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941.
Marieke Koopmans Dirk Martens Rene H. Wijffels 《Marine biotechnology (New York, N.Y.)》2010,12(3):340-349
To obtain more knowledge about carbon requirements for growth by sponges, the growth rate, respiration rate, and clearance
rate was measured in situ in Haliclona oculata. We found that only 34% of the particulate carbon pumped through the sponge was used for both respiration and growth. The
net growth efficiency, being the ratio of carbon incorporated in biomass and the total carbon used by the sponge for respiration
and growth, was found to be 0.099 ± 0.013. Thus, about 10% of the total used carbon was fixed in biomass, and over 90% was
used for generating energy for growth, maintenance, reproduction, and pumping. H. oculata had 2.5 μmol C available for every micromole O2 consumed. A value of 0.75 for respiratory quotient (RQ in micromole CO2 micromole O2−1) was used for H. oculata, which is the average value reported in literature for different marine invertebrates. Thus, carbon was available
in excess to meet the respiratory demand. Oxygen was found not to be the limiting factor for growth, since only 3.3% of the
oxygen pumped through the sponge body was used. Our results indicate that both oxygen and carbon availability are not limiting.
The low growth efficiency agrees with the low growth rates found for the species used in this study. 相似文献
942.
Naarmann IS Harnisch C Müller-Newen G Urlaub H Ostareck-Lederer A Ostareck DH 《RNA (New York, N.Y.)》2010,16(11):2189-2204
Erythroid precursor cells lose the capacity for mRNA synthesis due to exclusion of the nucleus during maturation. Therefore, the stability and translation of mRNAs that code for specific proteins, which function in late stages of maturation when reticulocytes become erythrocytes, are controlled tightly. Reticulocyte 15-lipoxygenase (r15-LOX) initiates the breakdown of mitochondria in mature reticulocytes. Through the temporal restriction of mRNA translation, the synthesis of r15-LOX is prevented in premature cells. The enzyme is synthesized only in mature reticulocytes, although r15-LOX mRNA is already present in erythroid precursor cells. Translation of r15-LOX mRNA is inhibited by hnRNP K and hnRNP E1, which bind to the differentiation control element (DICE) in its 3' untranslated region (3'UTR). The hnRNP K/E1-DICE complex interferes with the joining of the 60S ribosomal subunit to the 40S subunit at the AUG. We took advantage of the inducible human erythroid K562 cell system that fully recapitulates this process to identify so far unknown factors, which are critical for DICE-dependent translational regulation. Applying RNA chromatography with the DICE as bait combined with hnRNP K immunoprecipitation, we specifically purified the DEAD-box RNA helicase 6 (DDX6) that interacts with hnRNP K and hnRNP E1 in a DICE-dependent manner. Employing RNA interference and fluorescence in situ hybridization, we show that DDX6 colocalizes with endogenous human (h)r15-LOX mRNA to P-body-like RNP granules, from which 60S ribosomal subunits are excluded. Our data suggest that in premature erythroid cells translational silencing of hr15-LOX mRNA is maintained by DDX6 mediated storage in these RNP granules. 相似文献
943.
Dirk Repsilber Sabine Kern Anna Telaar Gerhard Walzl Gillian F Black Joachim Selbig Shreemanta K Parida Stefan HE Kaufmann Marc Jacobsen 《BMC bioinformatics》2010,11(1):27
Background
For heterogeneous tissues, such as blood, measurements of gene expression are confounded by relative proportions of cell types involved. Conclusions have to rely on estimation of gene expression signals for homogeneous cell populations, e.g. by applying micro-dissection, fluorescence activated cell sorting, or in-silico deconfounding. We studied feasibility and validity of a non-negative matrix decomposition algorithm using experimental gene expression data for blood and sorted cells from the same donor samples. Our objective was to optimize the algorithm regarding detection of differentially expressed genes and to enable its use for classification in the difficult scenario of reversely regulated genes. This would be of importance for the identification of candidate biomarkers in heterogeneous tissues. 相似文献944.
Jelley RA Elliott J Gibson KR Harrison T Beher D Clarke EE Lewis HD Shearman M Wrigley JD 《Bioorganic & medicinal chemistry letters》2006,16(14):3839-3842
Previously, chemistry effort on the gem-cyclohexane series of gamma-secretase inhibitors has focused on the 4-position of the cyclohexane ring. Recently chemistry has been directed towards the 3-position and substitution here has also provided compounds with high gamma-secretase activity. 相似文献
945.
Churcher I Beher D Best JD Castro JL Clarke EE Gentry A Harrison T Hitzel L Kay E Kerrad S Lewis HD Morentin-Gutierrez P Mortishire-Smith R Oakley PJ Reilly M Shaw DE Shearman MS Teall MR Williams S Wrigley JD 《Bioorganic & medicinal chemistry letters》2006,16(2):280-284
The protease gamma-secretase plays a pivotal role in the synthesis of pathogenic amyloid-beta in Alzheimer's disease (AD). Here, we report a further extension to a series of cyclohexyl sulfone-based gamma-secretase inhibitors which has allowed the preparation of highly potent compounds which also demonstrate robust Abeta(40) lowering in vivo (e.g., compound 32, MED 1mg/kg p.o. in APP-YAC mice). 相似文献
946.
Frederik A. R. Muylle Dirk Adriaensen Wim De Coen Jean-Pierre Timmermans Ronny Blust 《Biometals》2006,19(4):437-450
Intracellular zinc levels are homeostatically regulated and although most is bound, a pool of labile Zn(II) is present in
cells. We show here that the zinc probe FluoZin-3 is useful to monitor zinc fluxes during fluorescent imaging of the trout
hepatic cell line D11. Nuclei and bulk cytosol appeared to lack detectable labile zinc, while the punctuate staining pattern
colocalized with a lysosome-specific probe. Applying extracellular zinc alone resulted in vesicular sequestration of the metal
ion. Together with Na-pyrithione a delayed and toxic rise in cellular fluorescence was triggered. When using another ionophore,
4-Br A23187, a zinc buffering effect of the vesicular pools was evident. Secondly, N-ethylmaleimide induced a homogeneous fluorescence rise, which was strongly enhanced by addition of Zn-pyrithione and disappeared
after TPEN washing. This suggests the involvement of thiol residues in controlling available cytosolic zinc. Our observations
have implications for the interpretation of calculated intracellular Zn2+ concentrations. 相似文献
947.
The catalytic domain of a mosquitocidal toxin prolonged by a C-terminal 44 residue linker connecting to four ricin B-like domains was crystallized. Three crystal structures were established at resolutions between 2.5A and 3.0A using multi-wavelength and single-wavelength anomalous X-ray diffraction as well as molecular replacement phasing techniques. The chainfold of the toxin fragment corresponds to those of ADP-ribosylating enzymes. At pH 4.3 the fragment is associated in a C(7)-symmetric heptamer in agreement with an aggregate of similar size observed by size-exclusion chromatography. In two distinct crystal forms, the heptamers formed nearly spherical, D(7)-symmetric tetradecamers. Another crystal form obtained at pH 6.3 contained a recurring C(2)-symmetric tetramer, which, however, was not stable in solution. On the basis of the common chainfold and NAD(+)-binding site of all ADP-ribosyl transferases, the NAD(+)-binding site of the toxin was assigned at a high confidence level. In all three crystal forms the NAD(+) site was occupied by part of the 44 residue linker, explaining the known inhibitory effect of this polypeptide region. The structure showed that the cleavage site for toxin activation is in a highly mobile loop that is exposed in the monomer. Since it contains the inhibitory linker as a crucial part of the association contact, the observed heptamer is inactive. Moreover, the heptamer cannot be activated by proteolysis because the activation loop is at the ring center and not accessible for proteases. Therefore the heptamer, or possibly the tetradecamer, seems to represent an inactive storage form of the toxin. 相似文献
948.
Vercammen D Belenghi B van de Cotte B Beunens T Gavigan JA De Rycke R Brackenier A Inzé D Harris JL Van Breusegem F 《Journal of molecular biology》2006,364(4):625-636
Metacaspases are distant relatives of animal caspases found in plants, fungi and protozoa. We demonstrated previously that two type II metacaspases of Arabidopsis thaliana, AtMC4 and AtMC9 are Arg/Lys-specific cysteine-dependent proteases. We screened a combinatorial tetrapeptide library of 130,321 substrates with AtMC9. Here, we show that AtMC9 is a strict Arg/Lys-specific protease. Based on the position-specific scoring matrix derived from the substrate library results, the tetrapeptide Val-Arg-Pro-Arg was identified as an optimized substrate. AtMC9 had a kcat/KM of 4.6x10(5) M-1 s-1 for Ac-Val-Arg-Pro-Arg-amido-4-methyl-coumarin, representing a more than 10-fold improvement over existing fluorogenic substrates. A yeast two-hybrid screen with catalytically inactive AtMC9 as bait identified a serine protease inhibitor, designated AtSerpin1, which was found to be a potent inhibitor of AtMC9 activity in vitro through cleavage of its reactive center loop and covalent binding to AtMC9. On the basis of the substrate profiling of AtMC9 and confirmation through site-directed mutagenesis, the inhibitory P4-P1 cleavage site of AtSerpin1 was determined to be Ile-Lys-Leu-Arg351. Further mutagenesis of the AtSerpin1 inhibitory cleavage site modulated AtMC9 inhibition positively or negatively. Both AtMC9 and AtSerpin1 were localized in the extracellular space, suggesting an in vivo interaction as well. To our knowledge, this is the first report of plant protease inhibition by a plant serpin. 相似文献
949.
Liver transduction with recombinant adeno-associated virus is primarily restricted by capsid serotype not vector genotype 总被引:4,自引:0,他引:4 下载免费PDF全文
We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 x 10(10) to 1.8 x 10(12) particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in approximately 80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV. 相似文献
950.
Xu L Renaud L Müller JG Baicu CF Bonnema DD Zhou H Kappler CS Kubalak SW Zile MR Conway SJ Menick DR 《The Journal of biological chemistry》2006,281(45):34430-34440
The Na+-Ca2+ exchanger (NCX1) is up-regulated in hypertrophy and is often found up-regulated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. We have previously shown that the 1831-bp Ncx1 H1 (1831Ncx1) promoter directs cardiac-specific expression of the exchanger in both development and in the adult, and is sufficient for the up-regulation of Ncx1 in response to pressure overload. Here, we utilized adenoviral mediated gene transfer and transgenics to identify minimal regions and response elements that mediate Ncx1 expression in the heart. We demonstrate that the proximal 184 bp of the Ncx1 H1 (184Ncx1) promoter is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for up-regulation in response to pressure overload. Mutational analysis revealed that both the -80 CArG and the -50 GATA elements were required for expression in isolated adult cardiomyocytes. Chromatin immunoprecipitation assays in adult cardiocytes demonstrate that SRF and GATA4 are associated with the proximal region of the endogenous Ncx1 promoter. Transgenic lines were established for the 1831Ncx1 promoter-luciferase containing mutations in the -80 CArG or -50 GATA element. No luciferase activity was detected during development, in the adult, or after pressure overload in any of the -80 CArG transgenic lines. The Ncx1 -50 GATA mutant promoter was sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in development and for up-regulation in response to pressure overload but importantly, expression was no longer cardiac restricted. This work is the first in vivo study that demonstrates which cis elements are important for Ncx1 regulation. 相似文献