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131.
Franziska ExnowitzBernd Meyer Thomas Hackl 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(3):443-449
1H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, Km and Vmax, were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis-Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8 mM. Using the Lambert W function the parameters Km and Vmax were fitted to obtain the experimental progress curve and resulted in Km = 28 mM and Vmax = 13 μM/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding Km = 379 μM and kcat = 0.04 s− 1. The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function. 相似文献
132.
Cheng YK Beroukhim R Levine RL Mellinghoff IK Holland EC Michor F 《PLoS computational biology》2012,8(1):e1002337
Human cancer is caused by the accumulation of genetic alterations in cells. Of special importance are changes that occur early during malignant transformation because they may result in oncogene addiction and thus represent promising targets for therapeutic intervention. We have previously described a computational approach, called Retracing the Evolutionary Steps in Cancer (RESIC), to determine the temporal sequence of genetic alterations during tumorigenesis from cross-sectional genomic data of tumors at their fully transformed stage. Since alterations within a set of genes belonging to a particular signaling pathway may have similar or equivalent effects, we applied a pathway-based systems biology approach to the RESIC methodology. This method was used to determine whether alterations of specific pathways develop early or late during malignant transformation. When applied to primary glioblastoma (GBM) copy number data from The Cancer Genome Atlas (TCGA) project, RESIC identified a temporal order of pathway alterations consistent with the order of events in secondary GBMs. We then further subdivided the samples into the four main GBM subtypes and determined the relative contributions of each subtype to the overall results: we found that the overall ordering applied for the proneural subtype but differed for mesenchymal samples. The temporal sequence of events could not be identified for neural and classical subtypes, possibly due to a limited number of samples. Moreover, for samples of the proneural subtype, we detected two distinct temporal sequences of events: (i) RAS pathway activation was followed by TP53 inactivation and finally PI3K2 activation, and (ii) RAS activation preceded only AKT activation. This extension of the RESIC methodology provides an evolutionary mathematical approach to identify the temporal sequence of pathway changes driving tumorigenesis and may be useful in guiding the understanding of signaling rearrangements in cancer development. 相似文献
133.
Kriegenburg F 《The FEBS journal》2012,279(4):525
Here a brief introduction to the series is given, which highlights concepts, recent findings and current challenges in understanding chaperone function and quality control of proteins. 相似文献
134.
Franziska Theilig Irena Goranova Jochen R Hirsch Martin Wieske Semanur Unsal Sebastian Bachmann Rudiger W Veh Christian Derst 《Cellular physiology and biochemistry》2008,21(1-3):63-74
K(+)-channels fulfill several important functions in the mammalian kidney such as volume regulation, recirculation and secretion of K(+) ions, and maintaining the resting potential. In this study we used immunocytochemical methods, in situ hybridization, and nephron segment-specific RT-PCR to obtain a detailed picture of the cellular localization of two tandem pore domain potassium (K(2P)) channels, THIK-1 (K(2P)13.1, KCNK13) and THIK-2 (K(2P)12.1, KCNK12). Monospecific antibodies against C-terminal domains of rat THIK-1 and THIK-2 proteins (GST-fusion proteins) were raised in rabbits, freed from cross-reactivity, and affinity purified. All antibodies were validated by Western blot analysis, competitive ELISA, and preabsorption experiments. The expression of THIK channels in specific nephron segments was confirmed by double staining with marker proteins. Results indicate that in rat and mouse THIK-1 and THIK-2 were expressed in the proximal tubule (PT), thick ascending limb (TAL), connecting tubule (CNT), and cortical collecting duct (CCD). In human kidney THIK-1 and THIK-2 were localized in PT, TAL and CCD. Immunostaining of rat tissue revealed an intracellular expression of THIK-1 and THIK-2 throughout the identified nephron segments. However in mouse kidney THIK-2 was identified in basolateral membranes. Overall, the glomerulus, thin limbs and medullary collecting ducts were devoid of THIK-1 and THIK-2 signal. In summary, THIK-1 and THIK-2 are abundantly expressed in the proximal and distal nephron of the mammalian kidney. 相似文献
135.
Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase
Ulrich M Petre A Youhnovski N Prömm F Schirle M Schumm M Pero RS Doyle A Checkel J Kita H Thiyagarajan N Acharya KR Schmid-Grendelmeier P Simon HU Schwarz H Tsutsui M Shimokawa H Bellon G Lee JJ Przybylski M Döring G 《The Journal of biological chemistry》2008,283(42):28629-28640
Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO(-/-), gp91phox(-/-), and NOS(-/-) mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils. 相似文献
136.
Song T Mika F Lindmark B Liu Z Schild S Bishop A Zhu J Camilli A Johansson J Vogel J Wai SN 《Molecular microbiology》2008,70(1):100-111
We discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5' region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, sigma(E), suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract. 相似文献
137.
For both cells and tissues, shape is closely correlated with function presumably via geometry-dependent distribution of tension. In this study, we identify common shape determinants spanning cell and tissue scales. For cells whose sites of adhesion are restricted to small adhesive islands on a micropatterned substrate, shape resembles a sequence of inward-curved circular arcs. The same shape is observed for fibroblast-populated collagen gels that are pinned to a flat substrate. Quantitative image analysis reveals that, in both cases, arc radii increase with the spanning distance between the pinning points. Although the Laplace law for interfaces under tension predicts circular arcs, it cannot explain the observed dependence on the spanning distance. Computer simulations and theoretical modeling demonstrate that filamentous network mechanics and contractility give rise to a modified Laplace law that quantitatively explains our experimental findings on both cell and tissue scales. Our model in conjunction with actomyosin inhibition experiments further suggests that cell shape is regulated by two different control modes related to motor contractility and structural changes in the actin cytoskeleton. 相似文献
138.
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140.
We often form intentions but have to postpone them until the appropriate situation for retrieval and execution has come, an ability also referred to as event-based prospective memory. After intention completion, our cognitive system has to deactivate no-more-relevant intention representations from memory to avoid interference with subsequent tasks. In everyday life, we frequently rely on these abilities also in stressful situations. Surprisingly, little is known about potential stress effects on these functions. Therefore, the present study aimed to examine the reliability of event-based prospective memory and of intention deactivation in conditions of acute psychosocial stress. To this aim, eighty-two participants underwent the Trier Social Stress Test, a standardized stress protocol, or a standardized control situation. Following this treatment, participants performed a computerized event-based prospective memory task with non-salient and focal prospective memory cues in order to assess prospective memory performance and deactivation of completed intentions. Although the stress group showed elevated levels of salivary cortisol as marker of a stress-related increase in hypothalamus-pituitary-adrenal axis activity throughout the cognitive testing period compared to the no-stress group, prospective memory performance and deactivation of completed intentions did not differ between groups. Findings indicate that cognitive control processes subserving intention retrieval and deactivation after completion may be mostly preserved even under conditions of acute stress. 相似文献