margret E; Antonopoulos, Dionysios A; Rincon, Marco T; Band, Mark; Bari, Albert; Akraiko, Tatsiana; Hernandez, Alvaro; Thimmapuram, Jyothi; Henrissat, Bernard; Coutinho, Pedro M; Borovok, Ilya; Jindou, Sadanari; Lamed, Raphael; Flint, Harry J; Bayer, Edward A; White, Bryan A

2009-08-14

Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components.

A new low-B magnetar: Swift J1822.3-1606

NASA Astrophysics Data System (ADS)

Camero-Arranz, A.; Rea, N.; Israel, G. L.; Esposito, P.; Pons, J. A.; Mignani, R. P.; Turolla, R.; Zane, S.; Burgay, M.; Possenti, A.; Campana, S.; Enoto, T.; Gehrels, N.; Göğüş, E.; Götz, D.; Kouveliotou, C.; Makishima, K.; Mereghetti, S.; Oates, S. R.

2013-03-01

We report on the long term X-ray monitoring with Swift, RXTE, Suzaku, Chandra, and XMM-Newton of the outburst of the newly discovered magnetar Swift J1822.3-1606 (SGR 1822-1606), from the first observations soon after the detection of the short X-ray bursts which led to its discovery (July 2011), through the first stages of its outburst decay (April 2012). Our X-ray timing analysis finds the source rotating with a period of P = 8.43772016(2) s and a period derivative Ṗ = 8.3(2) × 10-14 ss-1, which entails an inferred dipolar surface magnetic field of B ≃ 2.7 × 1013 G at the equator. This measurement makes Swift J1822.3-1606 the second lowest magnetic field magnetar (after SGR 0418+5729; Rea et al. 2010). Following the flux and spectral evolution from the beginning of the outburst, we find that the flux decreased by about an order of magnitude, with a subtle softening of the spectrum, both typical of the outburst decay of magnetars. By modeling the secular thermal evolution of Swift J1822.3-1606, we find that the observed timing properties of the source, as well as its quiescent X-ray luminosity, can be reproduced if it was born with a poloidal and crustal toroidal fields of Bp ~ 1.5 × 1014 G and Btor ~ 7 × 1014 G, respectively, and if its current age is ~550 kyr (Rea et al. 2012).

Enzyme phylogenies as markers for the oxidation state of the environment: The case of respiratory arsenate reductase and related enzymes

PubMed Central

2008-01-01

Background Phylogenies of certain bioenergetic enzymes have proved to be useful tools for deducing evolutionary ancestry of bioenergetic pathways and their relationship to geochemical parameters of the environment. Our previous phylogenetic analysis of arsenite oxidase, the molybdopterin enzyme responsible for the biological oxidation of arsenite to arsenate, indicated its probable emergence prior to the Archaea/Bacteria split more than 3 billion years ago, in line with the geochemical fact that arsenite was present in biological habitats on the early Earth. Respiratory arsenate reductase (Arr), another molybdopterin enzyme involved in microbial arsenic metabolism, serves as terminal oxidase, and is thus situated at the opposite end of bioenergetic electron transfer chains as compared to arsenite oxidase. The evolutionary history of the Arr-enzyme has not been studied in detail so far. Results We performed a genomic search of genes related to arrA coding for the molybdopterin subunit. The multiple alignment of the retrieved sequences served to reconstruct a neighbor-joining phylogeny of Arr and closely related enzymes. Our analysis confirmed the previously proposed proximity of Arr to the cluster of polysulfide/thiosulfate reductases but also unravels a hitherto unrecognized clade even more closely related to Arr. The obtained phylogeny strongly suggests that Arr originated after the Bacteria/Archaea divergence in the domain Bacteria, and was subsequently laterally distributed within this domain. It further more indicates that, as a result of accumulation of arsenate in the environment, an enzyme related to polysulfide reductase and not to arsenite oxidase has evolved into Arr. Conclusion These findings are paleogeochemically rationalized by the fact that the accumulation of arsenate over arsenite required the increase in oxidation state of the environment brought about by oxygenic photosynthesis. PMID:18631373

The Use of Enzyme Hydrolysis to Assess the Seasonal Mobility and Bioavailability of Organic Phosphorus in Lake Sediments

NASA Astrophysics Data System (ADS)

Giles, C. D.; Lee, L. G.; Cade-Menun, B. J.; Rutila, B. C.; Schroth, A. W.; Xu, Y.; Hill, J. E.; Druschel, G.

2013-12-01

Lake sediments represent a significant internal source of phosphorus (P) in eutrophic freshwater systems during periods of high biological activity and oxygen depletion in sediments. Enzyme-labile and redox-sensitive P fractions may be a major component of the mobile sediment P pool which contributes to the development of harmful algal blooms. We present a high-through-put enzyme-based method for assessing potentially bioavailable (enzyme-labile) P in lake sediments and describe the relationship between enzyme-labile P, ascorbate-extractable (reactive) P and metals (Fe, Mn, Al, Ca), and P species identified using solution 31-P NMR spectroscopy. Sediment cores (0-10 cm) were collected from Lake Champlain over multiple years (Missisquoi Bay, VT, USA; 2007-2013). A principal components analysis of sediment properties suggests that enzyme-labile and reactive P, Mn, and Fe concentrations were more effective than the 31-P NMR methodology alone for differentiating algal bloom stage associated with periods of sediment anoxia. Bloom onset (July 2008) and peak bloom (August 2008, 2012) periods corresponded to the highest enzyme-labile P and lowest reactive P and metals proportions, despite 31-P NMR profiles which did not change significantly with respect to time and depth. High levels of reduced Fe and Mn ions were also detected in pore-water during this period, confirming previous reports that organic P bioavailability is linked to the redox status of sediments. High through-put analysis of enzyme-labile P fractions will provide spatially and temporally resolved information on bioavailable P pools at lower cost than traditional methods (i.e., 31-P NMR), and provide much-needed detail on aquatic P cycles during discrete stages of algal bloom development and sediment anoxia.

The Cytophaga hutchinsonii ChTPSP: First characterized bifunctional TPS-TPP protein as putative ancestor of all eukaryotic trehalose biosynthesis proteins.

PubMed

Avonce, Nelson; Wuyts, Jan; Verschooten, Katrien; Vandesteene, Lies; Van Dijck, Patrick

2010-02-01

The most widely distributed pathway to synthesize trehalose in nature consists of two consecutive enzymatic reactions with a trehalose-6-P (T6P)-synthase (TPS) enzyme, producing the intermediate T6P, and a T6P-phosphatase (TPP) enzyme, which dephosphorylates T6P to produce trehalose and inorganic phosphate. In plants, these enzymes are called Class I and Class II proteins, respectively, with some Class I proteins being active enzymes. The Class II proteins possess both TPS and TPP consensus regions but appear to have lost enzymatic activity during evolution. Plants also contain an extra group of enzymes of small protein size, of which some members have been characterized as functional TPPs. These Class III proteins have less sequence similarity with the Class I and Class II proteins. Here, we characterize for the first time, by using biochemical analysis and yeast growth complementation assays, the existence of a natural TPS-TPP bifunctional enzyme found in the bacterial species Cytophaga hutchinsonii. Through phylogenetic analysis, we show that prokaryotic genes such as ChTPSP might be the ancestor of the eukaryotic trehalose biosynthesis genes. Second, we show that plants have recruited during evolution, possibly by horizontal transfer from bacteria such as Rhodoferax ferrireducens, a new type of small protein, encoding TPP activity, which have been named Class III proteins. RfTPP has very high TPP activity upon expression in yeast. Finally, we demonstrate that TPS gene duplication, the recruitment of the Class III enzymes, and recruitment of an N-terminal regulatory element, which regulates the Class I enzyme activity in higher plants, were initiated very early in eukaryan evolution as the three classes of trehalose biosynthesis genes are already present in the alga Ostreococcus tauri.

Enzyme phylogenies as markers for the oxidation state of the environment: the case of respiratory arsenate reductase and related enzymes.

PubMed

Duval, Simon; Ducluzeau, Anne-Lise; Nitschke, Wolfgang; Schoepp-Cothenet, Barbara

2008-07-16

Phylogenies of certain bioenergetic enzymes have proved to be useful tools for deducing evolutionary ancestry of bioenergetic pathways and their relationship to geochemical parameters of the environment. Our previous phylogenetic analysis of arsenite oxidase, the molybdopterin enzyme responsible for the biological oxidation of arsenite to arsenate, indicated its probable emergence prior to the Archaea/Bacteria split more than 3 billion years ago, in line with the geochemical fact that arsenite was present in biological habitats on the early Earth. Respiratory arsenate reductase (Arr), another molybdopterin enzyme involved in microbial arsenic metabolism, serves as terminal oxidase, and is thus situated at the opposite end of bioenergetic electron transfer chains as compared to arsenite oxidase. The evolutionary history of the Arr-enzyme has not been studied in detail so far. We performed a genomic search of genes related to arrA coding for the molybdopterin subunit. The multiple alignment of the retrieved sequences served to reconstruct a neighbor-joining phylogeny of Arr and closely related enzymes. Our analysis confirmed the previously proposed proximity of Arr to the cluster of polysulfide/thiosulfate reductases but also unravels a hitherto unrecognized clade even more closely related to Arr. The obtained phylogeny strongly suggests that Arr originated after the Bacteria/Archaea divergence in the domain Bacteria, and was subsequently laterally distributed within this domain. It further more indicates that, as a result of accumulation of arsenate in the environment, an enzyme related to polysulfide reductase and not to arsenite oxidase has evolved into Arr. These findings are paleogeochemically rationalized by the fact that the accumulation of arsenate over arsenite required the increase in oxidation state of the environment brought about by oxygenic photosynthesis.

Investigation of the indigenous fungal community populating barley grains: Secretomes and xylanolytic potential.

PubMed

Sultan, Abida; Frisvad, Jens C; Andersen, Birgit; Svensson, Birte; Finnie, Christine

2017-10-03

The indigenous fungal species populating cereal grains produce numerous plant cell wall-degrading enzymes including xylanases, which could play important role in plant-pathogen interactions and in adaptation of the fungi to varying carbon sources. To gain more insight into the grain surface-associated enzyme activity, members of the populating fungal community were isolated, and their secretomes and xylanolytic activities assessed. Twenty-seven different fungal species were isolated from grains of six barley cultivars over different harvest years and growing sites. The isolated fungi were grown on medium containing barley flour or wheat arabinoxylan as sole carbon source. Their secretomes and xylanase activities were analyzed using SDS-PAGE and enzyme assays and were found to vary according to species and carbon source. Secretomes were dominated by cell wall degrading enzymes with xylanases and xylanolytic enzymes being the most abundant. A 2-DE-based secretome analysis of Aspergillus niger and the less-studied pathogenic fungus Fusarium poae grown on barley flour and wheat arabinoxylan resulted in identification of 82 A. niger and 31 F. poae proteins many of which were hydrolytic enzymes, including xylanases. The microorganisms that inhabit the surface of cereal grains are specialized in production of enzymes such as xylanases, which depolymerize plant cell walls. Integration of gel-based proteomics approach with activity assays is a powerful tool for analysis and characterization of fungal secretomes and xylanolytic activities which can lead to identification of new enzymes with interesting properties, as well as provide insight into plant-fungal interactions, fungal pathogenicity and adaptation. Understanding the fungal response to host niche is of importance to uncover novel targets for potential symbionts, anti-fungal agents and biotechnical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

PubMed

Henderson, Brittney R; Saeedi, Bejan J; Campagnola, Grace; Geiss, Brian J

2011-01-01

Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D) for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism.

PubMed

Watanabe, Seiya; Kodaki, Tsutomu; Kodak, Tsutomu; Makino, Keisuke

2006-02-03

Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.

Better postpartal performance in dairy cows supplemented with rumen-protected methionine compared with choline during the peripartal period.

PubMed

Zhou, Z; Vailati-Riboni, M; Trevisi, E; Drackley, J K; Luchini, D N; Loor, J J

2016-11-01

The onset of lactation in dairy cows is characterized by high output of methylated compounds in milk when sources of methyl group are in short supply. Methionine and choline (CHOL) are key methyl donors and their availability during this time may be limiting for milk production, hepatic lipid metabolism, and immune function. Supplementing rumen-protected Met and CHOL may improve overall performance and health of transition cows. The objective of this study was to evaluate the effect of supplemental rumen-protected Met and CHOL on performance and health of transition cows. Eighty-one multiparous Holstein cows were used in a randomized, complete, unbalanced block design with 2×2 factorial arrangement of Met (Smartamine M, Adisseo NA, Alpharetta, GA) and CHOL (ReaShure, Balchem Inc., New Hampton, NY) inclusion (with or without). Treatments (20 to 21 cows each) were control (CON), CON+Met (SMA), CON+CHOL (REA), and CON+Met+CHOL (MIX). From -50 to -21d before expected calving, all cows received the same diet (1.40Mcal of NE L /kg of DM) with no Met or CHOL. From -21d to calving, cows received the same close-up diet (1.52Mcal of NE L /kg of DM) and were assigned randomly to treatments (CON, SMA, REA, or MIX) supplied as top dresses. From calving to 30 DIM, cows were fed the same postpartal diet (1.71Mcal of NE L /kg of DM) and continued to receive the same treatments through 30 DIM. The Met supplementation was adjusted daily at 0.08% DM of diet and REA was supplemented at 60g/d. Incidence of clinical ketosis and retained placenta tended to be lower in Met-supplemented cows. Supplementation of Met (SMA, MIX) led to greater DMI compared with other treatments (CON, REA) in both close-up (14.3 vs. 13.2kg/d, SEM 0.3) and first 30d postpartum (19.2 vs. 17.2kg/d, SEM 0.6). Cows supplemented with Met (SMA, MIX) had greater yields of milk (44.2 vs. 40.4kg/d, SEM 1.2), ECM (44.6 vs. 40.5kg/d, SEM 1.0), and FCM (44.6 vs. 40.8kg/d, SEM 1.0) compared with other (CON, REA) treatments. Milk fat content did not differ in response to Met or CHOL. However, milk protein content was greater in Met-supplemented (3.32% vs. 3.14%, SEM 0.04%) but not CHOL-supplemented (3.27 vs. 3.19%, SEM 0.04%) cows. Supplemental CHOL led to greater blood glucose and insulin concentrations with lower glucose:insulin ratio. No Met or CHOL effects were detected for blood fatty acids or BHB, but a Met × time effect was observed for fatty acids due to higher concentrations on d 20. Results from the present study indicate that peripartal supplementation of rumen-protected Met but not CHOL has positive effects on cow performance. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

PubMed

Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

2014-03-03

Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Copyright © 2014 John Wiley & Sons, Inc. All rights reserved.

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

PubMed

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.

Proteomic analysis and food-grade enzymes of Moringa oleifer Lam. a Lam. flower.

PubMed

Shi, Yanan; Wang, Xuefeng; Huang, Aixiang

2018-08-01

Moringa oleifer Lam. flower contain high-proteins and function nutrients. Many advances have been made to it, but there is still no proteomic information of this species. Total protein from the flowers applied shotgun 2DLC-MS/MS proteomic identified 9443 peptides corresponding to 4004 high-confidence proteins by Proteome Discoverer™ Software 2.1. These proteins were mostly distributed ranging between 40 and 70 kDa. Gene Ontology (GO) analysis indicated that the largest of the proteins were cytoplasm 72.7%, catalytic activity 61.5% and macromolecule metabolism 43.7%, and KEGG analysis revealed that the largest group of 129 proteins was involved in Ribosome to directing protein synthesis (translation). Moreover, a number of commercially important food-grade enzymes were commented, 261 proteins were annotated as carbohydrate-active enzymes, 16 protease, 22 proteins are assigned to the citrate cycle, which the top proteins were assigned to GH family, cysteine synthase and serine/threonine-protein phosphatase. These enzymes indicated that is a new source with potential use for fermentation and brewing industry, fruit and vegetable storage and the development of function peptides. Copyright © 2018 Elsevier B.V. All rights reserved.

Uronic polysaccharide degrading enzymes.

PubMed

Garron, Marie-Line; Cygler, Miroslaw

2014-10-01

In the past several years progress has been made in the field of structure and function of polysaccharide lyases (PLs). The number of classified polysaccharide lyase families has increased to 23 and more detailed analysis has allowed the identification of more closely related subfamilies, leading to stronger correlation between each subfamily and a unique substrate. The number of as yet unclassified polysaccharide lyases has also increased and we expect that sequencing projects will allow many of these unclassified sequences to emerge as new families. The progress in structural analysis of PLs has led to having at least one representative structure for each of the families and for two unclassified enzymes. The newly determined structures have folds observed previously in other PL families and their catalytic mechanisms follow either metal-assisted or Tyr/His mechanisms characteristic for other PL enzymes. Comparison of PLs with glycoside hydrolases (GHs) shows several folds common to both classes but only for the β-helix fold is there strong indication of divergent evolution from a common ancestor. Analysis of bacterial genomes identified gene clusters containing multiple polysaccharide cleaving enzymes, the Polysaccharides Utilization Loci (PULs), and their gene complement suggests that they are organized to process completely a specific polysaccharide. Copyright © 2014 Elsevier Ltd. All rights reserved.

High frequency of X chromosome abnormalities in women with short stature and elevated liver enzymes.

PubMed

Roulot, Dominique; Malan, Valérie; Ziol, Marianne; Linglart, Agnès; Bourcier, Valérie; Beaugrand, Michel; Benzacken, Brigitte

2014-08-01

Paucisymptomatic forms of Turner's syndrome (TS), in which short stature is the predominant clinical abnormality, remain underdiagnosed. Abnormal liver tests are extremely frequent in adult TS patients reflecting various types of hepatic lesions. The objective of the study was to investigate whether unexplained elevated liver enzymes in women with short stature could reveal X chromosome abnormalities of undiagnosed TS. Thirty-one consecutive short stature women displaying elevated liver enzymes and no previous diagnosis of TS were compared with 31 age-matched controls in a prospective study. Liver biopsy was performed in 26 patients. Systematic karyotype analysis and fluorescence in situ hybridization. X chromosome abnormalities were found in 27 patients and one control (87.0% vs 3.2%, P < .0001), including a 45,X/46,XX mosaicism in 24 patients and isochromosome of the long arm in three. Liver histological analysis showed architectural changes in 17 patients with nodular regenerative hyperplasia in 12. Biliary lesions were present in 13 patients and liver steatosis in 20. X chromosome abnormalities indicative of cryptic TS are extremely frequent in short-stature women with unexplained elevated liver enzymes. In short-stature women, abnormal liver tests should lead to systematic karyotype analysis.

Genetic relationships within the genus Prevotella analyzed by multilocus enzyme electrophoresis and DNA-DNA hybridization.

PubMed

Combe, M L; Pons, J L

1999-12-01

The genetic diversity and relationships within the genus Prevotella were studied by analyzing twenty-five strains by multilocus enzyme electrophoresis (MLEE) at nine metabolic enzyme loci and DNA-DNA hybridization. MLEE revealed a high genetic diversity with 25 electrophoretic types (ETs) for the 25 strains studied, a mean number of alleles per enzyme locus of 6.8 and a mean genetic diversity per locus of 0.786. The index of association described by Maynard Smith et al. (1993) revealed a clonal structure within the genus Prevotella. A dendrogram generated by cluster analysis of a matrix of ETs showed that species like P. bivia, P. buccae, P. oris, P. oralis, P. nigrescens, and P. denticola form clusters that are consistent with DNA homologies. However, strains identified as P. melaninogenica or P. loescheii by DNA-DNA hybridization did not constitute distinct subpopulations in MLEE. MLEE analysis demonstrated its high power in differentiating closely related strains. It provides an alternative to 16S rRNA analysis for the study of phylogenetic relationships within the genus Prevotella, especially for differentiating strains with high DNA homology or high rRNA homology.

Evolutionary analysis of a novel zinc ribbon in the N-terminal region of threonine synthase.

PubMed

Kaur, Gurmeet; Subramanian, Srikrishna

2017-10-18

Threonine synthase (TS) catalyzes the terminal reaction in the biosynthetic pathway of threonine and requires pyridoxal phosphate as a cofactor. TSs share a common catalytic domain with other fold type II PALP dependent enzymes. TSs are broadly grouped into two classes based on their sequence, quaternary structure, and enzyme regulation. We report the presence of a novel zinc ribbon domain in the N-terminal region preceding the catalytic core in TS. The zinc ribbon domain is present in TSs belonging to both classes. Our sequence analysis reveals that archaeal TSs possess all zinc chelating residues to bind a metal ion that are lacking in the structurally characterized homologs. Phylogenetic analysis suggests that TSs with an N-terminal zinc ribbon likely represents the ancestral state of the enzyme while TSs without a zinc ribbon must have diverged later in specific lineages. The zinc ribbon and its N- and C-terminal extensions are important for enzyme stability, activity and regulation. It is likely that the zinc ribbon domain is involved in higher order oligomerization or mediating interactions with other biomolecules leading to formation of larger metabolic complexes.

Carbohydrate-active enzymes in Trichoderma harzianum: a bioinformatic analysis bioprospecting for key enzymes for the biofuels industry.

PubMed

Ferreira Filho, Jaire Alves; Horta, Maria Augusta Crivelente; Beloti, Lilian Luzia; Dos Santos, Clelton Aparecido; de Souza, Anete Pereira

2017-10-12

Trichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass. In this study, the CAZymes of T. harzianum were identified and classified using bioinformatic approaches after which the expression profiles of all annotated CAZymes were assessed via RNA-Seq, and a phylogenetic analysis was performed. A total of 430 CAZymes (3.7% of the total proteins for this organism) were annotated in T. harzianum, including 259 glycoside hydrolases (GHs), 101 glycosyl transferases (GTs), 6 polysaccharide lyases (PLs), 22 carbohydrate esterases (CEs), 42 auxiliary activities (AAs) and 46 carbohydrate-binding modules (CBMs). Among the identified T. harzianum CAZymes, 47% were predicted to harbor a signal peptide sequence and were therefore classified as secreted proteins. The GH families were the CAZyme class with the greatest number of expressed genes, including GH18 (23 genes), GH3 (17 genes), GH16 (16 genes), GH2 (13 genes) and GH5 (12 genes). A phylogenetic analysis of the proteins in the AA9/GH61, CE5 and GH55 families showed high functional variation among the proteins. Identifying the main proteins used by T. harzianum for biomass degradation can ensure new advances in the biofuel production field. Herein, we annotated and characterized the expression levels of all of the CAZymes from T. harzianum, which may contribute to future studies focusing on the functional and structural characterization of the identified proteins.

New Insights into the Phylogeny and Molecular Classification of Nicotinamide Mononucleotide Deamidases

PubMed Central

Sánchez-Carrón, Guiomar; Martínez-Moñino, Ana Belén; Sola-Carvajal, Agustín; Takami, Hideto; García-Carmona, Francisco; Sánchez-Ferrer, Álvaro

2013-01-01

Nicotinamide mononucleotide (NMN) deamidase is one of the key enzymes of the bacterial pyridine nucleotide cycle (PNC). It catalyzes the conversion of NMN to nicotinic acid mononucleotide, which is later converted to NAD+ by entering the Preiss-Handler pathway. However, very few biochemical data are available regarding this enzyme. This paper represents the first complete molecular characterization of a novel NMN deamidase from the halotolerant and alkaliphilic bacterium Oceanobacillus iheyensis (OiPncC). The enzyme was active over a broad pH range, with an optimum at pH 7.4, whilst maintaining 90 % activity at pH 10.0. Surprisingly, the enzyme was quite stable at such basic pH, maintaining 61 % activity after 21 days. As regard temperature, it had an optimum at 65 °C but its stability was better below 50 °C. OiPncC was a Michaelian enzyme towards its only substrate NMN, with a K m value of 0.18 mM and a kcat/K m of 2.1 mM-1 s-1. To further our understanding of these enzymes, a complete phylogenetic and structural analysis was carried out taking into account the two Pfam domains usually associated with them (MocF and CinA). This analysis sheds light on the evolution of NMN deamidases, and enables the classification of NMN deamidases into 12 different subgroups, pointing to a novel domain architecture never before described. Using a Logo representation, conserved blocks were determined, providing new insights on the crucial residues involved in the binding and catalysis of both CinA and MocF domains. The analysis of these conserved blocks within new protein sequences could permit the more efficient data curation of incoming NMN deamidases. PMID:24340054

Single-molecule paleoenzymology probes the chemistry of resurrected enzymes

PubMed Central

Perez-Jimenez, Raul; Inglés-Prieto, Alvaro; Zhao, Zi-Ming; Sanchez-Romero, Inmaculada; Alegre-Cebollada, Jorge; Kosuri, Pallav; Garcia-Manyes, Sergi; Kappock, T. Joseph; Tanokura, Masaru; Holmgren, Arne; Sanchez-Ruiz, Jose M.; Gaucher, Eric A.; Fernandez, Julio M.

2011-01-01

A journey back in time is possible at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx), dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32° C more stable than modern enzymes and the oldest show significantly higher activity than extant ones at pH 5. We probed their mechanisms of reduction using single-molecule force spectroscopy. From the force-dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs utilize chemical mechanisms of reduction similar to those of modern enzymes. While Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over a 4 Gyr time span to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth. PMID:21460845

Structural analysis of enzymes used for bioindustry and bioremediation.

PubMed

Tanokura, Masaru; Miyakawa, Takuya; Guan, Lijun; Hou, Feng

2015-01-01

Microbial enzymes have been widely applied in the large-scale, bioindustrial manufacture of food products and pharmaceuticals due to their high substrate specificity and stereoselectivity, and their effectiveness under mild conditions with low environmental burden. At the same time, bioremedial techniques using microbial enzymes have been developed to solve the problem of industrial waste, particularly with respect to persistent chemicals and toxic substances. And finally, structural studies of these enzymes have revealed the mechanistic basis of enzymatic reactions, including the stereoselectivity and binding specificity of substrates and cofactors. The obtained structural insights are useful not only to deepen our understanding of enzymes with potential bioindustrial and/or bioremedial application, but also for the functional improvement of enzymes through rational protein engineering. This review shows the structural bases for various types of enzymatic reactions, including the substrate specificity accompanying cofactor-controlled and kinetic mechanisms.

Improvement in Saccharification Yield of Mixed Rumen Enzymes by Identification of Recalcitrant Cell Wall Constituents Using Enzyme Fingerprinting.

PubMed

Badhan, Ajay; Wang, Yu-Xi; Gruninger, Robert; Patton, Donald; Powlowski, Justin; Tsang, Adrian; McAllister, Tim A

2015-01-01

Identification of recalcitrant factors that limit digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the efficiency of meat and milk production in ruminants. Enzyme fingerprinting of barley silage fed to heifers and total tract indigestible fibre residue (TIFR) collected from feces was used to identify cell wall components resistant to total tract digestion. Enzyme fingerprinting results identified acetyl xylan esterases as key to the enhanced ruminal digestion. FTIR analysis also suggested cross-link cell wall polymers as principal components of indigested fiber residues in feces. Based on structural information from enzymatic fingerprinting and FTIR, enzyme pretreatment to enhance glucose yield from barley straw and alfalfa hay upon exposure to mixed rumen-enzymes was developed. Prehydrolysis effects of recombinant fungal fibrolytic hydrolases were analyzed using microassay in combination with statistical experimental design. Recombinant hemicellulases and auxiliary enzymes initiated degradation of plant structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by mixed rumen enzymes. The validation results showed that microassay in combination with statistical experimental design can be successfully used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by mixed rumen enzymes.

α-l-rhamnosidase selective for rutin to isoquercitrin transformation from Penicillium griseoroseum MTCC-9224.

PubMed

Yadav, Sarita; Yadava, Sudha; Yadav, Kapil D S

2017-02-01

An α-l-rhamnosidase secreting fungal strain has been isolated from the decaying goose berry (Emblica officinalis) fruit peel. The fungal strain has been identified as Penicillium greoroseum MTCC-9224. The α-l-rhamnosidase of this fungal strain has been purified to homogeneity using a simple procedure involving concentration by ultra filtration and an anion exchange chromatography on DEAE-cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 97kDa in SDS-PAGE analysis. The native-PAGE analysis also gave a single protein band confirming the purity of the enzyme. Using p-nitrophenyl-α-l-rhamnopyranoside as the substrate, K m and k cat values of the enzyme were 0.65mM and 43.65s -1 , respectively. The pH and temperature optima of the enzyme were 6.5 and 57°C, respectively. The activation energy for the thermal denaturation of the enzyme was 27.9kJ/mol. The purified α-l-rhamnosidase hydrolyzed rutin to isoquercitrin and l-rhamnose but has no effect on naringin and hesperidin. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantifying enzymatic lysis: estimating the combined effects of chemistry, physiology and physics.

PubMed

Mitchell, Gabriel J; Nelson, Daniel C; Weitz, Joshua S

2010-10-04

The number of microbial pathogens resistant to antibiotics continues to increase even as the rate of discovery and approval of new antibiotic therapeutics steadily decreases. Many researchers have begun to investigate the therapeutic potential of naturally occurring lytic enzymes as an alternative to traditional antibiotics. However, direct characterization of lytic enzymes using techniques based on synthetic substrates is often difficult because lytic enzymes bind to the complex superstructure of intact cell walls. Here we present a new standard for the analysis of lytic enzymes based on turbidity assays which allow us to probe the dynamics of lysis without preparing a synthetic substrate. The challenge in the analysis of these assays is to infer the microscopic details of lysis from macroscopic turbidity data. We propose a model of enzymatic lysis that integrates the chemistry responsible for bond cleavage with the physical mechanisms leading to cell wall failure. We then present a solution to an inverse problem in which we estimate reaction rate constants and the heterogeneous susceptibility to lysis among target cells. We validate our model given simulated and experimental turbidity assays. The ability to estimate reaction rate constants for lytic enzymes will facilitate their biochemical characterization and development as antimicrobial therapeutics.

Antibody production of wild-type and enzyme V279F variants of PAF-AH as a risk factor for Cardiovascular disease

NASA Astrophysics Data System (ADS)

Ramadhani, Anggia N.; Puspitarini, Sapti; Sari, Anissa N.; Widodo

2017-11-01

Coronary artery disease (CAD) has emerged as a leading cause of death in Indonesia nowadays. WHO data in 2012 revealed that 37% of the Indonesian population died from this disease. CAD occurs because of endothelial dysfunction in the arteries. Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), is a phospholipase A2 enzyme, encoded by the PLA2G7 gene. This protein is predicted to be involved in inflammatory phospholipid metabolism so it can be used as a biomarker of CAD in the early phase. Thus, the purpose of this research is to discover the difference in antibody production between wild-type and mutant V279F. The PAF-AH enzyme was isolated from mice lymphocyte cells in order to develop this enzyme as a biomarker of cardiovascular disease. PAF-AH migrates at 55kDa according to SDS-PAGE analysis. Flow cytometry analysis showed that mutant PAF-AH (V279F) is more antigenic than wild-type PAF-AH. The missense mutation of V279F PAF-AH means this enzyme cannot catabolize the acetyl group at the sn-2 position of PAF.

Systematic inference of functional phosphorylation events in yeast metabolism.

PubMed

Chen, Yu; Wang, Yonghong; Nielsen, Jens

2017-07-01

Protein phosphorylation is a post-translational modification that affects proteins by changing their structure and conformation in a rapid and reversible way, and it is an important mechanism for metabolic regulation in cells. Phosphoproteomics enables high-throughput identification of phosphorylation events on metabolic enzymes, but identifying functional phosphorylation events still requires more detailed biochemical characterization. Therefore, development of computational methods for investigating unknown functions of a large number of phosphorylation events identified by phosphoproteomics has received increased attention. We developed a mathematical framework that describes the relationship between phosphorylation level of a metabolic enzyme and the corresponding flux through the enzyme. Using this framework, it is possible to quantitatively estimate contribution of phosphorylation events to flux changes. We showed that phosphorylation regulation analysis, combined with a systematic workflow and correlation analysis, can be used for inference of functional phosphorylation events in steady and dynamic conditions, respectively. Using this analysis, we assigned functionality to phosphorylation events of 17 metabolic enzymes in the yeast Saccharomyces cerevisiae , among which 10 are novel. Phosphorylation regulation analysis cannot only be extended for inference of other functional post-translational modifications but also be a promising scaffold for multi-omics data integration in systems biology. Matlab codes for flux balance analysis in this study are available in Supplementary material. yhwang@ecust.edu.cn or nielsenj@chalmers.se. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Discovery of novel hydroxamates as highly potent tumor necrosis factor-[alpha] converting enzyme inhibitors. Part II: Optimization of the S3′ pocket

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

Mazzola Jr., Robert D.; Zhu, Zhaoning; Sinning, Lisa

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.

Data-Driven Microbial Modeling for Soil Carbon Decomposition and Stabilization

NASA Astrophysics Data System (ADS)

Luo, Yiqi; Chen, Ji; Chen, Yizhao; Feng, Wenting

2017-04-01

Microorganisms have long been known to catalyze almost all the soil organic carbon (SOC) transformation processes (e.g., decomposition, stabilization, and mineralization). Representing microbial processes in Earth system models (ESMs) has the potential to improve projections of SOC dynamics. We have recently examined (1) relationships of microbial functions with environmental factors and (2) microbial regulations of decomposition and other key soil processes. According to three lines of evidence, we have developed a data-driven enzyme (DENZY) model to simulate soil microbial decomposition and stabilization. First, our meta-analysis of 64 published field studies showed that field experimental warming significantly increased soil microbial communities abundance, which is negatively correlated with the mean annual temperature. The negative correlation indicates that warming had stronger effects in colder than warmer regions. Second, we found that the SOC decomposition, especially the transfer between labile SOC and protected SOC, is nonlinearly regulated by soil texture parameters, such as sand and silt contents. Third, we conducted a global analysis of the C-degrading enzyme activities, soil respiration, and SOC content under N addition. Our results show that N addition has contrasting effects on cellulase (hydrolytic C-degrading enzymes) and ligninase (oxidative C-degrading enzymes) activities. N-enhanced cellulase activity contributes to the minor stimulation of soil respiration whereas N-induced repression on ligninase activity drives soil C sequestration. Our analysis links the microbial extracellular C-degrading enzymes to the SOC dynamics at ecosystem scales across scores of experimental sites around the world. It offers direct evidence that N-induced changes in microbial community and physiology play fundamental roles in controlling the soil C cycle. Built upon those three lines of empirical evidence, the DENZY model includes two enzyme pools and explicitly characterizes two classes of extracellular enzyme activities: one that degrades organic molecules containing both C and N (e.g., chitin or protein) and another that degrades only C (e.g., cellulose). The DENZY model assumes that the microbes allocate resources to different enzyme pools so as to exactly satisfy microbial CN ratio stoichiometry in response to changes in climate conditions and soil attributes. The DENZY model can simulate differential effects of nitrogen fertilization on the two groups of enzymes and thus soil respiration and SOC dynamics. We will select field experimental sites to test the DENZY model. With increasing amounts of available observations and data synthesis, this DENZY model will be better parameterized and have a potential to reveal how responses of microbial enzymes to environmental changes regulate soil carbon decomposition and stabilization.

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

PubMed

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 Oleuropein β-Glucosidase from Olive Fruit Is Involved in Determining the Phenolic Composition of Virgin Olive Oil

PubMed Central

Velázquez-Palmero, David; Romero-Segura, Carmen; García-Rodríguez, Rosa; Hernández, María L.; Vaistij, Fabián E.; Graham, Ian A.; Pérez, Ana G.; Martínez-Rivas, José M.

2017-01-01

Phenolic composition of virgin olive oil is determined by the enzymatic and/or chemical reactions that take place during olive fruit processing. Of these enzymes, β-glucosidase activity plays a relevant role in the transformation of the phenolic glycosides present in the olive fruit, generating different secoiridoid derivatives. The main goal of the present study was to characterize olive fruit β-glucosidase genes and enzymes responsible for the phenolic composition of virgin olive oil. To achieve that, we have isolated an olive β-glucosidase gene from cultivar Picual (OepGLU), expressed in Nicotiana benthamiana leaves and purified its corresponding recombinant enzyme. Western blot analysis showed that recombinant OepGLU protein is detected by an antibody raised against the purified native olive mesocarp β-glucosidase enzyme, and exhibits a deduced molecular mass of 65.0 kDa. The recombinant OepGLU enzyme showed activity on the major olive phenolic glycosides, with the highest levels with respect to oleuropein, followed by ligstroside and demethyloleuropein. In addition, expression analysis showed that olive GLU transcript level in olive fruit is spatially and temporally regulated in a cultivar-dependent manner. Furthermore, temperature, light and water regime regulate olive GLU gene expression in olive fruit mesocarp. All these data are consistent with the involvement of OepGLU enzyme in the formation of the major phenolic compounds present in virgin olive oil. PMID:29163620

The Peroxidase-Glucose Oxidase Enzyme System in the Undergraduate Laboratory.

ERIC Educational Resources Information Center

Woolridge, Elisa; And Others

1986-01-01

Offers a series of experiments which introduce students to the general principles of enzymology. The experiment demonstrates several basic enzyme properties and the chromatographic exercises provide an analysis of each enzymatic activity. Questions are also presented for extending discussion on the activities. (ML)

[Effect of Low-Intensity 900 MHz Frequency Electromagnetic Radiation on Rat Brain Enzyme Activities Linked to Energy Metabolism].

PubMed

Petrosyan, M S; Nersesova, L S; Gazaryants, M G; Meliksetyan, G O; Malakyan, M G; Bajinyan, S A; Akopian, J I

2015-01-01

The research deals with the effect of low-intensity 900 MHz frequency electromagnetic radiation (EMR), power density 25 μW/cm2, on the following rat brain and blood serum enzyme activities: creatine kinase (CK), playing a central role in the process of storing and distributing the cell energy, as well as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) that play a key role in providing the conjunction of carbohydrate and amino acid metabolism. The comparative analysis of the changes in the enzyme activity studied at different times following the two-hour single, as well as fractional, radiation equivalent of the total time showed that the most radiosensitive enzyme is the brain creatine kinase, which may then be recommended as a marker of the radio frequency radiation impact. According to the analysis of the changing dynamics of the CK, ALT and AST activity level, with time these changes acquire the adaptive character and are directed to compensate the damaged cell energy metabolism.

Recent Trends in Biosensors

NASA Astrophysics Data System (ADS)

Karube, Isao

The determination of organic compounds in foods is very important in food industries. A various compounds are contained in foods, selective determination methods are required for food processing and analysis. Electrochemical monitoring devices (biosensors) employing immobilized biocatalysts such as immobilized enzymes, organelles, microorganisms, and tissue have definite advantages. The enzyme Sensors consisted of immobilized enzymes and electrochemical devices. Enzyme sensors could be used for the determination of sugars, amino acids, organic acids, alcohols, lipids, nucleic acid derivatives, etc.. Furthermore, a multifunctional biosensor for the determination of several compounds has been developed for food processing. On the other hand, microbial sensors consisted of immobilized microorganisms and electrodes have been used for industrial and environmental analysis. Microbial sensors were applied for the determination of sugars, organic acids, alcohols, amino acids, mutagens, me thane, ammonia, and BOD. Furthermore, micro-biosensors using immobilized biocatalysts and ion sensitive field effect transistor or microelectrodes prepared by silicon fabrication technologies have been developed for medical ap. plication and food processing. This review summarizes the design and application of biosensors.

A gene horizontally transferred from bacteria protects arthropods from host plant cyanide poisoning

PubMed Central

Wybouw, Nicky; Dermauw, Wannes; Tirry, Luc; Stevens, Christian; Grbić, Miodrag; Feyereisen, René; Van Leeuwen, Thomas

2014-01-01

Cyanogenic glucosides are among the most widespread defense chemicals of plants. Upon plant tissue disruption, these glucosides are hydrolyzed to a reactive hydroxynitrile that releases toxic hydrogen cyanide (HCN). Yet many mite and lepidopteran species can thrive on plants defended by cyanogenic glucosides. The nature of the enzyme known to detoxify HCN to β-cyanoalanine in arthropods has remained enigmatic. Here we identify this enzyme by transcriptome analysis and functional expression. Phylogenetic analysis showed that the gene is a member of the cysteine synthase family horizontally transferred from bacteria to phytophagous mites and Lepidoptera. The recombinant mite enzyme had both β-cyanoalanine synthase and cysteine synthase activity but enzyme kinetics showed that cyanide detoxification activity was strongly favored. Our results therefore suggest that an ancient horizontal transfer of a gene originally involved in sulfur amino acid biosynthesis in bacteria was co-opted by herbivorous arthropods to detoxify plant produced cyanide. DOI: http://dx.doi.org/10.7554/eLife.02365.001 PMID:24843024

Bioinformatics approaches for structural and functional analysis of proteins in secondary metabolism in Withania somnifera.

PubMed

Sanchita; Singh, Swati; Sharma, Ashok

2014-11-01

Withania somnifera (Ashwagandha) is an affluent storehouse of large number of pharmacologically active secondary metabolites known as withanolides. These secondary metabolites are produced by withanolide biosynthetic pathway. Very less information is available on structural and functional aspects of enzymes involved in withanolides biosynthetic pathways of Withiana somnifera. We therefore performed a bioinformatics analysis to look at functional and structural properties of these important enzymes. The pathway enzymes taken for this study were 3-Hydroxy-3-methylglutaryl coenzyme A reductase, 1-Deoxy-D-xylulose-5-phosphate synthase, 1-Deoxy-D-xylulose-5-phosphate reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, and cycloartenol synthase. The prediction of secondary structure was performed for basic structural information. Three-dimensional structures for these enzymes were predicted. The physico-chemical properties such as pI, AI, GRAVY and instability index were also studied. The current information will provide a platform to know the structural attributes responsible for the function of these protein until experimental structures become available.

Portrait of an Enzyme, a Complete Structural Analysis of a Multimodular beta-N-Acetylglucosaminidase from Clostridium perfringens

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

Ficko-Blean, E.; Gregg, K; Adams, J

2009-01-01

Common features of the extracellular carbohydrate-active virulence factors involved in host-pathogen interactions are their large sizes and modular complexities. This has made them recalcitrant to structural analysis, and therefore our understanding of the significance of modularity in these important proteins is lagging. Clostridium perfringens is a prevalent human pathogen that harbors a wide array of large, extracellular carbohydrate-active enzymes and is an excellent and relevant model system to approach this problem. Here we describe the complete structure of C. perfringens GH84C (NagJ), a 1001-amino acid multimodular homolog of the C. perfringens ?-toxin, which was determined using a combination of smallmore » angle x-ray scattering and x-ray crystallography. The resulting structure reveals unprecedented insight into how catalysis, carbohydrate-specific adherence, and the formation of molecular complexes with other enzymes via an ultra-tight protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction.« less

Evaluation of microwave-assisted pretreatment of lignocellulosic biomass immersed in alkaline glycerol for fermentable sugars production.

PubMed

Diaz, Ana Belen; Moretti, Marcia Maria de Souza; Bezerra-Bussoli, Carolina; Carreira Nunes, Christiane da Costa; Blandino, Ana; da Silva, Roberto; Gomes, Eleni

2015-06-01

A pretreatment with microwave irradiation was applied to enhance enzyme hydrolysis of corn straw and rice husk immersed in water, aqueous glycerol or alkaline glycerol. Native and pretreated solids underwent enzyme hydrolysis using the extract obtained from the fermentation of Myceliophthora heterothallica, comparing its efficiency with that of the commercial cellulose cocktail Celluclast®. The highest saccharification yields, for both corn straw and rice husk, were attained when biomass was pretreated in alkaline glycerol, method that has not been previously reported in literature. Moreover, FTIR, TG and SEM analysis revealed a more significant modification in the structure of corn straw subjected to this pretreatment. Highest global yields were attained with the crude enzyme extract, which might be the result of its content in a great variety of hydrolytic enzymes, as revealed zymogram analysis. Moreover, its hydrolysis efficiency can be improved by its supplementation with commercial β-glucosidase. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sanfilippo syndrome: Overall review.

PubMed

Andrade, Fernando; Aldámiz-Echevarría, Luis; Llarena, Marta; Couce, María Luz

2015-06-01

Mucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) is a lysosomal storage disorder, caused by a deficiency in one of the four enzymes involved in the catabolism of glycosaminoglycan heparan sulfate. It is characterized by progressive cognitive decline and severe hyperactivity, with relatively mild somatic features. This review focuses on clinical features, diagnosis, treatment, and follow-up of MPS III, and provides information about supplementary tests and differential diagnosis. Given that few reviews of MPS III have been published, several studies were compiled to establish diagnostic recommendations. Quantitative urinary glycosaminoglycan analysis is strongly recommended, and measurement of disaccharides, heparin cofactor II-thrombin complex and gangliosides is also used. Enzyme activity of the different enzymes in blood serum, leukocytes or fibroblasts, and mutational analysis for SGSH, NAGLU, HGSNAT or GNS genes are required to confirm diagnosis and differentiate four subtypes of MPS III. Although there is no global consensus for treatment, enzyme replacement therapy and gene therapy can provide appropriate results. In this regard, recent publications on treatment and follow-up are discussed. © 2015 Japan Pediatric Society.

FT-Raman spectroscopic analysis of enhanced activity of supercritical carbon dioxide treated bacterial alpha-amylase.

PubMed

Paul, Kaninika; Dutta, Sayantani; Bhattacharjee, Paramita

2017-09-01

Our previous investigation on high pressure supercritical carbon dioxide treatment of a bacterial α-amylase had revealed enhanced activity of the same. 1 H NMR analysis of the activity enhanced enzyme led the authors to hypothesize that the enhancement was possibly owing to alterations in the active site of the enzyme. In the present study, the changes in the active site of the treated enzyme was analysed by Fourier-transform Raman (FT-Raman) spectroscopy. The spectra obtained revealed shifting of bands in the active site of α-amylase indicating a nudging effect of the bonds in this region consequent to high pressure treatment. Also, shifts in bands in the OH stretching vibration of water were observed in the enzyme spectra. These variations in the spectra confirmed changes in the active site as well as in the water associated with the same that perhaps had a concerted effect on the increased activity of α-amylase. Copyright © 2017 Elsevier Inc. All rights reserved.

Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.

PubMed

Sakamoto, Seiichi; Putalun, Waraporn; Vimolmangkang, Sornkanok; Phoolcharoen, Waranyoo; Shoyama, Yukihiro; Tanaka, Hiroyuki; Morimoto, Satoshi

2018-01-01

Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen-antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [1], a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977. Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article describes ELISA and its applications for the detection of plant secondary metabolites.

Possible NIR counterpart to the AXP 1E 1547.0-5408

NASA Astrophysics Data System (ADS)

Mignani, R. P.; Rea, N.; Israel, G. L.; Testa, V.; Esposito, P.

2008-10-01

On 2008 October 3rd, Swift-BAT triggered on several short bursts from the direction of the Anomalous X-ray Pulsar (AXP) 1E1547.0-5408 (Krimm et al. GCN 8311, 8312; Rea et al. Atel #1756). This is the second known transient radio AXP discovered to date (Gelfand & Gaensler 2007; Camilo et al. 2007), and it showed the previous X-ray outburst around June 2007 (Halpern et al. 2008). During the decay of the 2007 X-ray outburst we observed 1E 1547.0-5408 in the Ks band (on 2007 July 8th, 12th, and August 17th) with the NAOS -CONICA (NACO) instrument mounted at the ESO- VLT (Mignani, Rea, Testa, et al., 2008 submitted).

ANALYSIS OF OTORHINOLARYNGOLOGY, ORTHOPEDICS AND THORACIC SURGERY JOURNALS.

PubMed

Lima, Wilma Terezinha Anselmo

2015-01-01

To perform an extensive analysis of journals in Medicine III - CAPES, and specifically those in the areas of Otorhinolaryngology, Orthopedics and Traumatology and Chest Surgery. An active search for the impact factors in the Journal Citation Reports, Scimago, their indexation in Scielo, Lilacs, Scopus and Google Scholar, and their stratification in WebQualis was done. Forty-four journals with measured impact factors ranging from 3.006 to 0.128 were detected in the area of Otorhinolaryngology; however, only 26 of them (60%) had a Qualis measured by CAPES; in the stratification, no journal was detected in A1, three were A2 and nine B1. Three journals were located for Chest Surgery, with only one of them having a measured Qualis (A2) with a mean of 3.61. Sixty-seven journals were detected for Orthopedics and Traumatology, with an impact factor ranging from 4.699 to 0.156; Qualis was measured in only 38 of them (60%); there were three journal stratified as A1, seven as A2 and 25 as B1. The search for journals of higher impact induces authors to not publish in journals related to their area and facing more difficulties than investigators from other areas. Realizar análise ampla dos periódicos da Medicina III - CAPES e, especificamente, os pertencentes à Otorrinolaringologia, Ortopedia e Traumatologia, e Cirurgia Torácica. Busca ativa do fator de impacto dos periódicos das áreas citadas no Journal Citation Report e Scimago, sua indexação no Scielo, Lilacs, Scopus, Google Scholar e sua estratificação no WebQualis. Para a Otorrinolaringologia foram encontrados 44 periódicos, cujo fator de impacto variou de 3.006 a 0.128; entretanto, apenas 26 deles (60%) tinham Qualis medido pela CAPES; nas estratificações encontrou-se nenhuma revista em A1, três em A2 e nove em B1. Para a Cirurgia Torácica foram localizados três periódicos, sendo que apenas um tinha Qualis medido (A2) com média de 3.61. Os resultados da busca para a Ortopedia e Traumatologia permitiu encontrar 67 periódicos, cuja variação do fator de impacto foi de 4.699 a 0.156; apenas 38 deles (60%) tinham Qualis medido; três periódicos tinham estratificação A1, sete A2 e 25 em B1. A busca por revistas de maior impacto faz com que muitos especialistas deixem de publicar em revistas de sua área enfrentando mais dificuldades que pesquisadores das outras áreas.

Histopathological image analysis of chemical-induced hepatocellular hypertrophy in mice.

PubMed

Asaoka, Yoshiji; Togashi, Yuko; Mutsuga, Mayu; Imura, Naoko; Miyoshi, Tomoya; Miyamoto, Yohei

2016-04-01

Chemical-induced hepatocellular hypertrophy is frequently observed in rodents, and is mostly caused by the induction of phase I and phase II drug metabolic enzymes and peroxisomal lipid metabolic enzymes. Liver weight is a sensitive and commonly used marker for detecting hepatocellular hypertrophy, but is also increased by a number of other factors. Histopathological observations subjectively detect changes such as hepatocellular hypertrophy based on the size of a hepatocyte. Therefore, quantitative microscopic observations are required to evaluate histopathological alterations objectively. In the present study, we developed a novel quantitative method for an image analysis of hepatocellular hypertrophy using liver sections stained with hematoxylin and eosin, and demonstrated its usefulness for evaluating hepatocellular hypertrophy induced by phenobarbital (a phase I and phase II enzyme inducer) and clofibrate (a peroxisomal enzyme inducer) in mice. The algorithm of this imaging analysis was designed to recognize an individual hepatocyte through a combination of pixel-based and object-based analyses. Hepatocellular nuclei and the surrounding non-hepatocellular cells were recognized by the pixel-based analysis, while the areas of the recognized hepatocellular nuclei were then expanded until they ran against their expanding neighboring hepatocytes and surrounding non-hepatocellular cells by the object-based analysis. The expanded area of each hepatocellular nucleus was regarded as the size of an individual hepatocyte. The results of this imaging analysis showed that changes in the sizes of hepatocytes corresponded with histopathological observations in phenobarbital and clofibrate-treated mice, and revealed a correlation between hepatocyte size and liver weight. In conclusion, our novel image analysis method is very useful for quantitative evaluations of chemical-induced hepatocellular hypertrophy. Copyright © 2015 Elsevier GmbH. All rights reserved.

Biochemical analysis with microfluidic systems.

PubMed

Bilitewski, Ursula; Genrich, Meike; Kadow, Sabine; Mersal, Gaber

2003-10-01

Microfluidic systems are capillary networks of varying complexity fabricated originally in silicon, but nowadays in glass and polymeric substrates. Flow of liquid is mainly controlled by use of electroosmotic effects, i.e. application of electric fields, in addition to pressurized flow, i.e. application of pressure or vacuum. Because electroosmotic flow rates depend on the charge densities on the walls of capillaries, they are influenced by substrate material, fabrication processes, surface pretreatment procedures, and buffer additives. Microfluidic systems combine the properties of capillary electrophoretic systems and flow-through analytical systems, and thus biochemical analytical assays have been developed utilizing and integrating both aspects. Proteins, peptides, and nucleic acids can be separated because of their different electrophoretic mobility; detection is achieved with fluorescence detectors. For protein analysis, in particular, interfaces between microfluidic chips and mass spectrometers were developed. Further levels of integration of required sample-treatment steps were achieved by integration of protein digestion by immobilized trypsin and amplification of nucleic acids by the polymerase chain reaction. Kinetic constants of enzyme reactions were determined by adjusting different degrees of dilution of enzyme substrates or inhibitors within a single chip utilizing mainly the properties of controlled dosing and mixing liquids within a chip. For analysis of kinase reactions, however, a combination of a reaction step (enzyme with substrate and inhibitor) and a separation step (enzyme substrate and reaction product) was required. Microfluidic chips also enable separation of analytes from sample matrix constituents, which can interfere with quantitative determination, if they have different electrophoretic mobilities. In addition to analysis of nucleic acids and enzymes, immunoassays are the third group of analytical assays performed in microfluidic chips. They utilize either affinity capillary electrophoresis as a homogeneous assay format, or immobilized antigens or antibodies in heterogeneous assays with serial supply of reagents and washing solutions.

Effect of probiotics and synbiotics consumption on serum concentrations of liver function test enzymes: a systematic review and meta-analysis.

PubMed

Khalesi, Saman; Johnson, David Wayne; Campbell, Katrin; Williams, Susan; Fenning, Andrew; Saluja, Sonia; Irwin, Christopher

2017-11-08

The gut-liver interaction suggests that modification of gut bacterial flora using probiotics and synbiotics may improve liver function. This systematic review and meta-analysis aimed to clarify the effect of probiotics and synbiotics consumption on the serum concentration of liver function enzymes. PubMed (MEDLINE), Cumulative Index to Nursing and Allied Health Literature, and Cochrane Library (Central) were searched from 1980 to August 2017 for studies where adults consumed probiotics and/or synbiotics in controlled trials and changes in liver function enzymes were examined. A total of 17 studies (19 trials) were included in the meta-analysis. Random effects meta-analyses were applied. Probiotics and synbiotics significantly reduced serum alanine aminotransferase [- 8.05 IU/L, 95% confidence interval (CI) - 13.07 to - 3.04; p = 0.002]; aspartate aminotransferase (- 7.79 IU/L, 95% CI: - 13.93 to - 1.65; p = 0.02) and gamma-glutamyl transpeptidase (- 8.40 IU/L, 95% CI - 12.61 to - 4.20; p < 0.001). Changes in the serum concentration of alkaline phosphatase and albumin did not reach a statistically significant level. Changes to bilirubin levels were in favour of the control group (0.95 μmol/L, 95% CI 0.48-1.42; p < 0.001). Subgroup analysis suggested the existence of liver disease at baseline, synbiotics supplementation and duration of supplementation ≥ 8 weeks resulted in more pronounced improvement in liver function enzymes than their counterparts. Probiotics and synbiotics may be suggested as supplements to improve serum concentration of liver enzymes, especially when synbiotics administered for a period ≥ 8 weeks and in individuals with liver disease.

Structural modelling and comparative analysis of homologous, analogous and specific proteins from Trypanosoma cruzi versus Homo sapiens: putative drug targets for chagas' disease treatment

PubMed Central

2010-01-01

Background Trypanosoma cruzi is the etiological agent of Chagas' disease, an endemic infection that causes thousands of deaths every year in Latin America. Therapeutic options remain inefficient, demanding the search for new drugs and/or new molecular targets. Such efforts can focus on proteins that are specific to the parasite, but analogous enzymes and enzymes with a three-dimensional (3D) structure sufficiently different from the corresponding host proteins may represent equally interesting targets. In order to find these targets we used the workflows MHOLline and AnEnΠ obtaining 3D models from homologous, analogous and specific proteins of Trypanosoma cruzi versus Homo sapiens. Results We applied genome wide comparative modelling techniques to obtain 3D models for 3,286 predicted proteins of T. cruzi. In combination with comparative genome analysis to Homo sapiens, we were able to identify a subset of 397 enzyme sequences, of which 356 are homologous, 3 analogous and 38 specific to the parasite. Conclusions In this work, we present a set of 397 enzyme models of T. cruzi that can constitute potential structure-based drug targets to be investigated for the development of new strategies to fight Chagas' disease. The strategies presented here support the concept of structural analysis in conjunction with protein functional analysis as an interesting computational methodology to detect potential targets for structure-based rational drug design. For example, 2,4-dienoyl-CoA reductase (EC 1.3.1.34) and triacylglycerol lipase (EC 3.1.1.3), classified as analogous proteins in relation to H. sapiens enzymes, were identified as new potential molecular targets. PMID:21034488

Structural modelling and comparative analysis of homologous, analogous and specific proteins from Trypanosoma cruzi versus Homo sapiens: putative drug targets for chagas' disease treatment.

PubMed

Capriles, Priscila V S Z; Guimarães, Ana C R; Otto, Thomas D; Miranda, Antonio B; Dardenne, Laurent E; Degrave, Wim M

2010-10-29

Trypanosoma cruzi is the etiological agent of Chagas' disease, an endemic infection that causes thousands of deaths every year in Latin America. Therapeutic options remain inefficient, demanding the search for new drugs and/or new molecular targets. Such efforts can focus on proteins that are specific to the parasite, but analogous enzymes and enzymes with a three-dimensional (3D) structure sufficiently different from the corresponding host proteins may represent equally interesting targets. In order to find these targets we used the workflows MHOLline and AnEnΠ obtaining 3D models from homologous, analogous and specific proteins of Trypanosoma cruzi versus Homo sapiens. We applied genome wide comparative modelling techniques to obtain 3D models for 3,286 predicted proteins of T. cruzi. In combination with comparative genome analysis to Homo sapiens, we were able to identify a subset of 397 enzyme sequences, of which 356 are homologous, 3 analogous and 38 specific to the parasite. In this work, we present a set of 397 enzyme models of T. cruzi that can constitute potential structure-based drug targets to be investigated for the development of new strategies to fight Chagas' disease. The strategies presented here support the concept of structural analysis in conjunction with protein functional analysis as an interesting computational methodology to detect potential targets for structure-based rational drug design. For example, 2,4-dienoyl-CoA reductase (EC 1.3.1.34) and triacylglycerol lipase (EC 3.1.1.3), classified as analogous proteins in relation to H. sapiens enzymes, were identified as new potential molecular targets.

The dynamic basis of energy transduction in enzymes.

PubMed

Somogyi, B; Welch, G R; Damjanovich, S

1984-09-06

The most important idea underlying our treatment herein is the unity of the enzyme molecule and the medium. Appreciation of this relationship is vital, if enzymology is to graduate from its present reductionistic status to a more holistic posture. Enzymes are biological entities firstly, and isolated objects of physicochemical analysis secondly. Perhaps the most crucial 'biological lesson', particularly apropos of enzymes in intermediary metabolism, concerns the 'cytosociology' of enzyme action in vivo [94,128]. The natural habitat of many enzymes in the living cell is far different from that in bulk aqueous solution in vitro. In order to obtain a real grasp of the nature of enzyme function, one must ultimately couch enzymology in concepts emerging from contemporary cell biology [95]. Notwithstanding, analysis precedes synthesis; and one must needs begin with the individual enzyme molecule. The trenchant efforts of the physical chemist and the organic chemist have produced a wealth of information on the nature of the binding and catalytic events at the enzyme active site. While it is not yet possible to explain precisely the complete sequence of events in the catalytic process, nevertheless, the basic mechanisms by which enzymes effect catalysis (i.e., reduce activation energy) now seem apparent [81,129]. The new frontier is to be found, in exploring the dynamic role of the protein matrix [17]. Not only does the protein provide the 3-D scaffolding for active-site processes, but, more importantly, it serves as the local solvent for the bound chemical subsystem. Thus, the dynamical aspects of enzyme catalysis (for thermally based systems) must arise from the fluctuational properties of the protein molecule. This notion is the common denominator in all of the models in subsection IIC. It is the anisotropic nature of this fluctuational behavior, which would characterize the energy-transduction phenomenon leading to localized catalytic events at the active-site. In Section III we attempted to show that all of the various enzyme models contribute pieces to a single, all-embracing jig-saw puzzle. Some models focus on the dynamical properties of the protein per se, whereas others deal with the stochastic aspects of protein-solvent interaction. The two approaches are complementary, as are mutually interlocking pieces of a puzzle. The ultimate picture depicted by this 'jig-saw puzzle' is still somewhat vague--owing to the present paucity of empirical information on protein motions.(ABSTRACT TRUNCATED AT 400 WORDS)

Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in Corynebacterium glutamicum for lysine production.

PubMed

Chen, Zhen; Meyer, Weiqian; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

2011-07-01

Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study, we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another 14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays. The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter L-lysine within 30 h of fed-batch fermentation in a bioreactor.

Coevolutionary Analysis Enabled Rational Deregulation of Allosteric Enzyme Inhibition in Corynebacterium glutamicum for Lysine Production ▿

PubMed Central

Chen, Zhen; Meyer, Weiqian; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

2011-01-01

Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study, we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another 14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays. The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter l-lysine within 30 h of fed-batch fermentation in a bioreactor. PMID:21531824

Quantitative analysis of immobilized penicillinase using enzyme-modified AlGaN/GaN field-effect transistors.

PubMed

Müntze, Gesche Mareike; Baur, Barbara; Schäfer, Wladimir; Sasse, Alexander; Howgate, John; Röth, Kai; Eickhoff, Martin

2015-02-15

Penicillinase-modified AlGaN/GaN field-effect transistors (PenFETs) are utilized to systematically investigate the covalently immobilized enzyme penicillinase under different experimental conditions. We demonstrate quantitative evaluation of covalently immobilized penicillinase layers on pH-sensitive field-effect transistors (FETs) using an analytical kinetic PenFET model. This kinetic model is explicitly suited for devices with thin enzyme layers that are not diffusion-limited, as it is the case for the PenFETs discussed here. By means of the kinetic model it was possible to extract the Michaelis constant of covalently immobilized penicillinase as well as relative transport coefficients of the different species associated with the enzymatic reaction which, exempli gratia, give information about the permeability of the enzymatic layer. Based on this analysis we quantify the reproducibility and the stability of the analyzed PenFETs over the course of 33 days as well as the influence of pH and buffer concentration on the properties of the enzymatic layer. Thereby the stability measurements reveal a Michalis constant KM of (67 ± 13)μM while the chronological development of the relative transport coefficients suggests a detachment of physisorbed penicillinase during the first two weeks since production. Our results show that AlGaN/GaN PenFETs prepared by covalent immobilization of a penicillinase enzyme layer present a powerful tool for quantitative analysis of enzyme functionality. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of the Biotechnological Potential of a Lentinus crinitus Isolate in the Light of Its Secretome.

PubMed

Cambri, Geison; de Sousa, Mirta Mittelstedt Leal; Fonseca, Davi de Miranda; Marchini, Fabricio; da Silveira, Joana Lea Meira; Paba, Jaime

2016-12-02

Analysis of fungal secretomes is a prospection tool for the discovery of new catalysts with biotechnological applications. Since enzyme secretion is strongly modulated by environmental factors, evaluation of growth conditions is of utmost importance to achieve optimal enzyme production. In this work, a nonsequenced wood-rotting fungus, Lentinus crinitus, was used for secretome analysis by enzymatic assays and a proteomics approach. Enzyme production was assessed after the fungus was cultured in seven different carbon sources and three nitrogen-containing compounds. The biomass yields and secreted protein arrays differed drastically among growing conditions. A mixture of secreted extracts derived from solid and liquid cultures was inspected by shotgun mass spectrometry and two-dimensional gel electrophoresis (2-DE) prior to analysis via LC-MS/MS. Proteins were identified using mass spectrometry (MS)-driven BLAST. The spectrum of secreted proteins comprised CAZymes, oxidase/reductases, proteases, and lipase/esterases. Although preseparation by 2-DE improved the number of identifications (162) compared with the shotgun approach (98 identifications), the two strategies revealed similar protein patterns. Culture media with reduced water content stimulated the expression of oxidases/reductases, while hydrolases were induced during submerged fermentation. The diversity of proteins observed within both the CAZyme and oxidoreductase groups revealed in this fungus a powerful arsenal of enzymes dedicated to the breakdown and consumption of lignocellulose.

Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis.

PubMed

Dulermo, Thierry; Lazar, Zbigniew; Dulermo, Rémi; Rakicka, Magdalena; Haddouche, Ramedane; Nicaud, Jean-Marc

2015-09-01

The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica. Copyright © 2015. Published by Elsevier B.V.

Hepatic Xenobiotic Metabolizing Enzyme Gene Expression Through the Life Stages of the Mouse

EPA Science Inventory

BACKGROUND: Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). No comprehensive analysis of the mRNA expression of XMETs has been ca...

MODULATION OF EASTERN OYSTER HEMOCYTE ACTIVITIES BY PERKINSUS MARINUS EXTRACELLULAR PROTEINS

EPA Science Inventory

The oyster pathogen Perkinsus marinusproduces many extracellular proteins (ECP) in vitro. Analysis of this ECP revealed a battery of hydrolytic enzymes. Some of these enzymes are known to modulate the activity of host defense cells. Although information on the effects of P. marin...

Analysis of Structure and Specific Functional Groups Involved in Acetylcholinesterase Catalysis and Inhibition

DTIC Science & Technology

1992-12-15

et al., 1990). 2. SRpodoptera frugiperda (Sf9. Cells were typically grown in 250 mL of medium in a 500-mL spinner flask with slow stirring at 27"C in...reasonably good expression systems in Spodoptera for preparing large quantities of enzyme. The enzymes prepared from the baculovirus-Sjodo tera system were...4Standard Errors) for Wild-Type and Mutant Acetylcholinesterases Expressed in a Baculovirus- Spodoptera System’ enzyme 10’K, (M) Km tl/K. .. t 101K

Maintaining Genetic Integrity Under Extreme Conditions: Novel DNA Damage Repair Biology in the Archaea

DTIC Science & Technology

2013-11-23

Genetic analysis of Nre DNA repair function A4 Conclusions B. Widening the net in the search for new DNA-directed enzyme activities C. New tools for H...Figure 1) were hypothesised to be novel DNA repair enzymes . The stated aims of the proposal were to use a combination of genetic, biochemical and...in E.coli Almost all proteins that interact directly with PCNA are enzymes possessing DNA-directed activities such as nucleases, glycosylases

L-edge sum rule analysis on 3d transition metal sites: from d10 to d0 and towards application to extremely dilute metallo-enzymes.

PubMed

Wang, Hongxin; Friedrich, Stephan; Li, Lei; Mao, Ziliang; Ge, Pinghua; Balasubramanian, Mahalingam; Patil, Daulat S

2018-03-28

According to L-edge sum rules, the number of 3d vacancies at a transition metal site is directly proportional to the integrated intensity of the L-edge X-ray absorption spectrum (XAS) for the corresponding metal complex. In this study, the numbers of 3d holes are characterized quantitatively or semi-quantitatively for a series of manganese (Mn) and nickel (Ni) complexes, including the electron configurations 3d 10 → 3d 0 . In addition, extremely dilute (<0.1% wt/wt) Ni enzymes were examined by two different approaches: (1) by using a high resolution superconducting tunnel junction X-ray detector to obtain XAS spectra with a very high signal-to-noise ratio, especially in the non-variant edge jump region; and (2) by adding an inert tracer to the sample that provides a prominent spectral feature to replace the weak edge jump for intensity normalization. In this publication, we present for the first time: (1) L-edge sum rule analysis for a series of Mn and Ni complexes that include electron configurations from an open shell 3d 0 to a closed shell 3d 10 ; (2) a systematic analysis on the uncertainties, especially on that from the edge jump, which was missing in all previous reports; (3) a clearly-resolved edge jump between pre-L 3 and post-L 2 regions from an extremely dilute sample; (4) an evaluation of an alternative normalization standard for L-edge sum rule analysis. XAS from two copper (Cu) proteins measured using a conventional semiconductor X-ray detector are also repeated as bridges between Ni complexes and dilute Ni enzymes. The differences between measuring 1% Cu enzymes and measuring <0.1% Ni enzymes are compared and discussed. This study extends L-edge sum rule analysis to virtually any 3d metal complex and any dilute biological samples that contain 3d metals.

Brazilian spatial dynamics in the long term (1872-2000): ``path dependency'' or ``reversal of fortune''?

NASA Astrophysics Data System (ADS)

Monasterio, Leonardo Monteiro

2010-03-01

This paper analyzes the spatial dynamics of Brazilian regional inequalities between 1872 and 2000 using contemporary tools. The first part of the paper provides new estimates of income per capita in 1872 by municipality using census and electoral information on income by occupation. The level of analysis is the Minimum Comparable Areas 1872-2000 developed by Reis et al. (Áreas mínimas comparáveis para os períodos intercensitários de 1872 a 2000, 2007). These areas are the least aggregation of adjacent municipalities required to allow consistent geographic area comparisons between census years. In the second section of the paper, Exploratory Spatial Data Analysis, Markov chains and stochastic kernel techniques (spatially conditioned) are applied to the dataset. The results suggest that, in broad terms, the spatial pattern of income distribution in Brazil during that period of time has remained stable.

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

PubMed

Porter, Joanne L; Boon, Priscilla L S; Murray, Tracy P; Huber, Thomas; Collyer, Charles A; Ollis, David L

2015-02-20

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

A Quantitative Measure of Conformational Changes in Apo, Holo and Ligand-Bound Forms of Enzymes.

PubMed

Singh, Satendra; Singh, Atul Kumar; Wadhwa, Gulshan; Singh, Dev Bukhsh; Dwivedi, Seema; Gautam, Budhayash; Ramteke, Pramod W

2016-06-01

Determination of the native geometry of the enzymes and ligand complexes is a key step in the process of structure-based drug designing. Enzymes and ligands show flexibility in structural behavior as they come in contact with each other. When ligand binds with active site of the enzyme, in the presence of cofactor some structural changes are expected to occur in the active site. Motivation behind this study is to determine the nature of conformational changes as well as regions where such changes are more pronounced. To measure the structural changes due to cofactor and ligand complex, enzyme in apo, holo and ligand-bound forms is selected. Enzyme data set was retrieved from protein data bank. Fifteen triplet groups were selected for the analysis of structural changes based on selection criteria. Structural features for selected enzymes were compared at the global as well as local region. Accessible surface area for the enzymes in entire triplet set was calculated, which describes the change in accessible surface area upon binding of cofactor and ligand with the enzyme. It was observed that some structural changes take place during binding of ligand in the presence of cofactor. This study will helps in understanding the level of flexibility in protein-ligand interaction for computer-aided drug designing.

Genome wide comprehensive analysis and web resource development on cell wall degrading enzymes from phyto-parasitic nematodes.

PubMed

Rai, Krishan Mohan; Balasubramanian, Vimal Kumar; Welker, Cassie Marie; Pang, Mingxiong; Hii, Mei Mei; Mendu, Venugopal

2015-08-01

The plant cell wall serves as a primary barrier against pathogen invasion. The success of a plant pathogen largely depends on its ability to overcome this barrier. During the infection process, plant parasitic nematodes secrete cell wall degrading enzymes (CWDEs) apart from piercing with their stylet, a sharp and hard mouthpart used for successful infection. CWDEs typically consist of cellulases, hemicellulases, and pectinases, which help the nematode to infect and establish the feeding structure or form a cyst. The study of nematode cell wall degrading enzymes not only enhance our understanding of the interaction between nematodes and their host, but also provides information on a novel source of enzymes for their potential use in biomass based biofuel/bioproduct industries. Although there is comprehensive information available on genome wide analysis of CWDEs for bacteria, fungi, termites and plants, but no comprehensive information available for plant pathogenic nematodes. Herein we have performed a genome wide analysis of CWDEs from the genome sequenced phyto pathogenic nematode species and developed a comprehensive publicly available database. In the present study, we have performed a genome wide analysis for the presence of CWDEs from five plant parasitic nematode species with fully sequenced genomes covering three genera viz. Bursaphelenchus, Glorodera and Meloidogyne. Using the Hidden Markov Models (HMM) conserved domain profiles of the respective gene families, we have identified 530 genes encoding CWDEs that are distributed among 24 gene families of glycoside hydrolases (412) and polysaccharide lyases (118). Furthermore, expression profiles of these genes were analyzed across the life cycle of a potato cyst nematode. Most genes were found to have moderate to high expression from early to late infectious stages, while some clusters were invasion stage specific, indicating the role of these enzymes in the nematode's infection and establishment process. Additionally, we have also developed a Nematode's Plant Cell Wall Degrading Enzyme (NCWDE) database as a platform to provide a comprehensive outcome of the present study. Our study provides collective information about different families of CWDEs from five different sequenced plant pathogenic nematode species. The outcomes of this study will help in developing better strategies to curtail the nematode infection, as well as help in identification of novel cell wall degrading enzymes for biofuel/bioproduct industries.

Molecular characterization of genes encoding trypsin-like enzymes from Aedes aegypti larvae and identification of digestive enzymes.

PubMed

Soares, Tatiane S; Watanabe, Renata M O; Lemos, Francisco J A; Tanaka, Aparecida S

2011-12-10

Trypsin-like enzymes play an important role in the Aedes aegypti digestive process. The trypsin-like enzymes present in adults were characterized previously, but little is known about trypsins in larvae. In the present work, we identified one of the trypsin enzymes from Ae. aegypti larval midgut using a library of trypsin gene fragments, which was the sequence known as AAEL005607 from the Ae. aegypti genome. Quantitative PCR analysis showed that AAEL005607 was transcribed in all larval instars, but it was not present in adult midgut. In order to confirm transcription data, the trypsin-like enzymes from 4th instar larvae of Ae. aegypti midgut were purified and sequenced. Purified trypsin showed identity with the amino-terminal sequence of AAEL005607, AAEL005609 and AAEL005614. These three trypsins have high amino acids identity, and could all be used as a template for the design of inhibitors. In conclusion, for the first time, digestive enzymes of 4th larval instar of Ae. aegypti were purified and characterized. The knowledge of digestive enzymes present in Ae. aegypti larvae may be helpful in the development of a larvicide. Copyright © 2011 Elsevier B.V. All rights reserved.

Isolation and Compositional Analysis of a CP12-Associated Complex of Calvin Cycle Enzymes from Nicotiana tabacum

USDA-ARS?s Scientific Manuscript database

CP12 is a small intrinsically unstructured protein that forms a multiprotein complex with two Calvin Cycle enzymes, phosphoribulokinase (PRK) and NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The complex can be reconstituted in vitro from recombinant proteins under conditions t...

ANALYSIS OF SOIL AND DUST SAMPLES FOR POLYCHLORINATED BIPHENYLS BY ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

EPA Science Inventory

An inhibition enzyme-linked immunosorbent assay (ELISA) was used to determine polychlorinated biphenyls (PCBs) in house dust and soil. Soil and house dust samples were analyzed for PCB by both gas chromatography/electron capture detection (GC/ECD) and ELISA methods. A correlati...

Arrhenius analysis of the electrophorus electricus acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine.

PubMed

Oakes, Jesse; Nguyen, Tina; Britt, B Mark

2003-06-01

Ellman's method was used to determine the Michaelis-Menten parameters for the hydrolysis of acetylthiocholine by Electrophorus electricus acetylcholinesterase from 12 to 37 degrees C. Arrhenius analysis revealed that the activation energy for formation of the enzyme/substrate complex is 22.2 +/- 1.1 kJ/mole. The Arrhenius plot of k(cat) is markedly curved and attributed to comparable rates of acylation and deacylation due to the absence of evidence for a temperature-dependent enzyme conformational change by differential scanning calorimetry.

Um Breve Balanço dos Estudos em Astronomia e Educação no Brasil no Período de 2010 a 2013

NASA Astrophysics Data System (ADS)

Goncalves, Erica de Oliveira; Kern, C.

2014-10-01

No Brasil, as pesquisas em ensino de astronomia para a Educação Básica vem ganhando destaque. Posto como importante área do conhecimento para estudantes e professores, os estudos em astronomia conquistam espaços nos documentos oficiais da educação e nos currículos escolares. Diante desse cenário, fez-se, neste trabalho, um mapeamento no banco de dados da Biblioteca Digital Brasileira de Teses e Dissertações , com base nas palavras-chave "astronomia" e "educação" no período de 2010 a 2013. Para compor o que aqui denominamos de balanço da área de estudo, foram selecionados trabalhos e analisados os títulos, os resumos, as considerações finais e as referências, bem como identificamos as fontes epistemológicas correntes nas pesquisas de pós-graduação no período supracitado. Identificou-se, na maior parte dos trabalhos pesquisados, referenciais teóricos relacionados & agrave; área de física, ciências e astronomia que envolvem discussões sobre currículo e práticas pedagógicas vinculados ao ensino de astronomia no ensino fundamental e médio da Educação Bãsica e nos cursos de formação de professores.

Thermal inactivation of enzymes and pathogens in biosamples for MS analysis.

PubMed

Ahnoff, Martin; Cazares, Lisa H; Sköld, Karl

2015-01-01

Protein denaturation is the common basis for enzyme inactivation and inactivation of pathogens, necessary for preservation and safe handling of biosamples for downstream analysis. While heat-stabilization technology has been used in proteomic and peptidomic research since its introduction in 2009, the advantages of using the technique for simultaneous pathogen inactivation have only recently been addressed. The time required for enzyme inactivation by heat (≈1 min) is short compared with chemical treatments, and inactivation is irreversible in contrast to freezing. Heat stabilization thus facilitates mass spectrometric studies of biomolecules with a fast conversion rate, and expands the chemical space of potential biomarkers to include more short-lived entities, such as phosphorylated proteins, in tissue samples as well as whole-blood (dried blood sample) samples.

X-ray absorption spectroscopic studies of mononuclear non-heme iron enzymes

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

Westre, Tami E.

Fe-K-edge X-ray absorption spectroscopy (XAS) has been used to investigate the electronic and geometric structure of the iron active site in non-heme iron enzymes. A new theoretical extended X-ray absorption fine structure (EXAFS) analysis approach, called GNXAS, has been tested on data for iron model complexes to evaluate the utility and reliability of this new technique, especially with respect to the effects of multiple-scattering. In addition, a detailed analysis of the 1s→3d pre-edge feature has been developed as a tool for investigating the oxidation state, spin state, and geometry of iron sites. Edge and EXAFS analyses have then been appliedmore » to the study of non-heme iron enzyme active sites.« less

EnzDP: improved enzyme annotation for metabolic network reconstruction based on domain composition profiles.

PubMed

Nguyen, Nam-Ninh; Srihari, Sriganesh; Leong, Hon Wai; Chong, Ket-Fah

2015-10-01

Determining the entire complement of enzymes and their enzymatic functions is a fundamental step for reconstructing the metabolic network of cells. High quality enzyme annotation helps in enhancing metabolic networks reconstructed from the genome, especially by reducing gaps and increasing the enzyme coverage. Currently, structure-based and network-based approaches can only cover a limited number of enzyme families, and the accuracy of homology-based approaches can be further improved. Bottom-up homology-based approach improves the coverage by rebuilding Hidden Markov Model (HMM) profiles for all known enzymes. However, its clustering procedure relies firmly on BLAST similarity score, ignoring protein domains/patterns, and is sensitive to changes in cut-off thresholds. Here, we use functional domain architecture to score the association between domain families and enzyme families (Domain-Enzyme Association Scoring, DEAS). The DEAS score is used to calculate the similarity between proteins, which is then used in clustering procedure, instead of using sequence similarity score. We improve the enzyme annotation protocol using a stringent classification procedure, and by choosing optimal threshold settings and checking for active sites. Our analysis shows that our stringent protocol EnzDP can cover up to 90% of enzyme families available in Swiss-Prot. It achieves a high accuracy of 94.5% based on five-fold cross-validation. EnzDP outperforms existing methods across several testing scenarios. Thus, EnzDP serves as a reliable automated tool for enzyme annotation and metabolic network reconstruction. Available at: www.comp.nus.edu.sg/~nguyennn/EnzDP .

[Study on relationship between effective components and soil enzyme activity in different growth patterns of Panax ginseng].

PubMed

Yang, Yan-Wen; Jiang, Yuan-Tong

2016-08-01

Study on 5 effective components and 6 soil enzyme activities of 2 different growth patterns, analyse the dates with the canonical correlation analysis, In order to reveal the relations between the effective components and soil enzyme activities. The result showed that they had a great relation between the effective components and soil enzyme activities, the activity of the same enzyme in humus soil was higher than that in farmland soil. Growth pattern of farmland soil, if the invertase and phosphatase activity were too high, which would inhibit the accumulation of total ginsenoside, water-miscible total proteins and total amino acid; Growth pattern of humus soil, if the invertase, urease and phosphatase activity were too high, which would inhibit the accumulation of total ginsenoside and the total essential oils. Integral soil enzyme activity can be used as a index of soil quality, which, together with other growth factors. The appropriate enzyme activity can accelerate the circulation and transformation of all kinds of material in the soil, improve effectively components accumulation. Copyright© by the Chinese Pharmaceutical Association.

Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

PubMed Central

Valero, E; Varón, R; García-Carmona, F

1995-01-01

A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response. PMID:7619054

4-Chlorophenol biodegradation facilitator composed of recombinant multi-biocatalysts immobilized onto montmorillonite.

PubMed

Kwean, Oh Sung; Cho, Su Yeon; Yang, Jun Won; Cho, Wooyoun; Park, Sungyoon; Lim, Yejee; Shin, Min Chul; Kim, Han-Suk; Park, Joonhong; Kim, Han S

2018-07-01

A biodegradation facilitator which catalyzes the initial steps of 4-chlorophenol (4-CP) oxidation was prepared by immobilizing multiple enzymes (monooxygenase, CphC-I and dioxygenase, CphA-I) onto a natural inorganic support. The enzymes were obtained via overexpression and purification after cloning the corresponding genes (cphC-I and cphA-I) from Arthrobacter chlorophenolicus A6. Then, the recombinant CphC-I was immobilized onto fulvic acid-activated montmorillonite. The immobilization yield was 60%, and the high enzyme activity (82.6%) was retained after immobilization. Kinetic analysis indicated that the Michaelis-Menten model parameters for the immobilized CphC-I were similar to those for the free enzyme. The enzyme stability was markedly enhanced after immobilization. The immobilized enzyme exhibited a high level of activity even after repetitive use (84.7%) and powdering (65.8%). 4-CP was sequentially oxidized by a multiple enzyme complex, comprising the immobilized CphC-I and CphA-I, via the hydroquinone pathway: oxidative transformation of 4-CP to hydroxyquinol followed by ring fission of hydroxyquinol. Copyright © 2018 Elsevier Ltd. All rights reserved.

Toward finding a universally applicable parameterization of the β factor for Relaxed Eddy Accumulation applications

NASA Astrophysics Data System (ADS)

Vogl, Teresa; Hrdina, Amy; Thomas, Christoph

2016-04-01

The traditional eddy covariance (EC) technique requires the use of fast responding sensors (≥ 10 Hz) that do not exist for many chemical species found in the atmosphere. In this case, the Relaxed Eddy Accumulation (REA) method offers a means to calculate fluxes of trace gases and other scalar quantities (Businger and Oncley, 1990) and was originally derived from the eddy accumulation method (EA) first proposed by Desjardins (1972). While REA lessens the requirements for sensors and sampling and thus offers practical appeal, it introduces a dependence of the computed flux from a proportionality factor β. The accuracy of the REA fluxes hinges upon the correct determination of β, which was found to vary between 0.40 and 0.63 (Milne et al., 1999, Ammann and Meixner, 2002, Ruppert et al., 2006). However, formulating a universally valid parameterization for β instead of empirical evaluation has remained a conundrum and has been a main limitation for REA. In this study we take a fresh look at the dependencies and mathematical models of β by analyzing eddy covariance (EC) data and REA simulations for two field experiments in drastically contrasting environments: an exclusively physically driven environment in the Dry Valleys of Antarctica, and a biologically active system in a grassland in Germany. The main objective is to work toward a model parameterization for β that can be applied over wide range of surface conditions and forcings without the need for empirical evaluation, which is not possible for most REA applications. Our study discusses two different models to define β: (i) based upon scalar-scalar similarity, in which a different scalar is measured with fast-response sensors as a proxy for the scalar of interest, here referred to as β0; and (ii) computed solely from the vertical wind statistics, assuming a linear relationship between the scalar of interest and the vertical wind speed, referred to as βw. Results are presented for the carbon-dioxide, latent and sensible heat fluxes across the contrasting environments. First, the choice of an appropriate scalar to calculate β0 is discussed considering the sources and sinks of each scalar with an emphasis on the carbon dioxide flux, which shows strongly dissimilar dynamics between the Antarctic ecosystem and the grassland. Secondly, the impact of atmospheric stability on both β models is investigated. In a next step, we attempt to find a physically meaningful explanation for the overestimation of the REA scalar fluxes compared to those from EC for using βw. We do so by analyzing the probability density function (pdf) and its statistical moments for the vertical wind speed. We found its pdf to be non-Gaussian for the majority of cases, and detected a close to linear relationship of its kurtosis with βw. Finally, in an attempt to provide practical guidance for field measurements, we integrate our findings and propose an enhanced model parameterization, and evaluate the differences between our new model and a constant β. Ammann, C. and Meixner, F.X. (2002) Stability dependence of the relaxed eddy accumulation coefficient for various scalar quantities. J. Geophys. Res. 107. ACL7.1-ACL7.9 doi:10.1029/2001JD000649 Businger, J.A., Oncley, S.P. (1990) Flux measurement with conditional sampling. J. Atmos. Ocean. Tech. 7:349-352. Desjardins, R. L. (1972) A study of carbon-dioxide and sensible heat fluxes using the eddy correlation technique, Ph.D. dissertation, Cornell University, 189 pp. Desjardins, R.L. (1977) Description and evaluation of sensible heat flux detector. Boundary-Layer Meteorol. 11:147-154. Katul, G., Finkelstein, P. L., Clarke, J. F., and Ellestad, T. G. (1996) An Investigation of the Conditional Sampling Methods Used to Estimate Fluxes of Active, Reactive and Passive Scalars. J. Appl. Meteorol. 35: 1835-1845. Milne, R., Beverland, I. J., Hargreaves, K., and Moncrieff, J. B. (1999) Variation of the beta coefficient in the relaxed eddy accumulation method. Boundary-Layer Meteorol. 93: 211-225. Ruppert, J. ATEM software for atmospheric turbulent exchange measurements using eddy covariance and relaxed eddy accumulation systems: Bayreuth whole-air REA system setup, Universität Bayreuth, Abt. Mikrometeorologie, Print, ISSN 1614-8916, Arbeitsergebnisse 28, 29 S, 2005 Ruppert, J., Thomas, C., and Foken, T. (2006) scalar similarity for relaxed eddy accumulation methods. Boundary-Layer Meteorol. 120: 39-63.

Addressing Health Inequalities in the Delivery of the Human Papillomavirus Vaccination Programme: Examining the Role of the School Nurse

PubMed Central

Boyce, Tammy; Holmes, Alison

2012-01-01

Background HPV immunisation of adolescent girls is expected to have a significant impact in the reduction of cervical cancer. UK The HPV immunisation programme is primarily delivered by school nurses. We examine the role of school nurses in delivering the HPV immunisation programme and their impact on minimising health inequalities in vaccine uptake. Methods and Findings A rapid evidence assessment (REA) and semi-structured interviews with health professionals were conducted and analysed using thematic analysis. 80 health professionals from across the UK are interviewed, primarily school nurses and HPV immunisation programme coordinators. The REA identified 2,795 articles and after analysis and hand searches, 34 relevant articles were identified and analysed. Interviews revealed that health inequalities in HPV vaccination uptake were mainly related to income and other social factors in contrast to published research which emphasises potential inequalities related to ethnicity and/or religion. Most school nurses interviewed understood local health inequalities and made particular efforts to target girls who did not attend or missed doses. Interviews also revealed maintaining accurate and consistent records influenced both school nurses' understanding and efforts to target inequalities in HPV vaccination uptake. Conclusions Despite high uptake in the UK, some girls remain at risk of not being vaccinated with all three doses. School nurses played a key role in reducing health inequalities in the delivery of the HPV programme. Other studies identified religious beliefs and ethnicity as potentially influencing HPV vaccination uptake but interviews for this research found this appeared not to have occurred. Instead school nurses stated girls who were more likely to be missed were those not in education. Improving understanding of the delivery processes of immunisation programmes and this impact on health inequalities can help to inform solutions to increase uptake and address health inequalities in childhood and adolescent vaccination programmes. PMID:23028452

Molecular characterization of novel pyridoxal-5'-phosphate-dependent enzymes from the human microbiome.

PubMed

Fleischman, Nicholas M; Das, Debanu; Kumar, Abhinav; Xu, Qingping; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W; Klock, Heath E; Miller, Mitchell D; Elsliger, Marc-André; Godzik, Adam; Lesley, Scott A; Deacon, Ashley M; Wilson, Ian A; Toney, Michael D

2014-08-01

Pyridoxal-5'-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for ∼1.5% of most prokaryotic genomes and are estimated to be involved in ∼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases. © 2014 The Protein Society.

The inhibitory effect of metals and other ions on acid phosphatase activity from Vigna aconitifolia seeds.

PubMed

Srivastava, Pramod Kumar; Anand, Asha

2015-01-01

Sensitivity of acid phosphatase from Vigna aconitifolia seeds to metal ions, fluoride, and phosphate was examined. All the effectors had different degree of inhibitory effect on the enzyme. Among metal ions, molybdate and ferric ion were observed to be most potent inhibitors and both exhibited mixed type of inhibition. Acid phosphatase activity was inhibited by Cu2+ in a noncompetitive manner. Zn and Mn showed mild inhibition on the enzyme activity. Inhibition kinetics analysis explored molybdate as a potent inhibitor for acid phosphatase in comparison with other effectors used in this study. Fluoride was the next most strong inhibitor for the enzyme activity, and caused a mixed type of inhibition. Phosphate inhibited the enzyme competitively, which demonstrates that inhibition due to phosphate is one of the regulatory factors for enzyme activity.

Mold Pectinase Modified with Dialdehyde Derivatives of Dextran and Cellulose.

PubMed

Kobayashi, M; Chiba, Y; Funane, K; Ohya, S; Kato, Y

1996-01-01

Chemical modification of mold pectinase with dextran- and cellulose-dialdehydes was examined to improve the enzyme characteristics. The modified pectinase with dextran-dialdehyde retained about 50% of the original activity, and more than 80% of the total amino groups were modified. HPLC gel filtration analysis showed an increase in molecular weight of the reaction product. Reaction with cellulose-dialdehyde provided an immobilized form of pectinase. The immobilized pectinase was resistant to both acidic and alkaline pHs, and also acquired heat stability at 60°C. The optimum pH of the modified enzyme shifted from pH 4.5 to 5.0-5.5, and this enzyme had higher activity at neutral pH regions than the native enzyme. A rather low recovery of immobilized enzyme (14.5%) should be improved by the combination with various methods hitherto established.

Three-dimensional quantitative structure-activity relationship analysis for human pregnane X receptor for the prediction of CYP3A4 induction in human hepatocytes: structure-based comparative molecular field analysis.

PubMed

Handa, Koichi; Nakagome, Izumi; Yamaotsu, Noriyuki; Gouda, Hiroaki; Hirono, Shuichi

2015-01-01

The pregnane X receptor [PXR (NR1I2)] induces the expression of xenobiotic metabolic genes and transporter genes. In this study, we aimed to establish a computational method for quantifying the enzyme-inducing potencies of different compounds via their ability to activate PXR, for the application in drug discovery and development. To achieve this purpose, we developed a three-dimensional quantitative structure-activity relationship (3D-QSAR) model using comparative molecular field analysis (CoMFA) for predicting enzyme-inducing potencies, based on computer-ligand docking to multiple PXR protein structures sampled from the trajectory of a molecular dynamics simulation. Molecular mechanics-generalized born/surface area scores representing the ligand-protein-binding free energies were calculated for each ligand. As a result, the predicted enzyme-inducing potencies for compounds generated by the CoMFA model were in good agreement with the experimental values. Finally, we concluded that this 3D-QSAR model has the potential to predict the enzyme-inducing potencies of novel compounds with high precision and therefore has valuable applications in the early stages of the drug discovery process. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Soil ionomic and enzymatic responses and correlations to fertilizations amended with and without organic fertilizer in long-term experiments.

PubMed

Feng, Xumeng; Ling, Ning; Chen, Huan; Zhu, Chen; Duan, Yinghua; Peng, Chang; Yu, Guanghui; Ran, Wei; Shen, Qirong; Guo, Shiwei

2016-04-15

To investigate potential interactions between the soil ionome and enzyme activities affected by fertilization with or without organic fertilizer, soil samples were collected from four long-term experiments over China. Irrespective of variable interactions, fertilization type was the major factor impacting soil ionomic behavior and accounted for 15.14% of the overall impact. Sampling site was the major factor affecting soil enzymatic profile and accounted for 34.25% of the overall impact. The availabilities of Pb, La, Ni, Co, Fe and Al were significantly higher in soil with only chemical fertilizer than the soil with organic amendment. Most of the soil enzyme activities, including α-glucosidase activity, were significantly activated by organic amendment. Network analysis between the soil ionome and the soil enzyme activities was more complex in the organic-amended soils than in the chemical fertilized soils, whereas the network analysis among the soil ions was less complex with organic amendment. Moreover, α-glucosidase was revealed to generally harbor more corrections with the soil ionic availabilities in network. We concluded that some of the soil enzymes activated by organic input can make the soil more vigorous and stable and that the α-glucosidase revealed by this analysis might help stabilize the soil ion availability.

Soil ionomic and enzymatic responses and correlations to fertilizations amended with and without organic fertilizer in long-term experiments

PubMed Central

Feng, Xumeng; Ling, Ning; Chen, Huan; Zhu, Chen; Duan, Yinghua; Peng, Chang; Yu, Guanghui; Ran, Wei; Shen, Qirong; Guo, Shiwei

2016-01-01

To investigate potential interactions between the soil ionome and enzyme activities affected by fertilization with or without organic fertilizer, soil samples were collected from four long-term experiments over China. Irrespective of variable interactions, fertilization type was the major factor impacting soil ionomic behavior and accounted for 15.14% of the overall impact. Sampling site was the major factor affecting soil enzymatic profile and accounted for 34.25% of the overall impact. The availabilities of Pb, La, Ni, Co, Fe and Al were significantly higher in soil with only chemical fertilizer than the soil with organic amendment. Most of the soil enzyme activities, including α-glucosidase activity, were significantly activated by organic amendment. Network analysis between the soil ionome and the soil enzyme activities was more complex in the organic-amended soils than in the chemical fertilized soils, whereas the network analysis among the soil ions was less complex with organic amendment. Moreover, α-glucosidase was revealed to generally harbor more corrections with the soil ionic availabilities in network. We concluded that some of the soil enzymes activated by organic input can make the soil more vigorous and stable and that the α-glucosidase revealed by this analysis might help stabilize the soil ion availability. PMID:27079657

Molecular cloning, sequence and structural analysis of dehairing Mn(2+) dependent alkaline serine protease (MASPT) of Bacillus pumilus TMS55.

PubMed

Ibrahim, Kalibulla Syed; Muniyandi, Jeyaraj; Pandian, Shunmugiah Karutha

2011-10-01

Leather industries release a large amount of pollution-causing chemicals which creates one of the major industrial pollutions. The development of enzyme based processes as a potent alternative to pollution-causing chemicals is useful to overcome this issue. Proteases are enzymes which have extensive applications in leather processing and in several bioremediation processes due to their high alkaline protease activity and dehairing efficacy. In the present study, we report cloning, characterization of a Mn2+ dependent alkaline serine protease gene (MASPT) of Bacillus pumilus TMS55. The gene encoding the protease from B. pumilus TMS55 was cloned and its nucleotide sequence was determined. This gene has an open reading frame (ORF) of 1,149 bp that encodes a polypeptide of 383 amino acid residues. Our analysis showed that this polypeptide is composed of 29 residues N-terminal signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids. We performed bioinformatics analysis to compare MASPT enzyme with other proteases. Homology modeling was employed to model three dimensional structure for MASPT. Structural analysis showed that MASPT structure is composed of nine α-helices and nine β-strands. It has 3 catalytic residues and 14 metal binding residues. Docking analysis showed that residues S223, A260, N263, T328 and S329 interact with Mn2+. This study allows initial inferences about the structure of the protease and will allow the rational design of its derivatives for structure-function studies and also for further improvement of the enzyme.

Micromotors Powered by Enzyme Catalysis.

PubMed

Dey, Krishna K; Zhao, Xi; Tansi, Benjamin M; Méndez-Ortiz, Wilfredo J; Córdova-Figueroa, Ubaldo M; Golestanian, Ramin; Sen, Ayusman

2015-12-09

Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymatic reactions and their directed movement in substrate concentration gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the respective substrates, using optical microscopy and dynamic light scattering analysis. The diffusion of the particles was found to increase in a substrate concentration dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture.

ATP-dependent chromatin remodeling in T cells.

PubMed

Wurster, Andrea L; Pazin, Michael J

2012-02-01

One of the best studied systems for mammalian chromatin remodeling is transcriptional regulation during T cell development. The variety of these studies have led to important findings in T cell gene regulation and cell fate determination. Importantly, these findings have also advanced our knowledge of the function of remodeling enzymes in mammalian gene regulation. First we briefly present biochemical and cell-free analysis of 3 types of ATP dependent remodeling enzymes (SWI/SNF, Mi2, and ISWI) to construct an intellectual framework to understand how these enzymes might be working. Second, we compare and contrast the function of these enzymes during early (thymic) and late (peripheral) T cell development. Finally, we examine some of the gaps in our present understanding.

Stabilization of different types of transition states in a single enzyme active site: QM/MM analysis of enzymes in the alkaline phosphatase superfamily.

PubMed

Hou, Guanhua; Cui, Qiang

2013-07-17

The first step for the hydrolysis of a phosphate monoester (pNPP(2-)) in enzymes of the alkaline phosphatase (AP) superfamily, R166S AP and wild-type NPP, is studied using QM/MM simulations based on an approximate density functional theory (SCC-DFTBPR) and a recently introduced QM/MM interaction Hamiltonian. The calculations suggest that similar loose transition states are involved in both enzymes, despite the fact that phosphate monoesters are the cognate substrates for AP but promiscuous substrates for NPP. The computed loose transition states are clearly different from the more synchronous ones previously calculated for diester reactions in the same AP enzymes. Therefore, our results explicitly support the proposal that AP enzymes are able to recognize and stabilize different types of transition states in a single active site. Analysis of the structural features of computed transition states indicates that the plastic nature of the bimetallic site plays a minor role in accommodating multiple types of transition states and that the high degree of solvent accessibility of the AP active site also contributes to its ability to stabilize diverse transition-state structures without the need of causing large structural distortions of the bimetallic motif. The binding mode of the leaving group in the transition state highlights that vanadate may not always be an ideal transition state analog for loose phosphoryl transfer transition states.

Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis.

PubMed

Deng, Gejing; Gu, Rong-Fang; Marmor, Stephen; Fisher, Stewart L; Jahic, Haris; Sanyal, Gautam

2004-06-29

An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated. Copyright 2004 Elsevier B.V.

[Diversity and enzyme-producing activity of culturable halophilic bacteria in Daishan Saltern of East China].

PubMed

Yang, Dan-Dan; Li, Qian; Huang, Jing-Jing; Chen, Min

2012-11-01

Soil and saline water samples were collected from the Daishan Saltern of East China, and the halophilic bacteria were isolated and cultured by using selective media, aimed to investigate the diversity and enzyme-producing activity of culturable halophilic bacteria in saltern environment. A total of 181 strains were isolated by culture-dependent method. Specific primers were used to amplify the 16S rRNA gene of bacteria and archaea. The operation taxonomy units (OTUs) were determined by ARDRA method, and the representative strain of each OTU was sequenced. The phylogenetic position of all the isolated strains was determined by 16S rRNA sequencing. The results showed that the isolated 181 strains displayed 21 operational taxonomic units (OTUs), of which, 12 OTUs belonged to halophilic bacteria, and the others belonged to halophilic archaea. Phylogenetic analysis indicated that there were 7 genera presented among the halophilic bacteria group, and 4 genera presented among the halophilic archaea group. The dominant halophilic strains were of Halomonas and Haloarcula, with 46.8% in halophilic bacteria and 49.1% in halophilic archaea group, respectively. Enzyme-producing analysis indicated that most strains displayed enzyme-producing activity, including the activities of producing amylase, proteinase and lipase, and the dominant strains capable of enzyme-producing were of Haloarcula. Our results showed that in the environment of Daishan Saltern, there existed a higher diversity of halophilic bacteria, being a source sink for screening enzyme-producing bacterial strains.

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an [alpha]-Amino Acid Ester Hydrolase

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

Lai, Longsheng; Xu, Zhaohui; Zhou, Jiahai

2008-07-08

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of {alpha}-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar tomore » tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific {alpha}-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.« less

A 13-week research-based biochemistry laboratory curriculum.

PubMed

Lefurgy, Scott T; Mundorff, Emily C

2017-09-01

Here, we present a 13-week research-based biochemistry laboratory curriculum designed to provide the students with the experience of engaging in original research while introducing foundational biochemistry laboratory techniques. The laboratory experience has been developed around the directed evolution of an enzyme chosen by the instructor, with mutations designed by the students. Ideal enzymes for this curriculum are able to be structurally modeled, solubly expressed, and monitored for activity by UV/Vis spectroscopy, and an example curriculum for haloalkane dehalogenase is given. Unique to this curriculum is a successful implementation of saturation mutagenesis and high-throughput screening of enzyme function, along with bioinformatics analysis, homology modeling, structural analysis, protein expression and purification, polyacrylamide gel electrophoresis, UV/Vis spectroscopy, and enzyme kinetics. Each of these techniques is carried out using a novel student-designed mutant library or enzyme variant unique to the lab team and, importantly, not described previously in the literature. Use of a well-established set of protocols promotes student data quality. Publication may result from the original student-generated hypotheses and data, either from the class as a whole or individual students that continue their independent projects upon course completion. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):437-448, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach

DOE PAGES

Musumeci, Matias A.; Lozada, Mariana; Rial, Daniela V.; ...

2017-04-09

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putativemore » monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. As a result, this work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.« less

Molecular structure, spectroscopic and docking analysis of 1,3-diphenylpyrazole-4-propionic acid: A good prostaglandin reductase inhibitor

NASA Astrophysics Data System (ADS)

Kavitha, T.; Velraj, G.

2018-03-01

The molecule 1,3-diphenylpyrazole-4-propionic acid (DPPA) was optimized to its minimum energy level using density functional theory (DFT) calculations. The vibrational frequencies of DPPA were calculated along with their potential energy distribution (PED) and the obtained values are validated with the help of experimental calculations. The reactivity nature of the molecule was investigated with the aid of various DFT methods such as global reactivity descriptors, local reactivity descriptors, molecular electrostatic potential (MEP), natural bond orbitals (NBOs), etc. The prediction of activity spectra for substances (PASS) result forecast that, DPPA can be more active as a prostaglandin (PG) reductase inhibitor. The PGs are biologically synthesized by the cyclooxygenase (COX) enzyme which exists in COX1 and COX2 forms. The PGs produced by COX2 enzyme induces inflammation and fungal infections and hence the inhibition of COX2 enzyme is indispensable in anti-inflammation and anti-fungal activities. The docking analysis of DPPA with COX enzymes (both COX1 and COX2) were carried out and eventually, it was found that DPPA can selectively inhibit COX2 enzyme and can serve as a PG reductase inhibitor thereby acting as a lead compound for the treatment of inflammation and fungal diseases.

Morphological and physicochemical characterization of porous starches obtained from different botanical sources and amylolytic enzymes.

PubMed

Benavent-Gil, Yaiza; Rosell, Cristina M

2017-10-01

Porous starches might offer an attractive alternative as bio-adsorbents of a variety of compounds. However, morphology and physicochemical properties of starches must be understood before exploring their applications. Objective was to study the action of different amylolytic enzymes for producing porous starches. Wheat, rice, potato and cassava starches were treated with Amyloglucosidase (AMG), α-amylase (AM) and cyclodextrin-glycosyltransferase (CGTase). Morphological characteristics, chemical composition, adsorptive capacity and pasting/thermal properties were assessed. Scanning Electron Microscopy (SEM) showed porous structures with diverse pore size distribution, which was dependent on the enzyme type and starch source, but no differences were observed in the total granule surface occupied by pores. The adsorptive capacity analysis revealed that modified starches had high water absorptive capacity and showed different oil adsorptive capacity depending on the enzyme type. Amylose content analysis revealed different hydrolysis pattern of the amylases, suggesting that AMG mainly affected crystalline region meanwhile AM and CGTase attacked amorphous area. A heatmap illustrated the diverse pasting properties of the different porous starches, which also showed significant different thermal properties, with different behavior between cereal and tuber starches. Therefore, it is possible to modulate the properties of starches through the use of different enzymes. Copyright © 2017 Elsevier B.V. All rights reserved.

Reaction Mechanism and Molecular Basis for Selenium/Sulfur Discrimination of Selenocysteine Lyase*

PubMed Central

Omi, Rie; Kurokawa, Suguru; Mihara, Hisaaki; Hayashi, Hideyuki; Goto, Masaru; Miyahara, Ikuko; Kurihara, Tatsuo; Hirotsu, Ken; Esaki, Nobuyoshi

2010-01-01

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5′-phosphate-dependent removal of selenium from l-selenocysteine to yield l-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward l-selenocysteine and no activity to its cognate l-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with l-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with l-cysteine. Cys-375 on the flexible loop directed l-selenocysteine, but not l-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system. PMID:20164179

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach

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

Musumeci, Matias A.; Lozada, Mariana; Rial, Daniela V.

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putativemore » monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. As a result, this work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.« less

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach.

PubMed

Musumeci, Matías A; Lozada, Mariana; Rial, Daniela V; Mac Cormack, Walter P; Jansson, Janet K; Sjöling, Sara; Carroll, JoLynn; Dionisi, Hebe M

2017-04-09

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach

PubMed Central

Musumeci, Matías A.; Lozada, Mariana; Rial, Daniela V.; Mac Cormack, Walter P.; Jansson, Janet K.; Sjöling, Sara; Carroll, JoLynn; Dionisi, Hebe M.

2017-01-01

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer–Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments. PMID:28397770

Assessment of the potential suitability of selected commercially available enzymes for cleaning-in-place (CIP) in the dairy industry.

PubMed

Boyce, Angela; Piterina, Anna V; Walsh, Gary

2010-10-01

The potential suitability of 10 commercial protease and lipase products for cleaning-in-place (CIP) application in the dairy industry was investigated on a laboratory scale. Assessment was based primarily on the ability of the enzymes to remove an experimentally generated milk fouling deposit from stainless steel (SS) panels. Three protease products were identified as being most suitable for this application on the basis of their cleaning performance at 40 °C, which was comparable to that of the commonly used cleaning agent, 1% NaOH at 60 °C. This was judged by quantification of residual organic matter and protein on the SS surface after cleaning and analysis by laser scanning confocal microscopy (LSCM). Enzyme activity was removed/inactivated under conditions simulating those normally undertaken after cleaning (rinsing with water, acid circulation, sanitation). Preliminary process-scale studies strongly suggest that enzyme-based CIP achieves satisfactory cleaning at an industrial scale. Cost analysis indicates that replacing caustic-based cleaning procedures with biodegradable enzymes operating at lower temperatures would be economically viable. Additional potential benefits include decreased energy and water consumption, improved safety, reduced waste generation, greater compatibility with wastewater treatment processes and a reduction in the environmental impact of the cleaning process.

Cellulase assisted synthesis of nano-silver and gold: Application as immobilization matrix for biocatalysis.

PubMed

Mishra, Abhijeet; Sardar, Meryam

2015-01-01

In the present study, we report in vitro synthesis of silver and gold nanoparticles (NPs) using cellulase enzyme in a single step reaction. Synthesized nanoparticles were characterized by UV-VIS spectroscopy, Dynamic Light Spectroscopy (DLS), Transmission Electron Microscopy (TEM), Energy-dispersive X-ray Spectroscopy (EDX), X-ray Diffraction (XRD), Circular Dichroism (CD) and Fourier Transform Infrared Spectroscopy (FTIR). UV-visible studies shows absorption band at 415nm and 520nm for silver and gold NPs respectively due to surface plasmon resonance. Sizes of NPs as shown by TEM are 5-25nm for silver and 5-20nm for gold. XRD peaks confirmed about phase purity and crystallinity of silver and gold NPs. FTIR data shows presence of amide I peak on both the NPs. The cellulase assisted synthesized NPs were further exploited as immobilization matrix for cellulase enzyme. Thermal stability analysis reveals that the immobilized cellulase on synthesized NPs retained 77-80% activity as compared to free enzyme. While reusability data suggests immobilized cellulase can be efficiently used up to sixth cycles with minimum loss of enzyme activity. The secondary structural analysis of cellulase enzyme during the synthesis of NPs and also after immobilization of cellulase on these NPs was carried out by CD spectroscopy. Copyright © 2015 Elsevier B.V. All rights reserved.

A rapid and reliable PCR method for genotyping the ABO blood group.

PubMed

O'Keefe, D S; Dobrovic, A

1993-01-01

The ABO blood group has been used extensively as a marker in population studies, epidemiology, and forensic work. However, until the cloning of the gene, it was not possible to determine the genotype of group A and B individuals without recourse to family studies. We have developed a method to determine the ABO genotype directly from human DNA using multiplex PCR and restriction enzyme analysis. Two PCR fragments spanning positions 258 and 700 of the cDNA sequence are amplified. The site at position 258 allows us to differentiate the O allele from the A and B alleles. The site at position 700 allows us to distinguish the B allele from the A and O alleles. Analysis at the two sites thus allows us to distinguish the three alleles. The multiplex PCR product is digested separately with four enzymes, two for each of the sites. The pair of enzymes for each site cut in a reciprocal fashion. Whereas one enzyme for each site is theoretically sufficient for genotyping, the use of complementary pairs of enzymes prevents the assignment of a false genotype as a result of false negative or partial digestion. This method is fast and reliable, does not rely on probing of blots, and should be widely applicable.

Algorithm for Surface of Translation Attached Radiators (A-STAR). Volume III. Computer Codes.

DTIC Science & Technology

1982-05-01

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

Sweet, Marshall L.; Francisco, Abby; Roberts, Sydney G.

Rea Ventures Group, LLC (Rea Ventures) partnered with Southface Energy Institute (Southface)—a member of the U.S. Department of Energy’s Partnership for Home Innovation Building America research team—to rehabilitate 418 low-income multifamily rental apartments located at 14 properties in Georgia (International Energy Conservation Code Climate Zones 2–4). These 22-year-old units with individual utility meters were arranged in row house or townhouse style. Rehabilitation plans were developed using a process prescribed by the U.S. Department of Agriculture (USDA) Rural Development program, which partially funded the building upgrades. The USDA is responsible for building, upgrading, and subsidizing housing in rural areas nationwide; thismore » housing includes more than 14,000 existing multifamily housing developments. In 2012, more than $100 million in grants and loans were allocated for that purpose.« less

Four-year measurement of methane flux over a temperate forest with a relaxed eddy accumulation method

NASA Astrophysics Data System (ADS)

Sakabe, A.; Kosugi, Y.; Ueyama, M.; Hamotani, K.; Takahashi, K.; Iwata, H.; Itoh, M.

2013-12-01

Forests are generally assumed to be an atmospheric methane (CH4) sink (Le Mer and Roger, 2001). However, under Asian monsoon climate, forests are subject to wide spatiotemporal range in soil water status, where forest soils often became water-saturated condition heterogeneously. In such warm and humid conditions, forests may act as a CH4 source and/or sink with considerable spatiotemporal variations. Micrometeorological methods such as eddy covariance (EC) method continuously measure spatially-representative flux at a canopy scale without artificial disturbance. In this study, we measured CH4 fluxes over a temperate forest during four-year period using a CH4 analyzer based on tunable diode laser spectroscopy detection with a relaxed eddy accumulation (REA) method (Hamotani et al., 1996, 2001). We revealed the amplitude and seasonal variations of canopy-scale CH4 fluxes. The REA method is the attractive alternative to the EC method to measure trace-gas flux because it allows the use of analyzers with an optimal integration time. We also conducted continuous chamber measurements on forest floor to reveal spatial variations in soil CH4 fluxes and its controlling processes. The observations were made in an evergreen coniferous forest in central Japan. The site has a warm temperate monsoon climate with wet summer. Some wetlands were located in riparian zones along streams within the flux footprint area. For the REA method, the sonic anemometer (SAT-550, Kaijo) was mounted on top of the 29-m-tall tower and air was sampled from just below the sonic anemometer to reservoirs according to the direction of vertical wind velocity (w). After accumulating air for 30 minutes, the air in the reservoirs was pulled into a CO2/H2O gas analyzer (LI-840, Li-Cor) and a CH4 analyzer (FMA-200, Los Gatos Research). Before entering the analyzers, the sampled air was dried using a gas dryer (PD-50 T-48; Perma Pure Inc.). The REA flux is obtained from the difference in the mean concentrations of the reservoirs. In the chamber method, automated dynamic-closed chambers were located at three points of water-unsaturated forest floor. Soil CO2 and CH4 fluxes were measured using the same analyzers with the REA method. CH4 fluxes showed seasonal variations at both canopy and plot scales. Based on the chamber measurements, water-unsaturated forest floor mostly consumed CH4 throughout a year. In contrast, canopy-scale CH4 fluxes by the REA method seasonally fluctuated between emission and absorption. The seasonal variation of canopy-scale CH4 fluxes varied at years to years. Every year, no notable emission nor absorption was observed during winter when daily average air temperature was less than about 10°C. In this forest, the canopy-scale CH4 fluxes could be determined by a balance between sources by methanogens and sinks by methanotrophs. Since these two processes were influenced by soil conditions (e.g., soil temperature and soil moisture), canopy-scale CH4 fluxes were influenced by CH4 fluxes from wetlands within the forest, because magnitude of wetland emission was a few order larger than those of absorption. We will discuss the factors of interannual variation of the canopy- and plot-scale CH4 fluxes in terms of precipitation patterns.

Extracellular Enzyme Composition and Functional Characteristics of Aspergillus niger An-76 Induced by Food Processing Byproducts and Based on Integrated Functional Omics.

PubMed

Liu, Lin; Gong, Weili; Sun, Xiaomeng; Chen, Guanjun; Wang, Lushan

2018-02-07

Byproducts of food processing can be utilized for the production of high-value-added enzyme cocktails. In this study, we utilized integrated functional omics technology to analyze composition and functional characteristics of extracellular enzymes produced by Aspergillus niger grown on food processing byproducts. The results showed that oligosaccharides constituted by arabinose, xylose, and glucose in wheat bran were able to efficiently induce the production of extracellular enzymes of A. niger. Compared with other substrates, wheat bran was more effective at inducing the secretion of β-glucosidases from GH1 and GH3 families, as well as >50% of proteases from A1-family aspartic proteases. Compared with proteins induced by single wheat bran or soybean dregs, the protein yield induced by their mixture was doubled, and the time required to reach peak enzyme activity was shortened by 25%. This study provided a technical platform for the complex formulation of various substrates and functional analysis of extracellular enzymes.

Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation.

PubMed

El-Bessoumy, Ashraf A; Sarhan, Mohamed; Mansour, Jehan

2004-07-31

The L-asparaginase (E. C. 3. 5. 1. 1) enzyme was purified to homogeneity from Pseudomonas aeruginosa 50071 cells that were grown on solid-state fermentation. Different purification steps (including ammonium sulfate fractionation followed by separation on Sephadex G-100 gel filtration and CM-Sephadex C50) were applied to the crude culture filtrate to obtain a pure enzyme preparation. The enzyme was purified 106-fold and showed a final specific activity of 1900 IU/mg with a 43% yield. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed it was one peptide chain with M(r) of 160 kDa. A Lineweaver-Burk analysis showed a K(m) value of 0.147 mM and V(max) of 35.7 IU. The enzyme showed maximum activity at pH 9 when incubated at 37 degrees C for 30 min. The amino acid composition of the purified enzyme was also determined.

Microbial dextran-hydrolyzing enzymes: fundamentals and applications.

PubMed

Khalikova, Elvira; Susi, Petri; Korpela, Timo

2005-06-01

Dextran is a chemically and physically complex polymer, breakdown of which is carried out by a variety of endo- and exodextranases. Enzymes in many groups can be classified as dextranases according to function: such enzymes include dextranhydrolases, glucodextranases, exoisomaltohydrolases, exoisomaltotriohydrases, and branched-dextran exo-1,2-alpha-glucosidases. Cycloisomalto-oligosaccharide glucanotransferase does not formally belong to the dextranases even though its side reaction produces hydrolyzed dextrans. A new classification system for glycosylhydrolases and glycosyltransferases, which is based on amino acid sequence similarities, divides the dextranases into five families. However, this classification is still incomplete since sequence information is missing for many of the enzymes that have been biochemically characterized as dextranases. Dextran-degrading enzymes have been isolated from a wide range of microorganisms. The major characteristics of these enzymes, the methods for analyzing their activities and biological roles, analysis of primary sequence data, and three-dimensional structures of dextranases have been dealt with in this review. Dextranases are promising for future use in various scientific and biotechnological applications.

Charged Propargyl-Linked Antifolates Reveal Mechanisms of Antifolate Resistance and Inhibit Trimethoprim-Resistant MRSA Strains Possessing Clinically Relevant Mutations.

PubMed

Reeve, Stephanie M; Scocchera, Eric; Ferreira, Jacob J; G-Dayanandan, Narendran; Keshipeddy, Santosh; Wright, Dennis L; Anderson, Amy C

2016-07-14

Drug-resistant enzymes must balance catalytic function with inhibitor destabilization to provide a fitness advantage. This sensitive balance, often involving very subtle structural changes, must be achieved through a selection process involving a minimal number of eligible point mutations. As part of a program to design propargyl-linked antifolates (PLAs) against trimethoprim-resistant dihydrofolate reductase (DHFR) from Staphylococcus aureus, we have conducted a thorough study of several clinically observed chromosomal mutations in the enzyme at the cellular, biochemical, and structural levels. Through this work, we have identified a promising lead series that displays significantly greater activity against these mutant enzymes and strains than TMP. The best inhibitors have enzyme inhibition and MIC values near or below that of trimethoprim against wild-type S. aureus. Moreover, these studies employ a series of crystal structures of several mutant enzymes bound to the same inhibitor; analysis of the structures reveals a more detailed molecular understanding of drug resistance in this important enzyme.

Purification and characterization of a fibrinolytic enzyme from tempeh bongkrek as an alternative of thrombolytic agents

NASA Astrophysics Data System (ADS)

Sasmita, I. R. A.; Sutrisno, A.; Zubaidah, E.; Wardani, A. K.

2018-03-01

Tempeh is one of Indonesia’s traditional foods that contain fibrinolytic enzymes. Tempeh bongkrek shows very strong activity among various tempeh. The fibrinolytic enzymes of bongkrek tempeh are obtained by steps of purification i.e, ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The fibrinolytic enzymes has been successfully purified with a yield of 4.37%, specific activity of 3,361 U / mg and purification fold of 44.02. SDS PAGE analysis showed that the enzyme was purified in to single band with estimated molecular mass of 75.82 kDa. The purified enzyme has optimum pH of 7 and optimum temperature of 50°C and pH stability between pH 4 - 7 with temperature stability from 30°-50°C. The fibrinolytic activity is increased with addition of CaCl2 but inhibited with CuSO4, phenylmethylsulfonyl fluoride (PMSF), sodium dodecyl sulfate (SDS), and ethylenediaminetetraacetic acid (EDTA).

Transcriptome analysis of carbohydrate metabolism during bulblet formation and development in Lilium davidii var. unicolor.

PubMed

Li, XueYan; Wang, ChunXia; Cheng, JinYun; Zhang, Jing; da Silva, Jaime A Teixeira; Liu, XiaoYu; Duan, Xin; Li, TianLai; Sun, HongMei

2014-12-19

The formation and development of bulblets are crucial to the Lilium genus since these processes are closely related to carbohydrate metabolism, especially to starch and sucrose metabolism. However, little is known about the transcriptional regulation of both processes. To gain insight into carbohydrate-related genes involved in bulblet formation and development, we conducted comparative transcriptome profiling of Lilium davidii var. unicolor bulblets at 0 d, 15 d (bulblets emerged) and 35 d (bulblets formed a basic shape with three or four scales) after scale propagation. Analysis of the transcriptome revealed that a total of 52,901 unigenes with an average sequence size of 630 bp were generated. Based on Clusters of Orthologous Groups (COG) analysis, 8% of the sequences were attributed to carbohydrate transport and metabolism. The results of KEGG pathway enrichment analysis showed that starch and sucrose metabolism constituted the predominant pathway among the three library pairs. The starch content in mother scales and bulblets decreased and increased, respectively, with almost the same trend as sucrose content. Gene expression analysis of the key enzymes in starch and sucrose metabolism suggested that sucrose synthase (SuSy) and invertase (INV), mainly hydrolyzing sucrose, presented higher gene expression in mother scales and bulblets at stages of bulblet appearance and enlargement, while sucrose phosphate synthase (SPS) showed higher expression in bulblets at morphogenesis. The enzymes involved in the starch synthetic direction such as ADPG pyrophosphorylase (AGPase), soluble starch synthase (SSS), starch branching enzyme (SBE) and granule-bound starch synthase (GBSS) showed a decreasing trend in mother scales and higher gene expression in bulblets at bulblet appearance and enlargement stages while the enzyme in the cleavage direction, starch de-branching enzyme (SDBE), showed higher gene expression in mother scales than in bulblets. An extensive transcriptome analysis of three bulblet development stages contributes considerable novel information to our understanding of carbohydrate metabolism-related genes in Lilium at the transcriptional level, and demonstrates the fundamentality of carbohydrate metabolism in bulblet emergence and development at the molecular level. This could facilitate further investigation into the molecular mechanisms underlying these processes in lily and other related species.

Quantitative Enzymatic and Immunologic Histophotometry of Diseased Human Kid-Ney Tissues Using Tv-Camera and Computer Assisted Image Processing Systems.

NASA Astrophysics Data System (ADS)

Heinert, G.; Mondorf, W.

1982-11-01

High speed image processing was used to analyse morphologic and metabolic characteristics of clinically relevant kidney tissue alterations.Qualitative computer-assisted histophotometry was performed to measure alterations in levels of the enzymes alkaline phosphatase (Ap),alanine aminopeptidase (AAP),g-glutamyltranspepti-dase (GGTP) and A-glucuronidase (B-G1) and AAP and GGTP immunologically determined in prepared renal and cancer tissue sections. A "Mioro-Videomat 2" image analysis system with a "Tessovar" macroscope,a computer-assisted "Axiomat" photomicroscope and an "Interactive Image Analysis System (IBAS)" were employed for analysing changes in enzyme activities determined by changes in absorbance or transmission.Diseased kidney as well as renal neoplastic tissues could be distinguished by significantly (wilcoxon test,p<0,05) decreased enzyme concentrations as compared to those found in normal human kidney tissues.This image analysis techniques might be of potential use in diagnostic and prognostic evaluation of renal cancer and diseased kidney tissues.

Kinetics and inhibition of cyclomaltodextrinase from alkalophilic Bacillus sp. I-5.

PubMed

Kim, M J; Park, W S; Lee, H S; Kim, T J; Shin, J H; Yoo, S H; Cheong, T K; Ryu, S; Kim, J C; Kim, J W; Moon, T W; Robyt, J F; Park, K H

2000-01-01

The cyclomaltodextrinase from alkalophilic Bacillus sp. I-5 (CDase I-5) was expressed in Escherichia coli and the purified enzyme was used for characterization of the enzyme action. The hydrolysis products were monitored by both HPLC and high-performance ion chromatography analysis that enable the kinetic analysis of the cyclomaltodextrin (CD)-degrading reaction. Analysis of the kinetics of cyclomaltodextrin hydrolysis by CDase I-5 indicated that ring-opening of the cyclomaltodextrin was the major limiting step and that CDase I-5 preferentially degraded the linear maltodextrin chain by removing the maltose unit. The substrate binding affinity of the enzyme was almost same for those of cyclomaltodextrins while the rate of ring-opening was the fastest for cyclomaltoheptaose. Acarbose and methyl 6-amino-6-deoxy-alpha-d-glucopyranoside were relatively strong competitive inhibitors with K(i) values of 1.24 x 10(-3) and 8.44 x 10(-1) mM, respectively. Both inhibitors are likely to inhibit the ring-opening step of the CD degradation reaction. Copyright 2000 Academic Press.

Enhancing the cellular anti-proliferation activity of pyridazinones as c-met inhibitors using docking analysis.

PubMed

Xing, Weiqiang; Ai, Jing; Jin, Shiyu; Shi, Zhangxing; Peng, Xia; Wang, Lang; Ji, Yinchun; Lu, Dong; Liu, Yang; Geng, Meiyu; Hu, Youhong

2015-05-05

A series of 2, 6-disubstituted pyridazinone derivatives were evaluated and optimized for their c-Met inhibitory activity in enzyme and cellular assay. An analysis of the SAR results arising from computer modeling analysis of members of the library led to the proposal that in order to obtain optimal inhibitory activity in cellular systems the lipophilic/hydrophilic properties of individual structural fragments in the inhibitors need to match those of corresponding binding pockets in the enzyme. Guided by this proposal, the quinoline-pyridazinone 8a, containing hydrophobic 6-indolyl pyridazinone and quinoline moieties along with a hydrophilic morpholine terminal group, was designed and synthesized. The results of studies with this substance showed that it is a selective c-Met inhibitor with both a high enzyme inhibition IC50 value of 4.2 nM and a high EBC-1 cell proliferation inhibition IC50 value of 17 nM. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Impact of distal mutations on the network of coupled motions correlated to hydride transfer in dihydrofolate reductase.

PubMed

Wong, Kim F; Selzer, Tzvia; Benkovic, Stephen J; Hammes-Schiffer, Sharon

2005-05-10

A comprehensive analysis of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase is presented. Hybrid quantum/classical molecular dynamics simulations are combined with a rank correlation analysis method to extract thermally averaged properties that vary along the collective reaction coordinate according to a prescribed target model. Coupled motions correlated to hydride transfer are identified throughout the enzyme. Calculations for wild-type dihydrofolate reductase and a triple mutant, along with the associated single and double mutants, indicate that each enzyme system samples a unique distribution of coupled motions correlated to hydride transfer. These coupled motions provide an explanation for the experimentally measured nonadditivity effects in the hydride transfer rates for these mutants. This analysis illustrates that mutations distal to the active site can introduce nonlocal structural perturbations and significantly impact the catalytic rate by altering the conformational motions of the entire enzyme and the probability of sampling conformations conducive to the catalyzed reaction.

Role of genetic mutations in folate-related enzyme genes on Male Infertility

PubMed Central

Liu, Kang; Zhao, Ruizhe; Shen, Min; Ye, Jiaxin; Li, Xiao; Huang, Yuan; Hua, Lixin; Wang, Zengjun; Li, Jie

2015-01-01

Several studies showed that the genetic mutations in the folate-related enzyme genes might be associated with male infertility; however, the results were still inconsistent. We performed a meta-analysis with trial sequential analysis to investigate the associations between the MTHFR C677T, MTHFR A1298C, MTR A2756G, MTRR A66G mutations and the MTHFR haplotype with the risk of male infertility. Overall, a total of 37 studies were selected. Our meta-analysis showed that the MTHFR C677T mutation was a risk factor for male infertility in both azoospermia and oligoasthenoteratozoospermia patients, especially in Asian population. Men carrying the MTHFR TC haplotype were most liable to suffer infertility while those with CC haplotype had lowest risk. On the other hand, the MTHFR A1298C mutation was not related to male infertility. MTR A2756G and MTRR A66G were potential candidates in the pathogenesis of male infertility, but more case-control studies were required to avoid false-positive outcomes. All of these results were confirmed by the trial sequential analysis. Finally, our meta-analysis with trial sequential analysis proved that the genetic mutations in the folate-related enzyme genes played a significant role in male infertility. PMID:26549413

Lung function, atopy, specific hypersensitivity, and smoking of workers in the enzyme detergent industry over 11 years.

PubMed

Flood, D F; Blofeld, R E; Bruce, C F; Hewitt, J I; Juniper, C P; Roberts, D M

1985-01-01

A study of 2800 workers employed in three factories of the two major manufacturers of enzymatic products in the United Kingdom covering 11 years of operation from 1969 to 1980 showed that 2344 workers had sufficient lung function data to meet the operational criteria and these were analysed in three separate groups by factory locations. Spirometry and prick tests for specific skin reactions to standardised enzyme were performed at six monthly intervals for the first six years of the study and then annually. Factory enzyme dust and total dust measurements were made to determine the degree of dust exposure of the subjects. The lung function of the factory groups was analysed for the effects of working in the detergent industry, the degree of exposure to enzymes, skin prick test positivity to enzymes, atopicity, and smoking. The 4.5% of workers who had experienced respiratory effects from enzymes were analysed separately. Exposure to the enzyme allergen has had no significant long term effect on the lung function of the detergent workers. A higher proportion of atopics than non-atopics became skin test positive to the allergen and more smokers than non-smokers were sensitised. The overall lung function of detergent workers showed 39 ml/year loss in FEV1 on the 11 year longitudinal study and 51 ml/year loss on the lateral (cross sectional) analysis with better lung function in the south east than the north west of England. In the development of the methodology for the study several potential problems were discovered that could remain unrecognised in a cross sectional analysis performed in isolation.

Stereochemical Consequences of Vinylpyruvate Hydratase-Catalyzed Reactions.

PubMed

Johnson, William H; Stack, Tyler M M; Taylor, Stephanie M; Burks, Elizabeth A; Whitman, Christian P

2016-07-26

A stereochemical analysis has been carried out on two vinylpyruvate hydratases (VPH), which convert 2-hydroxy-2,4-pentadienoate to 2-keto-4S-hydroxypentanoate in meta-fission pathways. Bacterial strains with this pathway can use aromatic compounds as sole sources of energy and carbon. The analysis was carried out using the 5-methyl and 5-chloro derivatives of 2-hydroxy-2,4-pentadienoate with the enzymes from Pseudomonas putida mt-2 (Pp) and Leptothrix cholodnii SP-6 (Lc). In both organisms, VPH is in a complex with the preceding enzyme in the pathway, 4-oxalocrotonate decarboxylase (4-OD). In D2O, a deuteron is incorporated stereospecifically at the C-3 and C-5 positions of product by both Pp and Lc enzymes. Accordingly, the complexes generate (3S,5S)-3,5-[di-D]-2-keto-4S-hydroxyhexanoate and (3S,5R)-3,5-[di-D]-2-keto-4R-hydroxy-5-chloropentanoate (4R and 5R due to a priority numbering change). The substitution at C-5 (CH3 or Cl) or the source of the enzyme (Pp or Lc) does not change the stereochemical outcome. One mechanism that can account for the results is the ketonization of the 5-substituted dienol to the α,β-unsaturated ketone (placing a deuteron at C-5 in D2O), followed by the conjugate addition of water (placing a deuteron at C-3). The stereochemical outcome for VPH (from Pp and Lc) is the same as that reported for a related enzyme, 2-oxo-hept-4-ene-1,7-dioate hydratase, from Escherichia coli C. The combined observations suggest similar mechanisms for these three enzymes that could possibly be common to this group of enzymes.

Selection and molecular characterization of cellulolytic-xylanolytic fungi from surface soil-biomass mixtures from Black Belt sites.

PubMed

Okeke, Benedict C; Hall, Rosine W; Nanjundaswamy, Ananda; Thomson, M Sue; Deravi, Yasaman; Sawyer, Leah; Prescott, Andrew

2015-06-01

Plant biomass is an abundant renewable natural resource that can be transformed into chemical feedstocks. Enzymes used in saccharification of lignocellulosic biomass are a major part of biofuel production costs. A cocktail of cellulolytic and xylanolytic enzymes are required for optimal saccharification of biomass. Accordingly, thirty-two fungal pure cultures were obtained from surface soil-biomass mixtures collected from Black Belt sites in Alabama by culturing on lignocellulosic biomass medium. The fungal strains were screened for the coproduction of cellulolytic and xylanolytic enzymes. Strains that displayed promising levels of cellulolytic and xylanolytic enzymes were characterized by molecular analysis of DNA sequences from the large subunit and internal transcribed spacer (ITS) of their ribosomal RNA gene. Nucleotide sequence analysis revealed that two most promising isolates FS22A and FS5A were most similar to Penicillium janthinellum and Trichoderma virens. Production dynamics of cellulolytic and xylanolytic enzymes from these two strains were explored in submerged fermentation. Volumetric productivity after 120 h incubation was 121.08 units/L/h and 348 units/L/h for the filter paper cellulase and xylanase of strain FS22A, and 90.83 units/L/h and 359 units/L/h, respectively for strain FS5A. Assays with 10 times dilution of enzymes revealed that the activities were much higher than that observed in the crude culture supernatant. Additionally, both FS22A and FS5A also produced amylase in lignocellulose medium. The enzyme profiles of these strains and their activities suggest potential applications in cost effective biomass conversion and biodegradation. Copyright © 2015 Elsevier GmbH. All rights reserved.

Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus*

PubMed Central

Forsberg, Zarah; Nelson, Cassandra E.; Dalhus, Bjørn; Mekasha, Sophanit; Loose, Jennifer S. M.; Crouch, Lucy I.; Røhr, Åsmund K.; Gardner, Jeffrey G.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

2016-01-01

Cellvibrio japonicus is a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO, CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of the CjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and β-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show that CjLPMO10A is needed by C. japonicus to obtain efficient growth on both purified chitin and crab shell particles. PMID:26858252

Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus.

PubMed

Forsberg, Zarah; Nelson, Cassandra E; Dalhus, Bjørn; Mekasha, Sophanit; Loose, Jennifer S M; Crouch, Lucy I; Røhr, Åsmund K; Gardner, Jeffrey G; Eijsink, Vincent G H; Vaaje-Kolstad, Gustav

2016-04-01

Cellvibrio japonicusis a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO,CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of theCjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and β-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show thatCjLPMO10A is needed byC. japonicusto obtain efficient growth on both purified chitin and crab shell particles. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Selection and molecular characterization of cellulolytic–xylanolytic fungi from surface soil-biomass mixtures from Black Belt sites

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

Okeke, Benedict C.; Hall, Rosine W.; Nanjundaswamy, Ananda

Plant biomass is an abundant renewable natural resource that can be transformed into chemical feedstocks. Enzymes used in saccharification of lignocellulosic biomass is a major part of biofuel production costs. A cocktail of cellulolytic and xylanolytic enzymes are required for optimal saccharification of biomass. Accordingly, thirty-two fungal pure cultures were obtained from surface soil-biomass mixtures collected from Black Belt sites in Alabama by culturing on lignocellulosic biomass medium. The fungal strains were screened for the coproduction of cellulolytic and xylanolytic enzymes. Strains that displayed promising levels of cellulolytic and xylanolytic enzymes were characterized by molecular analysis of DNA sequences frommore » the large subunit and internal transcribed spacer (ITS) of their ribosomal RNA gene. Nucleotide sequence analysis revealed that two most promising isolates FS22A and FS5A were most similar to Penicillium janthinellum and Trichoderma virens. Production dynamics of cellulolytic and xylanolytic enzymes from these two strains were explored in submerged fermentation. Volumetric productivity after 120h incubation was 121.08 units/L/h and 348 units/L/h for the filter paper cellulase and xylanase of strain FS22A, and 90.83 units/L/h and 359 units/L/h, respectively for strain FS5A. Assays with 10 times dilution of enzymes revealed that the activities were much higher than that observed in the crude culture supernatant. Additionally, both FS22A and FS5A also produced amylase in lignocellulose medium. The enzyme profiles of these strains and their activities suggest potential applications in cost effective biomass conversion and biodegradation.« less

Selection and molecular characterization of cellulolytic–xylanolytic fungi from surface soil-biomass mixtures from Black Belt sites

DOE PAGES

Okeke, Benedict C.; Hall, Rosine W.; Nanjundaswamy, Ananda; ...

2015-03-10

Plant biomass is an abundant renewable natural resource that can be transformed into chemical feedstocks. Enzymes used in saccharification of lignocellulosic biomass is a major part of biofuel production costs. A cocktail of cellulolytic and xylanolytic enzymes are required for optimal saccharification of biomass. Accordingly, thirty-two fungal pure cultures were obtained from surface soil-biomass mixtures collected from Black Belt sites in Alabama by culturing on lignocellulosic biomass medium. The fungal strains were screened for the coproduction of cellulolytic and xylanolytic enzymes. Strains that displayed promising levels of cellulolytic and xylanolytic enzymes were characterized by molecular analysis of DNA sequences frommore » the large subunit and internal transcribed spacer (ITS) of their ribosomal RNA gene. Nucleotide sequence analysis revealed that two most promising isolates FS22A and FS5A were most similar to Penicillium janthinellum and Trichoderma virens. Production dynamics of cellulolytic and xylanolytic enzymes from these two strains were explored in submerged fermentation. Volumetric productivity after 120h incubation was 121.08 units/L/h and 348 units/L/h for the filter paper cellulase and xylanase of strain FS22A, and 90.83 units/L/h and 359 units/L/h, respectively for strain FS5A. Assays with 10 times dilution of enzymes revealed that the activities were much higher than that observed in the crude culture supernatant. Additionally, both FS22A and FS5A also produced amylase in lignocellulose medium. The enzyme profiles of these strains and their activities suggest potential applications in cost effective biomass conversion and biodegradation.« less

Investigation of the binding properties of a multi-modular GH45 cellulase using bioinspired model assemblies.

PubMed

Fong, Monica; Berrin, Jean-Guy; Paës, Gabriel

2016-01-01

Enzymes degrading plant biomass polymers are widely used in biotechnological applications. Their efficiency can be limited by non-specific interactions occurring with some chemical motifs. In particular, the lignin component is known to bind enzymes irreversibly. In order to determine interactions of enzymes with their substrates, experiments are usually performed on isolated simple polymers which are not representative of plant cell wall complexity. But when using natural plant substrates, the role of individual chemical and structural features affecting enzyme-binding properties is also difficult to decipher. We have designed and used lignified model assemblies of plant cell walls as templates to characterize binding properties of multi-modular cellulases. These three-dimensional assemblies are modulated in their composition using the three principal polymers found in secondary plant cell walls (cellulose, hemicellulose, and lignin). Binding properties of enzymes are obtained from the measurement of their mobility that depends on their interactions with the polymers and chemical motifs of the assemblies. The affinity of the multi-modular GH45 cellulase was characterized using a statistical analysis to determine the role played by each assembly polymer. Presence of hemicellulose had much less impact on affinity than cellulose and model lignin. Depending on the number of CBMs appended to the cellulase catalytic core, binding properties toward cellulose and lignin were highly contrasted. Model assemblies bring new insights into the molecular determinants that are responsible for interactions between enzymes and substrate without the need of complex analysis. Consequently, we believe that model bioinspired assemblies will provide relevant information for the design and optimization of enzyme cocktails in the context of biorefineries.

Purification and characterization of a protease produced by Bacillus megaterium RRM2: application in detergent and dehairing industries.

PubMed

Rajkumar, Renganathan; Jayappriyan, Kothilmozhian Ranishree; Rengasamy, Ramasamy

2011-12-01

An alkaline serine protease produced by Bacillus megaterium RRM2 isolated from the red alga, Kappaphycus alvarezii (Doty) Doty ex Silva was studied for the first time and the same analyzed for the production of protease in the present study. Identification of the bacterium was done on the basis of both biochemical analysis and by 16S rDNA sequence analysis. The extracellular protease obtained from B. megaterium RRM2 was purified by a three-step process involving ammonium sulphate precipitation, gel filtration (Sephadex G100) and Q-Sepharose column chromatography. The purity was found to be 30.6-fold with a specific activity of 3591.5 U/mg protein with a molecular weight of 27 kDa. The metal ions Ca(2+), Mg(2+), K(+) and Na(+) marginally enhanced the activity of the purified enzyme while Hg(2+), Cu(2+), Fe(2+), CO(2+) and Zn(2+), had reduced the activity. The enzyme was found to be active in the pH range of 9.0-10.0 and remained active up to 60 °C. Phenyl Methyl Sulfonyl Fluoride (PMSF) inhibited the enzyme activity, thus, confirming that this enzyme is an alkaline serine protease. Likewise, DTT also inhibited the enzyme thus confirming the disulfide nature of the enzyme. The enzyme exhibited a high degree of tolerance to Sodium Dodecyl Sulphate (SDS). The partially purified protease when used as an additive in the commercial detergents was found to be a suitable source for washing clothes especially those stained with blood. Further, it showed good dehairing activity within a short duration in goat skin without affecting its collagen component. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of drug binding pockets and repurposing opportunities for twelve essential enzymes of ESKAPE pathogens

PubMed Central

Naz, Sadia; Ngo, Tony; Farooq, Umar

2017-01-01

Background The rapid increase in antibiotic resistance by various bacterial pathogens underlies the significance of developing new therapies and exploring different drug targets. A fraction of bacterial pathogens abbreviated as ESKAPE by the European Center for Disease Prevention and Control have been considered a major threat due to the rise in nosocomial infections. Here, we compared putative drug binding pockets of twelve essential and mostly conserved metabolic enzymes in numerous bacterial pathogens including those of the ESKAPE group and Mycobacterium tuberculosis. The comparative analysis will provide guidelines for the likelihood of transferability of the inhibitors from one species to another. Methods Nine bacterial species including six ESKAPE pathogens, Mycobacterium tuberculosis along with Mycobacterium smegmatis and Eschershia coli, two non-pathogenic bacteria, have been selected for drug binding pocket analysis of twelve essential enzymes. The amino acid sequences were obtained from Uniprot, aligned using ICM v3.8-4a and matched against the Pocketome encyclopedia. We used known co-crystal structures of selected target enzyme orthologs to evaluate the location of their active sites and binding pockets and to calculate a matrix of pairwise sequence identities across each target enzyme across the different species. This was used to generate sequence maps. Results High sequence identity of enzyme binding pockets, derived from experimentally determined co-crystallized structures, was observed among various species. Comparison at both full sequence level and for drug binding pockets of key metabolic enzymes showed that binding pockets are highly conserved (sequence similarity up to 100%) among various ESKAPE pathogens as well as Mycobacterium tuberculosis. Enzymes orthologs having conserved binding sites may have potential to interact with inhibitors in similar way and might be helpful for design of similar class of inhibitors for a particular species. The derived pocket alignments and distance-based maps provide guidelines for drug discovery and repurposing. In addition they also provide recommendations for the relevant model bacteria that may be used for initial drug testing. Discussion Comparing ligand binding sites through sequence identity calculation could be an effective approach to identify conserved orthologs as drug binding pockets have shown higher level of conservation among various species. By using this approach we could avoid the problems associated with full sequence comparison. We identified essential metabolic enzymes among ESKAPE pathogens that share high sequence identity in their putative drug binding pockets (up to 100%), of which known inhibitors can potentially antagonize these identical pockets in the various species in a similar manner. PMID:28948099

Analysis of drug binding pockets and repurposing opportunities for twelve essential enzymes of ESKAPE pathogens.

PubMed

Naz, Sadia; Ngo, Tony; Farooq, Umar; Abagyan, Ruben

2017-01-01

The rapid increase in antibiotic resistance by various bacterial pathogens underlies the significance of developing new therapies and exploring different drug targets. A fraction of bacterial pathogens abbreviated as ESKAPE by the European Center for Disease Prevention and Control have been considered a major threat due to the rise in nosocomial infections. Here, we compared putative drug binding pockets of twelve essential and mostly conserved metabolic enzymes in numerous bacterial pathogens including those of the ESKAPE group and Mycobacterium tuberculosis . The comparative analysis will provide guidelines for the likelihood of transferability of the inhibitors from one species to another. Nine bacterial species including six ESKAPE pathogens, Mycobacterium tuberculosis along with Mycobacterium smegmatis and Eschershia coli , two non-pathogenic bacteria, have been selected for drug binding pocket analysis of twelve essential enzymes. The amino acid sequences were obtained from Uniprot, aligned using ICM v3.8-4a and matched against the Pocketome encyclopedia. We used known co-crystal structures of selected target enzyme orthologs to evaluate the location of their active sites and binding pockets and to calculate a matrix of pairwise sequence identities across each target enzyme across the different species. This was used to generate sequence maps. High sequence identity of enzyme binding pockets, derived from experimentally determined co-crystallized structures, was observed among various species. Comparison at both full sequence level and for drug binding pockets of key metabolic enzymes showed that binding pockets are highly conserved (sequence similarity up to 100%) among various ESKAPE pathogens as well as Mycobacterium tuberculosis . Enzymes orthologs having conserved binding sites may have potential to interact with inhibitors in similar way and might be helpful for design of similar class of inhibitors for a particular species. The derived pocket alignments and distance-based maps provide guidelines for drug discovery and repurposing. In addition they also provide recommendations for the relevant model bacteria that may be used for initial drug testing. Comparing ligand binding sites through sequence identity calculation could be an effective approach to identify conserved orthologs as drug binding pockets have shown higher level of conservation among various species. By using this approach we could avoid the problems associated with full sequence comparison. We identified essential metabolic enzymes among ESKAPE pathogens that share high sequence identity in their putative drug binding pockets (up to 100%), of which known inhibitors can potentially antagonize these identical pockets in the various species in a similar manner.

iSCHRUNK--In Silico Approach to Characterization and Reduction of Uncertainty in the Kinetic Models of Genome-scale Metabolic Networks.

PubMed

Andreozzi, Stefano; Miskovic, Ljubisa; Hatzimanikatis, Vassily

2016-01-01

Accurate determination of physiological states of cellular metabolism requires detailed information about metabolic fluxes, metabolite concentrations and distribution of enzyme states. Integration of fluxomics and metabolomics data, and thermodynamics-based metabolic flux analysis contribute to improved understanding of steady-state properties of metabolism. However, knowledge about kinetics and enzyme activities though essential for quantitative understanding of metabolic dynamics remains scarce and involves uncertainty. Here, we present a computational methodology that allow us to determine and quantify the kinetic parameters that correspond to a certain physiology as it is described by a given metabolic flux profile and a given metabolite concentration vector. Though we initially determine kinetic parameters that involve a high degree of uncertainty, through the use of kinetic modeling and machine learning principles we are able to obtain more accurate ranges of kinetic parameters, and hence we are able to reduce the uncertainty in the model analysis. We computed the distribution of kinetic parameters for glucose-fed E. coli producing 1,4-butanediol and we discovered that the observed physiological state corresponds to a narrow range of kinetic parameters of only a few enzymes, whereas the kinetic parameters of other enzymes can vary widely. Furthermore, this analysis suggests which are the enzymes that should be manipulated in order to engineer the reference state of the cell in a desired way. The proposed approach also sets up the foundations of a novel type of approaches for efficient, non-asymptotic, uniform sampling of solution spaces. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Dipeptidyl peptidase-II from probiotic Pediococcus acidilactici: Purification and functional characterization.

PubMed

Gandhi, Dimpi; Chanalia, Preeti; Attri, Pooja; Dhanda, Suman

2016-12-01

Dipeptidylpeptidase-II (DPP-II, E.C. 3.4.14.2), an exopeptidase was purified 15.4 fold with specific activity and yield of 15.4U/mg/mL and 14.68% respectively by a simple two step procedure from a probiotic Pediococcus acidilactici. DPP-II is 38.7KDa homodimeric serine peptidase with involvement of His and subunit mass of 18.9KDa. The enzyme exhibited optimal activity at pH 7.0 and 37°C with activation energy of 24.97kJ/mol. The enzyme retained more than 90% activity upto 50°C thus adding industrial importance. DPP-II hydrolysed Lys-Ala-4mβNA with K M of 50μM and V max of 30.8nmol/mL/min. In-silico characterization studies of DPP-II on the basis of peptide fragments obtained by MALDI-TOF revealed an evolutionary relationship between DPP-II of prokaryotes and phosphate binding proteins. Secondary and three-dimensional structure of enzyme was also deduced by in-silico approach. Functional studies of DPP-II by TLC and HPLC-analysis of collagen degraded products revealed that enzyme action released free amino acids and other metabolites. Microscopic and SDS-PAGE analysis of enzyme treated analysis of chicken's chest muscle (meat) hydrolysis revealed change and hydrolysis of myofibrils. This may affect the flavor and texture of meat thereby suggesting its role in meat tenderization. Being a protein of LAB (Lactic acid bacteria), it is also expected to be safe. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular docking studies to map the binding site of squalene synthase inhibitors on dehydrosqualene synthase of Staphylococcus aureus.

PubMed

Kahlon, Amandeep Kaur; Roy, Sudeep; Sharma, Ashok

2010-10-01

Dehydrosqualene synthase of Staphylococcus aureus is involved in the synthesis of golden carotenoid pigment staphyloxanthin. This pigment of S. aureus provides the antioxidant property to this bacterium to survive inside the host cell. Dehydrosqualene synthase (CrtM) is having structural similarity with the human squalene synthase enzyme which is involved in the cholesterol synthesis pathway in humans (Liu et al., 2008). Cholesterol lowering drugs were found to have inhibitory effect on dehydrosqualene synthase enzyme of S. aureus. The present study attempts to focus on squalene synthase inhibitors, lapaquistat acetate and squalestatins reported as cholesterol lowering agents in vitro and in vivo but not studied in context to dehydrosqualene synthase of S. aureus. Mode of binding of lapaquistat acetate and squalestatin analogs on dehydrosqualene synthase (CrtM) enzyme of S. aureus was identified by performing docking analysis with Scigress Explorer Ultra 7.7 docking software. Based on the molecular docking analysis, it was found that the His18, Arg45, Asp48, Asp52, Tyr129, Gln165, Asn168 and Asp172 residues interacted with comparatively high frequency with the inhibitors studied. Comparative docking study with Discovery studio 2.0 also confirmed the involvement of these residues of dehydrosqualene synthase enzyme with the inhibitors studied. This further confirms the importance of these residues in the enzyme function. In silico ADMET analysis was done to predict the ADMET properties of the standard drugs and test compounds. This might provide insights to develop new drugs to target the virulence factor, dehydrosqualene synthase of S. aureus.

Molecular basis of HHQ biosynthesis: molecular dynamics simulations, enzyme kinetic and surface plasmon resonance studies

PubMed Central

2013-01-01

Background PQS (PseudomonasQuinolone Signal) and its precursor HHQ are signal molecules of the P. aeruginosa quorum sensing system. They explicate their role in mammalian pathogenicity by binding to the receptor PqsR that induces virulence factor production and biofilm formation. The enzyme PqsD catalyses the biosynthesis of HHQ. Results Enzyme kinetic analysis and surface plasmon resonance (SPR) biosensor experiments were used to determine mechanism and substrate order of the biosynthesis. Comparative analysis led to the identification of domains involved in functionality of PqsD. A kinetic cycle was set up and molecular dynamics (MD) simulations were used to study the molecular bases of the kinetics of PqsD. Trajectory analysis, pocket volume measurements, binding energy estimations and decompositions ensured insights into the binding mode of the substrates anthraniloyl-CoA and β-ketodecanoic acid. Conclusions Enzyme kinetics and SPR experiments hint at a ping-pong mechanism for PqsD with ACoA as first substrate. Trajectory analysis of different PqsD complexes evidenced ligand-dependent induced-fit motions affecting the modified ACoA funnel access to the exposure of a secondary channel. A tunnel-network is formed in which Ser317 plays an important role by binding to both substrates. Mutagenesis experiments resulting in the inactive S317F mutant confirmed the importance of this residue. Two binding modes for β-ketodecanoic acid were identified with distinct catalytic mechanism preferences. PMID:23916145

76 FR 4281 - Recreation Resource Advisory Committees Charter Reestablishment

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-25

..., 2010. FOR FURTHER INFORMATION CONTACT: Julie Cox, National Recreation RAC Coordinator, USDA Forest... the purposes stated in REA. Dated: December 13, 2010. Pearlie S. Reed, Assistant Secretary for...

Diversity and Strain Specificity of Plant Cell Wall Degrading Enzymes Revealed by the Draft Genome of Ruminococcus flavefaciens FD-1

PubMed Central

Berg Miller, Margret E.; Antonopoulos, Dionysios A.; Rincon, Marco T.; Band, Mark; Bari, Albert; Akraiko, Tatsiana; Hernandez, Alvaro; Thimmapuram, Jyothi; Henrissat, Bernard; Coutinho, Pedro M.; Borovok, Ilya; Jindou, Sadanari; Lamed, Raphael; Flint, Harry J.; Bayer, Edward A.; White, Bryan A.

2009-01-01

Background Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. Methodology/Principal Findings The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. Conclusions/Significance The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components. PMID:19680555

An Optimized Microplate Assay System for Quantitative Evaluation of Plant Cell Wall Degrading Enzyme Activity of Fungal Culture Extracts

USDA-ARS?s Scientific Manuscript database

Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospe...

Simulated Analysis of Linear Reversible Enzyme Inhibition with SCILAB

ERIC Educational Resources Information Center

Antuch, Manuel; Ramos, Yaquelin; Álvarez, Rubén

2014-01-01

SCILAB is a lesser-known program (than MATLAB) for numeric simulations and has the advantage of being free software. A challenging software-based activity to analyze the most common linear reversible inhibition types with SCILAB is described. Students establish typical values for the concentration of enzyme, substrate, and inhibitor to simulate…

Characterization of the Impact of Life Stage on Xenobiotic Metabolizing Enzyme Expression and Gene -Chemical Interactions in the Liver

EPA Science Inventory

Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). We have carried out a comprehensive analysis of the mRNA expression of XMETs thro...

Appearance and accumulation of C/sub 4/ carbon pathway enzymes in developing maize leaves and differentiating maize A188 callus

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

Aoyagi, K.; Bassham, J.A.

1986-02-01

Regenerating maize A188 tissue cultures were examined for the presence of enzymes involved in C/sub 4/ photosynthesis, for cell morphology, and for /sup 14/C labeling kinetics to study the implementation of this pathway during plant development. For comparison, sections of maize seedling leaves were examined. Protein blot analysis using antibodies to leaf enzymes showed a different profile of these enzymes during the early stages of shoot regeneration from callus from the closely-coordinated profile observed in seedling leaves. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) and phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) were found in nonchlorophyllous callus while ribulose 1,5-bisphosphate carboxylase (RuBPC, ECmore » 4.1.1.39) and malic enzyme, NADP-specific (ME-NADP) (EC 1.3.1.37) were not detectable until later. Enzyme activity assays showed the presence of ME-NADP as well as PEPC and PPDK in nonchlorophyllous callus. However, the activities of ME-NADP and PEPC had properties similar to those of the enzymes from C/sub 3/ leaves and from etiolated C/sub 4/ leaf tissues, but differing from the corresponding enzymes in the mature leaf. Immunoprecipitation of in vitro translation products of poly(A)RNA extracted from embryoid-forming callus showed both the 110 kilodalton precursor to chloroplast PPDK and the 94 kilodalton polypeptide. Therefore, the chloroplast tye of PPDK mRNA is present prior to the appearance of leaf morphology. Analysis of the labeled products of /sup 14/CO/sub 2/ fixation by nonchlorophyllous calli indicated ..beta..-carboxylation to give acids of the tricarboxylic acid cycle, but no incorporation into phosphoglycerate. With greening of the callus, some incorporation into phosphoglycerate and sugar phosphates occurred, and this increased in shoots as they developed, although with older shoots the increase in ..beta..-carboxylation products was even greater.« less

Metatranscriptomic analysis of lignocellulolytic microbial communities involved in high-solids decomposition of rice straw

DOE PAGES

Simmons, Christopher W.; Reddy, Amitha P.; D’haeseleer, Patrik; ...

2014-12-31

New lignocellulolytic enzymes are needed that maintain optimal activity under the harsh conditions present during industrial enzymatic deconstruction of biomass, including high temperatures, the absence of free water, and the presence of inhibitors from the biomass. Enriching lignocellulolytic microbial communities under these conditions provides a source of microorganisms that may yield robust lignocellulolytic enzymes tolerant to the extreme conditions needed to improve the throughput and efficiency of biomass enzymatic deconstruction. Identification of promising enzymes from these systems is challenging due to complex substrate-enzyme interactions and requirements to assay for activity. In this study, metatranscriptomes from compost-derived microbial communities enriched onmore » rice straw under thermophilic and mesophilic conditions were sequenced and analyzed to identify lignocellulolytic enzymes overexpressed under thermophilic conditions. To determine differential gene expression across mesophilic and thermophilic treatments, a method was developed which pooled gene expression by functional category, as indicated by Pfam annotations, since microbial communities performing similar tasks are likely to have overlapping functions even if they share no specific genes. Differential expression analysis identified enzymes from glycoside hydrolase family 48, carbohydrate binding module family 2, and carbohydrate binding module family 33 domains as significantly overexpressed in the thermophilic community. Overexpression of these protein families in the thermophilic community resulted from expression of a small number of genes not currently represented in any protein database. Genes in overexpressed protein families were predominantly expressed by a single Actinobacteria genus, Micromonospora. In conclusion, coupling measurements of deconstructive activity with comparative analyses to identify overexpressed enzymes in lignocellulolytic communities provides a targeted approach for discovery of candidate enzymes for more efficient biomass deconstruction. Furthermore, glycoside hydrolase family 48 cellulases and carbohydrate binding module family 33 polysaccharide monooxygenases with carbohydrate binding module family 2 domains may improve saccharification of lignocellulosic biomass under high-temperature and low moisture conditions relevant to industrial biofuel production.« less

Magnetic Fe3O4@MCM-41 core-shell nanoparticles functionalized with thiol silane for efficient l-asparaginase immobilization.

PubMed

Ulu, Ahmet; Noma, Samir Abbas Ali; Koytepe, Suleyman; Ates, Burhan

2018-06-06

l-Asparaginase (l-ASNase) is a vital enzyme for medical treatment and food industry. Here, we assessed the use of Fe 3 O 4 @Mobil Composition of Matter No. 41 (MCM-41) magnetic nanoparticles as carrier matrix for l-ASNase immobilization. In addition, surface of Fe 3 O 4 @MCM-41 magnetic nanoparticles was functionalized with 3-mercaptopropyltrimethoxysilane (MPTMS) to enhance stability of l-ASNase. The chemical structure, thermal properties, magnetic profile and morphology of the thiol-functionalized Fe 3 O 4 @MCM-41 magnetic nanoparticles were characterized with Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential thermal analysis (DTA), differential scanning calorimetry (DSC), vibrating sample magnetometer (VSM), scanning electron microscope (SEM), energy dispersive X-ray (EDX) spectroscopy and zeta-potential measurement. l-ASNase was covalently immobilized onto the thiol-functionalized Fe 3 O 4 @MCM-41 magnetic nanoparticles. The properties of the immobilized enzyme, including optimum pH, temperature, kinetic parameters, thermal stability, reusability and storage stability were investigated and compared to free one. Immobilized enzyme was found to be stable over a wide range of pH and temperature range than free enzyme. The immobilized l-ASNase also showed higher thermal stability after 180 min incubation at 50 °C. The immobilized enzyme still retained 63% of its original activity after 16 times of reuse. The Km value for the immobilized enzyme was 1.15-fold lower than the free enzyme, which indicates increased affinity for the substrate. Additionally, the immobilized enzyme was active over 65% and 53% after 30 days of storage at 4 °C and room temperature (∼25 °C), respectively. Thereby, the results confirmed that thiol-functionalized Fe 3 O 4 @MCM-41 magnetic nanoparticles had high efficiency for l-ASNase immobilization and improved stability of L-ASNase.

Metatranscriptomic analysis of lignocellulolytic microbial communities involved in high-solids decomposition of rice straw

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

Simmons, Christopher W.; Reddy, Amitha P.; D’haeseleer, Patrik

New lignocellulolytic enzymes are needed that maintain optimal activity under the harsh conditions present during industrial enzymatic deconstruction of biomass, including high temperatures, the absence of free water, and the presence of inhibitors from the biomass. Enriching lignocellulolytic microbial communities under these conditions provides a source of microorganisms that may yield robust lignocellulolytic enzymes tolerant to the extreme conditions needed to improve the throughput and efficiency of biomass enzymatic deconstruction. Identification of promising enzymes from these systems is challenging due to complex substrate-enzyme interactions and requirements to assay for activity. In this study, metatranscriptomes from compost-derived microbial communities enriched onmore » rice straw under thermophilic and mesophilic conditions were sequenced and analyzed to identify lignocellulolytic enzymes overexpressed under thermophilic conditions. To determine differential gene expression across mesophilic and thermophilic treatments, a method was developed which pooled gene expression by functional category, as indicated by Pfam annotations, since microbial communities performing similar tasks are likely to have overlapping functions even if they share no specific genes. Differential expression analysis identified enzymes from glycoside hydrolase family 48, carbohydrate binding module family 2, and carbohydrate binding module family 33 domains as significantly overexpressed in the thermophilic community. Overexpression of these protein families in the thermophilic community resulted from expression of a small number of genes not currently represented in any protein database. Genes in overexpressed protein families were predominantly expressed by a single Actinobacteria genus, Micromonospora. In conclusion, coupling measurements of deconstructive activity with comparative analyses to identify overexpressed enzymes in lignocellulolytic communities provides a targeted approach for discovery of candidate enzymes for more efficient biomass deconstruction. Furthermore, glycoside hydrolase family 48 cellulases and carbohydrate binding module family 33 polysaccharide monooxygenases with carbohydrate binding module family 2 domains may improve saccharification of lignocellulosic biomass under high-temperature and low moisture conditions relevant to industrial biofuel production.« less

Assessing directed evolution methods for the generation of biosynthetic enzymes with potential in drug biosynthesis

PubMed Central

Nannemann, David P; Birmingham, William R; Scism, Robert A; Bachmann, Brian O

2011-01-01

To address the synthesis of increasingly structurally diverse small-molecule drugs, methods for the generation of efficient and selective biological catalysts are becoming increasingly important. ‘Directed evolution’ is an umbrella term referring to a variety of methods for improving or altering the function of enzymes using a nature-inspired twofold strategy of mutagenesis followed by selection. This article provides an objective assessment of the effectiveness of directed evolution campaigns in generating enzymes with improved catalytic parameters for new substrates from the last decade, excluding studies that aimed to select for only improved physical properties and those that lack kinetic characterization. An analysis of the trends of methodologies and their success rates from 81 qualifying examples in the literature reveals the average fold improvement for kcat (or Vmax), Km and kcat/Km to be 366-, 12- and 2548-fold, respectively, whereas the median fold improvements are 5.4, 3 and 15.6. Further analysis by enzyme class, library-generation methodology and screening methodology explores relationships between successful campaigns and the methodologies employed. PMID:21644826

Enzyme-directed assembly of nanoparticles in tumors monitored by in vivo whole animal imaging and ex vivo super-resolution fluorescence imaging.

PubMed

Chien, Miao-Ping; Carlini, Andrea S; Hu, Dehong; Barback, Christopher V; Rush, Anthony M; Hall, David J; Orr, Galya; Gianneschi, Nathan C

2013-12-18

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO/DMF to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micrometer-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super-resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.

A novel, extremely alkaliphilic and cold-active esterase from Antarctic desert soil.

PubMed

Hu, Xiao Ping; Heath, Caroline; Taylor, Mark Paul; Tuffin, Marla; Cowan, Don

2012-01-01

A novel, cold-active and highly alkaliphilic esterase was isolated from an Antarctic desert soil metagenomic library by functional screening. The 1,044 bp gene sequence contained several conserved regions common to lipases/esterases, but lacked clear classification based on sequence analysis alone. Moderate (<40%) amino acid sequence similarity to known esterases was apparent (the closest neighbour being a hypothetical protein from Chitinophaga pinensis), despite phylogenetic distance to many of the lipolytic "families". The enzyme functionally demonstrated activity towards shorter chain p-nitrophenyl esters with the optimal activity recorded towards p-nitrophenyl propionate (C3). The enzyme possessed an apparent T(opt) at 20°C and a pH optimum at pH 11. Esterases possessing such extreme alkaliphily are rare and so this enzyme represents an intriguing novel locus in protein sequence space. A metagenomic approach has been shown, in this case, to yield an enzyme with quite different sequential/structural properties to known lipases. It serves as an excellent candidate for analysis of the molecular mechanisms responsible for both cold and alkaline activity and novel structure-function relationships of esterase activity.

Design Strategy of Multi-electron Transfer Catalysts Based on a Bioinformatic Analysis of Oxygen Evolution and Reduction Enzymes.

PubMed

Ooka, Hideshi; Hashimoto, Kazuhito; Nakamura, Ryuhei

2018-05-14

Understanding the design strategy of photosynthetic and respiratory enzymes is important to develop efficient artificial catalysts for oxygen evolution and reduction reactions. Here, based on a bioinformatic analysis of cyanobacterial oxygen evolution and reduction enzymes (photosystem II: PS II and cytochrome c oxidase: COX, respectively), the gene encoding the catalytic D1 subunit of PS II was found to be expressed individually across 38 phylogenetically diverse strains, which is in contrast to the operon structure of the genes encoding major COX subunits. Selective synthesis of the D1 subunit minimizes the repair cost of PS II, which allows compensation for its instability by lowering the turnover number required to generate a net positive energy yield. The different bioenergetics observed between PS II and COX suggest that in addition to the catalytic activity rationalized by the Sabatier principle, stability factors have also provided a major influence on the design strategy of biological multi-electron transfer enzymes. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A preliminary crystallographic analysis of the putative mevalonate diphosphate decarboxylase from Trypanosoma brucei

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

Byres, Emma; Martin, David M. A.; Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk

2005-06-01

The gene encoding the putative mevalonate diphosphate decarboxylase, an enzyme from the mevalonate pathway of isoprenoid precursor biosynthesis, has been cloned from T. brucei. Recombinant protein has been expressed, purified and highly ordered crystals obtained and characterized to aid the structure–function analysis of this enzyme. Mevalonate diphosphate decarboxylase catalyses the last and least well characterized step in the mevalonate pathway for the biosynthesis of isopentenyl pyrophosphate, an isoprenoid precursor. A gene predicted to encode the enzyme from Trypanosoma brucei has been cloned, a highly efficient expression system established and a purification protocol determined. The enzyme gives monoclinic crystals in spacemore » group P2{sub 1}, with unit-cell parameters a = 51.5, b = 168.7, c = 54.9 Å, β = 118.8°. A Matthews coefficient V{sub M} of 2.5 Å{sup 3} Da{sup −1} corresponds to two monomers, each approximately 42 kDa (385 residues), in the asymmetric unit with 50% solvent content. These crystals are well ordered and data to high resolution have been recorded using synchrotron radiation.« less

Alteration of gene expression by restriction enzymes electroporated into plant cells.

PubMed

Ashraf, M; Altschuler, M; Galasinski, S; Griffiths, T D

1993-06-01

The alteration in the expression of a beta-glucuronidase (GUS) reporter gene was used to monitor the effect of restriction endonucleases electroporated into the tobacco (Nicotiana tabacum L.) protoplasts. Restriction enzyme (RE) Hind III which does not have a recognition site within the gene cassette, had little effect on enzyme activity. In contrast restriction endonucleases Hae III and Sau3A1 which possess 8 and 16 recognition sites in the GUS cassette, were found to reduce the enzyme activity by 89% and 94% respectively when compared to control electroporations. Restriction-site mutation analysis (RSM) and Southern blot analysis indicated the enzymatic degradation of GUS coding sequence by the REs Hae III and Sau3A1. Results of this study suggest that on electroporation, REs can enter into plant cells and alter the expression of the GUS gene. The alteration of gene expression is thus correlated with the digestion of GUS template DNA. Future applications of this technique could include addressing fundamental questions with regard to DNA repair, site-specific recombination, identifying mutations, insertional mutagenesis, enhancement of stable transformation and gene tagging in plants.

Combination analysis in genetic polymorphisms of drug-metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population.

PubMed

Ota, Tomoko; Kamada, Yuka; Hayashida, Mariko; Iwao-Koizumi, Kyoko; Murata, Shigenori; Kinoshita, Kenji

2015-01-01

The Cytochrome P450 is the major enzyme involved in drug metabolism. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is one important factor that contributes to drug therapy failure. We have developed a new straightforward TaqMan PCR genotyping assay to investigate the prevalence of the most common allelic variants of polymorphic CYP enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population. Moreover, we focused on the combination of each genotype for clinical treatment. The genotype analysis identified a total of 139 out of 483 genotype combinations of five genes in the 1,003 Japanese subjects. According to our results, most of subjects seemed to require dose modification during clinical treatment. In the near future, modifications should be considered based on the individual patient genotype of each treatment.

[Biodiversity and enzymes of culturable facultative-alkaliphilic actinobacteria in saline-alkaline soil in Fukang, Xinjiang].

PubMed

Zhang, Yongguang; Liu, Qing; Wang, Hongfei; Zhang, Daofeng; Chen, Jiyue; Zhang, Yuanming; Li, Wenjun

2014-02-04

In order to analyze the biodiversity of cultivable facultative-alkaliphilic actinobacteria and the enzymes they produced. Total 10 soil samples were collected from saline-alkaline environments of Fukang, Xinjiang province. Facultative-alkaliphilic actinobacteria strains were isolated and identified by 16S rRNA gene sequence analysis. Enzymes including amylase, proteinase, xylanase, and cellulase were detected. Total 116 facultative-alkaliphilic actinobacterial strains and 4 alkali-tolerant actinobacterial strains were isolated from the samples, and those strains were distributed within 22 genera in 13 families and 8 orders of actinobacteria based on their 16S rRNA gene sequence analysis. The ratio of non-predominant Streptomyces and Nocardiopsis strains were 53.3%. The positive rates of amylase, proteinase, xylanase and cellulase were 35.8, 37.6, 28.3 and 17.5%, respectively. Diverse facultative-alkaliphilic actinobacteria were discovered from saline-alkaline environments of Fukang. Facultative-alkaliphilic actinobacteria are a potential source for enzymes. The study would facilitate the knowledge of the diversity of facultative-alkaliphilic actinobacteria, and provide the technical basis for exploration of facultative-alkaliphilic actinobacteria resources.

SACCHARIS: an automated pipeline to streamline discovery of carbohydrate active enzyme activities within polyspecific families and de novo sequence datasets.

PubMed

Jones, Darryl R; Thomas, Dallas; Alger, Nicholas; Ghavidel, Ata; Inglis, G Douglas; Abbott, D Wade

2018-01-01

Deposition of new genetic sequences in online databases is expanding at an unprecedented rate. As a result, sequence identification continues to outpace functional characterization of carbohydrate active enzymes (CAZymes). In this paradigm, the discovery of enzymes with novel functions is often hindered by high volumes of uncharacterized sequences particularly when the enzyme sequence belongs to a family that exhibits diverse functional specificities (i.e., polyspecificity). Therefore, to direct sequence-based discovery and characterization of new enzyme activities we have developed an automated in silico pipeline entitled: Sequence Analysis and Clustering of CarboHydrate Active enzymes for Rapid Informed prediction of Specificity (SACCHARIS). This pipeline streamlines the selection of uncharacterized sequences for discovery of new CAZyme or CBM specificity from families currently maintained on the CAZy website or within user-defined datasets. SACCHARIS was used to generate a phylogenetic tree of a GH43, a CAZyme family with defined subfamily designations. This analysis confirmed that large datasets can be organized into sequence clusters of manageable sizes that possess related functions. Seeding this tree with a GH43 sequence from Bacteroides dorei DSM 17855 (BdGH43b, revealed it partitioned as a single sequence within the tree. This pattern was consistent with it possessing a unique enzyme activity for GH43 as BdGH43b is the first described α-glucanase described for this family. The capacity of SACCHARIS to extract and cluster characterized carbohydrate binding module sequences was demonstrated using family 6 CBMs (i.e., CBM6s). This CBM family displays a polyspecific ligand binding profile and contains many structurally determined members. Using SACCHARIS to identify a cluster of divergent sequences, a CBM6 sequence from a unique clade was demonstrated to bind yeast mannan, which represents the first description of an α-mannan binding CBM. Additionally, we have performed a CAZome analysis of an in-house sequenced bacterial genome and a comparative analysis of B. thetaiotaomicron VPI-5482 and B. thetaiotaomicron 7330, to demonstrate that SACCHARIS can generate "CAZome fingerprints", which differentiate between the saccharolytic potential of two related strains in silico. Establishing sequence-function and sequence-structure relationships in polyspecific CAZyme families are promising approaches for streamlining enzyme discovery. SACCHARIS facilitates this process by embedding CAZyme and CBM family trees generated from biochemically to structurally characterized sequences, with protein sequences that have unknown functions. In addition, these trees can be integrated with user-defined datasets (e.g., genomics, metagenomics, and transcriptomics) to inform experimental characterization of new CAZymes or CBMs not currently curated, and for researchers to compare differential sequence patterns between entire CAZomes. In this light, SACCHARIS provides an in silico tool that can be tailored for enzyme bioprospecting in datasets of increasing complexity and for diverse applications in glycobiotechnology.

Mitochondrial and nuclear phylogenetic analysis with Sanger and next-generation sequencing shows that, in Área de Conservación Guanacaste, northwestern Costa Rica, the skipper butterfly named Urbanus belli (family Hesperiidae) comprises three morphologically cryptic species

PubMed Central

2014-01-01

Background Skipper butterflies (Hesperiidae) are a relatively well-studied family of Lepidoptera. However, a combination of DNA barcodes, morphology, and natural history data has revealed several cryptic species complexes within them. Here, we investigate three DNA barcode lineages of what has been identified as Urbanus belli (Hesperiidae, Eudaminae) in Área de Conservación Guanacaste (ACG), northwestern Costa Rica. Results Although no morphological traits appear to distinguish among the three, congruent nuclear and mitochondrial lineage patterns show that “Urbanus belli” in ACG is a complex of three sympatric species. A single strain of Wolbachia present in two of the three cryptic species indicates that Urbanus segnestami Burns (formerly Urbanus belliDHJ01), Urbanus bernikerni Burns (formerly Urbanus belliDHJ02), and Urbanus ehakernae Burns (formerly Urbanus belliDHJ03) may be biologically separated by Wolbachia, as well as by their genetics. Use of parallel sequencing through 454-pyrosequencing improved the utility of ITS2 as a phylogenetic marker and permitted examination of the intra- and interlineage relationships of ITS2 variants within the species complex. Interlineage, intralineage and intragenomic compensatory base pair changes were discovered in the secondary structure of ITS2. Conclusion These findings corroborate the existence of three cryptic species. Our confirmation of a novel cryptic species complex, initially suggested by DNA barcode lineages, argues for using a multi-marker approach coupled with next-generation sequencing for exploration of other suspected species complexes. PMID:25005355

Multiscale pore structure and constitutive models of fine-grained rocks

NASA Astrophysics Data System (ADS)

Heath, J. E.; Dewers, T. A.; Shields, E. A.; Yoon, H.; Milliken, K. L.

2017-12-01

A foundational concept of continuum poromechanics is the representative elementary volume or REV: an amount of material large enough that pore- or grain-scale fluctuations in relevant properties are dissipated to a definable mean, but smaller than length scales of heterogeneity. We determine 2D-equivalent representative elementary areas (REAs) of pore areal fraction of three major types of mudrocks by applying multi-beam scanning electron microscopy (mSEM) to obtain terapixel image mosaics. Image analysis obtains pore areal fraction and pore size and shape as a function of progressively larger measurement areas. Using backscattering imaging and mSEM data, pores are identified by the components within which they occur, such as in organics or the clastic matrix. We correlate pore areal fraction with nano-indentation, micropillar compression, and axysimmetic testing at multiple length scales on a terrigenous-argillaceous mudrock sample. The combined data set is used to: investigate representative elementary volumes (and areas for the 2D images); determine if scale separation occurs; and determine if transport and mechanical properties at a given length scale can be statistically defined. Clear scale separation occurs between REAs and observable heterogeneity in two of the samples. A highly-laminated sample exhibits fine-scale heterogeneity and an overlapping in scales, in which case typical continuum assumptions on statistical variability may break down. Sandia National Laboratories is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia LLC, a wholly owned subsidiary of Honeywell International Inc. for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-NA0003525.

Drug & Gene Interaction Risk Analysis With & Without Genetic Testing Among Patients Undergoing MTM

ClinicalTrials.gov

2017-02-22

Cytochrome P450 CYP2D6 Enzyme Deficiency; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Ultrarapid Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Extensive Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Cytochrome P450 CYP2C9 Enzyme Deficiency; Cytochrome P450 CYP2C19 Enzyme Deficiency; Drug Metabolism, Poor, CYP2D6-RELATED; Drug Metabolism, Poor, CYP2C19-RELATED; CYP2D6 Polymorphism

Nonlinear estimation of parameters in biphasic Arrhenius plots.

PubMed

Puterman, M L; Hrboticky, N; Innis, S M

1988-05-01

This paper presents a formal procedure for the statistical analysis of data on the thermotropic behavior of membrane-bound enzymes generated using the Arrhenius equation and compares the analysis to several alternatives. Data is modeled by a bent hyperbola. Nonlinear regression is used to obtain estimates and standard errors of the intersection of line segments, defined as the transition temperature, and slopes, defined as energies of activation of the enzyme reaction. The methodology allows formal tests of the adequacy of a biphasic model rather than either a single straight line or a curvilinear model. Examples on data concerning the thermotropic behavior of pig brain synaptosomal acetylcholinesterase are given. The data support the biphasic temperature dependence of this enzyme. The methodology represents a formal procedure for statistical validation of any biphasic data and allows for calculation of all line parameters with estimates of precision.

Regional modeling of tropospheric NO2 vertical column density over East Asia during the period 2000-2010: comparison with multisatellite observations

NASA Astrophysics Data System (ADS)

Itahashi, S.; Uno, I.; Irie, H.; Kurokawa, J.-I.; Ohara, T.

2014-04-01

Satellite observations of the tropospheric NO2 vertical column density (VCD) are closely correlated to, and thus can be used to estimate, surface NOx emissions. In this study, the NO2 VCD simulated by a regional chemical transport model with emissions data from the updated Regional Emission inventory in ASia (REAS) version 2.1 were validated through comparison with multisatellite observations during the period 2000-2010. Rapid growth in NO2 VCD (~11% year-1) driven by the expansion of anthropogenic NOx emissions was identified above the central eastern China (CEC) region, except for the period during the economic downturn. In contrast, slightly decreasing trends (~2% year-1) were identified above Japan accompanied by a decline in anthropogenic emissions. To systematically compare the modeled NO2 VCD, we estimated sampling bias and the effect of applying the averaging kernel information, with particular focus on the SCanning Imaging Absorption spectroMeter for Atmospheric CHartographY (SCIAMACHY) data. Using the updated REAS, the modeled NO2 VCD reasonably reproduced annual trends observed by multisatellites, suggesting that the rate of increase of NOx emissions estimated by the updated REAS inventory would be robust. Province-scale revision of emissions above CEC is needed to further refine emission inventories. Based on the close linear relationship between modeled and observed NO2 VCD and anthropogenic NOx emissions, NOx emissions in 2009 and 2010, which were not covered by the updated REAS inventory, were estimated. NOx emissions from anthropogenic sources in China in 2009 and 2010 were determined to be 26.4 and 28.5 Tg year-1, respectively, indicating that NOx emissions increased more than twofold between 2000 and 2010. This increase reflected the strong growth of anthropogenic emissions in China following the rapid recovery from the economic downturn from late 2008 until mid-2009. Our method consists of simple estimations from satellite observations and provides results that are consistent with the most recent inventory of emissions data for China.

Modelling the role of electron attachment rates on column density ratios for C n H-/C n H (n=4,6,8) in dense molecular clouds

NASA Astrophysics Data System (ADS)

Gianturco, F. A.; Grassi, T.; Wester, R.

2016-10-01

The fairly recent detection of a variety of anions in the interstellar molecular clouds have underlined the importance of realistically modelling the processes governing their abundance. To pursue this task, our earlier calculations for the radiative electron attachment (REA) rates for C4H-, C6H-, and C8H- are employed in the present work, within a broad network of other concurrent reactions, to generate the corresponding column density ratios of anion/neutral (A/N) relative abundances. The latter are then compared with those obtained in recent years from observational measurements. The calculations involved the time-dependent solutions of a large network of chemical processes over an extended time interval and included a series of runs in which the values of REA rates were repeatedly scaled over several orders of magnitude. Macroscopic parameters for the Clouds’ modelling were also varied to cover a broad range of physical environments. It was found that, within the range and quality of the processes included in the present network,and selected from state-of-the-art astrophysical databases, the REA values required to match the observed A/N ratios needed to be reduced by orders of magnitude for C4H- case, while the same rates for C6H- and C8H- only needed to be scaled by much smaller factors. The results suggest that the generally proposed formation of interstellar anions by REA mechanism is overestimated by current models for the C4H- case, for which is likely to be an inefficient path to formation. This path is thus providing a rather marginal contribution to the observed abundances of C4H-, the latter being more likely to originate from other chemical processes in the network, as we discuss in some detail in the present work. Possible physical reasons for the much smaller differences against observations found instead for the values of the (A/N) ratios in two other, longer members of the series are put forward and analysed within the evolutionary modelling discussed in the present work.

Sex differences in language asymmetry are age-dependent and small: a large-scale, consonant-vowel dichotic listening study with behavioral and fMRI data.

PubMed

Hirnstein, Marco; Westerhausen, René; Korsnes, Maria S; Hugdahl, Kenneth

2013-01-01

Men are often believed to have a functionally more asymmetrical brain organization than women, but the empirical evidence for sex differences in lateralization is unclear to date. Over the years we have collected data from a vast number of participants using the same consonant-vowel dichotic listening task, a reliable marker for language lateralization. One dataset comprised behavioral data from 1782 participants (885 females, 125 non-right-handers), who were divided in four age groups (children <10 yrs, adolescents = 10-15 yrs, younger adults = 16-49 yrs, and older adults >50 yrs). In addition, we had behavioral and functional imaging (fMRI) data from another 104 younger adults (49 females, aged 18-45 yrs), who completed the same dichotic listening task in a 3T scanner. This database allowed us to comprehensively test whether there is a sex difference in functional language lateralization. Across all participants and in both datasets a right ear advantage (REA) emerged, reflecting left-hemispheric language lateralization. Accordingly, the fMRI data revealed a leftward asymmetry in superior temporal lobe language processing areas. In the N = 1782 dataset no main effect of sex but a significant sex by age interaction emerged: the REA increased with age in both sexes but as a result of an earlier onset in females the REA was stronger in female than male adolescents. In turn, male younger adults showed greater asymmetry than female younger adults (accounting for <1% of variance). There were no sex differences in children and older adults. The males in the fMRI dataset (N = 104) also had a greater REA than females (accounting for 4% of variance), but no sex difference emerged in the neuroimaging data. Handedness did not affect these findings. Taken together, our findings suggest that sex differences in language lateralization as assessed with dichotic listening exist, but they are (a) not necessarily reflected in fMRI data, (b) age-dependent and (c) relatively small. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed

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.

Differential signatures of bacterial and mammalian IMP dehydrogenase enzymes.

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

Zhang, R.; Evans, G.; Rotella, F.

1999-06-01

IMP dehydrogenase (IMPDH) is an essential enzyme of de novo guanine nucleotide synthesis. IMPDH inhibitors have clinical utility as antiviral, anticancer or immunosuppressive agents. The essential nature of this enzyme suggests its therapeutic applications may be extended to the development of antimicrobial agents. Bacterial IMPDH enzymes show bio- chemical and kinetic characteristics that are different than the mammalian IMPDH enzymes, suggesting IMPDH may be an attractive target for the development of antimicrobial agents. We suggest that the biochemical and kinetic differences between bacterial and mammalian enzymes are a consequence of the variance of specific, identifiable amino acid residues. Identification ofmore » these residues or combination of residues that impart this mammalian or bacterial enzyme signature is a prerequisite for the rational identification of agents that specifically target the bacterial enzyme. We used sequence alignments of IMPDH proteins to identify sequence signatures associated with bacterial or eukaryotic IMPDH enzymes. These selections were further refined to discern those likely to have a role in catalysis using information derived from the bacterial and mammalian IMPDH crystal structures and site-specific mutagenesis. Candidate bacterial sequence signatures identified by this process include regions involved in subunit interactions, the active site flap and the NAD binding region. Analysis of sequence alignments in these regions indicates a pattern of catalytic residues conserved in all enzymes and a secondary pattern of amino acid conservation associated with the major phylogenetic groups. Elucidation of the basis for this mammalian/bacterial IMPDH signature will provide insight into the catalytic mechanism of this enzyme and the foundation for the development of highly specific inhibitors.« less

Remarkable Reproducibility of Enzyme Activity Profiles in Tomato Fruits Grown under Contrasting Environments Provides a Roadmap for Studies of Fruit Metabolism1[W][OPEN

PubMed Central

Biais, Benoît; Bénard, Camille; Beauvoit, Bertrand; Colombié, Sophie; Prodhomme, Duyên; Ménard, Guillaume; Bernillon, Stéphane; Gehl, Bernadette; Gautier, Hélène; Ballias, Patricia; Mazat, Jean-Pierre; Sweetlove, Lee; Génard, Michel; Gibon, Yves

2014-01-01

To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum ‘Moneymaker’) plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels. PMID:24474652

Structural similarities and functional differences clarify evolutionary relationships between tRNA healing enzymes and the myelin enzyme CNPase.

PubMed

Muruganandam, Gopinath; Raasakka, Arne; Myllykoski, Matti; Kursula, Inari; Kursula, Petri

2017-05-16

Eukaryotic tRNA splicing is an essential process in the transformation of a primary tRNA transcript into a mature functional tRNA molecule. 5'-phosphate ligation involves two steps: a healing reaction catalyzed by polynucleotide kinase (PNK) in association with cyclic phosphodiesterase (CPDase), and a sealing reaction catalyzed by an RNA ligase. The enzymes that catalyze tRNA healing in yeast and higher eukaryotes are homologous to the members of the 2H phosphoesterase superfamily, in particular to the vertebrate myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). We employed different biophysical and biochemical methods to elucidate the overall structural and functional features of the tRNA healing enzymes yeast Trl1 PNK/CPDase and lancelet PNK/CPDase and compared them with vertebrate CNPase. The yeast and the lancelet enzymes have cyclic phosphodiesterase and polynucleotide kinase activity, while vertebrate CNPase lacks PNK activity. In addition, we also show that the healing enzymes are structurally similar to the vertebrate CNPase by applying synchrotron radiation circular dichroism spectroscopy and small-angle X-ray scattering. We provide a structural analysis of the tRNA healing enzyme PNK and CPDase domains together. Our results support evolution of vertebrate CNPase from tRNA healing enzymes with a loss of function at its N-terminal PNK-like domain.

Molecular and biochemical characterization of natural and recombinant phosphoglycerate kinase B from Trypanosoma rangeli.

PubMed

Villafraz, O; Rondón-Mercado, R; Cáceres, A J; Concepción, J L; Quiñones, W

2018-04-01

T. rangeli epimastigotes contain only a single detectable phosphoglycerate kinase (PGK) enzyme in their cytosol. Analysis of this parasite's recently sequenced genome showed a gene predicted to code for a PGK with the same molecular mass as the natural enzyme, and with a cytosolic localization as well. In this work, we have partially purified the natural PGK from T. rangeli epimastigotes. Furthermore, we cloned the predicted PGK gene and expressed it as a recombinant active enzyme. Both purified enzymes were kinetically characterized and displayed similar substrate affinities, with Km ATP values of 0.13 mM and 0.5 mM, and Km 3PGA values of 0.28 mM and 0.71 mM, for the natural and recombinant enzyme, respectively. The optimal pH for activity of both enzymes was in the range of 8-10. Like other PGKs, TrPGK is monomeric with a molecular mass of approximately 44 kDa. The enzyme's kinetic characteristics are comparable with those of cytosolic PGK isoforms from related trypanosomatid species, indicating that, most likely, this enzyme is equivalent with the PGKB that is responsible for generating ATP in the cytosol of other trypanosomatids. This is the first report of a glycolytic enzyme characterization from T. rangeli. Copyright © 2018 Elsevier Inc. All rights reserved.

Integrating Proteomics and Enzyme Kinetics Reveals Tissue-Specific Types of the Glycolytic and Gluconeogenic Pathways.

PubMed

Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

2015-08-07

Glycolysis is the core metabolic pathway supplying energy to cells. Whereas the vast majority of studies focus on specific aspects of the process, global analyses characterizing simultaneously all enzymes involved in the process are scarce. Here, we demonstrate that quantitative label- and standard-free proteomics allows accurate determination of titers of metabolic enzymes and enables simultaneous measurements of titers and maximal enzymatic activities (Amax) of all glycolytic enzymes and the gluconeogenic fructose 1,6-bisphosphatase in mouse brain, liver and muscle. Despite occurrence of tissue-specific isoenzymes bearing different kinetic properties, the enzyme titers often correlated well with the Amax values. To provide a more general picture of energy metabolism, we analyzed titers of the enzymes in additional 7 mouse organs and in human cells. Across the analyzed samples, we identified two basic profiles: a "fast glucose uptake" one in brain and heart, and a "gluconeogenic rich" one occurring in liver. In skeletal muscles and other organs, we found intermediate profiles. Obtained data highlighted the glucose-flux-limiting role of hexokinase which activity was always 10- to 100-fold lower than the average activity of all other glycolytic enzymes. A parallel determination of enzyme titers and maximal enzymatic activities allowed determination of kcat values without enzyme purification. Results of our in-depth proteomic analysis of the mouse organs did not support the concepts of regulation of glycolysis by lysine acetylation.

Enzymatic Removal of Diacetyl from Beer 1

PubMed Central

Thompson, Janet W.; Shovers, J.; Sandine, W. E.; Elliker, P. R.

1970-01-01

Use of diacetyl reductase, a reduced nicotinamide adenine dinucleotide (NADH)-requiring enzyme, to eliminate diacetyl off-flavor in beer was studied. The crude enzyme was extracted from Aerobacter aerogenes and partially purified by ammonium sulfate precipitation or Sephadex chromatography. In the semipure state, the enzyme was inactivated by lyophilization; in a crude state, the lyophilized extract remained stable for at least 4 months at — 20 C. A 50% reduction in specific activity within 5 min was observed when crude diacetyl reductase was suspended (5 mg of protein/ml) in phosphate buffer at pH 5.5 or below; a similar inactivation rate was observed when the crude enzyme was dissolved in a 5% aqueous ethyl alcohol solution. Effective crude enzyme activity in beer at a natural pH of 4.1 required protection of the enzyme in 10% gelatin. Incorporation of yeast cells with the gel-protected enzyme provided regeneration of NADH. Combinations of yeast, enzyme, and gelatin were tested to obtain data analyzed by regression analysis to determine the optimal concentration of each component of the system required to reduce the level of diacetyl in spiked (0.5 ppm) beer to less than 0.12 ppm within 48 hr at 5 C. The protected enzyme system was also effective in removing diacetyl from orange juice (pH 3.8) and some distilled liquors. PMID:4318450

In vitro analysis of protection of the enzyme bile salt hydrolase against enteric conditions by whey protein-gum arabic microencapsulation.

PubMed

Lambert, J M; Weinbreck, F; Kleerebezem, M

2008-09-24

The interest in efficient intestinal delivery of health-promoting substances is increasing. However, the delivery of vulnerable substances such as enzymes requires specific attention. The transit through the stomach, where the pH is very low, can be detrimental to the enzymatic activity of the protein to be delivered. Here, we describe the microencapsulation of the model enzyme bile salt hydrolase (Bsh) using whey protein-gum arabic microencapsulates for food-grade and targeted enzyme delivery in the proximal region of the small intestine. Furthermore, the efficacy of enteric coating microencapsulates for site-specific enzyme delivery was compared in vitro with living Lactobacillus plantarum WCFS1 bacteria that endogenously produce the Bsh enzyme. Microencapsulates allowed highly effective protection of the enzyme under gastric conditions. Moreover, Bsh release under intestinal conditions appeared to be very efficient, although in the presence of pancreatin, the Bsh activity decreased in time due to proteolytic degradation. In comparison, L. plantarum appeared to be capable to withstand gastric conditions as well as pancreatin challenge. Delivery using encapsulates and live bacteria each have different (dis)advantages that are discussed. In conclusion, live bacteria and food-grade microencapsulates provide alternatives for dedicated enteric delivery of specific enzymes, and the choice of enzyme to be delivered may determine which mode of delivery is most suitable.

Sequencing of oligosaccharides using enzyme array digestion with electrochemical and fluorescent detections

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

Sun, M.; Lee, C.S.

1997-12-31

The objective of this study is to develop a rapid and sensitive method for oligosaccharide sequencing. The oligosaccharides are subjected to the enzyme array digestion with exoglycosidases of known and well-defined specificities. The enzyme array method involves the division of oligosaccharide sample into aliquots, and the incubation of each aliquot with a precisely defined mixture of exoglycosidases. In the enzyme array method, the presence of a specific linkage anywhere in the oligosaccharide is determined by the inability of an enzyme mixture lacking a given enzyme to cleave that linkage ( a stop point) and the ability of the other enzymesmore » to cleave the linkage up to that point. The direct quantification of released monosaccharides from the enzyme array can be achieved by using pulsed amperometric detection (PAD) or by fluorescent derivatization with a fluorophoric agent. The measured monosaccharide concentrations in combination with the enzyme array analysis provide detail characterization of oligosaccharides with their sugar composition, configuration, and linkage information, The released monosaccharides are further quantified by anion exchange chromatography and capillary electrophoresis for the comparison with the results obtained from PAD and fluorescence measurements. Our enzyme array-electrochemical (or fluorescent) detection method does not require any separation procedure and any prior labeling of oligosaccharide and have several practical advantages over the current carbohydrate sequencing techniques including simplicity, speed, and the ability to use small amounts of starting material.« less

Effect of neohesperidin dihydrochalcone on the activity and stability of alpha-amylase: a comparative study on bacterial, fungal, and mammalian enzymes.

PubMed

Kashani-Amin, Elaheh; Ebrahim-Habibi, Azadeh; Larijani, Bagher; Moosavi-Movahedi, Ali Akbar

2015-10-01

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha-amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha-amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha-amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha-amylase surface in domain B. This domain shows differences in various alpha-amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact. Copyright © 2015 John Wiley & Sons, Ltd.

Studies on the enzymes of Sarcocystis suicanis: purification and characterization of an acid phosphatase.

PubMed

Farooqui, A A; Adams, D D; Hanson, W L; Prestwood, A K

1987-08-01

Percoll density gradient centrifugation was used for isolating large quantities of bradyzoites of Sarcocystis suicanis, which were used for enzymatic analysis. Crude extracts of bradyzoites contained activities suggestive of several acid hydrolases. Levels of acid and alkaline phosphatase were higher than those of beta-N-acetylhexosaminidase and beta-galactosidase. Acid phosphatase was purified 156-fold with an overall recovery of 54% using DEAE-Sepharose 4B and Sephadex G-200 chromatography. The partially purified enzyme was not a glycoprotein and had a molecular weight of approximately 170,000. The enzyme was markedly inhibited by Cu++, Hg++, and iodoacetamide, suggesting the presence of a sulfhydryl group. Sodium tartrate caused strong inhibition of the enzyme. The acid phosphatase of S. suicanis appears to be a unique enzyme that cannot be classified under high or low molecular weight acid phosphatases of widely diverse origin.

Covalent enzyme immobilization onto carbon nanotubes using a membrane reactor

NASA Astrophysics Data System (ADS)

Voicu, Stefan Ioan; Nechifor, Aurelia Cristina; Gales, Ovidiu; Nechifor, Gheorghe

2011-05-01

Composite porous polysulfone-carbon nanotubes membranes were prepared by dispersing carbon nanotubes into a polysulfone solution followed by the membrane formation by phase inversion-immersion precipitation technique. The carbon nanotubes with amino groups on surface were functionalized with different enzymes (carbonic anhydrase, invertase, diastase) using cyanuric chloride as linker between enzyme and carbon nanotube. The composite membrane was used as a membrane reactor for a better dispersion of carbon nanotubes and access to reaction centers. The membrane also facilitates the transport of enzymes to active carbon nanotubes centers for functionalization (amino groups). The functionalized carbon nanotubes are isolated by dissolving the membranes after the end of reaction. Carbon nanotubes with covalent immobilized enzymes are used for biosensors fabrications. The obtained membranes were characterized by Scanning Electron Microscopy, Thermal analysis, FT-IR Spectroscopy, Nuclear Magnetic Resonance, and functionalized carbon nanotubes were characterized by FT-IR spectroscopy.

Krebs cycle metabolon: structural evidence of substrate channeling revealed by cross-linking and mass spectrometry.

PubMed

Wu, Fei; Minteer, Shelley

2015-02-02

It has been hypothesized that the high metabolic flux in the mitochondria is due to the self-assembly of enzyme supercomplexes (called metabolons) that channel substrates from one enzyme to another, but there has been no experimental confirmation of this structure or the channeling. A structural investigation of enzyme organization within the Krebs cycle metabolon was accomplished by in vivo cross-linking and mass spectrometry. Eight Krebs cycle enzyme components were isolated upon chemical fixation, and interfacial residues between mitochondrial malate dehydrogenase, citrate synthase, and aconitase were identified. Using constraint protein docking, a low-resolution structure for the three-enzyme complex was achieved, as well as the two-fold symmetric octamer. Surface analysis showed formation of electrostatic channeling upon protein-protein association, which is the first structural evidence of substrate channeling in the Krebs cycle metabolon. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.