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Functional Expression (functional + expression)
Selected AbstractsFunctional Expression, Targeting and Ca2+ Signaling of a Mouse Melanopsin-eYFP Fusion Protein in a Retinal Pigment Epithelium Cell Line,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Maikel E. Giesbers Melanopsin, first discovered in Xenopus melanophores, is now established as a functional sensory photopigment of the intrinsically photosensitive retinal ganglion cells. These ganglion cells drive circadian rhythm and pupillary adjustments through projection to the brain. Melanopsin shares structural similarities with all known opsins. Comprehensive characterization of melanopsin with respect to its spectral properties, photochemical cascade and signaling partners requires a suitable recombinant system and high expression levels. This combination has not yet been described. To address this issue, we have expressed recombinant mouse melanopsin in several cell lines. Using enhanced yellow fluorescent protein (eYFP) as a visualization tag, expression was observed in all cell lines. Confocal microscopy revealed that melanopsin was properly routed to the plasma membrane only in retinal pigment epithelium (RPE)-derived D407 cells and in human embryonic kidney (HEK) cells. Further, we performed intracellular calcium measurements in order to probe the melanopsin signaling activity of this fusion protein. Transfected cells were loaded with the calcium indicator Fura2-AM. Upon illumination, an immediate but transient calcium response was observed in HEK as well as in D407 cells, while mock-transfected cells showed no calcium response under identical conditions. Supplementation with 11- cis retinal or all- trans retinal enhanced the response. After prolonged illumination the cells became desensitized. Thus, RPE-derived cells expressing recombinant melanopsin may constitute a suitable system for the study of the structural and functional characteristics of melanopsin. [source] Structural and functional comparison of 15S - and 15R -specific cyclooxygenases from the coral Plexaura homomallaFEBS JOURNAL, Issue 17 2004Karin Valmsen It has been known for 30 years that the gorgonian coral Plexaura homomalla contains either 15S- or 15R -configuration prostaglandins (PGs), depending on its location in the Caribbean. Recently we showed that the 15R -PGs in the R -variety of P. homomalla are formed by a unique cyclooxygenase (COX) with 15R oxygenation specificity [Valmsen, K., Järving, I., Boeglin, W.E., Varvas, K., Koljak, R., Pehk, T., Brash, A.R. & Samel, N. (2001) Proc. Natl. Acad. Sci. USA98, 7700]. Here we describe the cloning and characterization of a closely related COX protein (97% amino acid sequence identity) from the S -variety of P. homomalla. Functional expression of the S -variant COX cDNA in Sf9 insect cells followed by incubation with exogenous arachidonic acid resulted in formation of PG products with > 98% 15S -configuration. Mutational analysis was performed on a suggested active site determinant of C-15 oxygenation specificity, position 349 (Val in all S -specific COX, Ile in 15R -COX). The 15S -COX Val349 to Ile mutant formed 35% 15R- PGs, while the reverse mutation in the 15R -COX (Ile349Val) led to formation of 70% 15S- products. This establishes position 349 as an important determinant of the product stereochemistry at C-15. Our characterization of the enzyme variants demonstrates that very minor sequence divergence accounts for the content of epimeric PGs in the two variants of P. homomalla and that the differences do not arise by isomerization of the products. [source] Functional expression of a locust tyramine receptor in murine erythroleukaemia cellsINSECT MOLECULAR BIOLOGY, Issue 6 2001J. Poels Abstract The LCR/MEL system (Locus Control Region/Murine Erythroleukaemia cells) was employed to express and characterize the Locusta migratoria tyramine receptor (TyrLoc), an insect G protein-coupled receptor. Functional agonist-dependent responses were recorded in stable, tyramine receptor expressing cell clones (MEL-TyrLoc). Tyramine elicited a dose-dependent increase of cytosolic Ca2+ -ions and an attenuation of forskolin-induced cyclic adenosine monophosphate (AMP) production. Octopamine was shown to be a weak agonist for both responses. In addition, yohimbine proved to be a potent tyramine receptor antagonist. This study reports the first application of the LCR/MEL expression system in functional assays for G protein-coupled receptors and therefore expands the capabilities of this system by exploiting the functionality of the signal transduction pathways. [source] Functional expression of corticotropin-releasing hormone (CRH) receptor 1 in cultured rat microgliaJOURNAL OF NEUROCHEMISTRY, Issue 2 2002Wei Wang Abstract Corticotropin-releasing hormone (CRH), known as a key regulator of the hypothalamic,pituitary,adrenal axis response to stress, elicits its biological effects by binding to two membrane receptors (CRH-R1 and CRH-R2). The present studies examined the presence of functional expression of CRH receptors in cultured microglia of rat. CRH-R1 mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR), western blotting and receptor chemical cross-linking assay in cultured microglia. CRH-R2 mRNA was undectable by RT-PCR. The radioligand binding analysis using [125I]Tyr-rat/human CRH revealed a high affinity binding site (Kd of 1.2 nm and Bmax of 84 fmol/mg of protein). Competition studies using CRH and related peptides indicated kinetic and pharmacological characteristics consistent with the CRH-R1 receptor subtype. Receptor chemical cross-linking assay demonstrated a single band of CRH receptor with a molecular weight of ,77 kDa, which was inhibited in the presence of excess unlabeled rat/human CRH in a dose-dependent manner and inhibited by a CRH receptor,antagonist astressin. Functional coupled cAMP production in cultured microglia was stimulated by exogenous addition of CRH and related peptides in a dose-dependent manner and blocked by astressin. Our findings suggest the functional expression of CRH-R1 receptor in rat microglia, indicating an important mechanism of interaction between immune and neuroendocrine systems in brain physiological and,pathological conditions. [source] Functional expression of the hyperpolarization-activated, non-selective cation current If in immortalized HL-1 cardiomyocytesTHE JOURNAL OF PHYSIOLOGY, Issue 1 2002Laura Sartiani HL-1 cells are adult mouse atrial myocytes induced to proliferate indefinitely by SV40 large T antigen. These cells beat spontaneously when confluent and express several adult cardiac cell markers including the outward delayed rectifier K+ channel. Here, we examined the presence of a hyperpolarization-activated If current in HL-1 cells using the whole-cell patch-clamp technique on isolated cells enzymatically dissociated from the culture at confluence. Cell membrane capacitance (Cm) ranged from 5 to 53 pF. If was detected in about 30 % of the cells and its occurrence was independent of the stage of the culture. If maximal slope conductance was 89.7 ± 0.4 pS pF,1 (n= 10). If current in HL-1 cells showed typical characteristics of native cardiac If current: activation threshold between ,50 and ,60 mV, half-maximal activation potential of ,83.1 ± 0.7 mV (n= 50), reversal potential at ,20.8 ± 1.5 mV (n= 10), time-dependent activation by hyperpolarization and blockade by 4 mm Cs+. In half of the cells tested, activation of adenylyl cyclase by the forskolin analogue L858051 (20 ,m) induced both a ,6 mV positive shift of the half-activation potential and a ,37 % increase in the fully activated If current. RT-PCR analysis of the hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) expressed in HL-1 cells demonstrated major contributions of HCN1 and HCN2 channel isoforms to If current. Cytosolic Ca2+ oscillations in spontaneously beating HL-1 cells were measured in Fluo-3 AM-loaded cells using a fast-scanning confocal microscope. The oscillation frequency ranged from 1.3 to 5 Hz and the spontaneous activity was stopped in the presence of 4 mm Cs+. Action potentials from HL-1 cells had a triangular shape, with an overshoot at +15 mV and a maximal diastolic potential of ,69 mV, i.e. more negative than the threshold potential for If activation. In conclusion, HL-1 cells display a hyperpolarization-activated If current which might contribute to the spontaneous contractile activity of these cells. [source] Sodium channel inactivation defects are associated with acetazolamide-exacerbated hypokalemic periodic paralysisANNALS OF NEUROLOGY, Issue 3 2001Saïd Bendahhou PhD A novel mutation in a family with hypokalemic periodic paralysis is described. The mutation R672S is located in the voltage sensor segment S4 of domain II in the SCN4A gene encoding the human skeletal muscle voltage-gated sodium channel. Functional expression of the R672S channels in human embryonic kidney 293 cells revealed a small but significant hyperpolarizing shift in the steady-state fast inactivation, and a dramatic enhancement in channel slow inactivation. These two defects are mainly due to a slow recovery of the mutant channels from fast and/or slow inactivation. Our data may help explain the mechanism underlying hypokalemic periodic paralysis and the patient's worsening from acetazolamide. [source] Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiaeBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2001Birgit Morawski Abstract Biotechnology applications of horseradish peroxidase (HRP) would benefit from access to tailor-made variants with greater specific activity, lower Km for peroxide, and higher thermostability. Starting with a mutant that is functionally expressed in Saccharomyces cerevisiae, we used random mutagenesis, recombination, and screening to identify HRP-C mutants that are more active and stable to incubation in hydrogen peroxide at 50°C. A single mutation (N175S) in the HRP active site was found to improve thermal stability. Introducing this mutation into an HRP variant evolved for higher activity yielded HRP 13A7-N175S, whose half-life at 60°C and pH 7.0 is three times that of wild-type (recombinant) HRP and a commercially available HRP preparation from Sigma (St. Louis, MO). The variant is also more stable in the presence of H2O2, SDS, salts (NaCl and urea), and at different pH values. Furthermore, this variant is more active towards a variety of small organic substrates frequently used in diagnostic applications. Site-directed mutagenesis to replace each of the four methionine residues in HRP (M83, M181, M281, M284) with isoleucine revealed no mutation that significantly increased the enzyme's stability to hydrogen peroxide. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 99,107, 2001. [source] RhoA/ROCK and Cdc42 regulate cell-cell contact and N-cadherin protein level during neurodetermination of P19 embryonal stem cellsDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2004Isabel Laplante Abstract RhoGTPases regulate actin-based signaling cascades and cellular contacts. In neurogenesis, their action modulates cell migration, neuritogenesis, and synaptogenesis. Murine P19 embryonal stem cells differentiate to neurons upon aggregation in the presence of retinoic acid, and we previously showed that RhoA and Cdc42 RhoGTPases are sequentially up-regulated during neuroinduction, suggesting a role at this very early developmental stage. In this work, incubation of differentiating P19 cells with C3 toxin resulted in decreased aggregate cohesion and cadherin protein level. In contrast, C3 effects were not observed in cells overexpressing recombinant dominant active RhoA. On the other hand, C3 did not affect cadherin in uninduced cells and their postmitotic neuronal derivatives, respectively expressing E- and N-cadherin. RhoA is thus influential on cell aggregation and cadherin expression during a sensitive time window that corresponds to the switch of E- to N-cadherin. Cell treatment with Y27632 inhibitor of Rho-associated-kinase ROCK, or advanced overexpression of Cdc42 by gene transfer of a constitutively active form of the protein reproduced C3 effects. RhoA-antisense RNA also reduced cadherin level and the size of cell aggregates, and increased the generation of fibroblast-like cells relative to neurons following neuroinduction. Colchicin, a microtubule disrupter, but not cytochalasin B actin poison, importantly decreased cadherin in neurodifferentiating cells. Overall, our results indicate that the RhoA/ROCK pathway regulates cadherin protein level and cell-cell interactions during neurodetermination, with an impact on the efficiency of the process. The effect on cadherin seems to involve microtubules. The importance of correct timing of RhoA and Cdc42 functional expression in neurogenesis is also raised. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 289,307, 2004 [source] Neuropeptide Y in the olfactory microvillar cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2006Giorgia Montani Abstract This paper examines a possible role of microvillar cells in coordinating cell death and regeneration of olfactory epithelial neurons. The olfactory neuroepithelium of mammals is a highly dynamic organ. Olfactory neurons periodically degenerate by apoptosis and as a consequence of chemical or physical damage. To compensate for this loss of cells, the olfactory epithelium maintains a lifelong ability to regenerate from a pool of resident multipotent stem cells. To assure functional continuity and histological integrity of the olfactory epithelium over a period of many decades, apoptosis and regeneration require to be precisely coordinated. Among the factors that have been implicated in mediating this regulation is the neuropeptide Y (NPY). Knockout mice that lack functional expression of this neurogenic peptide show defects in embryonic development of the olfactory epithelium and in its ability to regenerate in the adult. Here we show that, in postnatal olfactory epithelia, NPY is exclusively expressed by a specific population of microvillar cells. We previously characterized these cells as a novel type of putative chemosensory cells, which are provided with a phosphatidyl-inositol-mediated signal transduction cascade. Our findings allow for the first time to suggest that microvillar cells are involved in connecting apoptosis to neuronal regeneration by stimulus-induced release of NPY. [source] Postnatal maturation of GABAA and GABAC receptor function in the mammalian superior colliculusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2001Mathias Boller Abstract In the stratum griseum superficiale (SGS) of the mammalian superior colliculus, GABAC receptors seem to control the excitability of projection neurons by selective inactivation of local GABAergic interneurons. As the onset of visual responses to SC begins well after birth in the rat, it is possible to study developmental changes in GABAergic mechanisms that are linked to the onset of visual information processing. In order to analyse postnatal changes in inhibitory mechanisms that involve GABA receptor function, we used extracellular field potential (FP) recordings and single cell patch-clamp techniques in slices from postnatal day 4 (P4) to P32 and examined the effects of GABA and muscimol on electrically evoked SGS cell activity. While GABAA receptor activation affected FP amplitudes throughout postnatal development, GABAC receptor activation did not significantly change FP amplitudes until the third postnatal week. Results from patch-clamping single cells, however, clearly demonstrate that GABAC receptors are already functional at P4 , similar to GABAA receptors. Throughout postnatal development, activation of GABAC receptors leads to a strong inhibition of inhibitory postsynaptic activity, indicating that GABAC receptors are expressed by inhibitory interneurons. Furthermore, the proportion of neurons that show decreased excitatory postsynaptic activity during GABAC receptor activation correlates with the proportion of GABAergic interneurons in SGS. Our patch-clamp results indicate that the functional expression of GABAC receptors by GABAergic interneurons does not change significantly during postnatal development. However, our measurements of FP amplitudes indicate that the maturation of the efferent connections of these GABAergic neurons within SGS during the third postnatal week strongly changes GABAC receptor function. [source] Functional angiotensin-converting enzyme 2 is expressed in human cardiac myofibroblastsEXPERIMENTAL PHYSIOLOGY, Issue 5 2008Jodie L. Guy The renin,angiotensin system (RAS), in particular angiotensin II, plays an important role in cardiac remodelling. Angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) are key players in the RAS and act antagonistically to regulate the levels of angiotensin II. In this study, we reveal the functional expression of ACE2 in human cardiac myofibroblasts, cells that are essential to the maintenance of normal cardiac architecture and also play a key role in myocardial remodelling. The observed reciprocal expression of ACE and ACE2 in these cells may reflect the possible opposing activity of these two enzymes. In this study, we demonstrate the presence of ACE2 as an ectoenzyme and reveal that ACE2 undergoes phorbol-12-myristate-13-acetate-inducible ectodomain shedding from the membrane. When cells were exposed to a number of pathophysiological stimuli, modulation of ACE2 levels was not detected. Importantly, whilst we found ACE2 to be expressed constitutively in cardiac myofibroblasts there were no detectable levels in either vascular smooth muscle cells or vascular endothelium, indicating that ACE2 expression is not ubiquitous. In paraffin sections of atrial appendage tissue, we observed a distinct staining pattern for ACE2 which appeared different from that of ACE. In conclusion, this study is the first to report co-expression of ACE and ACE2 in human cardiac myofibroblasts and may therefore present a model primary system for study of the comparative cell biology of ACE2 and ACE and their potentially opposing roles in myocardial remodelling. [source] Molecular cloning and functional expression of a gene encoding an antiarrhythmia peptide derived from the scorpion toxinFEBS JOURNAL, Issue 18 2002Fang Peng From a cDNA library of Chinese scorpion Buthus martensii Karsch, full-length cDNAs of 351 nucleotides encoding precursors (named BmKIM) that contain signal peptides of 21 amino acid residues, a mature toxin of 61 residues with four disulfide bridges, and an extra Gly-Lys-Lys tail, were isolated. The genomic sequence of BmKIM was cloned and sequenced; it consisted of two exons disrupted by an intron of 1622 bp, the largest known in scorpion toxin genomes, inserted in the region encoding the signal peptide. The cDNA was expressed in Escherichia coli. The recombinant BmKIM was toxic to both mammal and insects. This is the first report that a toxin with such high sequence homology with an insect-specific depressant toxin group exhibits toxicity to mammals. Using whole cell patch-clamp recording, it was discovered that the recombinant BmKIM inhibited thesodium current in rat dorsal root ganglion neurons andventricular myocytes and protected against aconitine- induced cardiac arrhythmia. [source] Isolated human astrocytes are not susceptible to infection by M- and T-tropic HIV-1 strains despite functional expression of the chemokine receptors CCR5 and CXCR4 ,GLIA, Issue 3 2001Agnès Boutet Abstract Within the brain, HIV-1 targets the microglia and astrocytes. Previous studies have reported that viral entry into astrocytes is independent of CD4, in contrast to microglia. We aimed to determine whether chemokine receptors play a role in mediating CD4-independent HIV-1 entry into astrocytes. We found that embryonic astrocytes and microglial cells express CCR5, CCR3, and CXCR4 transcripts. Intracellular calcium levels in astrocytes were found to increase following application of RANTES, MIP-1, (CCR5-agonist), SDF-1, (CXCR4-agonist), but not eotaxin (CCR3-agonist). In microglial cells, eotaxin was also able to modulate internal calcium homeostasis. CD4 was not present at the cell surface of purified astrocytes but CD4 mRNA could be detected by RT-PCR. Neither HIV-19533 (R5 isolate) nor HIV-1LAI (X4 isolate) penetrated into purified astrocytes. In contrast, mixed CNS cell cultures were infected by HIV-19533 and this was inhibited by anti-CD4 mAb in 4/4 tested cultures and by anti-CCR5 mAb in 2/4. Thus, the HIV-1 R5 strain requires CD4 to penetrate into brain cells, suggesting that CCR5 cannot be used as the primary receptor for M-tropic HIV-1 strains in astrocytes. Moreover, inconstant inhibition of HIV-1 entry by anti-CCR5 mAb supports the existence of alternative coreceptors for penetration of M-tropic isolates into brain cells. GLIA 34:165,177, 2001. © 2001 Wiley-Liss, Inc. [source] Sensitivity of the 2-oxoglutarate carrier to alcohol intake contributes to mitochondrial glutathione depletionHEPATOLOGY, Issue 3 2003Olga Coll The mitochondrial pool of reduced glutathione (mGSH) is known to play a protective role against liver injury and cytokine-mediated cell death. However, the identification of the mitochondrial carriers involved in its transport in hepatocellular mitochondria remains unestablished. In this study, we show that the functional expression of the 2-oxoglutarate carrier from HepG2 cells in mitochondria from Xenopus laevis oocytes conferred a reduced glutathione (GSH) transport activity that was inhibited by phenylsuccinate, a specific inhibitor of the carrier. In addition, the mitochondrial transport of GSH and 2-oxoglutarate in isolated mitochondria from rat liver exhibited mutual competition and sensitivity to glutamate and phenylsuccinate. Interestingly, the kinetics of 2-oxoglutarate transport in rat liver mitochondria displayed a single Michaelis-Menten component with a Michaelis constant of 3.1 ± 0.3 mmol/L and maximum velocity of 1.9 ± 0.1 nmol/mg protein/25 seconds. Furthermore, the initial rate of 2-oxoglutarate was reduced in mitochondria from alcohol-fed rat livers, an effect that was not accompanied by an alcohol-induced decrease in the 2-oxoglutarate messenger RNA levels but rather by changes in mitochondrial membrane dynamics induced by alcohol. The fluidization of mitochondria by the fluidizing agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) (A2C) restored the initial transport rate of both GSH and 2-oxoglutarate. Finally, these changes were reproduced in normal liver mitochondria enriched in cholesterol where the fluidization of cholesterol-enriched mitochondria with A2C restored the order membrane parameter and the mitochondrial 2-oxoglutarate uptake. In conclusion, these findings provide unequivocal evidence for 2-oxoglutarate as a GSH carrier and its sensitivity to membrane dynamics perturbation contributes in part to the alcohol-induced mGSH depletion. [source] cDNA cloning, functional expression and characterization of kynurenine 3-hydroxylase of Anopheles stephensi (Diptera: Culicidae)INSECT MOLECULAR BIOLOGY, Issue 5 2002M. Hirai Abstract Kynurenine 3-hydroxylase (K3H) is a NADPH-dependent flavin monooxygenase involved in the tryptophan pathway. Xanthurenic acid (XA) is a metabolite of this pathway and has recently been identified as a gamete activating factor (GAF) of the malarial parasite. We cloned K3H cDNA from Anopheles stephensi (AsK3H), because anopheline mosquitoes are a vector of the human malaria parasite, Plasmodium falciparum and the catalytic function of AsK3H in XA production. Recombinant AsK3H protein was expressed in Sf-9 cells using the baculovirus system and its enzymatic properties were characterized. The specific activities of crude cell lysate and affinity purified protein were 94.9 ± 6.2 and 865.6 ± 10.5 nmol/min/mg protein, respectively. The optimum pH of AsK3H was 7.0. Analysis of AsK3H gene expression using RT-PCR revealed that AsK3H was constitutively expressed in egg, larva, pupa and adult. [source] Electroporation-mediated muscarinic M3 receptor gene transfer into rat urinary bladderINTERNATIONAL JOURNAL OF UROLOGY, Issue 11 2004MASAYUKI OTANI Abstract Background: Muscarinic M3 (M3) receptor has been recognized as a major muscarinic receptor for smooth muscle contractions of the urinary bladder. Under the hypothesis that overexpression of M3 receptor in the urinary bladder would enhance urinary bladder contractions, we have transferred the M3 receptor gene into rat bladders using electroporation (EP) and evaluated the functional expression of the transferred gene. Methods: Plasmids expressing luciferase, a green fluorescence protein and M3 receptor were injected into the rat bladder and square-wave electric pulses were immediately applied. Two days after gene transfer, we analyzed gene expression. Immunohistochemical staining was performed and the contractile responses from isolated bladder strips, which were induced KCl, carbachol and electrical field stimulation (EFS), were evaluated. Results: The optimal conditions of electroporation were 8 pulses, 45 voltages, 50 milliseconds/pulses and 1 Hz. Under these conditions, luciferase gene expression was enhanced approximately 300-fold, compared to an injection of DNA only. Regarding immunohistochemistry with an anti-M3 receptor, an increase in immunoactivity was observed in the M3 receptor gene transferred rat bladder, compared to the bladder of the control rat. In rats with the transferred M3 receptor gene, carbachol- and EFS-induced maximum contractile responses of bladder smooth muscle strips significantly increased. Conclusions: These findings suggest that an in vivo EP procedure is an useful method for gene transfer into the bladder and that an overexpression of M3 receptor in the rat bladder enhances bladder contractility. This technique may become a new treatment modality for detrusor underactivity. [source] Impaired cell cycle regulation of the osteoblast-related heterodimeric transcription factor Runx2-Cbf, in osteosarcoma cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009Inga A. San Martin Bone formation and osteoblast differentiation require the functional expression of the Runx2/Cbf, heterodimeric transcription factor complex. Runx2 is also a suppressor of proliferation in osteoblasts by attenuating cell cycle progression in G1. Runx2 levels are modulated during the cell cycle, which are maximal in G1 and minimal beyond the G1/S phase transition (S, G2, and M phases). It is not known whether Cbf, gene expression is cell cycle controlled in preosteoblasts nor how Runx2 or Cbf, are regulated during the cell cycle in bone cancer cells. We investigated Runx2 and Cbf, gene expression during cell cycle progression in MC3T3-E1 osteoblasts, as well as ROS17/2.8 and SaOS-2 osteosarcoma cells. Runx2 protein levels are reduced as expected in MC3T3-E1 cells arrested in late G1 (by mimosine) or M phase (by nocodazole), but not in cell cycle arrested osteosarcoma cells. Cbf, protein levels are cell cycle independent in both osteoblasts and osteosarcoma cells. In synchronized MC3T3-E1 osteoblasts progressing from late G1 or mitosis, Runx2 levels but not Cbf, levels are cell cycle regulated. However, both factors are constitutively elevated throughout the cell cycle in osteosarcoma cells. Proteasome inhibition by MG132 stabilizes Runx2 protein levels in late G1 and S in MC3T3-E1 cells, but not in ROS17/2.8 and SaOS-2 osteosarcoma cells. Thus, proteasomal degradation of Runx2 is deregulated in osteosarcoma cells. We propose that cell cycle control of Runx2 gene expression is impaired in osteosarcomas and that this deregulation may contribute to the pathogenesis of osteosarcoma. J. Cell. Physiol. 221: 560,571, 2009. © 2009 Wiley-Liss, Inc. [source] The history of sweet taste: not exactly a piece of cakeJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2006Pierandrea Temussi Abstract Understanding the molecular bases of sweet taste is of crucial importance not only in biotechnology but also for its medical implications, since an increasing number of people is affected by food-related diseases like, diabetes, hyperlipemia, caries, that are more or less directly linked to the secondary effects of sugar intake. Despite the interest paid to the field, it is only through the recent identification and functional expression of the receptor for sweet taste that new perspectives have been opened, drastically changing our approach to the development of new sweeteners. We shall give an overview of the field starting from the early days up to discussing the newest developments. After a review of early models of the active site, the mechanisms of interaction of small and macromolecular sweet molecules will be examined in the light of accurate modeling of the sweet taste receptor. The analysis of the homology models of all possible dimers allowed by combinations of the human T1R2 and T1R3 sequences of the sweet receptor and the closed (A) and open (B) conformations of the mGluR1 glutamate receptor shows that only ,type B' sites, either T1R2(B) and T1R3(B), can host the majority of small molecular weight sweeteners. Simultaneous binding to the A and B sites is not possible with two large sweeteners but is possible with a small molecule in site A and a large one in site B. This observation accounted for the first time for the peculiar phenomenon of synergy between some sweeteners. In addition to these two sites, the models showed an external binding site that can host sweet proteins. Copyright © 2006 John Wiley & Sons, Ltd. [source] HIV-1 viral envelope glycoprotein gp120 produces oxidative stress and regulates the functional expression of multidrug resistance protein-1 (Mrp1) in glial cellsJOURNAL OF NEUROCHEMISTRY, Issue 3 2008Patrick T. Ronaldson Abstract Brain human immunodeficiency virus type-1 (HIV-1) infection is associated with oxidative stress, which may lead to HIV-1 encephalitis, a chronic neurodegenerative condition. In vitro, oxidative stress can be induced in glial cells by exposure to HIV-1 envelope protein glycoprotein (gp120). Multidrug resistance proteins (Mrps) are known to efflux endogenous substrates (i.e. GSH and GSSG) involved in cellular defense against oxidative stress. Altered GSH/GSSG export may contribute to oxidative damage during HIV-1 encephalitis. At present, it is unknown if gp120 exposure can alter the functional expression of Mrp isoforms. Heat-shock protein 70, inducible nitric oxide synthase, intracellular GSSG, 2,,7,-dichlorofluorescein fluorescence, and extracellular nitrite were increased in primary cultures of rat astrocytes triggered with gp120, suggesting an oxidative stress response. RT-PCR and immunoblot analysis demonstrated increased Mrp1 mRNA (2.3-fold) and protein (2.2-fold), respectively, in gp120 treated astrocytes while Mrp4 mRNA or protein expression was not changed. Cellular retention of 2,,7,-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, an established Mrp substrate, was reduced (twofold) in gp120-treated astrocytes, suggesting increased Mrp-mediated transport. In addition, GSH and GSSG export were enhanced in gp120-triggered cells. These data suggest that gp120 can up-regulate Mrp1, but not Mrp4, functional expression in cultured astrocytes. Our observation of increased GSH/GSSG efflux in response to gp120 treatment implies that Mrp isoforms may be involved in regulating the oxidative stress response in glial cells. [source] Group III metabotropic glutamate receptor activation suppresses self-replication of undifferentiated neocortical progenitor cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2008Noritaka Nakamichi§ Abstract We evaluated the possible functional expression of metabotropic glutamate receptors (mGluRs) by neural progenitors from embryonic mouse neocortex. Constitutive expression was seen with group I, II, and III mGluRs in undifferentiated cells and neurospheres formed by clustered cells during culture with epidermal growth factor. The group III mGluR agonist, l -2-amino-4-phosphonobutyrate, drastically reduced proliferation activity at 1,100 ,M without inducing cell death, with group I and group II mGluR agonists being ineffective, in these neurospheres. Both forskolin and a group III mGluR antagonist significantly increased the proliferation alone, but significantly prevented the suppression by l -2-amino-4-phosphonobutyrate. Activation of group III mGluR significantly decreased mRNA expression of the cell cycle regulator cyclinD1, in addition to inhibiting the transactivation mediated by cAMP of cyclinD1 gene in the pluripotent P19 progenitor cells. Prior activation of group III mGluR led to a significant decrease in the number of cells immunoreactive for a neuronal marker, with an increase in that for an astroglial marker irrespective of differentiation inducers. These results suggest that group III mGluR may be functionally expressed to suppress self-renewal capacity through a mechanism related to cAMP formation with promotion of subsequent differentiation into astroglial lineage in neural progenitors. [source] Functional expression of corticotropin-releasing hormone (CRH) receptor 1 in cultured rat microgliaJOURNAL OF NEUROCHEMISTRY, Issue 2 2002Wei Wang Abstract Corticotropin-releasing hormone (CRH), known as a key regulator of the hypothalamic,pituitary,adrenal axis response to stress, elicits its biological effects by binding to two membrane receptors (CRH-R1 and CRH-R2). The present studies examined the presence of functional expression of CRH receptors in cultured microglia of rat. CRH-R1 mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR), western blotting and receptor chemical cross-linking assay in cultured microglia. CRH-R2 mRNA was undectable by RT-PCR. The radioligand binding analysis using [125I]Tyr-rat/human CRH revealed a high affinity binding site (Kd of 1.2 nm and Bmax of 84 fmol/mg of protein). Competition studies using CRH and related peptides indicated kinetic and pharmacological characteristics consistent with the CRH-R1 receptor subtype. Receptor chemical cross-linking assay demonstrated a single band of CRH receptor with a molecular weight of ,77 kDa, which was inhibited in the presence of excess unlabeled rat/human CRH in a dose-dependent manner and inhibited by a CRH receptor,antagonist astressin. Functional coupled cAMP production in cultured microglia was stimulated by exogenous addition of CRH and related peptides in a dose-dependent manner and blocked by astressin. Our findings suggest the functional expression of CRH-R1 receptor in rat microglia, indicating an important mechanism of interaction between immune and neuroendocrine systems in brain physiological and,pathological conditions. [source] Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood-brain barrierJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 11 2001Mark Gumbleton Abstract A relatively simple, widely applicable, and robust in vitro method of predicting blood-brain barrier (BBB) permeability to central nervous system-acting drugs is an increasing need. A cell-based model offers the potential to account for transcellular and paracellular drug diffusional processes, metabolism, and active transport processes, as well as nondefined interactions between a drug and cellular material that may impact upon a membrane's overall permeability profile. Any in vitro BBB cell model to be utilized for the transendothelial BBB permeability screening of potential central nervous system drugs must display reproducible solute permeability, and a number of other general criteria including: a restrictive paracellular barrier; a physiologically realistic cell architecture; the functional expression of key transporter mechanisms; and allow ease of culture to meet the technical and time constraints of a screening program. This article reviews the range of in vitro cell-based BBB models available, including the primary/low passage bovine and porcine brain endothelial cultures as well as the spectrum of immortalized brain endothelial cell lines that have been established. The article further discusses the benefits and limitations of exploiting such systems as in vitro BBB permeability screens. © 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1681,1698, 2001 [source] Plasmodium falciparum- infected erythrocytes induce tissue factor expression in endothelial cells and support the assembly of multimolecular coagulation complexesJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2007I. M. B. FRANCISCHETTI Summary.,Background:,Plasmodium falciparum malaria infects 300,500 million people every year, causing 1,2 million deaths annually. Evidence of a coagulation disorder, activation of endothelial cells (EC) and increase in inflammatory cytokines are often present in malaria. Objectives:,We have asked whether interaction of parasitized red blood cells (pRBC) with EC induces tissue factor (TF) expression in vitro and in vivo. The role of phosphatidylserine-containing pRBC to support the assembly of blood coagulation complexes was also investigated. Results:,We demonstrate that mature forms of pRBC induce functional expression of TF by EC in vitro with productive assembly of the extrinsic Xnase complex and initiation of the coagulation cascade. Late-stage pRBC also support the prothrombinase and intrinsic Xnase complex formation in vitro, and may function as activated platelets in the amplification phase of the blood coagulation. Notably, post-mortem brain sections obtained from P. falciparum -infected children who died from cerebral malaria and other causes display a consistent staining for TF in the EC. Conclusions:,These findings place TF expression by endothelium and the amplification of the coagulation cascade by pRBC and/or activated platelets as potentially critical steps in the pathogenesis of malaria. Furthermore, it may allow investigators to test other therapeutic alternatives targeting TF or modulators of EC function in the treatment of malaria and/or its complications. [source] Odor discrimination by G protein-coupled olfactory receptorsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2002Kazushige Touhara Abstract The vertebrate olfactory system possesses a remarkable capacity to recognize and discriminate a variety of odorants by sending the coding information from peripheral olfactory sensory neurons in the olfactory epithelium to the olfactory bulb of the brain. The recognition of odorants appear to be mediated by a G protein-coupled receptor superfamily that consists of ,1% of total genes in vertebrates. Since the first discovery of the olfactory receptor gene superfamily in the rat, similar chemosensory receptors have been found in various species across different phyla. The functions of these receptors, however, had been uncharacterized until the recently successful functional expression and ligand screening of some olfactory receptors in various cell expression systems. The functional cloning of odorant receptors from single olfactory neurons allowed for the identification of multiple receptors that recognized a particular odorant of interest. Reconstitution of the odorant responses demonstrated that odorant receptors recognized various structurally-related odorant molecules with a specific molecular receptive range, and that odor discrimination is established based on a combinatorial receptor code model in which the identities of different odorants are encoded by a combination of odorant receptors. The receptor code for an odorant changes at different odorant concentrations, consistent with our experience that perceived quality of an odorant changes at different concentrations. The molecular bases of odor discrimination at the level of olfactory receptors appear to correlate well with the receptive field in the olfactory bulb where the input signal is further processed to create the specific odor maps. Microsc. Res. Tech. 58:135,141, 2002. © 2002 Wiley-Liss, Inc. [source] Nature, significance, and mechanisms of electrical heterogeneities in ventricleTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 2 2004Steven Poelzing Abstract Previously, dispersion of repolarization (DOR) has been extensively linked to the development of arrhythmias and sudden cardiac death. The electrical heterogeneities that cause DOR between transmural myocyte layers have been reported in a wide variety of animals and humans. The underlying causes of transmural electrical heterogeneities are in part due to heterogeneous functional expression of proteins responsible for ion handling. Recently, we found that electrophysiologic heterogeneities between subepicardial and midmyocardial cells can form a substrate for reentrant ventricular arrhythmias. However, cell-to-cell coupling through gap junctions is expected to attenuate transmural heterogeneities between cell types spanning the ventricular wall. In this article we review a hypothesis that regional uncoupling resulting from expression patterns of gap junctions across the ventricular wall underlies DOR, and DOR can be amplified under disease conditions which remodel gap junctions. We find the principle gap junction protein, connexin43 (Cx43), is selectively reduced in the subepicardium (by 24%) compared to deeper layers of normal canine left ventricle. Additionally, the greatest DOR occurs within the subepicardial-midmyocardial interface, precisely where Cx43 expression is reduced. The present data suggests that ion channel and gap junction heterogeneities act in conjunction to form and maintain transmural DOR. Importantly, both ion channel and gap junction remodeling occurs during many disease states such as heart failure. Importantly, in the absence of ion channel remodeling, pharmacological uncoupling increases transmural DOR, particularly within the epicardial-midmyocardial interface, to values observed in heart failure. Therefore, these data suggest that heterogeneous Cx43 expression produces functionally significant electrophysiologic heterogeneities across the ventricular wall and may be a mechanism for promoting DOR which underlie arrhythmias in heart failure. © 2004 Wiley-Liss, Inc. [source] A conserved cysteine residue in the third transmembrane domain is essential for homomeric 5-HT3 receptor functionTHE JOURNAL OF PHYSIOLOGY, Issue 4 2010Dai-Fei Wu The cysteine (Cys) residue at position 312 in the third transmembrane domain (M3) is conserved among 5-hydroxytryptamine type 3 (5-HT3) receptor subunits and many other subunits of the nicotinic acetylcholine (nACh) related Cys-loop receptor family, including most of the ,-aminobutyric acid type A (GABAA) and glycine receptor subunits. To elucidate a possible role for the Cys-312 in human 5-HT3A receptors, we replaced it with alanine and expressed the 5-HT3A(C312A) mutant in HEK293 cells. The mutation resulted in an absence of 5-HT-induced whole-cell current without reducing homopentamer formation, surface expression or 5-HT binding. The 5-HT3A(C312A) mutant, when co-expressed with the wild-type 5-HT3A subunit, did not affect functional expression of receptors, suggesting that the mutant is not dominant negative. Interestingly, co-expression of 5-HT3A(C312A) with 5-HT3B led to surface expression of heteropentamers that mediated small 5-HT responses. This suggests that the Cys-312 is essential for homomeric but not heteromeric receptor gating. To further investigate the relationship between residue 312 and gating we replaced it with amino acids located at the equivalent position within other Cys-loop subunits that are either capable or incapable of forming functional homopentamers. Replacement of 5-HT3A Cys-312 by Gly or Leu (equivalent residues in the nACh receptor , and , subunits) abolished and severely attenuated function, respectively, whereas replacement by Thr or Ser (equivalent residues in nACh receptor ,7 and GABAA, subunits) supported robust function. Thus, 5-HT3A residue 312 and equivalent polar residues in the M3 of other Cys-loop subunits are essential determinants of homopentameric gating. [source] De novo expression of Kv6.3 contributes to changes in vascular smooth muscle cell excitability in a hypertensive mice strainTHE JOURNAL OF PHYSIOLOGY, Issue 3 2009Alejandro Moreno-Domínguez Essential hypertension involves a gradual and sustained increase in total peripheral resistance, reflecting an increased vascular tone. This change associates with a depolarization of vascular myocytes, and relies on a change in the expression profile of voltage-dependent ion channels (mainly Ca2+ and K+ channels) that promotes arterial contraction. However, changes in expression and/or modulation of voltage-dependent K+ channels (Kv channels) are poorly defined, due to their large molecular diversity and their vascular bed-specific expression. Here we endeavor to characterize the molecular and functional expression of Kv channels in vascular smooth muscle cells (VSMCs) and their regulation in essential hypertension, by using VSMCs from resistance (mesenteric) or conduit (aortic) arteries obtained from a hypertensive inbred mice strain, BPH, and the corresponding normotensive strain, BPN. Real-time PCR reveals a differential distribution of Kv channel subunits in the different vascular beds as well as arterial bed-specific changes under hypertension. In mesenteric arteries, the most conspicuous change was the de novo expression of Kv6.3 (Kcng3) mRNA in hypertensive animals. The functional relevance of this change was studied by using patch-clamp techniques. VSMCs from BPH arteries were more depolarized than BPN ones, and showed significantly larger capacitance values. Moreover, Kv current density in BPH VSMCs is decreased mainly due to the diminished contribution of the Kv2 component. The kinetic and pharmacological profile of Kv2 currents suggests that the expression of Kv6.3 could contribute to the natural development of hypertension. [source] Activity-dependent regulation of voltage-gated Na+ channel expression in Mat-LyLu rat prostate cancer cell lineTHE JOURNAL OF PHYSIOLOGY, Issue 2 2006William J. Brackenbury We have shown previously that voltage-gated Na+ channels (VGSCs) are up-regulated in human metastatic disease (prostate, breast and small-cell lung cancers), and that VGSC activity potentiates metastatic cell behaviours. However, the mechanism(s) regulating functional VGSC expression in cancer cells remains unknown. We investigated the possibility of activity-dependent (auto)regulation of VGSC functional expression in the strongly metastatic Mat-LyLu model of rat prostate cancer. Pretreatment with tetrodotoxin (TTX) for 24,72 h subsequently suppressed peak VGSC current density without affecting voltage dependence. The hypothesis was tested that the VGSC auto-regulation occurred via VGSC-mediated Na+ influx and subsequent activation of protein kinase A (PKA). Indeed, TTX pretreatment reduced the level of phosphorylated PKA, and the PKA inhibitor KT5720 decreased, whilst the adenylate cyclase activator forskolin and the Na+ ionophore monensin both increased the peak VGSC current density. TTX reduced the mRNA level of Nav1.7, predominant in these cells, and VGSC protein expression at the plasma membrane, although the total VGSC protein level remained unchanged. TTX pretreatment eliminated the VGSC-dependent component of the cells' migration in Transwell assays. We concluded that the VGSC activity in Mat-LyLu rat prostate cancer cells was up-regulated in steady-state via a positive feedback mechanism involving PKA, and this enhanced the cells' migratory potential. [source] A novel Nav1.7 mutation producing carbamazepine-responsive erythromelalgia,ANNALS OF NEUROLOGY, Issue 6 2009Tanya Z. Fischer MD Objective Human and animal studies have shown that Nav1.7 sodium channels, which are preferentially expressed within nociceptors and sympathetic neurons, play a major role in inflammatory and neuropathic pain. Inherited erythromelalgia (IEM) has been linked to gain-of-function mutations of Nav1.7. We now report a novel mutation (V400M) in a three-generation Canadian family in which pain is relieved by carbamazepine (CBZ). Methods We extracted genomic DNA from blood samples of eight members of the family, and the sequence of SCN9A coding exons was compared with the reference Nav1.7 complementary DNA. Wild-type Nav1.7 and V400M cell lines were then analyzed using whole-cell patch-clamp recording for changes in activation, deactivation, steady-state inactivation, and ramp currents. Results Whole-cell patch-clamp studies of V400M demonstrate changes in activation, deactivation, steady-state inactivation, and ramp currents that can produce dorsal root ganglia neuron hyperexcitability that underlies pain in these patients. We show that CBZ, at concentrations in the human therapeutic range, normalizes the voltage dependence of activation and inactivation of this inherited erythromelalgia mutation in Nav1.7 but does not affect these parameters in wild-type Nav1.7. Interpretation Our results demonstrate a normalizing effect of CBZ on mutant Nav1.7 channels in this kindred with CBZ-responsive inherited erythromelalgia. The selective effect of CBZ on the mutant Nav1.7 channel appears to explain the ameliorative response to treatment in this kindred. Our results suggest that functional expression and pharmacological studies may provide mechanistic insights into hereditary painful disorders. Ann Neurol 2009;65:733,741 [source] Enhancement of Mussel Adhesive Protein Production in Escherichia coli by Co-expression of Bacterial HemoglobinBIOTECHNOLOGY PROGRESS, Issue 3 2008Doil Kim Mussel adhesive proteins (MAPs) have been considered as potential underwater and medical bioadhesives. Previously, we reported a functional expression of recombinant MAP hybrid fp-151, which is a fusion protein with six type 1 (fp-1) decapeptide repeats at each type 5 (fp-5) terminus, with practical properties in Escherichia coli. In the present work, we introduced the Vitreoscilla hemoglobin (VHb) co-expression strategy to enhance the production levels of hybrid fp-151 since VHb has been successfully used for efficient oxygen utilization in several expression systems, including E. coli. In both batch-type flask and fed-batch-type bioreactor cultures, we found that co-expression of VHb conferred higher cell growth and hybrid fp-151 production. Its positive effects were significantly increased in high cell density bioreactor cultures as the microaerobic environment was more quickly and severely formed. We obtained a ,1.9-fold higher (,1 g/L) production of MAP fp-151 from VHb co-expressing cells in fed-batch bioreactor cultures as compared to that from VHb non-expressing cells. Collectively and regardless of the culture type, VHb co-expression strategy was successful in enhancing the production of recombinant mussel adhesive proteins in the E. coli expression system. [source] | |||||||||||||||||||||||||||||||