We have determined, for the first time, the enthalpic contributions to the energy change associated with ligand reorganization (LR) upon the binding of the same ligand to multiple sites within human serum albumine (HSA). Quantum mechanics based density functional theory (DFT) has been used for the LR calculations, which provides much better accuracy than previously used molecular mechanics methods (MM). Our findings show that for some ligands these enthalpic contributions can be attributed to specific structural and conformational changes. 相似文献
For many types of cells, heat stress leads to an increase in intracellular free calcium concentration ([Ca2+](i)) that has been shown to trigger a wide variety of cellular responses. In T lymphocytes, for example, heat stress stimulates pathways that make them more susceptible to Fas/CD95-mediated apoptosis. Because of our interest in understanding more about the response of lymphocytes to various stressors, we used human peripheral and Jurkat T lymphocytes to investigate the effect of heat stress on calcium homeostasis. We found that peripheral and Jurkat T cells both exhibit cNOs activity but not iNOs activity. Heat stress increased NO production, which was inhibited by LNNA (a cNOs inhibitor) but not L-NIL (an iNOs inhibitor). Heat stress increased [Ca2+](i) in Jurkat T cells by decreasing the K(m) of the cell surface membrane Na+/Ca2+ exchanger for extracellular Ca2+. Heating also increased cNOs phosphorylation at tyrosine residues. In cells incubated with LNNA, heat stress promoted an increase in [Ca2+](i) and a decrease in [Na+](i) greater than in cells heated without LNNA, a larger decrease in K(m) of the Na+/Ca2+ exchanger for extracellular Ca2+, and decreased phosphorylation of cNOs. Our results suggest that cNOs plays an important regulatory role after heat stress. Heating appears to increase the phosphorylation of cNOs that is complexed with the Na+/Ca2+ exchanger to decrease its activity. This process is related to increased expression of Fas/CD95 on the cell surface, which might explain the apoptotic diathesis of lymphocytes after heat stress. 相似文献
Fas/Fas ligand (Fas L) death pathway is an important mediator of apoptosis. Deregulation of Fas pathway is reported to be involved in the immune escape of breast cancer and the resistance to anti-cancer drugs. In this study, we demonstrated that conditioned medium by normal breast epithelial cells (NBEC-CM) induced apoptosis of MCF-7 and T-47D Fas-sensitive cells but had no effect on MDA-MB-231 Fas-resistant cells. Inhibition of PI3 kinase or NF-kappaB by specific inhibitors or transient transfections restored the sensitivity of MDA-MB-231 cells to NBEC-induced apoptosis. Moreover, the constitutive activation of NF-kappaB was controlled by PI3 kinase because inhibition of PI3 kinase reduced NF-kappaB activity. Inducible activation of NF-kappaB rendered MCF-7 cells resistant to NBEC-CM- and Fas agonist antibody-triggered apoptosis. Therefore, constitutive or inducible activation of PI3 kinase and/or NF-kappaB in breast cancer cells rendered them resistant to NBEC-triggered apoptosis. In addition, Fas neutralizing antibody and dominant negative Fas abolished NBEC-triggered apoptosis. Western blot and confocal microscopy analysis showed an increase of membrane Fas/Fas L when cells were induced into apoptotis by NBEC-CM. Taken together, these data show that NBEC induced apoptosis in breast cancer cells via Fas signaling. 相似文献
The application of Ernst angle pulses in multidimensional NMR spectroscopy is theoretically and experimentally investigated. Theory shows that only for a few pulse sequences employed at high repetition rate, a remarkable gain in sensitivity is possible using Ernst angle pulses. As an example, a new variant of the heteronuclear multiple quantum coherence (HMQC) experiment, the fast-(1H,15N)-HMQC, is described. This sequence allows, with a 1 mM protein sample in H2O, the acquisition of a highly resolved two-dimensional (1H,15N) correlated spectrum within 37 s. The high efficiency of the fast-HMQC to detect ligand binding to a target protein is demonstrated. 相似文献
Large-ligand adsorption to membranes or cells is considered in the absence of cooperative interactions. For the low-saturation regime, a general and exact treatment is given by means of the concept of excluded areas. With the help of this formalism, shape dependence of the adsorption behavior can be discussed quantitatively. In addition, a formalism is presented which allows to calculate binding curves at arbitrary saturation for ligands having a symmetric shape (disks, regular polygons). The underlying model is a modified version of the hard core fluid theory of Andrews (Andrews, F.C. (1976) J. Chem. Phys. 64, 1941–1947). Apart from applications to symmetric ligands, the results can be used to derive limiting conditions for ligands of any shape. 相似文献
The idea that the mechanism of the very low energy fluxional behaviour of [Fe3(CO)12] may be based on the movement of the ligand icosahedron about the central Fe3 triangle which corresponds to a rotational symmetry operation of the CO ligand icosahedron is discussed and rejected. 相似文献
Aim: Stimulation of Fas death receptor is introduced as a major cause of non-alcoholic steatohepatitis (NASH) progression through suppression of cell viability. Therefore, the blocking of death pathways is hypothesised to be express new approaches to NASH therapy. For this purpose, current experiment applied synthetic small interference RNA (SiRNA) to trigger Fas death receptor and to show its potential therapeutic role in designed NASH model.
Methods: Male mice were placed on a western diet (WD) for 8 weeks and exposed to cigarette smoke during the last 4 weeks of feeding to induce NASH model. In the next step, Fas SiRNA was injected to mice aiming to examine specific Fas gene silencing, after 8 weeks. As a control, mice received scrambled SiRNA. Reversible possibility of disease was examined by 3 weeks of recovery.
Results: Analysis of data is accompanied with the significant histopathological changes (steatosis, ballooning and inflammation), increased lipid profile and hepatic enzyme activities (AST, ALT, ALP) plus TBARS as well as decreased antioxidants levels in NASH model. Upon Fas-SiRNA injection, almost all measured parameters of NASH such as overexpression of Fas receptor, caspase3, NF-kB genes and marked increase of hepatic TNF-α were significantly restored and were remained nearly unchanged following recovery liking as scrambled groups.
Conclusions: The suppression of Fas receptor signalling subsequent RNAi therapy may represent an applicable strategy to decline hepatocyte damages and so NASH progression in mice. 相似文献
Nitrilotriacetate (NTA)-mediated capture of a histidine-tagged protein is widely used as an easy and simple method to reversibly immobilize the protein onto a sensor chip for surface plasmon resonance (SPR). However, in spite of its advantages, the NTA-capturing strategy is rarely employed for ligand screening experiments using SPR, because it was thought to cause substantial errors in binding responses, due to the inevitable protein dissociation during the monitoring period. In this study, as demonstrated in a ligand screening for the histidine-tagged SH3 domain of the human phosphatidylinositol 3-kinase p85alpha subunit, false responses after adhesion of undesirable compounds to a target protein could be minimized with the NTA strategy, while binding responses of a positive control peptide still stayed within a 1%-deviation against the theoretical binding capacity. 相似文献