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1.
Reinout Heijungs Sangwon Suh René Kleijn 《The International Journal of Life Cycle Assessment》2005,10(2):103-112
Goal, Scope and Background To strengthen the evaluative power of LCA, life cycle interpretation should be further developed. A previous contribution (Heijungs & Kleijn 2001) elaborated five examples of concrete methods within the subset of numerical approaches towards interpretation. These methods were: contribution analysis, perturbation analysis, uncertainty analysis, comparative analysis, and discernibility analysis. Developments in software have enabled the possibility to apply the five example methods to explore the much-used Ecoinvent”96 database.Discussion of Methods The numerical approaches implemented in this study include contribution analysis, perturbation analysis, uncertainty analysis, comparative analysis, discernibility analysis and the newly developed key issue analysis. The data used comes from a very large process database: Ecoinvent’96, containing 1163 processes, 1181 economic flows and 571 environmental flows. Conclusions Results are twofold: they serve as a benchmark to the usefulness and feasibility of these numerical approaches, and they shed light on the question of stability and structure in an often-used large system of interconnected processes. Most of the approaches perform quite well: computation time on a moderate PC is between a few seconds a few minutes. Only Monte Carlo analyses may require much longer, but even then it appears that most questions can be answered within a few hours. Moreover, analytical expressions for error propagation are much faster than Monte Carlo analyses, while giving almost identical results. Despite the fact that many processes are connected to each other, leading to the possibility of a very unstable system and very sensitive coefficients, the overall results show that most results are not extremely uncertain. There are, however, some exceptions to this positive message. 相似文献
2.
3.
David Alsteens Vishukumar Aimanianda Pushpa Hegde Stéphane Pire Rémi Beau Jagadeesh Bayry Jean-Paul Latgé Yves?F. Dufrêne 《Biophysical journal》2013,105(2):320-327
Understanding the surface properties of the human opportunistic pathogen Aspergillus fumigatus conidia is essential given the important role they play during the fungal interactions with the human host. Although chitin synthases with myosin motor-like domain (CSM) play a major role in cell wall biosynthesis, the extent to which deletion of the CSM genes alter the surface structural and biophysical-biological properties of conidia is not fully characterized. We used three complementary atomic force microscopy techniques—i.e., structural imaging, chemical force microscopy with hydrophobic tips, and single-molecule force spectroscopy with lectin tips—to gain detailed insights into the nanoscale surface properties (ultrastructure, hydrophobicity) and polysaccharide composition of the wild-type and the chitin synthase mutant (ΔcsmA, ΔcsmB, and ΔcsmA/csmB) conidia of A. fumigatus. Wild-type conidia were covered with a highly hydrophobic layer of rodlet nanostructures. By contrast, the surface of the ΔcsmA mutant was almost completely devoid of rodlets, leading to loss of hydrophobicity and exposure of mannan and chitin polysaccharides. The ΔcsmB and ΔcsmA/csmB mutants showed a different behavior, i.e., the surfaces featured poorly organized rodlet layers, yet with a low hydrophobicity and substantial amounts of exposed mannan and chitin at the surface. As the rodlet layer is important for masking recognition of immunogenic fungal cell wall components by innate immune cells, disappearance of rodlet layers in all three chitin synthase mutant conidia was associated with an activation of human dendritic cells. These nanoscale analyses emphasize the important and distinct roles that the CSMA and CSMB genes play in modulating the surface properties and immune interactions of A. fumigatus and demonstrate the power of atomic force microscopy in fungal genetic studies for assessing the phenotypic characteristics of mutants altered in cell surface organization. 相似文献
4.
Hai-Li Cheng Rui-Yu Zhao Tian-Jiao Chen Wen-Bo Yu Fen Wang Ke-Di Cheng Ping Zhu 《Molecular & cellular proteomics : MCP》2013,12(8):2236-2248
Paclitaxel, a natural antitumor compound, is produced by yew trees at very low concentrations, causing a worldwide shortage of this important anticancer medicine. These plants also produce significant amounts of 7-β-xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for the semi-synthesis of paclitaxel. Some microorganisms can convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol, but the bioconversion yield needs to be drastically improved for industrial applications. In addition, the related β-xylosidases of these organisms have not yet been defined. We set out to discover an efficient enzyme for 10-deacetyltaxol production. By combining the de novo sequencing of β-xylosidase isolated from Lentinula edodes with RT-PCR and the rapid amplification of cDNA ends, we cloned two cDNA variants, Lxyl-p1–1 and Lxyl-p1–2, which were previously unknown at the gene and protein levels. Both variants encode a specific bifunctional β-d-xylosidase/β-d-glucosidase with an identical ORF length of 2412 bp (97% identity). The enzymes were characterized, and their 3.6-kb genomic DNAs (G-Lxyl-p1–1, G-Lxyl-p1–2), each harboring 18 introns, were also obtained. Putative substrate binding motifs, the catalytic nucleophile, the catalytic acid/base, and potential N-glycosylation sites of the enzymes were predicted. Kinetic analysis of both enzymes showed kcat/Km of up to 1.07 s−1mm−1 against 7-β-xylosyl-10-deacetyltaxol. Importantly, at substrate concentrations of up to 10 mg/ml (oversaturated), the engineered yeast could still robustly convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol with a conversion rate of over 85% and a highest yield of 8.42 mg/ml within 24 h, which is much higher than those reported previously. Therefore, our discovery might lead to significant progress in the development of new 7-β-xylosyl-10-deacetyltaxol-converting enzymes for more efficient use of 7-β-xylosyltaxanes to semi-synthesize paclitaxel and its analogues. This work also might lead to further studies on how these enzymes act on 7-β-xylosyltaxanes and contribute to the growing database of glycoside hydrolases.The protection and sustainable utilization of natural resources are among the most important and global problems of the 21st century. Paclitaxel (Taxol®) is mainly isolated from slow-growing yew trees (genus Taxus, family Taxaceae) and is known as a “blockbuster drug ” showing unique active mechanisms (1), with prominent activity against various cancers (including ovarian, breast, lung, head, and neck carcinomas and the AIDS-related Kaposi sarcoma) (2). However, the source of paclitaxel has always been a top concern, because its content in the plant is extremely low, and it is isolated in “large ” amounts (∼0.02%) only from the bark of the tree (3). A 100-year-old tree might yield 3 kg of bark, which provides enough paclitaxel for one 300-mg dose (4). To preserve the Taxus resource and alleviate some of the pressure on the source, several approaches have been employed to prepare paclitaxel or its analog Taxotere, including chemical semi-synthesis from the precursor 10-deacetylbaccatin III (DB),1 which is readily available from the twigs of yew trees such as Taxus baccata (5, 6); isolation from the twigs of nursery trees including T. chinensis var. mairei and T. media (hybrid); paclitaxel-producing endophytic strain fermentation (7, 8); and Taxus cell and tissue culture (9). The first two approaches might partially relieve this pressure, but they still cannot meet the growing market demand.7-β-xylosyltaxanes are much more abundant and are extracted simultaneously with paclitaxel and DB from various species of yew (10–12), but generally they are dealt with as byproducts. Among these analogues, 7-β-xylosyl-10-deacetyltaxol (XDT) can be obtained with a yield of as much as 0.5% (from dried stem bark) (13). These 7-β-xylosyltaxanes can be hydrolyzed via chemical or biological methods to give the corresponding 7-hydroxyltaxanes, including 10-deacetyltaxol (DT) and DB, for the semi-synthesis of paclitaxel. In contrast to the chemical approach, which utilizes periodate or other oxidizing agents and a substituted hydrazine in the reactions to remove the sugar, the biological approach is an enzymatic process that releases the d-xylose from 7-xylosyltaxanes through the specific β-xylosidase and is therefore considered to be environmentally friendly.β-xylosidases (EC3.2.1.37) belong to glycoside hydrolase (GH) or glycosidase (EC3.2.1.X) families 3, 30, 39, 43, 52, and 54 (14). However, the filamentous fungal β-xylosidases have hitherto been described as belonging only to GH families 3, 43, and 54 (15). Many kinds of β-xylosidases have been purified from different organisms, such as bacteria (16–19), fungi (20–23), and plants (24, 25). Some β-xylosidase genes, such as those from bacteria (18, 19, 26, 27) or from fungi (23, 28–30), have been cloned and characterized. However, none of these enzymes have been reported to be active against 7-β-xylosyltaxanes. In fact, a lot of commercially available xylosidases, xylanases, and other glycosidases do not have any activity specific for removing xylose from 7-β-xylosyltaxanes (31). Some bacterial isolates, such as Moraxella sp. (ATCC 55475) (31, 32), Cellulosimicrobium cellulans XZ-5 (CCTCC No. M207130) (33), and Enterobacter sp. (CGMCC 2487) (34), have been reported to have the ability to convert XDT to DT. But these strains gave low yields of DT (0.23, 0.4, and 0.76 mg/ml, respectively (31–34)), which is probably due to the ubiquitous low enzyme levels in the native organisms. The related β-xylosidases of these organisms have not yet been defined. Our lab discovered that a fungal species, Lentinula edodes, could transform XDT into DT, but a similarly low yield was also observed (supplemental Fig. S1). Thus, cloning and characterization of the specific enzyme from the fungus might lead to a new biocatalytic route of preparation for 7-hylosyltaxanes for the semi-synthesis of paclitaxel or its analogues. Here, we present a strategy in which we combine protein de novo sequencing with RT-PCR and the rapid amplification of cDNA ends (RACE) to mine the targeted β-xylosidase gene from this fungus. Moreover, yeast engineered with such a heterologous gene can robustly convert 7-β-xylosyltaxanes into 7-hydroxyltaxanes. 相似文献
5.
Nidhi Ahuja Dmitry Korkin Rachna Chaba Brent O. Cezairliyan Robert T. Sauer Kyeong Kyu Kim Carol A. Gross 《The Journal of biological chemistry》2009,284(8):5403-5413
The Escherichia coli envelope stress response is controlled by the
alternative sigma factor, σE, and is induced when unfolded
outer membrane proteins accumulate in the periplasm. The response is initiated
by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB
is an important negative regulator of envelope stress response that exerts its
negative effects onσE activity through its binding to RseA.
In this study, we analyze the interaction between RseA and RseB. We found that
tight binding of RseB to RseA required intact RseB. Using programs that
performed global and local sequence alignment of RseB and RseA, we found
regions of high similarity and performed alanine substitution mutagenesis to
test the hypothesis that these regions were functionally important. This
protocol is based on the hypothesis that functionally dependent regions of two
proteins co-evolve and therefore are likely to be sequentially conserved. This
procedure allowed us to identify both an N-terminal and C-terminal region in
RseB important for binding to RseA. We extensively analyzed the C-terminal
region, which aligns with a region of RseA coincident with the major RseB
binding determinant in RseA. Both allele-specific suppression analysis and
cysteine-mediated disulfide bond formation indicated that this C-terminal
region of similarity of RseA and RseB identifies a contact site between the
two proteins. We suggest a similar protocol can be successfully applied to
pairs of non-homologous but functionally linked proteins to find specific
regions of the protein sequences that are important for establishing
functional linkage.The Escherichia coli σE-mediated envelope stress
response is the major pathway to ensure homeostasis in the envelope
compartment of the cell
(1-3).
σE regulon members encode periplasmic chaperones and
proteases, the machinery for inserting β-barrel proteins into the outer
membrane and components controlling the synthesis and assembly of LPS
(4-6).
This pathway is highly conserved among γ-proteobacteria
(6).The σE response is initiated when periplasmic protein
folding and assembly is compromised
(7-9).
During steady state growth, σE is inhibited by its antisigma
factor, RseA, a membrane-spanning protein whose cytoplasmic domain binds to
σE with picomolar affinity
(10-13).
Accumulation of unassembled porin monomers serves as a signal to activate the
DegS protease to cleave RseA in its periplasmic domain
(14,
15). This initiates a
proteolytic cascade in which RseP cleaves periplasmically truncated RseA near
or within the cytoplasmic membrane to release the
RseAcytoplasmic-σE complex, and cytoplasmic
ATP-dependent proteases complete the degradation of RseA thereby releasing
active σE
(16-19).RseB, a second negative regulator of the envelope stress response
(11,
20,
21), binds to the periplasmic
domain of RseA with nanomolar affinity. RseB is an important regulator of the
response (2,
22,
23). It prevents RseP from
degrading intact RseA, thereby ensuring that proteolysis is initiated only
when the DegS protease is activated by a stress signal
(21). Additionally, RseB
prevents activated DegS from cleaving RseA, suggesting that interaction of
RseB with RseA must be altered before the signal transduction cascade is
activated (23).The goal of the present studies was to explore how RseB binds to RseA. The
interaction partner of RseB is the unstructured periplasmic domain of RseA
(RseA-peri). Within RseA-peri, amino acids ∼169-186 constitute a major
binding determinant to RseB
(23,
24). This peptide alone binds
RseB with 6 μm affinity, and deleting this region abrogates
binding to RseB (23).
Additional regions of RseA-peri also contribute to RseB binding, as intact
RseA-peri binds with 20 nm affinity to RseB
(23). Much less is known about
the regions of RseB required for interaction with RseA. RseB is homodimeric
two-domain protein, whose large N-terminal domain shares structural homology
with LolA, a protein that transports lipoproteins to outer membrane
(24,
25). The smaller C-terminal
domain is connected to the N-terminal domain by a linker, and the two domains
share a large interface, which may facilitate interdomain signaling.
Glutaraldehyde cross-linking studies indicate that the C-terminal domain
interacts with RseA, but the regions of interaction were not identified
(25).In the present report, we study the interaction of RseB and RseA. We
establish that both domains of RseB interact with RseA-peri. Using a global
sequence alignment, we discovered several regions in RseA and RseB that had
high sequence similarity, despite the low overall sequence similarity between
these two proteins, a finding that was independently confirmed by a local
sequence similarity algorithm. This suggested that these regions were
functionally dependent, and we performed a set of mutagenesis experiments
designed to test this idea. Our studies of the binding properties of these
mutants revealed that regions in both the N terminus and C terminus of RseB
modulate interaction with RseA. Moreover, genetic suppression analysis and
cysteine-mediated disulfide bond formation suggest that the region of RseA/B
with highest similarity (RseA residues 165-191 (major binding determinant in
RseA) and RseB residues 233-258) are interacting partners. 相似文献
6.
Background
Life cycles of medusozoan cnidarians vary widely, and have been difficult to document, especially in the most recently proposed class Staurozoa. However, molecular data can be a useful tool to elucidate medusozoan life cycles by tying together different life history stages.Methodology/Principal Findings
Genetic data from fast-evolving molecular markers (mitochondrial 16S, nuclear ITS1, and nuclear ITS2) show that animals that were presumed to be a hydrozoan, Microhydrula limopsicola (Limnomedusae, Microhydrulidae), are actually an early stage of the life cycle of the staurozoan Haliclystus antarcticus (Stauromedusae, Lucernariidae).Conclusions/Significance
Similarity between the haplotypes of three markers of Microhydrula limopsicola and Haliclystus antarcticus settles the identity of these taxa, expanding our understanding of the staurozoan life cycle, which was thought to be more straightforward and simple. A synthetic discussion of prior observations makes sense of the morphological, histological and behavioral similarities/congruence between Microhydrula and Haliclystus. The consequences are likely to be replicated in other medusozoan groups. For instance we hypothesize that other species of Microhydrulidae are likely to represent life stages of other species of Staurozoa. 相似文献7.
The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability. 相似文献
8.
Although penguins are key marine predators from the Southern Ocean, their migratory behaviour during the inter-nesting period remains widely unknown. Here, we report for the first time, to our knowledge, the winter foraging movements and feeding habits of a penguin species by using geolocation sensors fitted on penguins with a new attachment method. We focused on the macaroni penguin Eudyptes chrysolophus at Kerguelen, the single largest consumer of marine prey among all seabirds. Overall, macaroni penguins performed very long winter trips, remaining at sea during approximately six months within the limits of the Southern Ocean. They departed from Kerguelen in an eastward direction and distributed widely, over more than 3.106 km2. The penguins spent most of their time in a previously unrecognized foraging area, i.e. a narrow latitudinal band (47–49° S) within the central Indian Ocean (70–110° E), corresponding oceanographically to the Polar Frontal Zone. There, their blood isotopic niche indicated that macaroni penguins preyed mainly upon crustaceans, but not on Antarctic krill Euphausia superba, which does not occur at these northern latitudes. Such winter information is a crucial step for a better integrative approach for the conservation of this species whose world population is known to be declining. 相似文献
9.
10.
Rhynostelis Moure & Urban is a monotypic cleptoparasitic neotropical anthidiine genus currently known from two females. Herein, we describe and illustrate for the first time the male and its genitalia and it is confirmed that Rhynostelis parasitizes nests of Eufriesea. An identification key to the genera of cleptoparasitic anthidiine from the Neotropical region is also presented. 相似文献
11.
Fabrice N. Gravelat Anne Beauvais Hong Liu Mark J. Lee Brendan D. Snarr Dan Chen Wenjie Xu Ilia Kravtsov Christopher M. Q. Hoareau Ghyslaine Vanier Mirjam Urb Paolo Campoli Qusai Al Abdallah Melanie Lehoux Josée C. Chabot Marie-Claude Ouimet Stefanie D. Baptista J?rg H. Fritz William C. Nierman Jean Paul Latgé Aaron P. Mitchell Scott G. Filler Thierry Fontaine Donald C. Sheppard 《PLoS pathogens》2013,9(8)
12.
Fran?ois Hug Paul W. Hodges Timothy J. Carroll Enrico De Martino Justine Magnard Kylie Tucker 《PloS one》2016,11(4)
During a force-matched bilateral task, when pain is induced in one limb, a shift of load to the non-painful leg is classically observed. This study aimed to test the hypothesis that this adaptation to pain depends on the mechanical efficiency of the non-painful leg. We studied a bilateral plantarflexion task that allowed flexibility in the relative force produced with each leg, but constrained the sum of forces from both legs to match a target. We manipulated the mechanical efficiency of the non-painful leg by imposing scaling factors: 1, 0.75, or 0.25 to decrease mechanical efficiency (Decreased efficiency experiment: 18 participants); and 1, 1.33 or 4 to increase mechanical efficiency (Increased efficiency experiment: 17 participants). Participants performed multiple sets of three submaximal bilateral isometric plantarflexions with each scaling factor during two conditions (Baseline and Pain). Pain was induced by injection of hypertonic saline into the soleus. Force was equally distributed between legs during the Baseline contractions (laterality index was close to 1; Decreased efficiency experiment: 1.16±0.33; Increased efficiency experiment: 1.11±0.32), with no significant effect of Scaling factor. The laterality index was affected by Pain such that the painful leg contributed less than the non-painful leg to the total force (Decreased efficiency experiment: 0.90±0.41, P<0.001; Increased efficiency experiment: 0.75±0.32, P<0.001), regardless of the efficiency (scaling factor) of the non-painful leg. When compared to the force produced during Baseline of the corresponding scaling condition, a decrease in force produced by the painful leg was observed for all conditions, except for scaling 0.25. This decrease in force was correlated with a decrease in drive to the soleus muscle. These data highlight that regardless of the overall mechanical cost, the nervous system appears to prefer to alter force sharing between limbs such that force produced by the painful leg is reduced relative to the non-painful leg. 相似文献
13.
Hosts either tolerate avian brood parasitism or reject it by ejecting parasitic eggs, as seen in most rejecter hosts of common cuckoos, Cuculus canorus, or by abandoning parasitized clutches, as seen in most rejecter hosts of brown‐headed cowbirds, Molothrus ater. What explains consistent variation between alternative rejection behaviours of hosts within the same species and across species when exposed to different types of parasites? Life history theory predicts that when parasites decrease the fitness of host offspring, but not the future reproductive success of host adults, optimal clutch size should decrease. Consistent with this prediction, evolutionarily old cowbird hosts, but not cuckoo hosts, have lower clutch sizes than related rarely‐ or newly parasitized species. We constructed a mathematical model to calculate the fitness payoffs of egg ejector vs. nest abandoner hosts to determine if various aspects of host life history traits and brood parasites’ virulence on adult and young host fitness differentially influence the payoffs of alternative host defences. These calculations showed that in general egg ejection was a superior anti‐parasite strategy to nest abandonment. Yet, increasing parasitism rates and increasing fitness values of hosts’ eggs in both currently parasitized and future replacement nests led to switch points in fitness payoffs in favour of nest abandonment. Nonetheless, nest abandonment became selectively more favourable only at lower clutch sizes and only when hosts faced parasitism by a cowbird‐ rather than a cuckoo‐type brood parasite. We suggest that, in addition to evolutionary lag and gape‐size limitation, our estimated fitness differences based on life history trait variation provide new insights for the consistent differences observed in the anti‐parasite rejection strategies between many cuckoo‐ and cowbird‐hosts. 相似文献
14.
With the advance of genome-wide association studies and newly identified SNP (single-nucleotide polymorphism) associations with complex disease, important discoveries have emerged focusing not only on individual genes but on disease-associated pathways and gene sets. The authors used prospective myocardial infarction case-control studies nested in the Nurses’ Health and Health Professionals Follow-Up Studies to investigate genetic variants associated with myocardial infarction or LDL, HDL, triglycerides, adiponectin and apolipoprotein B (apoB). Using these case-control studies to illustrate an integrative systems biology approach, the authors applied SNP set enrichment analysis to identify gene sets where expression SNPs representing genes from these sets show enrichment in their association with endpoints of interest. The authors also explored an aggregate score approach. While power limited one’s ability to detect significance for association of individual loci with myocardial infarction, the authors found significance for loci associated with LDL, HDL, apoB and triglycerides, replicating previous observations. Applying SNP set enrichment analysis and risk score methods, the authors also found significance for three gene sets and for aggregate scores associated with myocardial infarction as well as for loci-related to cardiovascular risk factors, supporting the use of these methods in practice. 相似文献
15.
Hardeep Kaur Chitranshu Kumar Christophe Junot Michel B. Toledano Anand K. Bachhawat 《The Journal of biological chemistry》2009,284(21):14493-14502
GSH metabolism in yeast is carried out by the γ-glutamyl cycle as
well as by the DUG complex. One of the last steps in the
γ-glutamyl cycle is the cleavage of Cys-Gly by a peptidase to the
constitutent amino acids. Saccharomyces cerevisiae extracts carry
Cys-Gly dipeptidase activity, but the corresponding gene has not yet been
identified. We describe the isolation and characterization of a novel Cys-Gly
dipeptidase, encoded by the DUG1 gene. Dug1p had previously been
identified as part of the Dug1p-Dug2p-Dug3p complex that operates as an
alternate GSH degradation pathway and has also been suggested to function as a
possible di- or tripeptidase based on genetic studies. We show here that Dug1p
is a homodimer that can also function in a Dug2-Dug3-independent manner as a
dipeptidase with high specificity for Cys-Gly and no activity toward tri- or
tetrapeptides in vitro. This activity requires zinc or manganese
ions. Yeast cells lacking Dug1p (dug1Δ) accumulate Cys-Gly.
Unlike all other Cys-Gly peptidases, which are members of the metallopeptidase
M17, M19, or M1 families, Dug1p is the first to belong to the M20A family. We
also show that the Dug1p Schizosaccharomyces pombe orthologue
functions as the exclusive Cys-Gly peptidase in this organism. The human
orthologue CNDP2 also displays Cys-Gly peptidase activity, as seen by
complementation of the dug1Δ mutant and by biochemical
characterization, which revealed a high substrate specificity and affinity for
Cys-Gly. The results indicate that the Dug1p family represents a novel class
of Cys-Gly dipeptidases.GSH is a thiol-containing tripeptide
(l-γ-glutamyl-l-cysteinyl-glycine) present in
almost all eukaryotes (barring a few protozoa) and in a few prokaryotes
(1). In the cell, glutathione
exists in reduced (GSH) and oxidized (GSSG) forms. Its abundance (in the
millimolar range), a relatively low redox potential (-240 mV), and a high
stability conferred by the unusual peptidase-resistant γ-glutamyl bond
are three of the properties endowing GSH with the attribute of an important
cellular redox buffer. GSH also contributes to the scavenging of free radicals
and peroxides, the chelation of heavy metals, such as cadmium, the
detoxification of xenobiotics, the transport of amino acids, and the
regulation of enzyme activities through glutathionylation and serves as a
source of sulfur and nitrogen under starvation conditions
(2,
3). GSH metabolism is carried
out by the γ-glutamyl cycle, which coordinates its biosynthesis,
transport, and degradation. The six-step cycle is schematically depicted in
Fig. 1
(2).Open in a separate windowFIGURE 1.γ-Glutamyl cycle of glutathione metabolism.
γ-Glutamylcysteine synthetase and GSH synthetase carry out the first two
steps in glutathione biosynthesis. γ-glutamyltranspeptidase,
γ-glutamylcyclotransferase, 5-oxoprolinase, and Cys-Gly dipeptidase are
involved in glutathione catabolism. Activities responsible for
γ-glutamylcyclotransferase and 5-oxoprolinase have not been detected in
S. cerevisiae.In Saccharomyces cerevisiae, γ-glutamyl cyclotransferase and
5-oxoprolinase activities have not been detected, which has led to the
suggestion of the presence of an incomplete, truncated form of the
γ-glutamyl cycle (4) made
of γ-glutamyl transpeptidase
(γGT)4 and
Cys-Gly dipeptidase and only serving a GSH catabolic function. Although
γGT and Cys-Gly dipeptidase activities were detected in S.
cerevisiae cell extracts, only the γGT gene (ECM38) has
been identified so far. Cys-Gly dipeptidase activity has been identified in
humans (5,
6), rats
(7–10),
pigs (11,
12), Escherichia coli
(13,
14), and other organisms
(15,
16), and most of them belong
to the M17 or the M1 and M19 metallopeptidases gene families
(17).S. cerevisiae has an alternative γGT-independent GSH
degradation pathway (18) made
of the Dug1p, Dug2p, and Dug3p proteins that function together as a complex.
Dug1p also seem to carry nonspecific di- and tripeptidase activity, based on
genetic studies (19).We show here that Dug1p is a highly specific Cys-Gly dipeptidase, as is its
Schizosaccharomyces pombe homologue. We also show that the mammalian
orthologue of DUG1, CNDP2, can complement the defective utilization
of Cys-Gly as sulfur source of an S. cerevisiae strain lacking
DUG1 (dug1Δ). Moreover, CNDP2 has Cys-Gly dipeptidase
activity in vitro, with a strong preference for Cys-Gly over all
other dipeptides tested. CNDP2 and its homologue CNDP1 are members of the
metallopeptidases M20A family and have been known to carry carnosine
(β-alanyl-histidine) and carnosine-like (homocarnosine and anserine)
peptidase activity (20,
21). This study thus reveals
that the metallopeptidase M20A family represents a novel Cys-Gly peptidase
family, since only members of the M19, M1, and M17 family were known to carry
this function. 相似文献
16.
The DSM-IV major depression "bereavement exclusion" (BE), which recognizes that depressive symptoms are sometimes normal in recently bereaved individuals, is proposed for elimination in DSM-5. Evidence cited for the BE's invalidity comes from two 2007 reviews purporting to show that bereavement-related depression is similar to other depression across various validators, and a 2010 review of subsequent research. We examined whether the 2007 and 2010 reviews and subsequent relevant literature support the BE's invalidity. Findings were: a) studies included in the 2007 reviews sampled bereavement-related depression groups most of whom were not BE-excluded, making them irrelevant for evaluating BE validity; b) three subsequent studies cited by the 2010 review as supporting BE elimination did examine BE-excluded cases but were in fact inconclusive; and c) two more recent articles comparing recurrence of BE-excluded and other major depressive disorder cases both support the BE's validity. We conclude that the claimed evidence for the BE's invalidity does not exist. The evidence in fact supports the BE's validity and its retention in DSM-5 to prevent false positive diagnoses. We suggest some improvements to increase validity and mitigate risk of false negatives. 相似文献
17.
The three key players in the exocytotic release of neurotransmitters from
synaptic vesicles are the SNARE (soluble N-ethylmaleimide-sensitive
factor attachment protein receptor) proteins synaptobrevin 2, syntaxin 1a, and
SNAP-25. Their assembly into a tight four-helix bundle complex is thought to
pull the two membranes into close proximity. It is debated, however, whether
the energy generated suffices for membrane fusion. Here, we have determined
the thermodynamic properties of the individual SNARE assembly steps by
isothermal titration calorimetry. We found extremely large favorable enthalpy
changes counterbalanced by positive entropy changes, reflecting the major
conformational changes upon assembly. To circumvent the fact that ternary
complex formation is essentially irreversible, we used a stabilized
syntaxin-SNAP-25 heterodimer to study synaptobrevin binding. This strategy
revealed that the N-terminal synaptobrevin coil binds reversibly with
nanomolar affinity. This suggests that individual, membrane-bridging SNARE
complexes can provide much less pulling force than previously claimed.The molecular machinery that drives the Ca2+-dependent release
of neurotransmitters from synaptic vesicles is studied intensively. Three key
players in the underlying exocytotic fusion of the vesicle with the plasma
membrane are the proteins synaptobrevin 2/VAMP2 (vesicle-associated membrane
protein), syntaxin 1a, and
SNAP-253 (for review,
see Refs.
1-7).
They belong to the so-called SNARE protein family, the members of which are
involved in all vesicle fusion steps in the endocytic and secretory pathway.
In general, SNARE proteins are relatively small, tail-anchored membrane
proteins. Their key characteristic is the so-called SNARE motif, an extended
stretch of heptad repeats that is usually connected to a single transmembrane
domain by a short linker. Syntaxin and synaptobrevin each contain a single
SNARE motif, whereas SNAP-25 contains two SNARE motifs connected by a
palmitoylated linker region serving as a membrane anchor. The SNARE motifs of
the three proteins assemble into a very tight four-helix bundle between
opposing membranes; during this process the plasma membrane proteins syntaxin
and SNAP-25 provide the binding site for the vesicular synaptobrevin.
Formation of this complex is accompanied by extensive structural
rearrangements
(8-10).
Based on these findings, it was put forward that the formation of the SNARE
bundle provides the energy that drives membrane fusion. As the bundle is
oriented in parallel, it is thought that formation of this complex starts from
the membrane-proximal N termini and proceeds toward the C-terminal membrane
anchors, effectively pulling the membranes together (the “zipper”
model) (11). Although the
zipper scenario is intuitive, it has been difficult to demonstrate
directly.A decade ago it was shown that the three neuronal SNARE proteins are
sufficient to fuse artificial vesicles
(12). However, this
reductionist approach yields rather slow fusion rates
(12-14).
Over the years various different end products of SNARE catalysis (complete
fusion, hemifusion, and only tethered membranes) have been reported
(15-19).
These unsatisfactory results have fueled the debate over whether the assembly
process indeed provides enough impetus to fuse bilayers. Not surprisingly, an
alternative scenario has been put forward in which repulsive forces between
membranes bring the SNARE assembly to a grinding halt. According to this idea,
other factors like the Ca2+ sensor synaptotagmin or the small
soluble protein complexin are needed to induce membrane merging
(20-22).In simple terms, to find out whether the SNARE complex assembly is enough
for membrane fusion, only the amount of energy released during complex
formation and the amount of energy needed for membrane fusion need to be
compared. However, the physics of membrane fusion are very complicated, and it
is even more challenging to understand how proteins modulate the process. The
free energy for bilayer fusion in an aqueous environment is not very high, but
fusion is thought to require a large activation energy of about 40
kBT, as two charged membranes have to be brought into close
apposition. According to a theoretical model, the apposing membranes then need
to be modified into a stalk-like configuration. Before fusion occurs, the
process is thought to pass through a hemifusion intermediate in which only the
outer monolayers are merged (for review, see Refs.
23 and
24). The role of fusion
proteins is thought to lower the energy barrier for membrane fusion, but
understanding how they modulate the lipid membrane and how their
conformational changes are translated into a mechanical force is still in its
infancy. It is not clear, for instance, whether SNARE-catalyzed fusion indeed
proceeds through a stalk-like structure or just locally alters the membranes,
a mechanism that might need much less activation energy.As the folding and unfolding transitions of the ternary SNARE complex
exhibit a marked hysteresis
(25), the question of how much
energy is released during complex formation has been difficult to answer as
well. To avoid the quasi-irreversibility of the process, the problem has been
elegantly tackled by atomic force microscopy by two different research groups
(26-28).
In these experiments individual complexes affixed to solid supports were
ruptured, yielding energy values of 43 and 33 kBT. In another
approach, which used a surface-force apparatus (SFA), a comparable energy of
35 kBT has recently been determined
(29). Strikingly, these values
appear to correspond closely with the activation energy needed to fuse two
membranes, substantiating the view that SNAREs are nano-fusion machineries.
However, one should be cautious about the conclusion that these sophisticated
procedures in fact yield the genuine SNARE assembly energy. For example, with
the SFA approach, the number of complexes had to be deduced rather indirectly
to estimate the free energy. Moreover, these approaches offered only indirect
information about the reaction pathway.In this study we set out to determine the SNARE assembly energy more
directly by using isothermal titration calorimetry (ITC) complemented by
kinetic measurements. ITC is a powerful technique for studying the
thermodynamics of macromolecular interactions by directly measuring the heat
changes associated with complex formation, which at constant pressure is equal
to the enthalpy change (ΔH). The titration approach also yields
the stoichiometry (n), the entropy change (ΔS), and
the association constant (KA) of the reaction. We studied
the consecutive reaction steps individually to gain deeper insights into the
rugged energy landscape of complex formation. To study synaptobrevin binding
in isolation, we used a stabilized syntaxin-SNAP-25 heterodimer, which has
been shown to greatly accelerate liposome fusion rates
(30). This strategy revealed
that the N-terminal coil of synaptobrevin binds reversibly, making it feasible
to access the free energy of SNARE assembly. Overall, our results suggest that
individual SNARE complexes might provide much less pulling energy than
previously claimed. 相似文献
18.
Viperin is an evolutionarily conserved interferon-inducible protein that
localizes to the endoplasmic reticulum (ER) and inhibits a number of DNA and
RNA viruses. In this study, we report that viperin specifically localizes to
the cytoplasmic face of the ER and that an amphipathic α-helix at its N
terminus is necessary for the ER localization of viperin and sufficient to
promote ER localization of a reporter protein, dsRed. Overexpression of intact
viperin but not the amphipathic α-helix fused to dsRed induced
crystalloid ER. Consistent with other proteins that induce crystalloid ER,
viperin self-associates, and it does so independently of the amphipathic
α-helix. Viperin expression also affected the transport of soluble but
not membrane-associated proteins. Expression of intact viperin or an
N-terminal α-helix-dsRed fusion protein significantly reduced secretion
of soluble alkaline phosphatase and reduced its rate of ER-to-Golgi
trafficking. Similarly, viperin expression inhibited bulk protein secretion
and secretion of endogenous α1-antitrypsin and serum albumin
from HepG2 cells. Converting hydrophobic residues in the N-terminal
α-helix to acidic residues partially or completely restored normal
transport of soluble alkaline phosphatase, suggesting that the extended
amphipathic nature of the N-terminal α-helical domain is essential for
inhibiting protein secretion.Type I interferons are the first line of defense against viral infections.
The significance of the interferon pathway is illustrated by the
susceptibility of interferon signaling mutants to infection and by viral
mechanisms that counteract this pathway
(1,
2). Although many genes are
induced upon interferon stimulation, very few of these genes have been
functionally characterized. Viperin is highly induced by both Type I and II
interferons and has a broad range of antiviral activity, inhibiting DNA
viruses, notably human cytomegalovirus
(3); RNA viruses such as
influenza, hepatitis C virus
(HCV),2 and
alphaviruses
(4-6);
and retroviruses such as human immunodeficiency virus
(7). Upon expression, viperin
localizes to the endoplasmic reticulum (ER), where it interacts with
farnesyl-diphosphate synthase, an enzyme involved in lipid biosynthesis. This
interaction appears to result in the disruption of lipid raft microdomains and
prevention of influenza virus from budding from the plasma membrane
(4).Although recent studies have explored the antiviral functions of viperin,
the general biochemical properties of this protein remain largely undefined.
Viperin is highly conserved across both mammals and lower vertebrates and
shares homology with the MoaA family of “radical
S-adenosylmethionine” enzymes that bind Fe-S clusters
(3,
8). In addition to a putative
Fe-S cluster-binding domain, viperin has a 42-amino acid residue N-terminal
amphipathic α-helix, and similar domains in other proteins have been
shown to bind membranes and induce membrane curvature
(9,
10).In this study, we examined the role of the viperin N-terminal
α-helical domain in both cellular localization and ER membrane
morphology and analyzed the biochemical properties of viperin. We discovered
that viperin forms dimers and induces a tightly ordered, visually striking
array of ER membranes, known as crystalloid
ER(11-13),
upon overexpression. In addition, viperin expression impedes the secretion of
a variety of soluble proteins. Although the N-terminal amphipathic
α-helix is not sufficient to induce crystalloid ER formation, it is both
necessary and sufficient to mediate ER localization and to inhibit protein
secretion. 相似文献
19.
Background
Recent neuroscientific evidence suggests that empathy for pain activates similar neural representations as the first-hand experience of pain. However, empathy is not an all-or-none phenomenon but it is strongly malleable by interpersonal, intrapersonal and situational factors. This study investigated how two different top-down mechanisms – attention and cognitive appraisal - affect the perception of pain in others and its neural underpinnings.Methodology/Principal Findings
We performed one behavioral (N = 23) and two functional magnetic resonance imaging (fMRI) experiments (N = 18). In the first fMRI experiment, participants watched photographs displaying painful needle injections, and were asked to evaluate either the sensory or the affective consequences of these injections. The role of cognitive appraisal was examined in a second fMRI experiment in which participants watched injections that only appeared to be painful as they were performed on an anesthetized hand. Perceiving pain in others activated the affective-motivational and sensory-discriminative aspects of the pain matrix. Activity in the somatosensory areas was specifically enhanced when participants evaluated the sensory consequences of pain. Perceiving non-painful injections into the anesthetized hand also led to signal increase in large parts of the pain matrix, suggesting an automatic affective response to the putatively harmful stimulus. This automatic response was modulated by areas involved in self/other distinction and valence attribution – including the temporo-parietal junction and medial orbitofrontal cortex.Conclusions/Significance
Our findings elucidate how top-down control mechanisms and automatic bottom-up processes interact to generate and modulate other-oriented responses. They stress the role of cognitive processing in empathy, and shed light on how emotional and bodily awareness enable us to evaluate the sensory and affective states of others. 相似文献20.
Investigating the range and population dynamics of introduced species provides insight into species behavior, habitat preferences, and potential of becoming established. Here, we show the current population status of the red‐necked wallaby (Notamacropus rufogriseus) in Britain based on records from an eleven‐year period (2008–2018). Records were obtained from Local Environmental Records Centres (LERCs), the National Biodiversity Network (NBN), and popular media. All records were mapped and compared to a historical distribution map (1940–2007), derived from published data. A total of 95 confirmed wallaby sightings were recorded between 2008 and 2018, of which 64 came from media sources, 18 from Local Environmental Records Centres (LERCs), seven from the National Biodiversity Network (NBN), and six from the published literature (Yalden, Br. Wildl., 24, 2013, 169). The greatest density of wallaby sightings was in southern England, with the Chiltern Hills Area of Outstanding Natural Beauty a particular hot spot (n = 11). More sightings were recorded in August than in any other month. Much of the species’ ecology and responses to British biota and anthropogenic pressures are unknown, and therefore, further research is warranted. The methods used here are widely applicable to other non‐native species, particularly those that the public are more likely to report and could be an important supplement to existing studies of conservation and management relevance. 相似文献