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81.
82.
Starch and α-glucan acting enzymes, modulating their properties by directed evolution 总被引:1,自引:0,他引:1
Starch is the major food reserve in plants and forms a large part of the daily calorie intake in the human diet. Industrially, starch has become a major raw material in the production of various products including bio-ethanol, coating and anti-staling agents. The complexity and diversity of these starch based industries and the demand for high quality end products through extensive starch processing, can only be met through the use of a broad range of starch and α-glucan modifying enzymes. The economic importance of these enzymes is such that the starch industry has grown to be the largest market for enzymes after the detergent industry. However, as the starch based industries expand and develop the demand for more efficient enzymes leading to lower production cost and higher quality products increases. This in turn stimulates interest in modifying the properties of existing starch and α-glucan acting enzymes through a variety of molecular evolution strategies. Within this review we examine and discuss the directed evolution strategies applied in the modulation of specific properties of starch and α-glucan acting enzymes and highlight the recent developments in the field of directed evolution techniques which are likely to be implemented in the future engineering of these enzymes. 相似文献
83.
Marie‐Agnès Travers Ronan Le Bouffant Carolyn S. Friedman Florence Buzin Bertrand Cougard Sylvain Huchette Marcel Koken Christine Paillard 《Journal of cellular biochemistry》2009,106(1):152-160
Vibrio harveyi is a marine bacterial pathogen responsible for episodic abalone epidemics associated with massive mortalities in France, Japan, and Australia. The aim of this study was the understanding of a possible role of the p38 MAPK in abalone haemocyte responses towards this bacterium. First, the pathogenicity of different V. harveyi strains was compared in both immersion and injection trials, and clear differences were detected. The three strains, ORM4, 04/092, and 05/053, all isolated from moribund abalone, induced up to 80% mortalities in immersion or injection challenges (LD50 (ORM4) = 2.5 × 102 CFU animal?1). The two strains, LMG 4044T and LMG 7890 were non‐pathogenic towards abalone in immersion trials, and needed very high numbers for killing by intramuscular injections (LD50 = 8.9 × 104 and 1.6 × 105 CFU animal?1, respectively). To start unraveling the mechanism explaining these differences, the p38‐MAPK, a keyplayer in antimicrobial immune response, was studied. The non‐pathogenic strain, LMG 7890 can be eliminated by abalone haemocytes and induces haemocyte phagocytosis and high ROS production. With different concentrations of a p38‐specific inhibitor, SB203580, p38 implication was shown. This inhibitor reduced phagocytosis and ROS induction leading to LMG 7890 proliferation. In the case of the pathogenic ORM4 which can not be eliminated by abalone haemocytes, no phagocytosis and ROS production was induced, and a retarded p38 activation was observed. Taken together, our results suggest that p38 MAPK modulation may be one of the ways of virulent V. harveyi to attack its host and escape abalone immune response. J. Cell. Biochem. 106: 152–160, 2009. © 2008 Wiley‐Liss, Inc. 相似文献
84.
Paul A. Fitzpatrick Anthony F. Guinan Tony G. Walsh Ronan P. Murphy Maria T. Killeen Nicholas P. Tobin Adrian R. Pierotti Philip M. Cummins 《The international journal of biochemistry & cell biology》2009,41(11):2287-2294
Neprilysin (NEP, neutral endopeptidase, EC3.4.24.11), a zinc metallopeptidase expressed on the surface of endothelial cells, influences vascular homeostasis primarily through regulated inactivation of natriuretic peptides and bradykinin. Earlier in vivo studies reporting on the anti-atherosclerotic effects of NEP inhibition and on the atheroprotective effects of flow-associated laminar shear stress (LSS) have lead us to hypothesize that the latter hemodynamic stimulus may serve to down-regulate NEP levels within the vascular endothelium. To address this hypothesis, we have undertaken an investigation of the effects of LSS on NEP expression in vitro in bovine aortic endothelial cells (BAECs), coupled with an examination of the signalling mechanism putatively mediating these effects. BAECs were exposed to physiological levels of LSS (10 dynes/cm2, 24 h) and harvested for analysis of NEP expression using real-time PCR, Western blotting, and immunocytochemistry. Relative to unsheared controls, NEP mRNA and protein were substantially down-regulated by LSS (≥50%), events which could be prevented by treatment of BAECs with either N-acetylcysteine, superoxide dismutase, or catalase, implicating reactive oxygen species (ROS) involvement. Employing pharmacological and molecular inhibition strategies, the signal transduction pathway mediating shear-dependent NEP suppression was also examined, and roles implicated for Gβγ, Rac1, and NADPH oxidase activation in these events. Treatment of static BAECs with angiotensin-II, a potent stimulus for NADPH oxidase activation, mimicked the suppressive effects of shear on NEP expression, further supporting a role for NADPH oxidase-dependent ROS production. Interestingly, inhibition of receptor tyrosine kinase signalling had no effect. In conclusion, we confirm for the first time that NEP expression is down-regulated in vascular endothelial cells by physiological laminar shear, possibly via a mechanotransduction mechanism involving NADPH oxidase-induced ROS production. 相似文献
85.
86.
The Family X DNA Polymerase from Deinococcus radiodurans Adopts
a Non-standard Extended Conformation
Nicolas Leulliot Lionel Cladière Fran?ois Lecointe Dominique Durand Ulrich Hübscher Herman van Tilbeurgh 《The Journal of biological chemistry》2009,284(18):11992-11999
Deinococcus radiodurans is an extraordinarily radioresistant
bacterium that is able to repair hundreds of radiation-induced double-stranded
DNA breaks. One of the players in this pathway is an X family DNA polymerase
(PolXDr). Deletion of PolXDr has been shown to decrease
the rate of repair of double-stranded DNA breaks and increase cell sensitivity
to gamma-rays. A 3′→5′ exonuclease activity that stops
cutting close to DNA loops has also been demonstrated. The present crystal
structure of PolXDr solved at 2.46-Å resolution reveals that
PolXDr has a novel extended conformation in stark contrast to the
closed “right hand” conformation commonly observed for DNA
polymerases. This extended conformation is stabilized by the C-terminal PHP
domain, whose putative nuclease active site is obstructed by its interaction
with the polymerase domain. The overall conformation and the presence of non
standard residues in the active site of the polymerase X domain makes
PolXDr the founding member of a novel class of polymerases involved
in DNA repair but whose detailed mode of action still remains enigmatic.DNA replication and repair are functions that are of vital importance for
the maintenance of cellular life. These functions are carried out by various
DNA replicating engines, most of them acting as multiprotein complexes.
Deinococcus radiodurans, a Gram-positive bacterium, is characterized
by an extraordinary resistance to ionizing radiation and desiccation. After
radiation induced cutting of its 3.28-megabase genome into hundreds of small
fragments, it is capable of reassembling it completely
(1). Different hypotheses have
been suggested to explain this radioresistance. A recently proposed mechanism
involves the creation of long linear DNA intermediates by an extended
synthesis-dependent strand annealing process, where overlapping chromosomal
fragments are used both as primers and as templates for synthesis of
complementary single strands
(2). Recircularization of
chromosomes would be assured by homologous recombination. Although DNA
polymerase I is one of the main enzymes involved in this process, it was shown
that other proteins affect double strand break repair efficiency in D.
radiodurans. One of these is an X family DNA polymerase
(PolXDr)5
(3). Cells devoid of
PolXDr protein show increased sensitivity to γ-irradiation
and a longer delay in the restoration of an intact genome after irradiation.
It was therefore proposed that PolXDr has an important role in
double strand break repair in D. radiodurans. The contribution of
PolXDr may become essential for instance when damage gets too
important or, alternatively, it may act in different repair pathways from
polymerase I. Indeed, some of the X DNA polymerases, such as Saccharomyces
cerevisiae Pol4 and human polymerase λ
(4) have been proposed to play
important roles in different DNA repair processes, including non-homologous
end-joining (5). It was shown
that PolXDr also has strong 3′→5′ exonuclease
activity that is stimulated by Mn2+
(6). This activity is
associated with proofreading mechanisms in other polymerase families and
encoded by protein domains or subunits distinct from the polymerase catalytic
domain (7). Curiously the
exonuclease activity of PolXDr is modulated upon encounter of a
stem-loop structure. The combination of both activities leads to the
hypothesis that PolXDr might be involved in DNA repair, potentially
non-homologous end-joining, by processing damaged DNA or repair intermediates,
thus generating substrates for other repair proteins
(6). Very recently an
orthologue of PolX from Bacillus subtilis was characterized. It was
shown that PolXBs is a template-directed DNA polymerase acting on
DNA gaps with a downstream 5′ phosphate group, suggesting it may play a
role in base excision repair
(8).DNA polymerases all combine a catalytic palm domain, a thumb domain,
binding double-stranded DNA, and a finger domain that fixes the incoming
nucleotide. The polymerase domain of the X family belongs to the
Polβ-like nucleotidyltransferase superfamily, sharing ∼25% amino acid
identity with the DNA polymerase domains of Polλ, Pol4, and Polβ.
PolXDr has a second domain at the C terminus called PHP, with
strong sequence identity with the histidinol phosphatase involved in histidine
transport in bacteria. Due to its similarity to histidinol phosphatase and the
presence of a trinuclear zinc site, the PolXDr PHP domain is
thought to function as phosphoesterase
(9). In the context of DNA
polymerases, this activity might be responsible for the degradation of
pyrophosphate, thus driving the polymerization reaction, or contributes to a
nuclease reaction that would be involved in proofreading the newly synthesized
strand. The deletion of the PHP domain also had a negative effect on survival
of γ-irradiated cells suggesting that this domain possesses a function
in DNA repair. Unexpectedly, deletion of the PHP domain destroys structure
modulated but not the general 3′→5′ exonuclease activity
(6). No activity could be
demonstrated for the PHP domain alone.In this report we present the crystal structure of PolXDr at
2.46-Å resolution. Surprisingly, PolXDr adopts a stretched
out conformation instead of the commonly observed closed right hand
conformation. In the active site of the polymerase catalytic domain, the two
universally conserved aspartates are replaced by two glutamates, whereas the
active site of the PHP domain is obstructed by its interaction with the
polymerase domain. 相似文献
87.
Ellen M Moran Ronan Mullan Jennifer McCormick Mary Connolly Owen Sullivan Oliver FitzGerald Barry Bresnihan Douglas J Veale Ursula Fearon 《Arthritis research & therapy》2009,11(4):R113-12
Introduction
The aim of this study was to examine IL-17A in patients, following anti-TNF-α therapy and the effect of IL-17A on matrix turnover and cartilage degradation. 相似文献88.
Thorsten Barnickel Jason Weston Ronan Collobert Hans-Werner Mewes Volker Stümpflen 《PloS one》2009,4(7)
To reduce the increasing amount of time spent on literature search in the life sciences, several methods for automated knowledge extraction have been developed. Co-occurrence based approaches can deal with large text corpora like MEDLINE in an acceptable time but are not able to extract any specific type of semantic relation. Semantic relation extraction methods based on syntax trees, on the other hand, are computationally expensive and the interpretation of the generated trees is difficult. Several natural language processing (NLP) approaches for the biomedical domain exist focusing specifically on the detection of a limited set of relation types. For systems biology, generic approaches for the detection of a multitude of relation types which in addition are able to process large text corpora are needed but the number of systems meeting both requirements is very limited. We introduce the use of SENNA (“Semantic Extraction using a Neural Network Architecture”), a fast and accurate neural network based Semantic Role Labeling (SRL) program, for the large scale extraction of semantic relations from the biomedical literature. A comparison of processing times of SENNA and other SRL systems or syntactical parsers used in the biomedical domain revealed that SENNA is the fastest Proposition Bank (PropBank) conforming SRL program currently available. 89 million biomedical sentences were tagged with SENNA on a 100 node cluster within three days. The accuracy of the presented relation extraction approach was evaluated on two test sets of annotated sentences resulting in precision/recall values of 0.71/0.43. We show that the accuracy as well as processing speed of the proposed semantic relation extraction approach is sufficient for its large scale application on biomedical text. The proposed approach is highly generalizable regarding the supported relation types and appears to be especially suited for general-purpose, broad-scale text mining systems. The presented approach bridges the gap between fast, cooccurrence-based approaches lacking semantic relations and highly specialized and computationally demanding NLP approaches. 相似文献
89.
Background
There exists a lack of knowledge regarding the quantity and quality of scientific yield in relation to individual cancer types. We aimed to measure the proportion, quality and relevance of oncology-related articles, and to relate this output to their associated disease burden. By incorporating the impact factor(IF) and Eigenfactor™(EF) into our analysis we also assessed the relationship between these indices and the output under study.Methods
All publications in 2007 were retrieved for the 26 most common cancers. The top 20 journals ranked by IF and EF in general medicine and oncology, and the presence of each malignancy within these titles was analysed. Journals publishing most prolifically on each cancer were identified and their impact assessed.Principal Findings
63260 (PubMed) and 126845 (WoS) entries were generated, respectively. 26 neoplasms accounted for 25% of total output from the top medical publications. 5 cancers dominated the first quartile of output in the top oncology journals; breast, prostate, lung, and intestinal cancer, and leukaemia. Journals associated with these cancers were associated with much higher IFs and EFs than those journals associated with the other cancer types under study, although these measures were not equivalent across all sub-specialties. In addition, yield on each cancer was related to its disease burden as measured by its incidence and prevalence.Conclusions
Oncology enjoys disproportionate representation in the more prestigious medical journals. 5 cancers dominate yield, although this attention is justified given their associated disease burden. The commonly used IF and the recently introduced EF do not correlate in the assessment of the preeminent oncology journals, nor at the level of individual malignancies; there is a need to delineate between proxy measures of quality and the relevance of output when assessing its merit. These results raise significant questions regarding the best method of assessment of research and scientific output in the field of oncology. 相似文献90.
Nengyi Zhang Amit Gur Yves Gibon Ronan Sulpice Sherry Flint-Garcia Michael D. McMullen Mark Stitt Edward S. Buckler 《PloS one》2010,5(4)