Chinese Hamster Ovary Cell Membranes Research Articles

Bioconjugation of a Near-Infrared DNA-Stabilized Silver Nanocluster to Peptides and Human Insulin by Copper-Free Click Chemistry.

DNA-stabilized silver nanoclusters (DNA-AgNCs) are biocompatible emitters with intriguing properties. However, they have not been extensively used for bioimaging applications due to the lack of structural information and hence predictable conjugation strategies. Here, a copper-free click chemistry method for linking a well-characterized DNA-AgNC to molecules of interest is presented. Three different peptides and a small protein, human insulin, were tested as labeling targets. The conjugation to the target compounds was verified by MS, HPLC, and time-resolved anisotropy measurements. Moreover, the spectroscopic properties of DNA-AgNCs were found to be unaffected by the linking reactions. For DNA-AgNC-conjugated human insulin, fluorescence imaging studies were performed on Chinese hamster ovary (CHO) cells overexpressing human insulin receptor B (hIR-B). The specific staining of the CHO cell membranes demonstrates that DNA-AgNCs are great candidates for bioimaging applications, and the proposed linking strategy is easy to implement when the DNA-AgNC structure is known.

In vitro assessment of the binding and functional responses of ozanimod and its plasma metabolites across human sphingosine 1-phosphate receptors

Ozanimod is approved in multiple countries for the treatment of adults with either relapsing multiple sclerosis or moderately to severely active ulcerative colitis. Ozanimod is metabolized in humans to form seven active plasma metabolites, including two major active metabolites CC112273 and CC1084037, and an inactive metabolite. Here, the binding and activity of ozanimod and its metabolites across human sphingosine 1-phosphate receptors were determined. Binding affinity was assessed in Chinese hamster ovary cell membranes expressing recombinant human sphingosine 1-phosphate receptors 1 and 5 via competitive radioligand binding using tritium-labeled ozanimod; selectivity via functional potency assessment was performed using [35S]-guanosine-5'-(γ-thio)-triphosphate binding assays. Receptor internalization was assessed in human embryonic kidney 293 cells overexpressing sphingosine 1-phosphate receptor 1–green fluorescent protein and Chinese hamster ovary cells overexpressing sphingosine 1-phosphate receptor 5–hemagglutinin via fluorescence activated cell sorting. Functional activity was assessed in primary cultures of human astrocytes via phosphorylation assays. Ozanimod and its functionally active metabolites bound to the same sites within sphingosine 1-phosphate receptors 1 and 5, with metabolites displaying the same selectivity profile as ozanimod. Agonism at sphingosine 1-phosphate receptor 1 induced receptor internalization, whereas sphingosine 1-phosphate receptor 5 did not. Ozanimod, CC112273, and CC1084037 elicited functional intracellular signaling in human astrocytes, pharmacologically characterized to be mediated by sphingosine 1-phosphate receptor 1. The active plasma metabolites of ozanimod bound to sphingosine 1-phosphate receptors 1 and 5 and displayed similar pharmacologic profiles as their parent compound, likely contributing to clinical efficacy in patients with relapsing multiple sclerosis or moderately to severely active ulcerative colitis.

Assessment of TiO2 Nanoparticle Impact on Surface Morphology of Chinese Hamster Ovary Cells.

The process of nanoparticles entering the cells of living organisms is an important step in understanding the influence of nanoparticles on biological processes. The interaction of nanoparticles with the cell membrane is the first step in the penetration of nanoparticles into cells; however, the penetration mechanism is not yet fully understood. This work reported the study of the interaction between TiO2 nanoparticles (TiO2-NPs) and Chinese hamster ovary (CHO) cells using an in vitro model. The characterization of crystalline phases of TiO2 NPs was evaluated by transmission electron microscopy (TEM), X-ray diffraction (XRD) spectrum, and atomic force microscopy (AFM). Interaction of these TiO2 nanoparticles (TiO2- NPs) with the CHO cell membrane was investigated using atomic force microscopy (AFM) and Raman spectroscopy. The XRD analysis result showed that the structure of the TiO2 particles was in the rutile phase with a crystallite size of 60 nm, while the AFM result showed that the particle size distribution had two peaks with 12.1 nm and 60.5 nm. The TEM analysis confirmed the rutile phase of TiO2 powder. Our study showed that exposure of CHO cells to TiO2-NPs caused morphological changes in the cell membranes and influenced the viability of cells. The TiO2-NPs impacted the cell membrane surface; images obtained by AFM revealed an ‘ultra structure‘ with increased roughness and pits on the surface of the membrane. The depth of the pits varied in the range of 40–80 nm. The maximal depth of the pits after the treatment with TiO2-NPs was 100% higher than the control values. It is assumed that these pits were caveolae participating in the endocytosis of TiO2-NPs. The research results suggest that the higher maximal depth of the pits after the exposure of TiO2-NPs was determined by the interaction of these TiO2-NPs with the cell’s plasma membrane. Moreover, some of pits may have been due to plasma membrane damage (hole) caused by the interaction of TiO2-NPs with membrane constituents. The analysis of AFM images demonstrated that the membrane roughness was increased with exposure time of the cells to TiO2-NPs dose. The average roughness after the treatment for 60 min with TiO2-NPs increased from 40 nm to 78 nm. The investigation of the membrane by Raman spectroscopy enabled us to conclude that TiO2-NPs interacted with cell proteins, modified their conformation, and potentially influenced the structural damage of the plasma membrane.

Design, Synthesis, and Biological Evaluation of 4,4'-Difluorobenzhydrol Carbamates as Selective M1 Antagonists.

Due to their important role in mediating a broad range of physiological functions, muscarinic acetylcholine receptors (mAChRs) have been a promising target for therapeutic and diagnostic applications alike; however, the list of truly subtype-selective ligands is scarce. Within this work, we have identified a series of twelve 4,4’-difluorobenzhydrol carbamates through a rigorous docking campaign leveraging commercially available amine databases. After synthesis, these compounds have been evaluated for their physico–chemical property profiles, including characteristics such as HPLC-logD, tPSA, logBB, and logPS. For all the synthesized carbamates, these characteristics indicate the potential for BBB permeation. In competitive radioligand binding experiments using Chinese hamster ovary cell membranes expressing the individual human mAChR subtype hM1-hM5, the most promising compound 2 displayed a high binding affinitiy towards hM1R (1.2 nM) while exhibiting modest-to-excellent selectivity versus the hM2-5R (4–189-fold). All 12 compounds were shown to act in an antagonistic fashion towards hM1R using a dose-dependent calcium mobilization assay. The structural eligibility for radiolabeling and their pharmacological and physico–chemical property profiles render compounds 2, 5, and 7 promising candidates for future position emission tomography (PET) tracer development.

Electrochemiluminescence Microscopy of Cells: Essential Role of Surface Regeneration.

Electrochemiluminescence (ECL) microscopy is successfully applied to image cells, micro-/nano-objects, and electrochemical processes at electrode surfaces. The classic ECL tandem system is composed of the [Ru(bpy)3]2+ luminophore with the very efficient tripropylamine (TPA) coreactant. The dramatic decrease of the ECL signal observed when recording successive ECL images constitutes a key limitation for the development of ECL microscopy. Herein, we investigated the progressive decrease of the ECL signal of Chinese hamster ovary (CHO) cells. The plasma membranes of CHO cells were labeled with a [Ru(bpy)3]2+ derivative, and the ECL images were recorded using the TPA coreactant. We demonstrate that the loss of the ECL signal is related to the electrochemical step because of a progressive lower TPA oxidation current. We tested a cathodic regenerative treatment of the electrode surface, which allowed us to restore the initial TPA oxidation intensity and thus to record a sequence of ECL images without any vanishing of the light signal. The electrochemical approach presented here is an essential step for the development of ECL microscopy of cells.

Synthesis, Biological, and Computational Evaluation of Antagonistic, Chiral Hydrobenzoin Esters of Arecaidine Targeting mAChR M1

Muscarinic acetylcholine receptors (mAChRs) are a pivotal constituent of the central and peripheral nervous system. Yet, therapeutic and diagnostic applications thereof are hampered by the lack of subtype selective ligands. Within this work, we synthesized and chemically characterized three different stereoisomers of hydrobenzoin esters of arecaidine by NMR, HR-MS, chiral chromatography, and HPLC-logP. All compounds are structurally eligible for carbon-11 labeling and show appropriate stability in Dulbecco’s phosphate-buffered saline (DPBS) and F12 cell culture medium. A competitive radioligand binding assay on Chinese hamster ovary cell membranes comprising the human mAChR subtypes M1-M5 showed the highest orthosteric binding affinity for subtype M1 and a strong influence of stereochemistry on binding affinity, which corresponds to in silico molecular docking experiments. Ki values toward M1 were determined as 99 ± 19 nM, 800 ± 200 nM, and 380 ± 90 nM for the (R,R)-, (S,S)-, and racemic (R,S)-stereoisomer, respectively, highlighting the importance of stereochemical variations in mAChR ligand development. All three stereoisomers were shown to act as antagonists toward mAChR M1 using a Fluo-4 calcium efflux assay. With respect to future positron emission tomography (PET) tracer development, the (R,R)-isomer appears especially promising as a lead structure due to its highest subtype selectivity and lowest Ki value.

Pharmacological properties of whale shark (Rhincodon typus) melanocortin-2 receptor and melancortin-5 receptor: Interaction with MRAP1 and MRAP2

In the current study, the whale shark (ws; Rhincodon typus) melanocortin-2 receptor (MC2R) co-expressed with wsMRAP1 in Chinese Hamster Ovary (CHO) Cells could be stimulated in a dose dependent manner by ACTH(1–24) with an EC50 of 2.6 × 10−10 M ± 9.7 × 10−11. When the receptor was expressed alone, stimulation was only observed at [10−6 M]. A comparable increase in sensitivity to stimulation by srDes-Ac-αMSH was also observed when the receptor was co-expressed with wsMRAP1. In addition, co-expression with wsMRAP1 significantly increased the trafficking of wsMC2R to the plasma membrane of CHO cells. Surprisingly, co-expression with wsMRAP2 also increased sensitivity to stimulation by ACTH(1–24) and srDes-Ac-αMSH, and increased trafficking of the receptor to the plasma membrane. These observations are in sharp contrast to the response of MC2R orthologs of bony vertebrates which have an obligate requirement for co-expression with MRAP1 for both trafficking to the plasma membrane and activation, and while co-expression with MRAP2 increases trafficking, it has minimal effects on activation. In addition, when comparing the activation features of wsMC2R with those of the elephant shark MC2R and red stingray MC2R orthologs, both similarities and differences are observed. The spectrum of features for cartilaginous fish MC2R orthologs will be discussed. A second objective of this study was to determine whether wsMC5R has features in common with wsMC2R in terms of ligand selectivity and interaction with wsMRAP paralogs. While wsMC5R can be activated by either srACTH(1–24) or srDes-Ac-αMSH, and co-expression with wsMRAP1 enhances this activation, wsMRAP1 had no effect on the trafficking of wsMC5R. Co-expression with wsMRAP2 had no positive or negative effect on either ligand sensitivity or trafficking of wsMC5R.

Indomethacin Enhances Type 1 Cannabinoid Receptor Signaling.

In addition to its known actions as a non-selective cyclooxygenase (COX) 1 and 2 inhibitor, we hypothesized that indomethacin can act as an allosteric modulator of the type 1 cannabinoid receptor (CB1R) because of its shared structural features with the known allosteric modulators of CB1R. Indomethacin enhanced the binding of [3H]CP55940 to hCB1R and enhanced AEA-dependent [35S]GTPγS binding to hCB1R in Chinese hamster ovary (CHO) cell membranes. Indomethacin (1 μM) also enhanced CP55940-dependent βarrestin1 recruitment, cAMP inhibition, ERK1/2 and PLCβ3 phosphorylation in HEK293A cells expressing hCB1R, but not in cells expressing hCB2R. Finally, indomethacin enhanced the magnitude and duration of CP55940-induced hypolocomotion, immobility, hypothermia, and anti-nociception in C57BL/6J mice. Together, these data support the hypothesis that indomethacin acted as a positive allosteric modulator of hCB1R. The identification of structural and functional features shared amongst allosteric modulators of CB1R may lead to the development of novel compounds designed for greater CB1R or COX selectivity and compounds designed to modulate both the prostaglandin and endocannabinoid systems.

Probing Deviation of Adhered Membrane Dynamics between Reconstituted Liposome and Cellular System.

The dynamics of cell-cell adhesion are complicated due to complexities in cellular interactions and intra-membrane interactions. In the present work, we have reconstituted a liposome-based model system to mimic the cell-cell adhesion process. Our model liposome system consists of one fluorescein-tagged and one TRITC (tetramethyl-rhodamine isothiocyanate)-tagged liposome, adhered through biotin-neutravidin interaction. We monitored the adhesion process in liposomes using Förster Resonance Energy Transfer (FRET) between fluorescein (donor) and TRITC (acceptor). Occurrence of FRET is confirmed by the decrease in donor lifetime as well as distinct rise time of the acceptor fluorescence. Interestingly, the acceptor's emission exhibits fluctuations in the range of ≈3±1 s. This may be attributed to structural oscillations associated in two adhered liposomes arising from the flexible nature of biotin-neutravidin interaction. We have compared the dynamics in a cell-mimicking liposome system with that in an in vitro live cell system. In the adhered live cell system, we used CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, donor) and nile red (acceptor), which are known to stain the membrane of CHO (Chinese Hamster Ovary) cells. The dynamics of the adhered membranes of two live CHO cells were observed through FRET between CPM and nile red. The acceptor fluorescence intensity exhibits an oscillation in the time-scale of ≈1±0.75 s, which is faster compared to the reconstituted liposome system, indicating the contributions and involvement of multiple dynamic protein complexes around the cell membrane. This study offers simple reconstituted model systems to understand the complex membrane dynamics using a FRET-based physical chemistry approach.

Muscarinic agonist, (±)-quinuclidin-3-yl-(4-fluorophenethyl)(phenyl)carbamate: High affinity, but low subtype selectivity for human M1 – M5 muscarinic acetylcholine receptors

Novel quinuclidinyl N-phenylcarbamate analogs were synthesized, and binding affinities at M1-M5 muscarinic acetylcholine receptor (mAChR) subtypes were determined using Chinese hamster ovary (CHO) cell membranes stably expressing one specific subtype of human mAChR. Although not subtype selective, the lead analog (±)-quinuclidin-3-yl-(4-fluorophenethyl)(phenyl)carbamate (3c) exhibited the highest affinity (Ki = 2.0, 13, 2.6, 2.2, 1.8 nM) at each of the M1-M5 mAChRs, respectively. Based on results from the [3H]dopamine release assay using rat striatal slices, 3c acted as an agonist at mAChRs. The effect of 3c was inhibited by the nonselective mAChR antagonist, scopolamine, and 3c augmented release evoked by oxotremorine. A potent analog from the same scaffold, (±)-quinuclidin-3-yl-(4-methoxyphenethyl)(phenyl)-carbamate (3b) exhibited the greatest selectivity (17-fold) at M3 over M2 mAChRs. These analogs could serve as leads for further discovery of novel subtype-selective muscarinic ligands with the goal of providing therapeutics for substance use disorders and chronic obstructive pulmonary disease.

Rimonabant, a potent CB1 cannabinoid receptor antagonist, is a Gαi/o protein inhibitor

Rimonabant is a potent and selective cannabinoid CB1 receptor antagonist widely used in animal and clinical studies. Besides its antagonistic properties, numerous studies have shown that, at micromolar concentrations rimonabant behaves as an inverse agonist at CB1 receptors. The mechanism underpinning this activity is unclear. Here we show that micromolar concentrations of rimonabant inhibited Gαi/o-type G proteins, resulting in a receptor-independent block of G protein signaling. Accordingly, rimonabant decreased basal and agonist stimulated [35S]GTPγS binding to cortical membranes of CB1- and GABAB-receptor KO mice and Chinese Hamster Ovary (CHO) cell membranes stably transfected with GABAB or D2 dopamine receptors. The structural analog of rimonabant, AM251, decreased basal and baclofen-stimulated GTPγS binding to rat cortical and CHO cell membranes expressing GABAB receptors. Rimonabant prevented G protein-mediated GABAB and D2 dopamine receptor signaling to adenylyl cyclase in Human Embryonic Kidney 293 cells and to G protein-coupled inwardly rectifying K+ channels (GIRK) in midbrain dopamine neurons of CB1 KO mice. Rimonabant suppressed GIRK gating induced by GTPγS in CHO cells transfected with GIRK, consistent with a receptor-independent action. Bioluminescent resonance energy transfer (BRET) measurements in living CHO cells showed that, in presence or absence of co-expressed GABAB receptors, rimonabant stabilized the heterotrimeric Gαi/o-protein complex and prevented conformational rearrangements induced by GABAB receptor activation. Rimonabant failed to inhibit Gαs-mediated signaling, supporting its specificity for Gαi/o-type G proteins. The inhibition of Gαi/o protein provides a new site of rimonabant action that may help to understand its pharmacological and toxicological effects occurring at high concentrations.

Quaternary structures of opsin in live cells revealed by FRET spectrometry.

Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that initiates phototransduction in the retina. The receptor consists of the apoprotein opsin covalently linked to the inverse agonist 11-cis retinal. Rhodopsin and opsin have been shown to form oligomers within the outer segment disc membranes of rod photoreceptor cells. However, the physiological relevance of the observed oligomers has been questioned since observations were made on samples prepared from the retina at low temperatures. To investigate the oligomeric status of opsin in live cells at body temperatures, we utilized a novel approach called Förster resonance energy transfer spectrometry, which previously has allowed the determination of the stoichiometry and geometry (i.e. quaternary structure) of various GPCRs. In the current study, we have extended the method to additionally determine whether or not a mixture of oligomeric forms of opsin exists and in what proportion. The application of this improved method revealed that opsin expressed in live Chinese hamster ovary (CHO) cells at 37°C exists as oligomers of various sizes. At lower concentrations, opsin existed in an equilibrium of dimers and tetramers. The tetramers were in the shape of a near-rhombus. At higher concentrations of the receptor, higher-order oligomers began to form. Thus, a mixture of different oligomeric forms of opsin is present in the membrane of live CHO cells and oligomerization occurs in a concentration-dependent manner. The general principles underlying the concentration-dependent oligomerization of opsin may be universal and apply to other GPCRs as well.

Polyethylene glycol-mediated fusion of herpes simplex type 1 virions with the plasma membrane of cells that support endocytic entry.

BackgroundMouse B78 cells and Chinese hamster ovary (CHO) cells are important to the study of HSV-1 entry because both are resistant to infection at the level of viral entry. When provided with a gD-receptor such as nectin-1, these cells support HSV-1 entry by an endocytosis pathway. Treating some viruses bound to cells with the fusogen polyethylene glycol (PEG) mediates viral fusion with the cell surface but is insufficient to rescue viral entry. It is unclear whether PEG-mediated fusion of HSV with the plasma membrane of B78 or CHO cells results in successful entry and infection.FindingsTreating HSV-1 bound to B78 or CHO cells with PEG allowed viral entry as measured by virus-induced beta-galactosidase activity. Based on the mechanism of PEG action, we propose that entry likely proceeds by direct fusion of HSV particles with the plasma membrane. Under the conditions tested, PEG-mediated infection of CHO cells progressed to the level of HSV late gene expression, while B78 cells supported HSV DNA replication. We tested whether proteolysis or acidification of cell-bound virions could trigger HSV fusion with the plasma membrane. Under the conditions tested, mildly acidic pH of 5–6 or the protease trypsin were not capable of triggering HSV-1 fusion as compared to PEG-treated cell-bound virions.ConclusionsB78 cells and CHO cells, which typically endocytose HSV prior to viral penetration, are capable of supporting HSV-1 entry via direct penetration. HSV capsids delivered directly to the cytosol at the periphery of these cells complete the entry process. B78 and CHO cells may be utilized to screen for factors that trigger entry as a consequence of fusion of virions with the cell surface, and PEG treatment can provide a necessary control.

Muscarinic acetylcholine receptor binding affinities of pethidine analogs.

A series of pethidine analogs were synthesized and their affinities for the [(3)H]N-methyl-scopolamine (NMS) binding site on muscarinic acetylcholine receptors (mAChRs) were determined using M1, M3 or M5 human mAChRs expressed by Chinese hamster ovary (CHO) cell membranes. Compound 6b showed the highest binding affinities at M1, M3 and M5 mAChRs (Ki=0.67, 0.37, and 0.38 μM, respectively).

Discovery of M5 Muscarinic Acetylcholine Receptor Antagonists: 1‐Methyl‐4‐Phenylpiperidine Analogs

Ventral tegmental area (VTA) dopamine (DA) neurons project to nucleus accumbens (NAc) and are believed to mediate the rewarding effects of abused drugs. Stimulation of M5 muscarinic acetylcholine receptors (mAChRs) activates VTA DA neurons (Omelchenko N et al., 2006). M5 knockout mice exhibit reduced cocaine and morphine self-administration (Fink-Jensen et al., 2003; Basile et al., 2002). Microinfusion of scopolamine (M1-M5 mAChRs antagonist) into VTA attenuates cocaine self-administration (Solecki et al., 2013). Thus, M5 mAChR antagonists may be novel treatments for drug abuse. We evaluated a series of 1-methyl-4-phenylpiperidine containing analogs as antagonists at M5 mAChRs. Affinity for 12 analogs at the [3H]N-methylscopolamine binding site was determined using Chinese hamster ovary cell membranes expressing human M1, M3 or M5 mAChRs. XZ-11341a exhibited the highest affinity at M1, M3 and M5 mAChRs (Ki = 0.67 ± 0.08, 0.37 ± 0.04, 0.38 ± 0.011 µM, respectively). Generally, replacing the ester in with an amide reduced affinity at all subtypes. For example, XZ-11343b (amide) had no affinity compared with XZ-11343a (ester, moderate affinity) at M3 mAChRs. Replacing the carbamate moiety with a carbamide also attenuated affinity at all subtypes. Current insights regarding the SAR will direct future synthetic approaches towards identifying potent selective M5 mAChR antagonists (Supported by DA030667).

Low dosage of rimonabant leads to anxiolytic-like behavior via inhibiting expression levels and G-protein activity of kappa opioid receptors in a cannabinoid receptor independent manner

What is knownThere is an increasing number of studies demonstrating the direct effect of the cannabinoid receptor 1 (CB1) antagonist/inverse agonist rimonabant on the opioid system. The kappa opioid receptors (KORs) are well known to mediate depression- and anxiety-like behavior. Clinical studies on chronic rimonabant administration have revealed that rimonabant leads to a very similar pathophysiology, suggesting a potential impact of rimonabant on KORs. ObjectivesOur objectives were to examine the putative effects of rimonabant on KOR ligand binding, G-protein activity, protein expression and how all these contribute to the development of depression- and anxiety-like behavior. ResultsIn Chinese hamster ovary (CHO) cell membranes transfected with rat KOR (CHO-rKOR) rimonabant inhibited KOR agonist [3H]U69593 binding in the micromolar range in competition binding experiments and specifically reduced KOR basal activity at lower micromolar concentrations in [35S]GTPγS binding assays. Rimonabant significantly inhibited dynorphin (1–11)-induced [35S]GTPγS binding in micromolar range in CHO-rKOR cells, CB1 knockout (CB1 K.O.) and CB1/CB2 double knockout mouse forebrain membranes. A single dose of i.p. 0.1 mg/kg rimonabant significantly reduced dynorphin (1–11)-induced KOR G-protein activity and KOR protein expression levels 24 h following the administration in both wild type and CB1 K.O. mice forebrain. Furthermore, in elevated plus maze mice showed an anxiolytic-like effect upon rimonabant injection that could be reversed by 1 mg/kg KOR antagonist norbinaltorphimine. The anxiolytic-like effects were further confirmed with the light–dark box test. ConclusionRimonabant reduced KOR ligand binding, receptor mediated G-protein activity and protein expression level, which overall leads to altered anxiety-like behavior.

Cytoskeleton Modification and Cholesterol Depletion Affect Membrane Properties and Caveolae Positioning of CHO Cells

The formation of protrusions is necessary for numerous biological processes. It involves extension of the plasma membrane, and the force needed for this is provided by the actin cytoskeleton. Tether pulling with optical tweezers can mimic the formation of a protrusion, so we used this method to investigate the effects of modifying not only actin (with latrunculin A) but also microtubules (with nocodazole) and the plasma membrane itself (with methyl-β-cyclodextrin) on the Chinese hamster ovary cell membrane. After these modifications, the membrane reservoir was supposed to redistribute. Caveolae constitute a small part of the reservoir, so the redistribution of caveolar proteins such as caveolin-1 and cavin-1 that represents caveolae per se was assessed. The main findings concerning protrusion force and membrane reservoir availability were as follows: (1) they correlated inversely, (2) their values underwent the greatest change after microtubule disruption, and (3) membrane composition had a major influence on the parameters studied. F-actin disruption and cholesterol depletion decreased, and microtubule disruption increased the amount of the caveolar proteins (caveolae). Caveolae presented just an example of the membrane reservoir, and from our findings, we suppose that the perturbations caused were too large to be related to caveolae redistribution alone. The integrity of the cytoskeleton and plasma membrane composition are important factors in the formation of protrusions and in determining the availability and distribution of the membrane reservoir.

GpA Dimerization in Plasma Membranes of CHO, HEK293T and A431 Cells

Glycophorin A is the major sialoglycoprotein of the human erythrocyte membrane. GpA dimerization has been extensively studied in detergents and in model lipid bilayers, but measurements in the plasma membrane of mammalian cells are rare. Using a FRET based technique, we have measured the free energy of GpA dimerization in plasma membrane vesicles derived from Chinese Hamster Ovary (CHO), Human Embryonic Kidney (HEK293T) and A431 (epidermoid carcinoma) cells, using three different vesiculation methods. These measurements provide new insights into the effect of the environment on membrane protein interactions.

The propeptide of human thyroid peroxidase is not required for its cellular, enzymatic or immunological activity

Thyroid Peroxidase (TPO), a dimeric, membrane-bound glycoprotein found in the thyroid follicular lumen, catalyses the biosynthesis of thyroxine, and is also a major autoantigen in autoimmune thyroid disease (AITD). TPO molecules undergo posttranslational modifications such as glycosylation, heme incorporation, dimer formation, and proteolytic processing in the N-terminal region. Due to propeptide deletion, in human thyroid cells the TPO protein loses 108 amino acids in the N-terminus to yield mature enzyme. During the course of studies to obtain homogenous preparations of recombinant TPO for structural studies, we investigated the role of the propeptide in TPO structure, function and trafficking. We engineered recombinant human TPO truncated at the N-terminal residue (TPO-pro) and subsequently expressed in Chinese hamster ovary (CHO) cells. Then we characterized TPO-pro and compared its properties with wild-type recombinant TPO (TPOwt) based upon its subcellular localisation, immunological and biochemical properties such as glycosylation and enzymatic activity. Molecular modeling and dynamics simulation of TPO-pro comprising a dimer of MPO-like domains were performed in order to investigate the impact of propeptide removal. Using immunodetection and densitometric analysis we showed that deletion of the propeptide did not influence expression level of TPO-pro in comparison with TPOwt. Upon SDS-PAGE under non-reducing conditions, we observed dimer formation for both TPOwt and TPO-pro. Furthermore, we tested by flow cytometry the reactivity of TPO constructs with anti-TPO antibodies and confirmed that the propeptide deletion did not disturb TPO transport to the membrane of CHO cells. This finding was further supported by immunocytochemistry. ELISA and flow cytometry analysis revealed that the conformation of epitopes recognized by a panel of human anti-TPO antibodies was not considerably affected in TPO-pro. Measurement of the Amplex Ultra Red oxidation of cells expressing TPOwt and TPO-pro confirmed faithful enzymatic activity of extracellular truncated TPO. Study of the carbohydrate content showed that TPO-pro is effectively N-glycosylated. Molecular modelling and dynamics simulations were consistent with our findings. In conclusion, the propeptide is not essential for the proper folding and transport of TPO produced in CHO cells. The successful expression in a membrane-anchored, active form that is insensitive to intramolecular proteolysis is a key advance for purification of substantial quantities of homogenous preparation of TPO for structural and crystallization studies.

Effects of geissoschizine methyl ether, an indole alkaloid in Uncaria hook, a constituent of yokukansan, on human recombinant serotonin 7 receptor.

Effects of seven alkaloids, geissoschizine methyl ether (GM), hirsutine, hirsuteine, rhynchophylline, isorhynchophylline, corynoxeine and isocorynoxeine, in Uncaria hook, a constituent of the kampo medicine yokukansan, on serotonin(7) (5-HT(7)) receptor were investigated using Chinese hamster ovary (CHO) cell membranes and human embryonic kidney 293 (HEK293) cells stably expressing the human recombinant 5-HT(7) receptor. A competitive binding assay using CHO membranes showed that GM (IC(50)=0.034μM) more strongly inhibited the binding of the radioligand [(3)H] LSD to 5-HT(7) receptor than the other alkaloids, suggesting that GM is bound to 5-HT(7) receptor. Agonistic/antagonistic effects of GM (1-50μM) on the receptor were evaluated by measuring intracellular cAMP levels in HEK239 cells. GM (IC(50)=6.0μM) inhibited 5-HT-induced cAMP production in a concentration-dependent manner, as well as the specific 5-HT(7) receptor antagonist SB-269970 (0.1-1μM). However, GM did not induce intracellular cAMP production as 5-HT did. These results suggest that GM has an antagonistic effect on 5-HT(7) receptor.