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Cell-surface Expression (cell-surface + expression)
Selected AbstractsCell-surface phytase on Pichia pastoris cell wall offers great potential as a feed supplementFEMS MICROBIOLOGY LETTERS, Issue 1 2010Piyanun Harnpicharnchai Abstract Cell-surface expression of phytase allows the enzyme to be expressed and anchored on the cell surface of Pichia pastoris. This avoids tedious downstream processes such as purification and separation involved with extracellular expression. In addition, yeast cells with anchored proteins can be used as a whole-cell biocatalyst with high value added. In this work, the phytase was expressed on the cell surface of P. pastoris with a glycosylphosphatidylinositol anchoring system. The recombinant phytase was shown to be located at the cell surface. The cell-surface phytase exhibited high activity with an optimal temperature at 50,55 °C and two optimal pH peaks of 3 and 5.5. The surface-displayed phytase also exhibited similar pH stability and pepsin resistance to the native and secreted phytase. In vitro digestibility test showed that P. pastoris containing cell-surface phytase released phosphorus from feedstuff at a level similar to secreted phytase. Yeast cells expressing phytase also provide additional nutrients, especially biotin and niacin. Thus, P. pastoris with phytase displayed on its surface has a great potential as a whole-cell supplement to animal feed. [source] Mechanisms determining cell membrane expression of different ,, TCR chain pairingsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2009Laurent Boucontet Abstract We investigated the ability of the most common TCR-, and , chains to express on the cell surface. V,1C,4 and V,7C,1 chains paired with all TCR-, chains tested, whereas V,4C,1 chains were found with V,4 and V,5, but not with V,2 or V,6 chains, and V,2C,2 chains were expressed only with V,5. Mapping studies showed that up to four polymorphic residues influence the different co-expressions of V,1 and V,2 chains with V, chains. Unexpectedly, these residues are not located in the canonical ,/, interface, but in the outer part of the ,, TCR complex exposed to the solvent. Expression of functional V,4 or V,6 chains in V,2/V,5+ cells or of functional V,2C,2 in V,1+ cells reduced cell-surface expression of the ,, TCR. Taken together, these data show that (i) the V,/V, repertoire of mouse ,, T cells is reduced by physical constraints in their associations. (ii) Lack of V,2/V, expression is due to the formation of aberrant TCR complexes, rather than to an intrinsic inability of the chains to pair and (iii) despite not being expressed at the cell surface, the presence of a functionally rearranged V,2 chain in ,, T cells results in reduced TCR levels. [source] Long-term potentiation of mGluR1 activity by depolarization-induced Homer1a in mouse cerebellar Purkinje neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2003Itsunari Minami Abstract Metabotropic glutamate receptor 1 (mGluR1) plays a crucial role in synaptic plasticity and motor learning in the cerebellum. We have studied activity-dependent changes in mGluR1 function in mouse cultured Purkinje neurons. Depolarizing stimulation potentiated Ca2+ and current responses to an mGluR1 agonist for several hours in the cultured Purkinje neurons. It also blocked internalization of mGluR1 and increased the number of mGluR1s on the cell membrane. We found that depolarization simultaneously increased transcription of Homer1a in Purkinje neurons. Homer1a inhibited internalization and increased cell-surface expression of mGluR1 when coexpressed in human embryonic kidney (HEK)-293 cells. Depolarization-induced Homer1a expression in Purkinje neurons was blocked by a mitogen-activated protein kinase (MAPK) inhibitor. Changes in internalization and mGluR1-mediated Ca2+ response were also blocked by inhibition of MAPK activity, suggesting that localization and activity of mGluR1 were regulated in the same signalling pathway as Homer1a expression. It is thus suggested that depolarization of the Purkinje neuron leads to the increment in mGluR1 responsiveness through MAPK activity and induction of Homer1a expression, which increases active mGluR1 on the cell surface by blocking internalization of mGluR1. [source] The lipopolysaccharide-recognition mechanism in cells expressing TLR4 and CD14 but lacking MD-2FEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2007Takahiro Ohnishi Abstract We analysed the lipopolysaccharide (LPS)-recognition mechanism in cells expressing TLR4 and CD14 but lacking MD-2. When TLR4 and CD14 were transiently expressed in HEK293 cells, cell-surface expression of TLR4 was observed, although the expression level was lower than that in cells coexpressing MD-2. We found that membrane CD14,TLR4 complexes were formed in these cells in response to LPS stimulation even in the absence of MD-2 expression, although NF-,B-dependent reporter activity was not induced. A strong activation of NF-,B was observed when these cells were stimulated with LPS followed by soluble MD-2 in this order, even when excess LPS was removed after formation of the CD14,TLR4 complex by washing cells prior to sMD-2 addition. From these results, we propose an additional LPS-recognition mechanism. In cells expressing TLR4 and CD14 but lacking MD-2, LPS is first transferred to membrane CD14 with the aid of LPS binding protein, which leads to the formation of the TLR4,CD14 complex. Then, the binding of soluble MD-2 to this complex triggers the transmembrane signal transduction. Cells expressing TLR4 and CD14 but lacking MD-2, such as airway epithelial cells, may be activated in response to LPS by this mechanism. [source] Insulin exerts neuroprotection by counteracting the decrease in cell-surface GABAA receptors following oxygen,glucose deprivation in cultured cortical neuronsJOURNAL OF NEUROCHEMISTRY, Issue 1 2005John G. Mielke Abstract A loss of balance between excitatory and inhibitory signaling leads to excitoxicity, and contributes to ischemic cell death. Reduced synaptic inhibition as a result of dysfunction of the ionotropic GABAA receptor has been suggested as one of the major causes for this imbalance, although the underlying mechanisms remain poorly understood. In the present study, we investigated whether oxygen,glucose deprivation (OGD), an ischemia-like challenge, alters cell-surface expression of GABAA receptors in cultured hippocampal neurons, and thereby leads to excitotoxic cell death. Using cell culture ELISA as a cell surface receptor assay, we found that OGD produced a marked decrease in cell surface GABAA receptors, without altering the total amount of receptors. Furthermore, the reduction could be prevented by inhibition of receptor endocytosis with hypertonic sucrose treatment. Notably, insulin significantly limited OGD-induced changes in cell-surface GABAA receptors. In parallel, insulin protected cultured neurons against both glutamate toxicity and OGD, as assayed by mitochondrial reduction of Alamar Blue. Importantly, insulin-mediated neuroprotection was eliminated when bicuculline, a GABAA receptor antagonist, was co-applied with insulin during OGD. Together, our results strongly suggest that ischemia-like insults decrease cell surface GABAA receptors in neurons via accelerated internalization, and that insulin provides neuroprotection by counteracting this reduction. [source] Effect of isoflurane on monocyte adhesion molecule expression in human whole blood,ACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 5 2003L. W. De Rossi Background: Recruitment of monocytes to inflamed tissue is a crucial step in the acute inflammatory reaction. Adherence of monocytes to endothelial cells followed by transmigration depends on monocyte surface adhesion molecules, inflammatory cytokines and chemoattractant chemokines. In the present study, we determined the effect of isoflurane on monocyte adhesion receptor expression in vitro. Methods: Citrated whole blood was incubated for 60 min with either 0.5 or 1 MAC isoflurane. In unstimulated blood samples and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) monocyte cell-surface expression of the selectins PSGL-1 and L-selectin, and the ,2 -integrins CD11a and CD11b were evaluated by flow cytometry. Results: Isoflurane reduced significantly the expression of PSGL-1 on unstimulated monocytes, whereas the remaining selectins and ,2 -integrins were not affected. At both concentrations, the FMLP-induced removal of PSGL-1 from the monocyte surface was increased. Furthermore, at 1 MAC isoflurane the FMLP-induced increase in CD11a expression was significantly inhibited. The surface expression of L-selectin and CD11b was not affected following exposure to isoflurane. Conclusion: Isoflurane increases the removal of the selectin PSGL-1 from the monocyte surface. Since PSGL-1 is important during the initial step of monocyte adhesion to endothelial P-selectin, the decrease in monocyte surface PSGL-1 may have profound effects on monocyte,endothelial interactions. Furthermore, the effects of isoflurane on monocyte adhesion molecule expression are different from those reported for neutrophils. [source] Identification of domains influencing assembly and ion channel properties in ,7 nicotinic receptor and 5-HT3 receptor subunit chimaerasBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2007V J Gee Background and purpose: Nicotinic acetylcholine receptors (nAChRs) and 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are members of the superfamily of neurotransmitter-gated ion channels. Both contain five subunits which assemble to form either homomeric or heteromeric subunit complexes. With the aim of identifying the influence of subunit domains upon receptor assembly and function, a series of chimaeras have been constructed containing regions of the neuronal nAChR ,7 subunit and the 5-HT3 receptor 3A subunit. Experimental approach: A series of subunit chimaeras containing ,7 and 5-HT3A subunit domains have been constructed and expressed in cultured mammalian cells. Properties of the expressed receptors have been examined by means of radioligand binding, agonist-induced changes in intracellular calcium and patch-clamp electrophysiology. Key results: Subunit domains which influence properties such as rectification, desensitization and conductance have been identified. In addition, the influence of subunit domains upon subunit folding, receptor assembly and cell-surface expression has been identified. Co-expression studies with the nAChR-associated protein RIC-3 revealed that, in contrast to the potentiating effect of RIC-3 on ,7 nAChRs, RIC-3 caused reduced levels of cell-surface expression of some ,7/5-HT3A chimaeras. Conclusions and implications: Evidence has been obtained which demonstrates that subunit transmembrane domains are critical for efficient subunit folding and assembly. In addition, functional characterization of subunit chimaeras revealed that both extracellular and cytoplasmic domains exert a dramatic and significant influence upon single-channel conductance. These data support a role for regions other than hydrophobic transmembrane domains in determining ion channel properties. British Journal of Pharmacology (2007) 152, 501,512; doi:10.1038/sj.bjp.0707429; published online 27 August 2007 [source] Differential responsiveness of human hepatoma cells versus normal hepatocytes to TRAIL in combination with either histone deacetylase inhibitors or conventional cytostaticsCANCER SCIENCE, Issue 8 2008Johanna Dzieran Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for the treatment of cancer because it elicits cell death in many tumor cells while sparing most normal cells. Liver cancer, however, is largely resistant to TRAIL and, thus, requires sensitization for TRAIL-mediated cytotoxicity. Sensitization may be achieved by cotreatment with chemotherapeutic agents. In this study, we comparatively investigated the treatment efficacy of TRAIL in combination with histone deacetylase inhibitors (HDI) versus TRAIL in combination with conventional cytostatics in the hepatocellular carcinoma cell line HepG2 and in the childhood hepatoblastoma cell line Huh6. We found that TRAIL resistance could be overcome by cotreatment with the HDI vorinostat, sodium butyrate and MS-275, but not by cotreatment with the cytostatics carboplatin and etoposide. However, TRAIL combination treatment bears the risk of sensitizing otherwise TRAIL-resistant normal cells. We thus explored a potential cytotoxic effect of combined HDI/TRAIL treatment in normal hepatocytes: TRAIL in conjunction with HDI did not impose any cytotoxicity on the non-malignant cells. In searching for the determinants of HDI-mediated TRAIL sensitization in hepatoma cells, we observed that HDI treatment did not increase cell-surface expression of proapoptotic TRAIL receptors. Instead, HDI treatment enhanced TRAIL-induced cleavage of Bid. In conclusion, our data suggest that HDI are potent sensitizers to TRAIL in hepatoma cells and that the combination of HDI and TRAIL is selectively active in hepatoma cells without affecting normal hepatocytes, indicating that the combination of HDI and TRAIL may be an effective approach for the treatment of advanced liver cancer. (Cancer Sci 2008; 99: 1685,1692) [source] Down-regulation of CXCR1 and CXCR2 expression on human neutrophils upon activation of whole blood by S. aureus is mediated by TNF-,CLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2001I. Tikhonov It was suggested that bacterial products can inhibit the expression of leucocyte chemokine receptors during sepsis and affect leucocyte functions in septic syndrome. Superantigens and toxins produced by Staphylococcus aureus are capable of activating leucocytes via binding to MHC-II antigens on monocytes and T-cell receptor molecules on T lymphocytes. It was recently shown that staphylococcal enterotoxins directly down-regulate the expression of CC chemokine receptors on monocytes through binding to MHC class II molecules. We studied the effects of killed S. aureus on the expression of interleukin-8 receptors, CXCR1 and CXCR2, on polymorphonuclear leucocytes (PMN), which are known to lack the expression of MHC-II antigens. It was shown that S. aureus down-regulated the cell-surface expression of CXCR1 and CXCR2 on PMN in the whole blood and total blood leucocyte fraction containing PMN and monocytes, but did not modulate IL-8 receptor expression in purified PMN suspension. Antibody to TNF-, abrogated down-regulation of IL-8 receptors induced by S. aureus. In contrast, LPS reduced CXCR1 and CXCR2 expression in purified PMN and whole blood in a TNF-,-independent manner. We further showed that TNF-,-induced decrease of CXCR1 and CXCR2 expression was associated with lower IL-8 binding and lower CXCR1 and CXCR2 mRNA levels, and was abrogated by protease inhibitors. We suggest that during septicemia, S. aureus may inhibit neutrophil responsiveness to IL-8 and other CXC chemokines via TNF-,- mediated down-regulation of CXCR1 and CXCR2. [source] Molecular Diversity Of Vascular Potassium Channel IsoformsCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 4 2002Victoria P Korovkina SUMMARY 1. One essential role for potassium channels in vascular smooth muscle is to buffer cell excitation and counteract vasoconstrictive influences. Several molecular mechanisms regulate potassium channel function. The interaction of these mechanisms may be one method for fine-tuning potassium channel activity in response to various physiological and pathological challenges. 2. The most prevalent K+ channels in vascular smooth muscle are large-conductance calcium- and voltage-sensitive channels (maxi-K channels) and voltage-gated channels (Kv channels). Both channel types are complex molecular structures consisting of a pore-forming , -subunit and an ancillary , -subunit. The maxi-K and Kv channel , -subunits assemble as tetramers and have S4 transmembrane domains that represent the putative voltage sensor. While most vascular smooth muscle cells identified to date contain both maxi-K and Kv channels, the expression of individual , -subunit isoforms and , -subunit association occurs in a tissue-specific manner, thereby providing functional specificity. 3. The maxi-K channel , -subunit derives its molecular diversity by alternative splicing of a single-gene transcript to yield multiple isoforms that differ in their sensitivity to intracellular Ca2+ and voltage, cell surface expression and post- translational modification. The ability of this channel to assemble as a homo- or heterotetramer allows for fine-tuning control to intracellular regulators. Another level of diversity for this channel is in its association with accessory , -subunits. Multiple , -subunits have been identified that can arise either from separate genes or alternative splicing of a , -subunit gene. The maxi-K channel , -subunits modulate the channel's Ca2+ and voltage sensitivity and kinetic and pharmacological properties. 4. The Kv channel , -subunit derives its diverse nature by the expression of several genes. Similar to the maxi-K channel, this channel has been shown to assemble as a homo- and heterotetramer, which can significantly change the Kv current phenotype in a given cell type. Association with a number of the ancillary , -subunits affects Kv channel function in several ways. Beta-subunits can induce inactivating properties and act as chaperones, thereby regulating channel cell-surface expression and current kinetics. [source] Molecular genetic study of congenital nephrogenic diabetes insipidus and rescue of mutant vasopressin V2 receptor by chemical chaperonesNEPHROLOGY, Issue 2 2007HAE IL CHEONG SUMMARY: Aim: X-linked nephrogenic diabetes insipidus is a rare disease caused by mutations in the arginine vasopressin V2 receptor (AVPR2) gene, which encodes vasopressin V2 receptor (V2R). More than a half of reported mutations in AVPR2 are missense mutations, and a large number of missense mutant receptors fail to fold properly and therefore are not routed to the cell surface. Methods: We analysed the AVPR2 gene in 14 unrelated patients with X-linked nephrogenic diabetes insipidus, and found 13 different mutations including eight missense point mutations. The cellular expression patterns of three missense mutant (A98P, L274P and R113W) and wild-type V2R were determined in transfected COS-7 cells. Results: In contrast to wild-type V2R, the cell-surface expressions of mutant receptors were totally (A98P and L274P) or partially (R113W) absent. Instead, they were retained intracellularly. However, treatment of cells with two chemical chaperones (100 mmol/L trimethylamine oxide or 2% dimethyl sulfoxide) or incubation at 26°C restored the cell-surface expressions of mutant receptors. Conclusion: These data show that some chemical chaperones correct the mistrafficking of misfolded A98P, L274P and R113W V2R. Thus, we believe that a therapeutic strategy based on chemical chaperones in patients with these mutations is worth trying. [source] | ||||||||||||||||||||||