Role of GSH in estrone sulfate binding and translocation by the multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.

Nuclear translocation of cytochrome c during apoptosis

Release of cytochrome c from mitochondria is a major event during apoptosis. Released cytochrome c has been shown to activate caspase-dependent apoptotic signals. In this report, we provide evidence for a novel role of cytochrome c in caspase-independent nuclear apoptosis. We showed that cytochrome c, released from mitochondria upon apoptosis induction, gradually accumulates in the nucleus as evidenced by both immunofluorescence and subcellular fractionation. Parallel to nuclear accumulation of cytochrome c, acetylated histone H2A, but not unmodified H2A, was released from the nucleus to the cytoplasm. Addition of purified cytochrome c to isolated nuclei recapitulated the preferential release of acetylated, but not deacetylated, histone H2A. Cytochrome c was also found to induce chromatin condensation. These results suggest that the nuclear accumulation of cytochrome c may be directly involved in the remodeling of chromatin. Our results provide evidence of a novel role for cytochrome c in inducing nuclear apoptosis.

Orexin-stimulated MAP kinase cascades are activated through multiple G-protein signalling pathways in human H295R adrenocortical cells: diverse roles for orexins A and B

Orexins A and B (ORA and ORB) are neuropeptide hormones found throughout the central nervous system and periphery. They are required for a host of physiological processes including mitogen-activated protein kinase (MAPK) regulation, steroidogenesis, appetite control and energy regulation. While some signalling mechanisms have been proposed for individual recombinant orexin receptors in generic mammalian cell types, it is clear that the peripheral effects of orexin are spatially and temporally complex. This study dissects the different G-protein signalling and MAPK pathways activated in a pluripotent human adrenal H295R cell line capable of all the physiological steps involved in steroidogenesis. Both extracellular receptor kinase 1/2 (ERK1/2) and p38 were phosphorylated rapidly with a subsequent decline, in a time- and dose-dependent manner, in response to both ORA and ORB. Conversely, there was little or no direct activation of the ERK5 or JNK pathway. Analysis using signalling and MAPK inhibitors as well as receptor-specific antagonists determined the precise mediators of the orexin response in these cells. Both ERK1/2 and p38 activation were predominantly Gq- and to a lesser extent Gs-mediated; p38 activation even had a small Gi-component. Effects were broadly comparable for both orexin sub-types ORA and ORB and although most of the effects were transmitted through the orexin receptor-1 subtype, we did observe a role for orexin receptor-2-mediated activation of both ERK1/2 and p38. Cortisol secretion also differed in response to ORA and ORB. These data suggest multiple roles for orexin-mediated MAPK activation in an adrenal cell-line, this complexity may help to explain the diverse biological actions of orexins with wide-ranging consequences for our understanding of the mechanisms initiated by these steroidogenic molecules.

Mechanism of the attenuation of proteolysis-inducing factor stimulated protein degradation in muscle by β-hydroxy-β-methylbutyrate

The leucine metabolite β-hydroxy-β-methylbutyrate (HMB) prevents muscle protein degradation in cancer-induced weight loss through attenuation of the ubiquitin-proteasome proteolytic pathway. To investigate the mechanism of this effect, the action of HMB on protein breakdown and intracellular signaling leading to increased proteasome expression by the tumor factor proteolysis-inducing factor (PIF) has been studied in vitro using murine myotubes as a surrogate model of skeletal muscle. A comparison has been made of the effects of HMB and those of eicosapentaenoic acid (EPA), a known inhibitor of PIF signaling. At a concentration of 50 μmol/L, EPA and HMB completely attenuated PIF-induced protein degradation and induction of the ubiquitin-proteasome proteolytic pathway, as determined by the "chymotrypsin-like" enzyme activity, as well as protein expression of 20S proteasome α- and β-subunits and subunit p42 of the 19S regulator. The primary event in PIF-induced protein degradation is thought to be release of arachidonic acid from membrane phospholipids, and this process was attenuated by EPA, but not HMB, suggesting that HMB might act at another step in the PIF signaling pathway. EPA and HMB at a concentration of 50 μmol/L attenuated PIF-induced activation of protein kinase C and the subsequent degradation of inhibitor κBα and nuclear accumulation of nuclear factor κB. EPA and HMB also attenuated phosphorylation of p42/44 mitogen-activated protein kinase by PIF, thought to be important in PIF-induced proteasome expression. These results suggest that HMB attenuates PIF-induced activation and increased gene expression of the ubiquitin-proteasome proteolytic pathway, reducing protein degradation.

New numerical model of stimulated Brillouin scattering in optical fibres with nonuniformity

A new numerical model which incorporates Brillouin shift frequency variations arising from fibre inhomogeneities has been developed for stimulated Brillouin scattering in optical fibres. This enables simulations of backscattered and transmitted power as functions of input power based only on known physical and material parameters as well as the polarisation factor and the measured Brillouin gain linewidth for the fibre. Agreement between modelled and experimental power characteristics for a CW input is excellent.

Simulation of optical fibre communication systems influenced by stimulated brillouin scattering

Boyd's SBS model which includes distributed thermal acoustic noise (DTAN) has been enhanced to enable the Stokes-spontaneous density depletion noise (SSDDN) component of the transmitted optical field to be simulated, probably for the first time, as well as the full transmitted field. SSDDN would not be generated from previous SBS models in which a Stokes seed replaces DTAN. SSDDN becomes the dominant form of transmitted SBS noise as model fibre length (MFL) is increased but its optical power spectrum remains independent of MFL. Simulations of the full transmitted field and SSDDN for different MFLs allow prediction of the optical power spectrum, or system performance parameters which depend on this, for typical communication link lengths which are too long for direct simulation. The SBS model has also been innovatively improved by allowing the Brillouin Shift Frequency (BS) to vary over the model fibre length, for the nonuniform fibre model (NFM) mode, or to remain constant, for the uniform fibre model (UFM) mode. The assumption of a Gaussian probability density function (pdf) for the BSF in the NFM has been confirmed by means of an analysis of reported Brillouin amplified power spectral measurements for the simple case of a nominally step-index single-mode pure silica core fibre. The BSF pdf could be modified to match the Brillouin gain spectra of other fibre types if required. For both models, simulated backscattered and output powers as functions of input power agree well with those from a reported experiment for fitting Brillouin gain coefficients close to theoretical. The NFM and UFM Brillouin gain spectra are then very similar from half to full maximum but diverge at lower values. Consequently, NFM and UFM transmitted SBS noise powers inferred for long MFLs differ by 1-2 dB over the input power range of 0.15 dBm. This difference could be significant for AM-VSB CATV links at some channel frequencies. The modelled characteristic of Carrier-to-Noise Ratio (CNR) as a function of input power for a single intensity modulated subcarrier is in good agreement with the characteristic reported for an experiment when either the UFM or NFM is used. The difference between the two modelled characteristics would have been more noticeable for a higher fibre length or a lower subcarrier frequency.

Rapid aquaporin translocation regulates cellular water flow:the mechanism of hypotonicity-induced sub-cellular localization of the aquaporin 1 water channel

The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The family's structural features and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this has only been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations, through transient receptor potential channels, that trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly-changing local cellular water availability. Moreover, since calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.

An emerging consensus on aquaporin translocation as a regulatory mechanism

Water passes through cell membranes relatively slowly by diffusion. In order to maintain water homeostasis, the rapid and specific regulation of cellular water flow is mediated by the aquaporin (AQP) family of membrane protein water channels. The wide range of tissues that are known to express AQPs is reflected by their involvement in many physiological processes and diseases; thirteen human AQPs have been identified to date and the majority are highly specific for water while others show selectivity for water, glycerol and other small solutes. Receptor mediated translocation, via hormone activation, is an established method of AQP regulation, especially for AQP2. There is now an emerging consensus that the rapid and reversible translocation of other AQPs from intracellular vesicles to the plasma membrane, triggered by a range of stimuli, confers altered membrane permeability thereby acting as a regulatory mechanism. This review examines the molecular components that may enable such AQP regulation; these include cytoskeletal proteins, kinases, calcium and retention or localization signals. Current knowledge on the dynamic regulation of sub-cellular AQP translocation in response to a specific trigger is explored in the context of the regulation of cellular water flow. © 2013 Informa UK, Ltd.

TATPred:a Bayesian method for the identification of twin arginine translocation pathway signal sequences

The twin arginine translocation (TAT) system ferries folded proteins across the bacterial membrane. Proteins are directed into this system by the TAT signal peptide present at the amino terminus of the precursor protein, which contains the twin arginine residues that give the system its name. There are currently only two computational methods for the prediction of TAT translocated proteins from sequence. Both methods have limitations that make the creation of a new algorithm for TAT-translocated protein prediction desirable. We have developed TATPred, a new sequence-model method, based on a Nave-Bayesian network, for the prediction of TAT signal peptides. In this approach, a comprehensive range of models was tested to identify the most reliable and robust predictor. The best model comprised 12 residues: three residues prior to the twin arginines and the seven residues that follow them. We found a prediction sensitivity of 0.979 and a specificity of 0.942.

The formation of phosphatidylcholine oxidation products by stimulated phagocytes

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by neutrophils and mononuclear cells, to provide a model of lipid oxidation in the absence of competing protein. Phorbol 12-myristate 13-acetate-stimulated neutrophils were incubated with phospholipid vesicles containing dipalmitoyl phosphatidylcholine, palmitoyl-arachidonoyl phosphatidylcholine (PAPC) and stearoyl-oleoyl phosphatidylcholine, before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry. The formation of monohydroperoxides (814 m/z) and bis-hydroperoxides (846 m/z) of PAPC was observed. However, the major oxidized product occurred at 828 m/z, and was identified as 1-palmitoyl-2-(5,6-epoxyisoprostane E-2)-sn-glycero-3-phosphocholine. These products were also formed in incubations where the neutrophils were replaced by mononuclear cells, and the amounts produced per million cells were similar. These results show that following oxidative attack by phagocytes stimulated by PMA, intact phospholipid oxidation products can be detected. The identification of an epoxyisoprostane phospholipid as the major product of phagocyte-induced phospholipid oxidation is novel, and in view of its inflammatory properties has implications for phagocyte involvement in atherogenesis.

Fiber-type transitions and satellite cell activation in low-frequency-stimulated muscles of young and aging rats

We examined satellite cell content and the activity of satellite cell progeny in tibialis anterior muscles of young (15 weeks) and aging (101 weeks) Brown Norway (BN) rats, after they were exposed for 50 days to a standardized and highly reproducible regime of chronic low-frequency electrical stimulation. Chronic low-frequency electrical stimulation was successful in inducing fast-to-slow fiber-type transformation, characterized by a 2.3-fold increase in the proportion of IIA fibers and fourfold and sevenfold decreases in the proportion of IID/X and IIB fibers in both young and aging BN rats. These changes were accompanied by a twofold increase in the satellite cell content in both the young and aging groups; satellite cell content reached a level that was significantly higher in the young group (p < .04). The total muscle precursor cell content (i.e., satellite cells plus progeny), however, did not differ between groups, because there was a greater number of satellite cell progeny passing through the proliferative and differentiative compartments of the aging group. The resulting 1.5-fold increase in myonuclear content was similar in the young and aging groups. We conclude that satellite cells and satellite cell progeny of aging BN rats possess an unaltered capacity to contribute to the adaptive response.