Identification of significant regions of transcription factor DP-1 (TFDP-1) involved in stability/instability of the protein

We previously reported the identification of DP-1 isoforms (α and β), which are structurally C-terminus-deleted ones, and revealed the low-level expression of these isoforms. It is known that wild-type DP-1 is degraded by the ubiquitin-proteasome system, but few details are known about the domains concerned with the protein stability/instability for the proteolysis of these DP-1 isoforms. Here we identified the domains responsible for the stability/instability of DP-1. Especially, the DP-1 “Stabilon” domain was a C-terminal acidic motif and was quite important for DP-1 stability. Moreover, we propose that this DP-1 Stabilon may be useful for the stability of other nuclear proteins when fused to them.     

Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.     

TRAIL Receptors Serve as Stress-Associated Molecular Patterns to Promote ER-Stress-Induced Inflammation

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Ultrastructural changes in isolated rat hepatocytes exposed to different CCl4 concentrations

Ultrastructural data are presented on time-course changes in isolated rat hepatocyte suspensions exposed either to 1.2 or 1.8 mM CCl4 for up to 1 h. The subcellular changes at the lower concentration, but not the higher, are shown to closely parallel those reported to occur in rat hepatocytes following ingestion of CCl4.     

The role of the lipid matrix in the biosynthesis of dolichyl-linked oligosaccharides

The enzymes in the dolichol pathway are membrane-proteins that utilize a combination of hydrophilic and extremely hydrophobic substrates. The enzymes in this pathway that have been purified and characterized to any extent have either been shown to be stabilized by mixed phospholipid/detergent micelles, or else require a lipid matrix for catalytic activity. Further understanding of the mechanisms of these essential enzymes may require developing methods for the reconstitution of the glycosyltransferases and their hydrophobic substrates in appropriate lipid matrices. Abbreviations: CHO, Chinese hamster ovary; Dol, dolichol; DAG, diacylglycerol; DOPC, dioleolylphosphatidylcholine; DOPE, dioleolyphosphatidylethanolamine; ER, endoplasmic reticulum; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol This revised version was published online in November 2006 with corrections to the Cover Date.     

Gramicidin A-phospholipid model systems

Gramicidin A forms ion-conducting channels which can traverse the hydrocarbon core of lipid bilayer membranes. The structures formed by gramicidin A are among the best characterized of all membrane-bound polypeptides or proteins. In this review a brief summary is given of the occurrence, conformation, and synthesis of gramicidin A, and of its use as a model for ion transport and the interaction of proteins and lipids in biological membranes.     

Single chloride-permeable channels of large conductance in cultured cardiac cells of new-born rats

Large conductance channels were observed in the membrane of cultured cardiac cells of newborn rats studied with the patch-clamp technique in cell-attached and inside-out configurations. These channels were observed in 4% of the patches. In the cell-attached configuration they exhibited outward rectification and partial inactivation. In the inside-out configuration no rectification occurred but inactivation was present, mainly during hyperpolarizations. Two channels with large single unit conductances (400–450 pS) and one with a smaller conductance (200–250 pS) were frequently observed in the same patch. The two large channels generally had different kinetics. Under steady-state conditions the opening probability of the faster channel appeared to be voltage-independent. The slower channel was activated by depolarization. In asymmetrical solutions the permeability ratios P _Na/P _Cl were 0.03 and 0.24 for the larger and smaller channels, respectively; corresponding values for P _Ba/P _Cl were 0.04 and 0.09. It is proposed that in cardiac membranes the chloride permeability system is composed of widely dispersed microclusters forming grouped channels of different types and sizes.     

Depolarized fluorescence photobleaching recovery

The effects of the fact that the laser sources typically used in fluorescence photobleaching recovery (FPR) experiments in the most commonly employed in-line microscope imaging geometries, are highly linearly polarized, are examined in some detail. The implications of the results, in particular for the interpretation of FPR data in complex cell membrane systems in terms of laterally mobile and immobile sub-populations of the labelled molecular species of concern, are discussed. Methods of experimentally eliminating the potentially major rotational diffusion-based artifacts, different from those appropriate to three-dimensional (solution or suspension) systems which require other than in-line geometries, are delineated.Abbreviations FPR fluorescence photobleaching recovery - FRAP fluorescence recovery after photobleaching - 2- and 3-D two- and three-dimensional     

Chain length and pressure dependence of lipid translational diffusion

The translational diffusion of pyrene, pyrene butyric acid and pyrene decanoic acid has been determined in phosphatidylcholine bilayers of different chain length and under pressure up to 200 bars. In the liquid crystalline phase and at a given temperature the diffusion decreases with increasing chain length. At a constant reduced temperature, T _red (about 10 K above the transition temperature), long chain lipids exhibit the fastest diffusion which is in disagreement with hydrodynamic models but favours free volume models for diffusion in lipid bilayers. The volume of activation, V _act, calculated from the decrease of the diffusion coefficient with pressure, ln D/P, depends on lipid chain length. V _act decreases with decreasing lipid chain length at a given temperature, T=65°C, and increases at the reduced temperature. These results are again in agreement with the dependence of the diffusion on lipid chain length and therefore with the free volume model.Abbreviations DLPC Dilauroylphosphatidylcholine - DMPC Dimyristoylphosphatidylcholine - DPPC Dipalmitoylphosphatidylcholine - DSPC Distearoylphosphatidylcholine - LUV Large unilamellar vesicles - SUV Small unilamellar vesicles - Tris Tris(hydroxymethyl)aminomethan     

Solubilisation of a Glutamate Binding Protein from Rat Brain

Rat brain synaptic plasma membranes were solubilised in either 1% Triton X-100 or potassium cholate and subjected to batch affinity adsorption on L-glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L-glutamate in 0.1% detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L-[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L-glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD = 1.0 and 1.8 microM for Triton X-100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X-100-solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L-glutamate, L-aspartate, and sulphur-containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists--quisqualate, N-methyl-D-aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since solubilisation with Triton X-100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.