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101.
Gromadski KB Schümmer T Strømgaard A Knudsen CR Kinzy TG Rodnina MV 《The Journal of biological chemistry》2007,282(49):35629-35637
The interactions of elongation factor 1A (eEF1A) from Saccharomyces cerevisiae with elongation factor 1Balpha (eEF1Balpha), guanine nucleotides, and aminoacyl-tRNA were studied kinetically by fluorescence stopped-flow. eEF1A has similar affinities for GDP and GTP, 0.4 and 1.1 microm, respectively. Dissociation of nucleotides from eEF1A in the absence of the guanine nucleotide exchange factor is slow (about 0.1 s(-1)) and is accelerated by eEF1Balpha by 320-fold and 250-fold for GDP and GTP, respectively. The rate constant of eEF1Balpha binding to eEF1A (10(7)-10(8) M (-1) s(-1)) is independent of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s(-1), which is compatible with the rate of protein synthesis in the cell. eEF1A.GTP binds Phe-tRNA(Phe) with a K(d) of 3 nm, whereas eEF1A.GDP shows no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu. 相似文献
102.
Unterreitmeier S Fuchs A Schäffler T Heym RG Frishman D Langosch D 《Journal of molecular biology》2007,374(3):705-718
Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context. 相似文献
103.
Hinney A Nguyen TT Scherag A Friedel S Brönner G Müller TD Grallert H Illig T Wichmann HE Rief W Schäfer H Hebebrand J 《PloS one》2007,2(12):e1361
Background
Obesity is a major health problem. Although heritability is substantial, genetic mechanisms predisposing to obesity are not very well understood. We have performed a genome wide association study (GWA) for early onset (extreme) obesity.Methodology/Principal Findings
a) GWA (Genome-Wide Human SNP Array 5.0 comprising 440,794 single nucleotide polymorphisms) for early onset extreme obesity based on 487 extremely obese young German individuals and 442 healthy lean German controls; b) confirmatory analyses on 644 independent families with at least one obese offspring and both parents. We aimed to identify and subsequently confirm the 15 SNPs (minor allele frequency ≥10%) with the lowest p-values of the GWA by four genetic models: additive, recessive, dominant and allelic. Six single nucleotide polymorphisms (SNPs) in FTO (fat mass and obesity associated gene) within one linkage disequilibrium (LD) block including the GWA SNP rendering the lowest p-value (rs1121980; log-additive model: nominal p = 1.13×10−7, corrected p = 0.0494; odds ratio (OR)CT 1.67, 95% confidence interval (CI) 1.22–2.27; ORTT 2.76, 95% CI 1.88–4.03) belonged to the 15 SNPs showing the strongest evidence for association with obesity. For confirmation we genotyped 11 of these in the 644 independent families (of the six FTO SNPs we chose only two representing the LD bock). For both FTO SNPs the initial association was confirmed (both Bonferroni corrected p<0.01). However, none of the nine non-FTO SNPs revealed significant transmission disequilibrium.Conclusions/Significance
Our GWA for extreme early onset obesity substantiates that variation in FTO strongly contributes to early onset obesity. This is a further proof of concept for GWA to detect genes relevant for highly complex phenotypes. We concurrently show that nine additional SNPs with initially low p-values in the GWA were not confirmed in our family study, thus suggesting that of the best 15 SNPs in the GWA only the FTO SNPs represent true positive findings. 相似文献104.
Schäfer C Steffen H Krzykowski KJ Göke B Groblewski GE 《American journal of physiology. Gastrointestinal and liver physiology》2003,285(4):G726-G734
Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112-2117, 2002). Investigation of the effects of phorbol ester (12-o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 microM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol. 相似文献
105.
Influence of hydrocortisone on the mechanical properties of the cerebral endothelium in vitro 总被引:4,自引:0,他引:4 下载免费PDF全文
Cerebral endothelial cells accomplish the barrier functions between blood and brain interstitium. Structural features are the tight junctions between adjacent endothelial cells and the formation of marginal folds at the cell-cell contacts. The glucocorticoid hydrocortisone (HC) has been reported to enforce the blood-brain-barrier in vitro measurable by an increase of the transendothelial electrical resistance. This study shows the impact of HC on the mechanical and morphological properties of confluent cell layers of brain microvascular endothelial cells. HC induces an increase in height of these marginal folds and a reduction of the intercellular contact surface. These morphological changes are accompanied by changes in cell elasticity. Staining of fibrous actin indicates that HC induces a reorganization of the actin cortex. The quantitative determination of the local elastic properties of cells reveals for the first time an HC-induced increase of the representative Young's modulus according to cytoskeletal rearrangements. For this study, cells of two different species, porcine brain capillary endothelial cells and murine brain capillary endothelial cells, were used yielding similar results, which clearly demonstrates that the HC effect on the cell elasticity is species independent. 相似文献
106.
Konstantin Lepikhov Michele Boiani Richard Reinhardt Hans Schöler Jörn Walter 《The EMBO journal》2010,29(11):1877-1888
In mammalian zygotes, the 5‐methyl‐cytosine (5mC) content of paternal chromosomes is rapidly changed by a yet unknown but presumably active enzymatic mechanism. Here, we describe the developmental dynamics and parental asymmetries of DNA methylation in relation to the presence of DNA strand breaks, DNA repair markers and a precise timing of zygotic DNA replication. The analysis shows that distinct pre‐replicative (active) and replicative (active and passive) phases of DNA demethylation can be observed. These phases of DNA demethylation are concomitant with the appearance of DNA strand breaks and DNA repair markers such as γH2A.X and PARP‐1, respectively. The same correlations are found in cloned embryos obtained after somatic cell nuclear transfer. Together, the data suggest that (1) DNA‐methylation reprogramming is more complex and extended as anticipated earlier and (2) the DNA demethylation, particularly the rapid loss of 5mC in paternal DNA, is likely to be linked to DNA repair mechanisms. 相似文献
107.
Mucosal T lymphocyte numbers are selectively reduced in integrin alpha E (CD103)-deficient mice. 总被引:8,自引:0,他引:8
M P Sch?n A Arya E A Murphy C M Adams U G Strauch W W Agace J Marsal J P Donohue H Her D R Beier S Olson L Lefrancois M B Brenner M J Grusby C M Parker 《Journal of immunology (Baltimore, Md. : 1950)》1999,162(11):6641-6649
The mucosal lymphocyte integrin alpha E(CD103)beta 7 is thought to be important for intraepithelial lymphocyte (IEL) localization or function. We cloned the murine integrin gene encoding alpha E, localized it to chromosome 11, and generated integrin alpha E-deficient mice. In alpha E-/- mice, intestinal and vaginal IEL numbers were reduced, consistent with the known binding of alpha E beta 7 to E-cadherin expressed on epithelial cells. However, it was surprising that lamina propria T lymphocyte numbers were diminished, as E-cadherin is not expressed in the lamina propria. In contrast, peribronchial, intrapulmonary, Peyer's patch, and splenic T lymphocyte numbers were not reduced in alpha E-deficient mice. Thus, alpha E beta 7 was important for generating or maintaining the gut and vaginal T lymphocytes located diffusely within the epithelium or lamina propria but not for generating the gut-associated organized lymphoid tissues. Finally, the impact of alpha E deficiency upon intestinal IEL numbers was greater at 3-4 wk of life than in younger animals, and affected the TCR alpha beta+ CD8+ T cells more than the gamma delta T cells or the TCR alpha beta+ CD4+CD8- population. These findings suggest that alpha E beta 7 is involved in the expansion/recruitment of TCR alpha beta+ CD8+ IEL following microbial colonization. Integrin alpha E-deficient mice will provide an important tool for studying the role of alpha E beta 7 and of alpha E beta 7-expressing mucosal T lymphocytes in vivo. 相似文献
108.
Georgakopoulos A Marambaud P Efthimiopoulos S Shioi J Cui W Li HC Schütte M Gordon R Holstein GR Martinelli G Mehta P Friedrich VL Robakis NK 《Molecular cell》1999,4(6):893-902
In MDCK cells, presenilin-1 (PS1) accumulates at intercellular contacts where it colocalizes with components of the cadherin-based adherens junctions. PS1 fragments form complexes with E-cadherin, beta-catenin, and alpha-catenin, all components of adherens junctions. In confluent MDCK cells, PS1 forms complexes with cell surface E-cadherin; disruption of Ca(2+)-dependent cell-cell contacts reduces surface PS1 and the levels of PS1-E-cadherin complexes. PS1 overexpression in human kidney cells enhances cell-cell adhesion. Together, these data show that PS1 incorporates into the cadherin/catenin adhesion system and regulates cell-cell adhesion. PS1 concentrates at intercellular contacts in epithelial tissue; in brain, it forms complexes with both E- and N-cadherin and concentrates at synaptic adhesions. That PS1 is a constituent of the cadherin/catenin complex makes that complex a potential target for PS1 FAD mutations. 相似文献
109.
Dr. A. Gebauer Dr. T. Scheper Prof. Dr. K. Schügerl 《Bioprocess and biosystems engineering》1987,2(1):13-23
E. coli ATCC 11105 was cultivated in a 10-1 stirred tank reactor and in a 60-1 tower loop reactor in batch and continuous operation. By on-line measurements of O2 and CO2 concentrations in the outlet gas, pH, temperature, cell mass concentration X as well as dissolved O2 concentration along the tower in the broth, gas holdup, broth recirculation rate through the loop and by offline measurements of substrate concentration DOC and cell mass concentration along the tower, the maximum specific growth rate
m
, yield coefficients Y
X/S. Y
X/DOC and
were evaluated in stirred tank and tower loop in batch and continuous cultures with and without motionless mixers in the tower and at different broth circulation rates through the loop. To control the accuracy of the measurements the C balance was calculated and 95% of the C content was covered.The biological parameters determined depend on the mode of operation as well as on the reactor used. Furthermore, they depend on the recirculation rate of the broth and built-ins in the tower. The unstructured cell and reactor models are unable to explain these differences. Obviously, structured cell and reactor models are needed. The cell mass concentration can be determined on line by NADH fluorescence in balanced growth, if the model parameters are determined under the same operational conditions in the same reactor.List of Symbols
a, b
empirical parameters in Eq. (1)
- CPR kg/(m3 h)
CO2 production rate
-
C kg/m3
concentration
-
D l/h
dilution rate
- DOC kg/m3
dissolved organic carbon
-
I
net. fluorescence intensity
-
K
S
kg/m3
Monod constant
-
k
L
a l/h
volumetric mass transfer coefficient
- OTR kg/(m3 h)
oxygen transfer rate
- OUR kg/(m3 h)
oxygen utilization rate
- RQ = CPR/OUR
respiratory quotient
-
S kg/m3
substrate concentration
-
t h,min, s
time
-
t
u
min
recirculation time
-
t
M
min
mixing time
-
v m3/h
volumetric flow rate through the loop
-
X kg/m3
(dry) cell mass concentration
-
Y
X/S
yield coefficient of cell mass with regard to the consumed substrate
-
Y
X/DOC
yield coefficient of the cell mass with regard to the consumed DOC
-
Y
X/O
yield coefficient of the cell mass with regard to the consumed oxygen
-
Z
relative distance in the tower from the aerator with regard to the height of the aerated broth
-
l/h
specific growth rate
-
m
l/h
maximum specific growth rate
Indices
f
feed
-
e
outlet 相似文献
110.
B. Müller E. Schlichting L. Bischoff K. Schügerl 《Applied microbiology and biotechnology》1987,26(3):206-210
Summary Penicillin G was extracted from mycelfree fermentation broths by means of the carrier (Amberlite LA-2) in n-butylacetate at pH 5 in a 7.6 m high pilot plant Karr-column with degrees of extraction E=98–99% and penicillin enrichments up to 3. The reextraction was carried out with phosphate buffer at pH-values above 7.5 with degree of extractions E=86–88% and penicillin enrichments up to 3. The penicillin and carrier losses were negligible. The influence of the process variables on the extraction degree was investigated. The penicillin extraction of the model medium and the fermentation broths were compared. Recommendations are given for the optimal penicillin recovery with reactive extraction.Symbols a
specific interfacial area with regard to the volume of the continuous phase
- cA
concentration of carrier
- cAHP,O
concentration of complex in feed
- cP,cP,O
concentration of penicillin acid anion in theaqueous phase, in the feed
-
d
32
Sauter droplet diameter
-
E
degree of extraction
-
f
stroke frequency
-
V
aq
throughput of the aqueous phase
-
V
0
throughput of the organic phase
-
Z
dimensionsless longitudinal coordinate of the column with regard to its active length (4m)
-
holdup of the organic phase 相似文献