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排序方式: 共有177条查询结果,搜索用时 203 毫秒
31.
Salman Can Adam Fiolka Dirk Wilhelm Maria Burian Stefan von Delius Alexander Meining Armin Schneider Hubertus Feussner 《Biomedizinische Technik》2008,53(4):185-189
Abstract One of the current main challenges in transluminal surgery is in obtaining sterile and secure access to the peritoneal cavity. Since the transgastric approach has not fulfilled these requirements up to now, a new transcolonic surgical approach was developed to achieve these objectives and enhance the potential of transluminal surgery. A new set of instruments comprising an endoscopic trocar, a flexible obturator and a modified transanal endoscopic microsurgery device was designed to permit sterile sigmoid access for transcolonic surgery. The set of instruments has already been successfully tested in an experimental in vivo survival study that confirmed safety and sterility as objectives during surgical intervention. The suitability of the instruments for use in the human anatomy was confirmed by a cadaveric study. 相似文献
32.
Marion Brodhagen Dimitrios I Tsitsigiannis Ellen Hornung Cornelia Goebel Ivo Feussner Nancy P Keller 《Molecular microbiology》2008,67(2):378-391
In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans, oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene (ZmLOX3) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild-type A. nidulans, and into a DeltappoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2-3 expression was decreased when infected by A. nidulansDeltappo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross-talk in the Aspergillus-seed pathosystem. 相似文献
33.
34.
Enzymatic methods are described for the analysis of ATP, ATP + ADP, total adenylates, or P-creatine in biological samples. The methods include (i) direct fluorometric procedures for the measurement of 0.1–10 nmol using hexokinase and glucose-6-P-dehydrogenase as the indicator step; (ii) an enzymatic cycling procedure with a sensitivity of 1–50 pmol; and (iii) the measurement of light emission in the luciferin-luciferase system with a sensitivity of 0.1–80 pmol. 相似文献
35.
Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2) and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2) was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity. 相似文献
36.
Daniel Maynard Sara Mareike Müller Monika Hahmeier Jana Löwe Ivo Feussner Harald Gröger Andrea Viehhauser Karl-Josef Dietz 《Bioorganic & medicinal chemistry》2018,26(7):1356-1364
Oxidation products of the poly-unsaturated fatty acids (PUFAs) arachidonic acid, α-linolenic acid and docosahexaenoic acid are bioactive in plants and animals as shown for the cyclopentenones prostaglandin 15d-PGJ2 and PGA2, cis-(+)-12-oxophytodienoic acid (12-OPDA), and 14-A-4 neuroprostane. In this study an inexpensive and simple enzymatic multi-step one-pot synthesis is presented for 12-OPDA, which is derived from α-linolenic acid, and the analogous docosahexaenoic acid (DHA)-derived cyclopentenone [(4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl]-cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid, OCPD]. The three enzymes utilized in this multi-step cascade were crude soybean lipoxygenase or a recombinant lipoxygenase, allene oxide synthase and allene oxide cyclase from Arabidopsis thaliana. The DHA-derived 12-OPDA analog OCPD is predicted to have medicinal potential and signaling properties in planta. With OCPD in hand, it is shown that this compound interacts with chloroplast cyclophilin 20-3 and can be metabolized by 12-oxophytodienoic acid reductase (OPR3) which is an enzyme relevant for substrate bioactivity modulation in planta. 相似文献
37.
Lipoxygenases (LOXs) consist of a class of enzymes that catalyze the regio- and stereospecific dioxygenation of polyunsaturated fatty acids. Current reports propose that a conserved glycine residue in the active site of R-lipoxygenases and an alanine residue at the corresponding position in S-lipoxygenases play a crucial role in determining the stereochemistry of the product. Recently, a bifunctional lipoxygenase with a linoleate diol synthase activity from Nostoc sp. PCC7120 with R stereospecificity and the so far unique feature of carrying an alanine instead of the conserved glycine in the position of the sequence determinant for chiral specificity was identified. The recombinant carboxy-terminal domain was purified after expression in Escherichia coli. The ability of the enzyme to use linoleic acid esterified to a bulky phosphatidylcholine molecule as a substrate suggested a tail-fist binding orientation of the substrate. Site directed mutagenesis of the alanine to glycine did not cause alterations in the stereospecificity of the products, while mutation of the alanine to valine or isoleucine modified both regio- and enantioselectivity of the enzyme. Kinetic measurements revealed that substitution of Ala by Gly or Val did not significantly influence the reaction characteristics, while the A162I mutant showed a reduced vmax. Based on the mutagenesis data obtained, we suggest that the existing model for stereocontrol of the lipoxygenase reaction may be expanded to include enzymes that seem to have in general a smaller amino acid in R and a bulkier one in S lipoxygenases at the position that controls stereospecificity. 相似文献
38.
Hornung E Kunze S Liavonchanka A Zimmermann G Kühn D Fritsche K Renz A Kühn H Feussner I 《Phytochemistry》2008,69(16):2774-2780
Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes, which catalyze specific dioxygenation of polyunsaturated fatty acids. According to their positional specificity of linoleic acid oxygenation plant LOX have been classified into linoleate 9- and linoleate 13-LOX and recent reports identified a critical valine at the active site of 9-LOX. In contrast, more bulky phenylalanine or histidine residues were found at this position in 13-LOX. We have recently cloned a LOX-isoform from Momordica charantia and multiple amino acid alignments indicated the existence of a glutamine (Gln599) at the position were 13-LOX usually carry histidine or phenylalanine residues. Analyzing the pH-dependence of the positional specificity of linoleic acid oxygenation we observed that at pH-values higher than 7.5 this enzyme constitutes a linoleate 13-LOX whereas at lower pH, 9-H(P)ODE was the major reaction product. Site-directed mutagenesis of glutamine 599 to histidine (Gln599His) converted the enzyme to a pure 13-LOX. These data confirm previous observation suggesting that reaction specificity of certain LOX-isoforms is not an absolute enzyme property but may be impacted by reaction conditions such as pH of the reaction mixture. We extended this concept by identifying glutamine 599 as sequence determinant for such pH-dependence of the reaction specificity. Although the biological relevance for this alteration switch remains to be investigated it is of particular interest that it occurs at near physiological conditions in the pH-range between 7 and 8. 相似文献
39.
Hans-Henning Kunz Michael Scharnewski Kirstin Feussner Ivo Feussner Ulf-Ingo Flügge Martin Fulda Markus Gierth 《The Plant cell》2009,21(9):2733-2749
Fatty acid β-oxidation is essential for seedling establishment of oilseed plants, but little is known about its role in leaf metabolism of adult plants. Arabidopsis thaliana plants with loss-of-function mutations in the peroxisomal ABC-transporter1 (PXA1) or the core β-oxidation enzyme keto-acyl-thiolase 2 (KAT2) have impaired peroxisomal β-oxidation. pxa1 and kat2 plants developed severe leaf necrosis, bleached rapidly when returned to light, and died after extended dark treatment, whereas the wild type was unaffected. Dark-treated pxa1 plants showed a decrease in photosystem II efficiency early on and accumulation of free fatty acids, mostly α-linolenic acid [18:3(n-3)] and pheophorbide a, a phototoxic chlorophyll catabolite causing the rapid bleaching. Isolated wild-type and pxa1 chloroplasts challenged with comparable α-linolenic acid concentrations both showed an 80% reduction in photosynthetic electron transport, whereas intact pxa1 plants were more susceptible to the toxic effects of α-linolenic acid than the wild type. Furthermore, starch-free mutants with impaired PXA1 function showed the phenotype more quickly, indicating a link between energy metabolism and β-oxidation. We conclude that the accumulation of free polyunsaturated fatty acids causes membrane damage in pxa1 and kat2 plants and propose a model in which fatty acid respiration via peroxisomal β-oxidation plays a major role in dark-treated plants after depletion of starch reserves. 相似文献
40.
Florian Brodhun Cornelia G?bel Ellen Hornung Ivo Feussner 《The Journal of biological chemistry》2009,284(18):11792-11805
The homothallic ascomycete Aspergillus nidulans serves as model
organism for filamentous fungi because of its ability to propagate with both
asexual and sexual life cycles, and fatty acid-derived substances regulate the
balance between both cycles. These so-called psi (precocious
sexual inducer) factors are produced by psi
factor-producing oxygenases (Ppo enzymes). Bioinformatic analysis predicted
the presence of two different heme domains in Ppo proteins: in the N-terminal
region, a fatty acid heme dioxygenase/peroxidase domain is predicted, whereas
in the C-terminal region, a P450 heme thiolate domain is predicted. To analyze
the reaction catalyzed by Ppo enzymes, PpoA was expressed in Escherichia
coli as an active enzyme. The protein was purified by 62-fold and
identified as a homotetrameric ferric heme protein that metabolizes mono- as
well as polyunsaturated C16 and C18 fatty acids at pH
∼7.25. The presence of thiolate-ligated heme was confirmed on the basis of
sequence alignments and the appearance of a characteristic 450 nm CO-binding
spectrum. Studies on its reaction mechanism revealed that PpoA uses different
heme domains to catalyze two separate reactions. Within the heme peroxidase
domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic
acid by abstracting a H-atom from C-8 of the fatty acid, yielding a
carbon-centered radical that reacts with molecular dioxygen. In the second
reaction step, 8-hydroperoxyoctadecadienoic acid is isomerized within the P450
heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. We identify PpoA as
a bifunctional P450 fusion protein that uses a previously unknown reaction
mechanism for forming psi factors.The fungus Aspergillus nidulans (teleomorph Emericella
nidulans) is a homothallic ascomycete that has a defined sexual and
asexual developmental cycle. Therefore, it serves as a model system for the
understanding of fungal development
(1). Oxidized unsaturated fatty
acids, so-called oxylipins, derived from endogenous fatty acids were found to
influence the development of the asexual conidiophores and sexual
cleistothecia
(2–6).
Moreover, they seem to regulate the secondary metabolism of the fungus
(7). These substances were
collectively named psi factors and are primarily a mixture of hydroxylated
oleic (18:1Δ9Z;
x:yΔz denotes a fatty
acid with x carbons and y double bonds in position
z counting from the carboxyl end), linoleic
(18:2Δ9Z,12Z),
and α-linolenic
(18:3Δ9Z,12Z,15Z)
acids. They are termed psiβ, psiα, and psiγ, respectively.
Psi factors can be further classified by the number and positioning of hydroxy
groups on the fatty acid backbone: psiB (OH at C-8, e.g.
(8R)-HODE),2
psiA (OH at C-5 and C-8, e.g. (5S,8R)-DiHODE), and
psiC (OH at C-8 and the δ-lactone ring)
(8,
9).The psi factor (8R)-HODE was first discovered in the fungus
Laetisaria arvalis
(10,
11); it was later also found
in Gaeumannomyces graminis
(12,
13), where the first enzyme,
which is responsible for production of (8R)-HPODE, 7,8-LDS, was
detected (13). This
heme-containing enzyme is bifunctional because it oxidizes
18:2Δ9Z,12Z
in a first reaction step to (8R)-HPODE and subsequently isomerizes
this intermediate compound to (7S,8S)-DiHODE
(13–15).After the genome of A. nidulans was available, Keller and
co-workers (6,
16,
17) found three genes that
share a high homology with the sequence of 7,8-LDS, namely ppoA,
ppoB, and ppoC. They showed that the deletion of these genes had
a significant effect (i) on the developmental ratio between the asexual
conidiospores and sexual ascospores; (ii) on the production of psi factors;
and (iii) on the production of secondary metabolites, the mycotoxins
(6,
7,
16,
17). Furthermore, the encoded
proteins showed remarkable sequence homology to both mammalian PGHS isoforms,
enzymes that are responsible for the synthesis of prostaglandins
(18). Using the NCBI conserved
domain search analysis tool, it turned out that ppoA amino acid
residues 210–580 contain a domain similar to mammalian heme peroxidases,
whereas residues 650–1050 contain a CYPX domain, similar to
P450 heme thiolate enzymes
(16). However, for 7,8-LDS
from G. graminis, only the mammalian heme peroxidase domain is
predicted. The identity of conserved catalytic domains between Ppo enzymes and
mammalian PGHS ranges from 25 to 29% for PGHS-2 and from 25 to 26% for PGHS-1
(19). PpoA and 7,8-LDS show
42% amino acid identity.Oliw and co-workers (20)
observed that incubation of homogenates of mycelia of A. nidulans
with
18:2Δ9Z,12Z
converted the fatty acid to (8R)-HODE and
(5S,8R)-DiHODE as the major products. (8R)-HPODE,
(10R)-HODE, and (10R)-HPODE were detected as minor products.
Incubation of mycelia of Aspergillus fumigatus with deuterium-labeled
18:2Δ9Z,12Z
revealed that the synthesis of (8R)-HPODE is accomplished via
pro-S-hydrogen abstraction at C-8 and antarafacial dioxygen
insertion. (5S,8R)-DiHODE is generated via an additional
pro-S-hydrogen abstraction at C-5 of the substrate
(20,
21).Additional studies with fungal knock-out strains led to the hypothesis that
PpoA may be responsible for the synthesis of (8R)-hydroperoxides,
which are partially reduced to (8R)-hydroxides
(20). It was suggested that,
analogous with 7,8-LDS, (8R)-hydroperoxides are then converted to
5,8-dihydroxides by PpoA. Furthermore, it was concluded that ppoC may
code for linoleate (10R)-DOX
(20). Analysis of Ppo enzymes
from A. nidulans in studies published so far has been performed
either by using knock-out mutants to demonstrate the absence of a subset of
psi factors or by using crude mycelial extracts; both experimental setups have
the disadvantage of observing multiple enzymatic reactions in parallel.To characterize the biochemical properties of PpoA in more detail, we
cloned and expressed recombinant PpoA in Escherichia coli. After
purification of the enzyme by up to 62-fold, biochemical characterization was
performed. The studies revealed mechanistic as well as structural similarities
to and differences from 7,8-LDS from G. graminis. Both enzymes were
found to be homotetrameric ferric heme proteins that catalyze the synthesis of
(8R)-HPODE. Whereas G. graminis 7,8-LDS converts the
intermediate formed to (7S,8S)-DiHODE, PpoA produces
5,8-DiHODE.Using site-directed mutagenesis, we provide evidence that there are
striking differences between both enzymes regarding the catalytic reaction
cycle. Thus, we found that PpoA uses different domains to catalyze the two
reaction steps. We suggest that the DOX reaction, yielding 8-HPODE, takes
place in the N-terminal heme peroxidase domain. The isomerization of this
intermediate product to the end product, 5,8-DiHODE, is accomplished, however,
independently by the C-terminal P450 heme thiolate domain in an
8-hydroperoxide isomerase reaction.In addition, we are able to provide evidence that, during the catalysis,
PpoA generates a carbon-centered radical presumably at C-8, like G.
graminis 7,8-LDS. Furthermore, we determined the kinetic parameters for
the first reaction step. 相似文献