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831.
In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus. 相似文献
832.
The allelopathic activity of the aquatic macrophyte, Stratiotes aloides, was determined with laboratory experiments. Active compounds exuded in the medium or present in plant tissue were extracted
using standard procedures and solid phase extraction (SPE). The activity towards various cyanobacteria and chlorophytes was
tested in two different bioassay systems using agar plates and liquid cultures of phytoplankton. Extracts and exudates of
S. aloides affected phytoplankton growth. SPE-enriched exudates and enriched water from a natural Stratiotes stand caused inhibition of target species, however, also some controls were active. Phytoplankton species exhibited differential
sensitivity to extracts of S. aloides. We observed inhibitory and stimulatory effects on phytoplankton. In general, more cyanobacteria than other phytoplankton
species were inhibited, and the inhibition of cyanobacteria was stronger. In most cases, nutrient (P or K) limitation of Synechococcus elongatus and Scenedesmus obliquus decreased the sensitivity of these species towards allelochemicals from Stratiotes aloides, except for P-limited cultures of Scenedesmus. The allelopathically active compound(s) from Stratiotes are moderately lipophilic and most likely no phenolic compounds. Our results indicate that allelopathy (besides nutrient
interference and shading) might also account for the low phytoplankton and filamentous algae densities in the vicinity of
Stratiotes plants, at least during certain phases of the life-cycle of Stratiotes. 相似文献
833.
Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from 14C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD+, indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD+/NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB. 相似文献
834.
Deschner J Rath-Deschner B Wypasek E Anghelina M Sjostrom D Agarwal S 《Journal of biomechanics》2007,40(7):1541-1549
We sought to examine whether cyclic tensile strain (CTS) regulates the gene expression of tumor necrosis factor (TNF)-alpha, its receptors TNFR1 and TNFR2, and inducible nitric oxide synthase (iNOS) under inflammatory conditions, and whether these effects of CTS are sustained. Rat temporomandibular joint disc cells (TDC) were exposed to CTS in the presence or absence of interleukin (IL)-1beta for 4 and 24h. Cells were also stimulated with IL-1beta for 24h while being subjected to CTS only for the initial 1, 2, 4, 8, and 12h or the entire 24h incubation time. Furthermore, cells were incubated with IL-1beta for 24, 36, or 48 h while being exposed to CTS only for the initial 8h. Gene expression of TNF-alpha, its receptors, and iNOS was analyzed by RT-PCR, whereas protein synthesis was determined by ELISA for TNF-alpha, immunofluorescence for TNFRs, and Griess reaction for nitric oxide. CTS inhibited the IL-1beta-stimulated synthesis of TNF-alpha, TNFR2, and iNOS. TNFR1 was constitutively expressed but not regulated by IL-1beta or CTS. Application of CTS for only 1 or 2h during a 24h incubation with IL-1beta was sufficient to inhibit IL-1beta-induced upregulation of TNF-alpha, TNFR2, and iNOS. However, for maximal inhibition of these genes a longer exposure of CTS was required. These findings are the first to show that biomechanical signals regulate the expression of TNFR2 but not TNFR1 under inflammatory conditions. Furthermore, the antiinflammatory effects of biomechanical signals on TDC are maintained for prolonged periods of time but are transient. 相似文献
835.
836.
Bacteria-derived peptidoglycans constitute pathogen-associated molecular patterns triggering innate immunity in Arabidopsis 总被引:2,自引:0,他引:2
Gust AA Biswas R Lenz HD Rauhut T Ranf S Kemmerling B Götz F Glawischnig E Lee J Felix G Nürnberger T 《The Journal of biological chemistry》2007,282(44):32338-32348
837.
Volatiles of Stenotrophomonas, Serratia, and Bacillus species inhibited mycelial growth of many fungi and Arabidopsis thaliana (40 to 98%), and volatiles of Pseudomonas species and Burkholderia cepacia retarded the growth to lesser extents. Aspergillus niger and Fusarium species were resistant, and B. cepacia and Staphylococcus epidermidis promoted the growth of Rhizoctonia solani and A. thaliana. Bacterial volatiles provide a new source of compounds with antibiotic and growth-promoting features. 相似文献
838.
Bacterial antagonists are bacteria that negatively affect the growth of other organisms. Many antagonists inhibit the growth
of fungi by various mechanisms, e.g., secretion of lytic enzymes, siderophores and antibiotics. Such inhibition of fungal
growth may indirectly support plant growth. Here, we demonstrate that small organic volatile compounds (VOCs) emitted from
bacterial antagonists negatively influence the mycelial growth of the soil-borne phytopathogenic fungus Rhizoctonia solani Kühn. Strong inhibitions (99–80%) under the test conditions were observed with Stenotrophomonas maltophilia R3089, Serratia plymuthica HRO-C48, Stenotrophomonas rhizophila P69, Serratia odorifera 4Rx13, Pseudomonas trivialis 3Re2-7, S. plymuthica 3Re4-18 and Bacillus subtilis B2g. Pseudomonas fluorescens L13-6-12 and Burkholderia cepacia 1S18 achieved 30% growth reduction. The VOC profiles of these antagonists, obtained through headspace collection and analysis
on GC-MS, show different compositions and complexities ranging from 1 to almost 30 compounds. Most volatiles are species-specific,
but overlapping volatile patterns were found for Serratia spp. and Pseudomonas spp. Many of the bacterial VOCs could not be identified for lack of match with mass-spectra of volatiles in the databases. 相似文献
839.
Rosker C Lohberger B Hofer D Steinecker B Quasthoff S Schreibmayer W 《American journal of physiology. Cell physiology》2007,293(2):C783-C789
The blocking efficacy of 4,9-anhydro-TTX (4,9-ah-TTX) and TTX on several isoforms of voltage-dependent sodium channels, expressed in Xenopus laevis oocytes, was tested (Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, and Nav1.8). Generally, TTX was 40–231 times more effective, when compared with 4,9-ah-TTX, on a given isoform. An exception was Nav1.6, where 4,9-ah-TTX in nanomole per liter concentrations sufficed to result in substantial block, indicating that 4,9-ah-TTX acts specifically at this peculiar isoform. The IC50 values for TTX/4,9-ah-TTX were as follows (in nmol/l): 7.8 ± 1.3/1,260 ± 121 (Nav1.2), 2.8 ± 2.3/341 ± 36 (Nav1.3), 4.5 ± 1.0/988 ± 62 (Nav1.4), 1,970 ± 565/78,500 ± 11,600 (Nav1.5), 3.8 ± 1.5/7.8 ± 2.3 (Nav1.6), 5.5 ± 1.4/1,270 ± 251 (Nav1.7), and 1,330 ± 459/>30,000 (Nav1.8). Analysis of approximal half-maximal doses of both compounds revealed minor effects on voltage-dependent activation only, whereas steady-state inactivation was shifted to more negative potentials by both TTX and 4,9-ah-TTX in the case of the Nav1.6 subunit, but not in the case of other TTX-sensitive ones. TTX shifted steady-state inactivation also to more negative potentials in case of the TTX-insensitive Nav1.5 subunit, where it also exerted profound effects on the time course of recovery from inactivation. Isoform-specific interaction of toxins with ion channels is frequently observed in the case of proteinaceous toxins. Although the sensitivity of Nav1.1 to 4,9-ah-TTX is not known, here we report evidence on a highly isoform-specific TTX analog that may well turn out to be an invaluable tool in research for the identification of Nav1.6-mediated function, but also for therapeutic intervention. sodium channel; tetrodotoxin 相似文献
840.
Localization of the iron-regulatory proteins hemojuvelin and transferrin receptor 2 to the basolateral membrane domain of hepatocytes 总被引:1,自引:1,他引:0
Merle U Theilig F Fein E Gehrke S Kallinowski B Riedel HD Bachmann S Stremmel W Kulaksiz H 《Histochemistry and cell biology》2007,127(2):221-226
The newly discovered proteins hemojuvelin (Hjv) and transferrin receptor type 2 (TfR2) are involved in iron metabolism. Mutations
in the Hjv and TfR2 gene cause hemochromatosis. We investigated the expression and cellular localization of Hjv and TfR2 in
rat and human liver. The expression of Hjv and TfR2 was shown on mRNA and protein level by RT–PCR and immunoblot experiments.
Their cellular localization was studied by immunofluorescence with antibodies raised against Hjv and TfR2. Hjv and TfR2 are
present in human and rat liver and in primary human hepatocytes. Antisera raised against Hjv identified immunoreactive proteins
with an apparent size of 44 and 46 kDa in immunoblot experiments of rat and human liver extracts, which are in accordance
with the putative membrane-bound and cleaved soluble forms of this protein, respectively. TfR2 was detected as a 105 kDa protein
corresponding to the predicted size of glycosylated TfR2 monomers. In immunofluorescence experiments, Hjv and TfR2 were found
in rat liver only in hepatocytes. At the subcellular level, both proteins were predominantly localized to the basolateral
membrane domain of hepatocytes. The localization of Hjv and TfR2 at the same membrane domain renders a functional interaction
of these two proteins in iron homeostasis possible.
Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .
Kulaksiz and Stremmel contributed equally to this work. 相似文献