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Oocyte Expression System (oocyte + expression_system)
Selected AbstractsThe taurine transporter: mechanisms of regulationACTA PHYSIOLOGICA, Issue 1-2 2006X. Han Abstract Taurine transport undergoes an adaptive response to changes in taurine availability. Unlike most amino acids, taurine is not metabolized or incorporated into protein but remains free in the intracellular water. Most amino acids are reabsorbed at rates of 98,99%, but reabsorption of taurine may range from 40% to 99.5%. Factors that influence taurine accumulation include ionic environment, electrochemical charge, and post-translational and transcriptional factors. Among these are protein kinase C (PKC) activation and transactivation or repression by proto-oncogenes such as WT1, c-Jun, c-Myb and p53. Renal adaptive regulation of the taurine transporter (TauT) was studied in vivo and in vitro. Site-directed mutagenesis and the oocyte expression system were used to study post-translational regulation of the TauT by PKC. Reporter genes and Northern and Western blots were used to study transcriptional regulation of the taurine transporter gene (TauT). We demonstrated that (i) the body pool of taurine is controlled through renal adaptive regulation of TauT in response to taurine availability; (ii) ionic environment, electrochemical charge, pH, and developmental ontogeny influence renal taurine accumulation; (iii) the fourth segment of TauT is involved in the gating of taurine across the cell membrane, which is controlled by PKC phosphorylation of serine 322 at the post-translational level; (iv) expression of TauT is repressed by the p53 tumour suppressor gene and is transactivated by proto-oncogenes such as WT1, c-Jun, and c-Myb; and (v) over-expression of TauT protects renal cells from cisplatin-induced nephrotoxicity. [source] Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2004Takahiro Yasuda Abstract The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel , and ,2,, subunits on expression levels and biophysical properties of three different types (Cav1.2, Cav2.1 and Cav2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Cav1.2 and Cav2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel , subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel ,1 subunit, and ,3 subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the ,2,, subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Cav2 channel family, appears to act synergistically with , subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by , and by ,2,, subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the ,2,,1 subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes. [source] Molecular mechanisms of intercellular communication in the hormonal and neural systemsIUBMB LIFE, Issue 5-6 2006Shigetada Nakanishi Abstract This paper reviews our studies that have addressed the molecular mechanisms underlying the biosynthesis and reception of extracellular signaling molecules and integrative mechanisms of extracellular-intracellular signaling transmission in biological systems. We introduced recombinant DNA technology into the neuroendocrine system and established the concept that a single peptide precursor encompasses multiple biologically active peptides and brings about coordinate functions in various biological systems. We then developed a novel functional cloning of membrane receptors and ion channels by combining an oocyte expression system with electrophysiology. We molecularly elucidated not only various peptide receptors, including the first demonstration of the molecular entity of a G protein-coupled peptide receptor (GPCR), substance K receptor, and also diverse members of both G protein-coupled metabotropic type and NMDA type of neurotransmitter glutamate receptors. We demonstrated many novel synaptic mechanisms involving distinct types of glutamate receptors in brain function and dysfunction. These include the mechanisms underlying segregation of light-dark signals in visual transmission, discrimination and memory formation in olfactory transmission, and motor co-ordination in the cerebellum, basal ganglia and the retinal network. iubmb Life, 58: 349-357, 2006 [source] CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytesTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005G. Nagel The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR,ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain. [source] Transport of di- and tripeptides in teleost fish intestineAQUACULTURE RESEARCH, Issue 5 2010Tiziano Verri Abstract The initial observation of peptide absorption in fish intestine dates back to 1981, when, in rainbow trout (Oncorhynchus mykiss), the rate of intestinal absorption of the dipeptide glycylglycine (Gly-Gly) was compared in vivo with the rate of absorption of its component amino acid glycine (Gly). The description of the identification of the underlying mechanisms that allow di- and tripeptide transport across the plasma membranes in fish was provided in 1991, when the first evidence of peptide transport activity was reported in brush-border membrane vesicles of intestinal epithelial cells of Mozambique tilapia (Oreochromis mossambicus) by monitoring uptake of radiolabelled glycyl- l -phenylalanine (Gly- l -Phe). Since then, the existence of a carrier-mediated, H+ -dependent transport of di- and tripeptides (H+/peptide cotransport) in the brush-border membrane of fish enterocytes has been confirmed in many teleost species by a variety of biochemical approaches, providing basic kinetics and substrate specificities of the transport activity. In 2003, the first peptide transporter from a teleost fish, i.e. the zebrafish (Danio rerio) PEPtide transporter 1 (PEPT1), was cloned and functionally characterized in the Xenopus laevis oocyte expression system as a low-affinity/high-capacity system. PEPT1 is the protein in brush-border membranes responsible for translocation of intact di- and tripeptides released from dietary protein by luminal and membrane-bound proteases and peptidases. The transporter possesses affinities for the peptide substrates in the 0.1,10 mM range, depending on the structure and physicochemical nature of the substrates. After the molecular and functional characterization of the zebrafish transporter, the interest in PEPT1 in teleost fish has increased and approaches for cloning and functional characterization of PEPT1 orthologues from other fish species, some of them of the highest commercial value, are now underway. In this paper, we provide a brief overview of the transport of di- and tripeptides in teleost fish intestine by recalling the bulk of biochemical, biophysical and physiological observations collected in the pre-cloning era and by recapitulating the more recent molecular and functional data. [source] | ||||||||||