Total RNA was extracted from cell membrane preparations, and labeled cDNA's representing the mRNA pool were reverse-transcribed directly from total RNA isolated for use in the microarray hybridizations. Two specific human GE Array Kits (Superarray Inc.) that both contain 23 marker genes, related to signal transduction pathways or cancer/tumor suppression, plus 2 housekeeping genes (β-actin and GAPDH), were utilized. Arrays from treated and control cells (n = 4 per group) were evaluated using a Student's t-test. Gene expression was significantly induced for tumor protein p53 (p53), cyclin-dependent kinase inhibitor 1C (p57 Kip2), breast cancer Type 2 early onset (BRCA2), cAMP responsive element binding protein 2 (ATF-2), interleukin 2 (IL-2), heat shock 27 KD protein (hsp27), and CYP19 (aromatase). Induction of p57 Kip2, p53, BRCA2, IL-2, and ATF-2 would be expected to decrease cellular proliferation and increase tumor suppression and/or apoptosis. PEITC treatment produced significant alterations in some genes involved in tumor suppression and cellular proliferation/apoptosis that may be important in explaining the chemopreventive effects of PEITC.Phenethyl isothiocyanate (PEITC), a component in cruciferous vegetables, can block chemical carcinogenesis in animal models. Our objective was to determine the effect of treatment with PEITC on gene expression changes in MCF-7 human breast cancer cells in order to evaluate potential mechanisms involved in its chemopreventive effects. MCF-7 cells were treated for 48 hours with either PEITC (3  Brassica and other vegetables of the family Cruciferae  and the genus Raphanus (radishes and daikons) [μmol/day) and tens of milligrams of ITCs are released following the consumption of vegetables in the diet [Diet and environmental factors may represent very significant factors in the genesis of breast cancer. Organic isothiocyanates , also known as mustard oils, are present in the form of glucosinolates in daikons) . When vedaikons) , 2. The the diet . AdditioBrassica vegetable consumption increases the ratio of 2-hydroxyestrone (a noncarcinogenic metabolite of estrogen) to 16α-hydroxyestrone (a carcinogenic metabolite of estrogen), in healthy postmenopausal women [More than 20 natural and synthetic ITCs have been shown to block chemical carcinogenesis induced by environmental carcinogens including polycyclic aromatic hydrocarbons and nitrosamines in animal models –6. It haal women . The mec50 value of PEITC for inhibition of cell growth of human breast cancer MCF-7 cells  is 6.51 ± 0.86 μM [α36 protein  in breast cancer cells [α36 protein is unknown [Phenethyl isothiocyanate PEITC,  is one o 0.86 μM . Interes 0.86 μM . In addier cells . The exa unknown . PEITC i unknown . PEITC i unknown .μM) of PEITC. Studies have examined the mechanisms underlying the cell growth inhibitory effects of PEITC in human leukemia [To better understand the precise molecular mechanism(s) by which PEITC exerts its effects on MCF-7 human breast cancer cells, we utilized cDNA gene arrays to assess the gene expression profiles of breast cancer cells treated with physiologically relevant levels  were purchased from Sigma-Aldrich . RPMI 1640 medium, fetal bovine serum, and PBS were from Invitrogen . MCF-7 cells were obtained from the National Cancer Institute. 2 cell culture flasks in RPMI 1640 culture media supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml of streptomycin, in a 37°C incubator in a humidified atmosphere of 5% CO2/95% air. MCF-7 cells, with passage numbers of 16 to 24, were used in the experiments. Cells were treated with either 3 μM PEITC or 0.015% DMSO (vehicle control) for 48 h. The rationale for choosing this time point was to capture gene expression profiles of genes involved during the onset of growth inhibition and apoptotic processes. The concentration of PEITC used is one that is achievable in plasma after the consumption of food or dietary supplements. Plasma concentration of up to 1 μM has been reported after the ingestion of watercress 100 g [MCF-7 cells were grown in 75 cmss 100 g .Total RNA from each sample was isolated using a SV Total RNA Isolation System , following the manufacturer's instructions. Total RNA was quantitated spectrophotometrically at 260 nm. β actin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH)) , were utilized. Each array consists of 23 genes in duplicate, as well as control spots ) . The genβ actin  and CYP19 using Stratagene's Mx4000 Multiplex Quantitative PCR System . The same total RNA prepared for the gene arrays was also used for RTQ RT-PCR. Total RNA (560 ng) from each sample was reverse transcribed into cDNA using a Superscript first strand cDNA synthesis kit  according to the manufacturer's protocol. PCR reactions for CYP19 and β actin were carried out by mixing 5 μL of cDNA, 5 μL of 10  × PCR buffer, 2 μL of deoxynucleoside triphosphate mix , 1 μL each of 10 μM primer, 0.5 μL reference dye rhodamine-X , 0.5 μL SYBR green I , 2 U Taq polymerase  and 34.75 μL H2O, and amplified for 40 cycles. Primers for β actin  were designed using the computer program Primer Express . Primers for CYP19  have been described [Real-time quantitative reverse transcriptase polymerase chain reaction was performed on escribed . The PCREscherichia coli cells . Cloned PCR products were confirmed by sequencing and used to construct standard curves for absolute quantification of copy number. The standard curves were run in triplicate concurrently on the same plate with samples, which were also run in triplicate. The reported copy number was estimated from the linear regression of the standard curve on the same plate. The PCR product of each gene was cloned into a pCR 2.1 TOPO vector  and transformed into One Shot chemically competent t-tests with P < .05 was used for statistical analysis for both arrays and RTQ RT-PCR.Student's μM) significantly altered the expression of seven of the 46 genes present on the two arrays: p53, ATF-2, hsp27, BRCA2, IL-2, p57, and CYP19 (Treatment of MCF-7 cells for 48 hours with PEITC (3 nd CYP19 . All 7 gμM) is consistent with the findings of a publication that reported the effects of PEITC in human nonsmall cell lung carcinoma A549 cells [μM), and Western blot analyses demonstrated that increased expression of p53 protein was associated with PEITC-induced apoptosis [μM did not increase the protein expression of p53 in MCF-7 cells [μM for 24 or 36 h) suppressed the expression of p53 [PEITC increased the expression of p53, a tumor suppressor gene, by 2.12 fold . Tumor s49 cells . In thespoptosis . c-Jun Npoptosis . In cont-7 cells . Howevern of p53 , suggestμM PEITC upregulated expression of GADD34 mRNA [Although we did not investigate the effect of PEITC on p53 activity, it is possible that PEITC also induces p53 activity through GADD34 (Growth Arrest and DNA Damage-Inducible Protein), a proapoptotic gene. In a previous study, 25 D34 mRNA , and GADD34 mRNA . In factD34 mRNA . In thisExpression of ATF-2 was increased by PEITC (2.60-fold). ATF-2 is a member of the ATF/CREB family of proteins. It is also known as a cAMP response element-binding protein (CREBP-1). It acts as a transcription factor which regulates transcription in response to the extracellular signals and has a decisive role in cell proliferation, tumorigenesis and apoptosis. Breast cancer frequently develops in mutant mice heterozygous for the ATF-2 gene . TherefoκB and p53 signaling pathways [PEITC exposure resulted in the upregulation of Hsp27 by 1.84-fold in MCF-7 cells in this study. The induction of Hsp27 in response to various types of stress correlates with increased resistance to subsequent cellular damage [pathways . Howeverpathways .p57 is a tight-binding inhibitor of several G1 cyclin/Cdk complexes and a negative regulator of cell proliferation . Even thPEITC upregulated BRCA2 by 4.22-fold in the current study. BRCA2 functions as a tumor suppressor gene which stabilizes DNA structures at stalled replication forks . MutatioInterleukin-2 (IL-2) was upregulated by PEITC in this investigation. IL-2 is a cytokine produced by T cells whose main function is to stimulate the growth and cytotoxic response of activated T lymphocytes . IL-2 haβ actin, the increase of CYP19 was 1.8-fold compared to controls (P = .060) by RTQ RT-PCR, compared to 5.22-fold from the gene array data. The AP-1 motif, which regulates the CYP19 promoter [μM PEITC has been reported to activate AP-1 activity in both prostate and bladder cancer cell lines [The gene CYP19 expression was significantly increased by PEITC exposure. We confirmed the upregulation of CYP19 observed in the gene array studies, using RTQ RT-PCR. After normalization to promoter , may be ll lines , 54.The induction of CYP19 (aromatase) which is a key enzyme involved in the conversion of androgens to estrogens, would be considered a negative effect with regards to cancer prevention . Since eμM) can produce significant alterations in genes involved in tumor suppression and cellular proliferation/apoptosis. We report for the first time transcriptional induction of p53, ATF-2, hsp27, BRCA2, IL-2, p57, and CYP19 by PEITC in the human breast cancer cells, and our results are supported by other studies which have reported increased expression [In conclusion, PEITC treatment (3 pression  and incrpression  of p53 ppression . These a


Calmodulin is an important multifunctional molecule that regulates the activities of a large number of proteins in the cell. Calcium binding induces conformational transitions in calmodulin that make it specifically active to particular target proteins. The precise mechanisms underlying calcium binding to calmodulin are still, however, quite poorly understood.In this study, we adopt a structural systems biology approach and develop a mathematical model to investigate various types of cooperative calcium-calmodulin interactions. We compare the predictions of our analysis with physiological dose-response curves taken from the literature, in order to provide a quantitative comparison of the effects of different mechanisms of cooperativity on calcium-calmodulin interactions. The results of our analysis reduce the gap between current understanding of intracellular calmodulin function at the structural level and physiological calcium-dependent calmodulin target activation experiments.2+ dissociation and cooperatively effected dissociation constants, and variations in the number of Ca2+ ions required to bind CaM for target activation.Our model predicts that the specificity and selectivity of CaM target regulation is likely to be due to the following factors: variations in the target-specific Ca CaMKII kinase, on the other hand, binds to the Ca2+-CaM complex rather than apo-CaM. Each kinase has a unique combination of K1 and K2 dissociation constants. The half length peptides of CaATPase and skMLCK appear to have an even more complex mechanism of binding. As mentioned earlier, each Ca2+-CaM complex (with variable numbers of Ca2+ ions bound) may have a unique set of K1 and K2 constants for a target protein or peptide. For simplicity, all possible CaM species were divided into two groups: i) those with less than 3 Ca2+ ions bound and ii) those with three and four Ca2+ ions bound to CaM. A comparison of the model predictions with the Ca2+-CaM-parts of skMLCK and CaATPase peptide binding data  Mathematical models for protein interactions are usually derived according to a number of assumptions which will inevitably be more or less applicable to each particular protein. The structure of CaM suggests that this molecule is very likely to have non-sequential Ca2+ binding to CaM and Ca2+-CaM-target peptides. This analysis allowed us to distinguish between (a) proteins that form a complex with CaM in its Ca2+ free state and then interact with Ca2+ ions and (b) other proteins which interact with Ca2+ bound CaM with variable numbers of Ca2+ ions. However, the transient kinetics has not been addressed in this study.(2) Our model suggests that the structural data alone cannot provide the required level of information and comparisons with the dose-response data are required. Predictions from the mathematical model used in this study were compared with the dose-response curves for Ca2+-CaM-target complex assembly and the number of Ca2+ ions required to bind CaM for target activation.(3) In addition to the diversity of interaction interfaces, the specificity and selectivity of CaM target activation may be achieved by variations in the target-specific dissociation and cooperatively effected dissociation constants, the order of Ca2+-CaM interactions in our study. To clarify how various factors contribute to the CaM-dependent regulation, we describe three models for CaM starting from a very basic approximation of completely independent Ca2+ binding sites and gradually progressing to more realistic models that take account of cooperativity mechanisms and the Ca2+-CaM-target peptide complex assembly.Here, we present the mathematical equations used to describe Ca2+-CaM interactions, Ca2+ binding sites can be considered independent. We therefore derive the equations for the case of independent binding. We assume that CaM undergoes a conformational transition upon Ca2+ binding and adopts a unique conformation according to the number of bound ions. We will denote CaM conformations by cmj, j = 0,1, ..., 4. cm0 is a conformation with no bound ligand molecules and cm1 is a conformation with one bound ligand molecule. The concentration of CaM conformation with a given number of bound ligand molecules as a function of ligand concentration is given by:In this model we assume that at very basic level of Cacm0 is the total concentration of CaM, u is normalised Ca2+ concentration,i not being occupied and i being bound. ci equals 1 if a binding site is occupied and 0 if it is not. The probability of CaM being in a particular bound state is equal to the product of the probabilities of each individual binding site.where 2+ concentration are given by:The probabilities for a binding site to be not occupied or occupied as a function of CaK and u are the microscopic equilibrium dissociation constant and the ligand concentration, respectively. Effectively these are Michaelis-Menten equations for a protein in a complex with and without a ligand molecule, but normalized by the total protein concentration.where the n binding sites:The multiplication of probabilities from (2) for occupied sites gives an equation for a fully bound protein with K and u are the equilibrium dissociation constant and the ligand concentration, respectively.where the 2+ binding to CaM are the Hill [Other well known models to describe Cathe Hill  and the the Hill ,52 equatA complete mathematical description of the relationship between macroscopic constants derived from the Adair equation and the proposed model is provided in the Supplementary Materials Section  with n ligand binding sites in a particular ligand-bound state is given by the multiplication of probabilities (2):In the most general case, the concentration of any multisite protein L0 is the total protein concentration and Li is the concentration of conformation i, Kj are the equilibrium dissociation constants of each binding site, and u is the ligand concentration.where If a protein has identical binding sites, equation 5 simplifies to the following formula:L1(u), ..., Ln-1(u) of a multisite protein are all bell-shaped curves, the conformation L0(u) is the apo state of a multisite protein, whereas the Ln(u) is the fully bound multisite protein. If equation (5) is divided by L0, then instead of predicting protein concentrations in specific ligand-bound conformations, it predicts the probability of a particular conformation to be in that state as a function of ligand concentration.The conformations N as a function of Ca2+ concentration is given by:The formula predicting the concentration of CaM in complex with target protein cmi(u) is substituted from equation 5, and Kd is the dissociation constant for CaM-target interactions, N0 is the total concentration of target protein.where 2+-CaM complexes as a function of Ca2+ concentration with a reasonable accuracy, it does not capture the effects of the cooperative influence of Ca2+ binding sites. There are several possible ways to incorporate these cooperative mechanisms into the model. In order to derive a model that illustrates what contribution cooperativity makes to the distribution of concentration profiles of Ca2+-CaM complexes, we assume that in the N-terminal domain, the first centre is cooperatively bound to the second, and in the C-terminal, the third is cooperatively bound to the fourth. In this case, we will define the dissociation constants as K1, K2, K3, K4, While the previous model provides predictions for the number of CaThe probabilities for the first binding site to be free K1 and u are the equilibrium dissociation constant for the first centre and the ligand concentration, respectively.where The probabilities for the second centre to be in a particular state are:K2 and 2+ binding site.where The probabilities for the third centre are:K3 is a dissociation of the third binding site.where The probabilities for the fourth site are given by:K4 and 2+ binding site.where cmi conformations to be in a particular conformation with i bound ligand molecules as a function of ligand concentration are:The probabilities of ki = 0, if the binding site i is not occupied and ki = 1 if the centre i is occupied by a ligand molecule.where 2+-CaM complexes with 1, 2 and 3 Ca2+ ions with and without cooperativity is shown in Figure The distribution of intermediate CaWe next compare the amount of bound ligand in the presence and in the absence of the cooperative mechanism. In the absence of any cooperativity the multisite protein binds ligand molecules according to the equation:In the case of pairs of cooperatively interacting centres, the amount of bound ligand is given by:Figure C-terminal domain. A more precise description would also assume the first Ca2+ binding site cooperatively depends on the second and the third Ca2+ binding site depends on the fourth binding sites. For two Ca2+ binding sites in the N-terminal domain, the more realistic case is described by the following system of differential equations:In the previous model we have assumed that the second binding site was cooperatively dependent on the first, but the first site was not dependent on the second. Similar assumptions were made for the cm00, cm01, cm10, cm11 are CaM molecules without Ca2+ ions, with one Ca2+ ion bound to the N-terminal domain, with one Ca2+ ion bound to the N-terminal domain, and CaM species with two bound Ca2+ ions at each terminal domain, respectively. k1 and k2 are the association constants and kc1 and kc2 are the cooperatively modified association constants for the N- terminal binding sites of CaM, respectively. Similarly, d1, d2, dc1, dc2 are the dissociation and cooperatively modified dissociation constants for the N- and terminal binding sites, respectively. Note that a similar system of differential equations can be developed for the C-terminal.where The conservation law gives:In steady-state, the matrix for the system (15) with the last equation substituted by (16) is given by:The matrix (17) can be solved using Cramer's method. The determinant of the matrix is given by:k1 = k2 = kc1 = kc2 is a simplifying assumption that allows analytical solution of the current system.where N-terminal domain are given by:The matrices for the individual species of the N00, N01, N10 and N11 correspond to the N-terminal domain without any Ca2+ ions bound, with one Ca2+ ion bound to one or another binding site and to the fully bound state, respectively.where The determinants of the matrices (19–22) are given by:N-terminal to be in a particular state with variable numbers of Ca2+ ions are given by:The probabilities for the N-terminal to be in a Ca2+ state, 2+ ion, and N-terminal to be occupied by Ca2+.where C-terminal state as a function of Ca2+ concentration, a similar procedure can be applied. Similarly to (17) and (18), the determinant for the C-terminal is given by:In order to derive the steady-state dependence of the Where C-terminal states is given by:Similarly to (19–23), the solution for the C-terminal to be in a particular state with variable number of Ca2+ ions are given by:The probabilities for the N-terminal to be in a Ca2+ state, 2+ ion, and N-terminal occupied by Ca2+.where 2+ ions:The combination of (24) and (27) provides the solution for individual CaM species with variable numbers of CaN- and C-terminals, respectively. 2+ ion bound to N- and C-terminals, respectively. Comparison of numerical solutions of this system with the available experimental data [N- and C-terminal globular domains, each containing two symmetrical and cooperatively bound EF-hand Ca2+ binding sites.where tal data ,15,33 al2+-CaM interactions and incorporate the pairwise cooperative interactions between the EF-hand binding sites within the N- and C-terminals, whereas the N- and C-terminal domains are considered to be independent of each other. Note that the equations developed here for the Ca2+-CaM complexes are essentially different from the Michelis-Menten, Hill and Adair models, and also differ from models with independent binding sites (5) or with limited amounts of cooperativity (11) and (12).The equations (28) are specific to Ca2+-CaM complexes (28) have been further applied to calculate binding with the target peptides and proteins:The equations for the CaT0 is the total concentration of a target protein or a peptide, Kd is the equilibrium dissociation constant between CaM and a target protein, and 2+ complex or the sum of several CaM complexes.where 2+-CaM complexes have been varied simultaneously with the dissociation constants to fit the experimental data. This analysis allows us to predict the Ca2+-CaM complexes required for activation of specific protein targets. The fitting of dissociation constants of Ca2+ binding sites on CaM molecules to the experimental dose-response curves reveals the impact of the target protein on the Ca2+-CaM interactions. The dissociation constants calculated based on the cooperative Model 3, which also takes into account the impact of target proteins, are compared with the dissociation constants calculated using the Hill and modified Adair equation in the original experimental publications for the same data in Table The combinations of CaNVV developed and implemented the project under the supervision of DGB and NVK. All authors contributed to the analysis of the model, and PH–H provided biological interpretations of the results. All authors contributed to the writing of the final manuscript.2+-CaM binding model with free Ca2+ access.The relationship between the Adair and independent binding models. The relationship between macroscopic constants derived from the Adair equation and the proposed CaClick here for file


Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN) in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS) and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD) imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS.G93A associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage – dependent cytosolic Ca2+ elevations and mitochondria – controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca2+ elevations in WT (SH-SY5YWT) and G93A (SH-SY5YG93A) expressing cells were observed. In contrast, mitochondrial intracellular Ca2+ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation.To gain insights into the molecular mechanisms of SOD12+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells an established cellular model of ALS. The primary goals of this paper are (1) method development, and (2) screening for deficits in mutant cells on the single cell level. On the technological level, our method promises to serve as a valuable tool to identify mitochondrial and Ca2+-related defects during G93A-mediated MN degeneration. In addition, our experiments support a model where a specialized interplay between cytosolic calcium profiles and mitochondrial mechanisms contribute to the selective degeneration of neurons in ALS.In this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca SOD1 with high inter-subject variation of progression, including the point mutation G93A i) signaling in cells transfected with wild-type and G93A-mutant SOD1 varies at physiological levels, and if there are functional consequences of changes in mitochondrial function on Ca2+ homeostasis in the presence of mtSOD1G93A. To elucidate the underlying molecular events and cellular alterations involved in oxidative stress induced by the aberrant Cu-Zn chemistry and the roles of impaired Ca2+ handling and oxidative stress induced by ROS in fALS, we used SH-SY5Y neuroblastoma cells transfected with the G93A mutant form of SOD1 typical for fALS (SH-SY5YG93A or G93A) and SH-SY5Y cells transfected with wild-type (WT) human SOD1 (SH-SY5YWT or WT) and mimic the situation present in heterozygous patients, previously established as an in-vitro cell culture model of ALS, resembling the situation of heterozygous patients .2 i were measured in non-transfected SH-SY5Y cells  and SH-SY5Y cells expressing WT or G93A attached to glass cover slips after 2–5 days in culture. Cell layers were incubated with RPMI-1640  containing 10% FCS  and 0.846 mM Ca2+ (supplier's data). Cells were rinsed with RPMI -1640 and incubated for 20 min at 37°C to allow for complete de-esterification.The calcium-sensitive fluorescent dye, Fura-2, was introduced into cells by loading the membrane-permeable AM-ester forms .Changes in [Cantration . Therefo2+]i and was represented as F/F0. Further analysis was performed off-line with the IGOR software  and Origin software, version 7.5.Fura-2 (Kd ~ 0.2 μM) was monitored by a computer-controlled monochromator , which was connected to an Axioscope microscope  via quartz fiber optics and a minimum number of optical components for maximum fluorescence excitation . The apparatus was equipped with a CCD camera system ,52, whic2+]i and mitochondrial calcium ([Ca2+]mito) were performed utilizing calcium-sensitive fluorescent dyes . SH-SY5Y cells expressing either WT or G93A were attached to glass cover slips and loaded initially with 5 μM Fura-2 AM at 37°C for 30 min as described above. Subsequently, cells were washed with RPMI-1640 for 20 min and further incubated with 10 μM Rhod-2 AM for 20 min at 37°C (5% CO2 and 95% O2). The coverslips were rinsed with RPMI-1640 and further incubated for 20 min at 37°C to allow for complete de-esterification.Simultaneous fluorometric measurements of [Ca2+]i and [Ca2+]mito profiles with a temporal resolution in the millisecond time domain, we utilized a computer-controlled monochromator that rapidly switched between excitation wavelengths of 390 nm (Fura-2) and 550 nm (Rhod-2) and collected wavelengths above 565 nm for both excitation conditions. Schematic representation of the CCD-imaging setup used for conventional calcium imaging and simultaneous imaging of [Ca2+]i and [Ca2+]mito signals in the cell line preparation are illustrated in Additional file To clearly separate and simultaneously monitor the dynamic i and [Ca2+]mito signals to remove the noise.Each cover slip was used for a single experiment and included more than 4 cells for each imaging experiment. Unless otherwise indicated, values in the text are given as mean ± standard deviation (SD) and the error bars indicate SD too. All values represent at least three separate experiments. Significance was calculated using the two tailed unpaired Student t-test. A 2+ influx in WT and G93A transfected cells was done in the presence of FCCP. For these experiments the SH-SY5Y cells were loaded with 5 μM Fura-2 AM through the incubation method and [Ca2+]i transients were triggered once Fura-2 AM concentration was equilibrated after washing. Ca2+ release from intracellular stores by inhibition of the sarcoplasmic/endoplasmic reticulm Ca2+-dependent ATPase pump and inhibition of F1, F0-ATP Synthase was done by intervention of thapsi, and oligo, respectively.Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), thapsigargin, and oligomycin were purchased from Sigma-Aldrich Chemie . FCCP was bath applied at a dose of 2 μM. A comparative analysis of the Ca2+ release, caffeine has been established as a specific activator of Ca2+ influx from the RyR-dependent Ca2+ stores [2+]i regulation together with the association on MN degeneration in G93A transfected SH-SY5Y cells, we used pharmacological agents as a specific activator of the ER-dependent Ca2+ release response i measured for both WT and G93A-transfected SH-SY5Y cells , after peak FCCP challenge (3 min), and after FCCP wash in WT-SOD1 (a-c) and G93A (d-f) transfected cells are shown in Fig. 2+]i as cytoplasmic calcium versus time curves in 6 representative cells during FCCP challenge. The mitochondrial uncoupler induces a fast transient elevation in [Ca2+]i and a fast recovery to baseline in WT transfected cells .The transfected cells were treated with FCCP (3 min) and the [Calls Fig. . A CCD i2+ may have a perceptible influence on the existence of a [Ca2+]i transient. This effect was studied by following the Ca2+ signal under healthy and deleterious mitochondrial conditions in the presence of FCPP. We found that, in G93A transfected SH-SY5Y cells, the Ca2+ influx (peak fluorescence) was diminished compared to WT transfected SH-SY5Y cells, which, peaked within 2–3 min and was followed by a baseline recovery in approximately 2 min. The G93A transfected cells took almost 5–6 min to achieve a baseline recovery. However, the cells transfected with G93A suffered from reduced [Ca2+]mito after depletion of mitochondrial Ca2+ stores by FCCP  located in the inner mitochondrial membrane . The accCCP Fig. .2+ sequestering organelle, FCCP was applied to evacuate mitochondrial Ca2+after the WT and G93A transfected cells had been exposed to an evoked Ca2+ load through a depolarizing stimulus of K+. First, the SH-SY5Y cells transfected with WT or G93A were stained with Fura-2 AM and exposed to 30 mM K+ for 30 sec, which was followed by a 3 min challenge with FCCP  and 0.1497 ± 0.0362  for the WT and G93A transfected cells, respectively. Fig. To analyze the comparative efficiency of mitochondria as a Ca2+-ATPase inhibitor thapsi, an extremely tight-binding inhibitor of intracellular Ca2+ pumps which induces rapid Ca2+ release from intracellular stores by inhibition of the sarcoplasmic/endoplasmic reticulm Ca2+-dependent ATPase pump without inositol phosphate formation mito increase while measuring an increase in [Ca2+]i, we were able to efficiently separate cytosolic and mitochondrial Ca2+ at a temporal resolution in the millisecond time domain. As shown in Fig. 2+ release in G93A transfected cells is smaller in amplitude and exhibits slower kinetics compared to WT transfected cells. To confirm that there are separate Ca2+ uptake and release events from the cytosol and mitochondria, the transfected cells were treated with FCCP and the separate Ca2+ levels measured simultaneously  compared with mitochondria after Ca2+ elevation by FCCP (3 min) indicating its low efficiency to sequester Ca2+ in the WT and G93A transfected SH-SY5Y cells, which was slightly higher in WT  was added before FCCP application. Oligo does not influence [Ca2+]i heavily and left basal Ca2+ levels, as well as recovery times of Ca2+ transients, unaffected for upto several minutes  phenylhydrazone; FCS: fetal calf serum; FMNs: facial motoneurons; HMNs: hypoglossal motoneurons; hALS: human amyotrophic lateral sclerosis; MCU: mitochondrial calcium uniporter; MN: motoneuron; mtSOD1: mutant Cu/Zn superoxide dismutase1; oligo: oligomycin; ROI: regions of interest; ROS: reactive oxygen species; RPMI 1640: Roswell Park Memorial Institute medium; RyR: ryanodine receptor; SD: standard deviation; SOD1: Cu/Zn superoxide dismutase1; TPPThe authors declare that they have no competing interests.MKJ performed the experiments, analyzed the data, and wrote the initial version of the manuscript. WDZ, MG and RN established the primary cultured human SH-SH5Y cell line and maintained them during the course of the project. Cell lines expressing either wild-type (Wt) human SOD1 or the G93A mutants of this enzyme were produced by AF and MTC. CL, AZ, MTC and RN participated in the design of the study and participated in the organization of financial support. BUK is the principal investigator of the project. Besides providing laboratory support and supervision, he is primarily responsible for financial support and project management. All authors read and approved the final manuscript.Schematic representation of method and spectra. Schematic representation of the (A) CCD-imaging setup used for calcium imaging and (B) simultaneous measurements of [Ca2+]i and [Ca2+]mito in SH-SY5Y cell line preparations. C) Spectral view of calcium imaging. The fluorescence excitation at 510 nm and emission detected at 340 nm are shown for Ca2+-saturated (A) and Ca2+-free (B) Fura-2 in pH 7.2 buffer. D) Spectral view of simultaneous [Ca2+]i and [Ca2+]mito measurements generated by the Molecular Probes spectral view program. Fluorescence excitation of fura-2 was done at 390 nm and 550 nm of rhod-2, respectively; emission was at 510 nm  and at 600 nm (rhod-2) separated by a 565 nm dichroic mirror.Click here for fileMitochondria-dependent responses of cytosolic calcium in SH-SY5Y neuroblastoma cells  loaded with Fura-2 AM and superfused with DMEM medium. A) Representative CCD camera photomicrograph of Fura-2 AM loaded SH-SY5Y cells. Scale bar is 10 μm. B) FCCP-evoked mitochondria-dependent responses of cytosolic calcium.Click here for file


Using human lymphocytes, we studiedcytosolic calcium (Cai) and various Ca2+ transportmechanisms in subjects with Type 2 diabetesmellitus and control subjects. Ca2+-specific fluorescentprobes (Fura-2 and Fluo-3) were used tomonitor the Ca2+ signals. Thapsigargin, a potent andspecific inhibitor of the sarco(endo)plasmic reticulumCa2+-ATPase (SERCA), was used to study Ca2+-store dependent Ca2+ fluxes. Significant (P < 0.05)elevation of basal Cai levels was observed inlymphocytes from diabetic subjects. Cai levels werepositively correlated with fasting, plasma glucoseand HbAlc. There was also a significant (P < 0.05)reduction in plasma membrane calcium (PMCA)ATPase activity in diabetic subjects compared tocontrols. Cells from Type 2 diabetics exhibited anincreased Ca2+ influx (as measured both by Fluo-3fliorescence and C45a assays) as a consequence ofof thapsigargin-mediated Ca2+ store depletion. Uponaddition of Mn2+ (a surrogate of Ca2+), the fura-2fluorescence decayed in an exponential fashion andthe rate and extent of this decline was steeper andgreater in cells from type 2 diabetic patients. Therewas also a significant (P < 0.05) difference in theNa+/Ca2+ exchange activity in Type 2 diabeticpatients, both under resting conditions and after challenging the cells with thapsigargin, when theinternal store Ca2+ sequestration was circumvented.Pharmacological activation of protein kinase C(PKC) in cells from patients resulted in only partialinhibition of Ca2+ entry. We conclude that cellularCa2+ accumulation in cells from Type 2 diabetesresults from (a) reduction in PMCA ATPase activity,(b) modulation of Na+/Ca2+ exchange and (3)increased Ca2+ influx across the plasma membrane.Altered cytosolic Ca


Vibrio cholerae is the causative agent of cholera. Extensive studies reveal that complicated regulatory cascades regulate expression of virulence genes, the products of which are required for V. cholerae to colonize and cause disease. In this study, we investigated the expression of the key virulence regulator ToxR under different conditions.toxR expression was lower in an aphB mutant strain. AphB has been previously shown to be a key virulence regulator that is required to activate the expression of tcpP. When expressed constitutively, AphB is able to activate the toxR promoter. Furthermore, gel shift analysis indicates that AphB binds toxR promoter region directly. We also characterize the effect of AphB on the levels of the outer membrane porins OmpT and OmpU, which are known to be regulated by ToxR.We found that compared to that of wild type grown to stationary phase, the V. cholerae possesses an additional regulatory loop that use AphB to activate the expression of two virulence regulators, ToxR and TcpP, which together control the expression of the master virulence regulator ToxT.Our data indicate that  Vibrio cholerae is the etiologic agent of cholera. The ability of V. cholerae to colonize and cause disease in hosts requires production of a number of virulence factors during infection. The two major virulence determinants of V. cholerae are encoded by two separate genetic elements: cholera toxin (CT), which causes the diarrhea characteristic of cholera, and the toxin-coregulated pilus (TCP), which is essential for attachment and colonization of intestinal epithelia ,,29,toxT,ondition . As expeype Fig. , howeverV. cholerae. In this pathway, AphA and AphB activate tcpP transcriptional expression directly by binding to different promoter regions of tcpP. ToxR and TcpP cooperate in turn by binding different sites of the toxT promoter to activate transcription, leading to the production of the virulence factors TCP and CT. However, the full ToxR regulon is more complex than previously thought. In this paper, we showed that AphA and AphB are also necessary for full ToxR production at the stationary phase. Furthermore, we demonstrated that AphB is sufficient for toxR transcriptional activation in the heterogenic host E. coli through binding of the toxR promoter region. Thus, the effect of AphB on ToxR levels propagates further in the transcription cascade, increasing the transcription of a key gene in V. cholerae pathogenesis, toxT. We have therefore identified another factor responsible for altering end product levels in the V. cholerae virulence axis. Since AphB is at the top of a virulence cascade with multiple end pathways, it appears now that AphB is a central factor in switching the cell from an environmental state to a virulent one. Since it activates ToxR in addition to TcpP, and further influences porin expression, AphB is a divergence point at which nonlinearity is introduced into the V. cholerae virulence pathway. Eukaryotic cells have extremely complex networks of protein and DNA interactions leading to precise control of protein expression levels. Having a more complex network of transcriptional activation and repression in the V. cholerae virulence cascade could enable the bacterial cell to fine-tune its expression levels to optimize its ability to colonize the intestine and spread to other hosts.The ToxR regulon is the classic virulence gene regulation pathway in Vibrio cholerae C6706 dATP and the Klenow fragment of DNA polymerase I. Binding reactions contained 0.1 ng of DNA and MBP-AphB proteins in a buffer consisting of 10 mM Tris-HCl (pH 7.9), 1 mM EDTA, 1 mM dithiothreitol, 60 mM KCl, and 30 mg of calf thymus DNA/ml. After 20 minutes of incubation at 25°C, samples were size-fractionated using 5% polyacrylamide gels in 1× TAE buffer . The radioactivity of free DNA and AphB-DNA complexes was visualized by using a Typhoon 9410 PhosphorImager (Molecular Dynamics).MBP-AphB protein was purified through amylose columns according to the manufacturer's instructions (New England Biolabs). PCR products of the different lengths of XX, AS, ZL, BK, and JZ designed research; XX, AS, and ZL performed research; XX, AS, and JZ analyzed data, XX, AS, ZL, BK, and JZ wrote the paper. All authors read and approved the final manuscript.


It exerts its effects through interactions with a range of target proteins one of which is interleukin receptor accessory protein like-1 (IL1RAPL1) protein. Mutations in IL1RAPL1 have recently been associated with autism spectrum disorders and a missense mutation (R102Q) on NCS-1 has been found in one individual with autism. We have examined the effect of this mutation on the structure and function of NCS-1. From use of NMR spectroscopy, it appeared that the R102Q affected the structure of the protein particularly with an increase in the extent of conformational exchange in the C-terminus of the protein. Despite this change NCS-1(R102Q) did not show changes in its affinity for Ca2+ or binding to IL1RAPL1 and its intracellular localisation was unaffected. Assessment of NCS-1 dynamics indicated that it could rapidly cycle between cytosolic and membrane pools and that the cycling onto the plasma membrane was specifically changed in NCS-1(R102Q) with the loss of a Ca2+ -dependent component. From these data we speculate that impairment of the normal cycling of NCS-1 by the R102Q mutation could have subtle effects on neuronal signalling and physiology in the developing and adult brain.Neuronal calcium sensor-1 (NCS-1) is a Ca The Neuronal Calcium Sensor (NCS) proteins are a family of such proteins that are predominantly expressed in neurones and are characterised by their structural similarities and a high (low micromolar) affinity for Ca2+2+-binding domains of which 2–3 have the capacity to bind Ca2+, and many are N-terminally myristoylated Calcium has effects on multiple aspects of neuronal function through the action of CaC. elegans, and enhanced when it is over-expressed NCS-1 is the most conserved of the NCS protein family, with an orthologue, Frq1, found in yeast Amongst the proteins that interact with NCS-1 is interleukin receptor assessory protein like-1 (IL1RAPL1) In previous studies linking NCS-1 to disease states 1H/15N heteronuclear single quantum coherence (1H/15N HSQC) NMR spectra were acquired for the wild-type and mutant proteins. Unmyristoylated, 15N-labeled NCS-1 and NCS-1(R102Q) were prepared and spectra collected for the Ca2+ loaded samples. Resonance assignments of the 1H/15N HSQC spectrum of wild-type NCS-1 were made through comparison to published data 2+ loaded NCS-1 . Resonances corresponding to all amino acid residues were assigned, with the exception of N-terminal residues 1–5, which are unstructured 1H/15N HSQC spectra resonances corresponding to the majority of residues coincided with those found in the spectrum of the wild type protein, indicating that most of the mutant is structurally identical to wild type protein  with respect to NCS-1 amino acid sequence. Resonance changes were seen throughout the polypeptide sequence with the number of changes more marked around the site of mutation in helix F (residues 99–109). Interestingly, many of the chemical shift perturbations also occurred at the C-terminus with the resonances of 10 of the C-terminal 21 residues being undetectable in the 1H/15N HSQC spectrum of NCS-1(R102Q). It appears that the R102Q mutation has given rise to long-range effects, leading to an increase in the dynamics of the C-terminus region. These residues correspond to a region in the crystal structure that is stabilised by hydrogen bonding 2+-binding characteristics of the protein. We used changes in intrinsic tryptophan fluorescence to report the conformational change in NCS-1 and NCS-1(R102Q) in response to Ca2+ binding, as this approach has been successfully used previously 2+ concentrations in the buffer were incrementally increased. Saturating concentrations of free Mg2+ were present in the buffer to insure that this ion did not influence the results 2+  fluorescence did not fall to the same extent, but this may be because one of the only two tryptophan residues in NCS-1(R102Q), W103, is in a position adjacent to the mutated residue. Importantly, changes in wild-type and mutant NCS-1 fluorescence followed a similar pattern, suggesting that the proteins underwent conformational change at the same Cation see .2+  to GST-IL1RAPL1(569–644) were identified nor were any differences in Ca2+-dependency observed. In a separate experiment to quantify binding to GST-IL1RAPL1(569–644), the level of binding of NCS-1(R102Q) was no different  from that for wild-type NCS-1 at 1 µM free Ca2+ concentration after correction for levels of input protein  corresponds almost exactly to the minimal NCS-1(174–190) domain required for its binding to IL1RAPL1 2+ . Specifi protein .In vitro, it was decided to examine their expression, localisation and characteristics in cells. Since the NMR data indicated that the R102Q mutation affected the IL1RAPL1-binding domain of NCS-1, and since we had not ruled out the possibility of a cellular effect on the NCS-1-IL1RAPL1 interaction, we chose to use cells that endogenously express both NCS-1 and IL1RAPL1 in this study. Previous studies on NCS-1 and IL1RAPL1 have been conducted using PC12 cells, but endogenous expression of IL1RAPL1 has not been detected in these cells Since no functional differences between NCS-1 and NCS-1(R102Q) proteins were identified NCS-1 associates with the plasma membrane and Golgi complex as a result of its N-terminal myristoylation To identify any difference in the localisation of NCS-1 and NCS-1(R102Q), N2A cells were alternately cotransfected with NCS-1-ECFP and NCS-1(R102Q)-mCherry, and NCS-1(R102Q)-ECFP and NCS-1-mCherry. When the lysates from individually transfected wells were subjected to SDS-PAGE and Western blotting, and the blots were probed with an antibody against GFP, it was found that the proteins were expressed at the same levels  indicatiin vitro, or in its localisation in N2A cells, we proceeded to examine whether the mutation might affect the capacity of NCS-1 to modulate intracellular Ca2+ signalling. The involvement of NCS-1 in Ca2+ signalling is complex, as it has been suggested to act via various target proteins. It has been reported to interact with InsP3 receptors, and to enhance their activity in a Ca2+-dependent manner 2+ channels (VGCCs), and to be required for the negative regulation of N-type channels by IL1RAPL1 2+ in the present study, we used the SERCA pump inhibitor thapsigargin to release Ca2+ from the endoplasmic reticulum followed by Ca2+ addition to the external medium to determine general effects of NCS-1 over-expression on Ca2+ release and entry. N2A cells were examined in a protocol in which they were exposed to 1 µM thapsigargin in the absence of extracellular Ca2+ in order to release calcium from internal stores, and then extracellular Ca2+ was added to a concentration of 3 mM in order to provoke store-operated calcium entry. The resulting changes in intracellular Ca2+ concentration were monitored using the Ca2+ indicator Fluo-4, and the responses of untransfected and transfected cells in the same dishes were directly compared. No significant difference was identified between the responses of untransfected cells and cells transfected to express mCherry as a control, and in both cases, there was an initial increase in intracellular Ca2+ concentration after addition of thapsigargin, and this was followed by a larger rise in intracellular Ca2+ concentration after addition of extracellular Ca2+  calcium-binding lar Ca2+ . In contlar Ca2+ . Expresslar Ca2+ . There w2+ concentrations 2+ concentrations, and only interacts with membranes transiently. This possibility has not, however, been investigated. In order to establish whether NCS-1 associates with membranes in a transient or a stable manner, fluorescence loss in photobleaching (FLIP) experiments were carried out. The rationale behind this technique is that where a partially cytosolic fluorescent protein associates transiently with membranes, progressive bleaching of the cytosolic pool leads to parallel loss of cytosolic and membrane fluorescence. Whereas, when a protein is more stably associated with membranes, membrane fluorescence is lost at a much slower rate. Parts A and B of Previous work has shown that unlike the situation with a number of other NCS family members, the myristoyl group of NCS-1 is continually exposed, and NCS-1 is membrane-associated, at resting CaTo compare the dynamics of NCS-1- and NCS-1(R102Q)-membrane interactions at the TGN in more detail, fluorescence recovery after photobleaching (FRAP) experiments were conducted. The small GTPase ARF-1, which has also been shown to interact with Golgi membranes in a transient manner It was important to continue characterisation of the association/dissociation kinetics of NCS-1 and NCS-1(R102Q), as although the kinetics were no different at the TGN, some of the functions of NCS-1 are restricted to the plasma membrane. FRAP was used to assess the dynamics of NCS-1 and NCS-1(R102Q)-EGFP. In these experiments, cells expressing each of the proteins were tested on the same day with measurements alternating between wild-type and mutant expressing cells so that the data for each protein could be directly compared. Upon examination of plasma membrane association/dissociation kinetics, it was found that the recovery of NCS-1-EGFP and NCS-1(R102Q)-EGFP fluorescence differed .Under resting conditions, recovery was fast with both proteins and best fit by two-phase association kinetics, but while NCS-1-EGFP fluorescence recovered with halftimes of 1.64 s and 14.23 s, NCS-1(R102Q)-EGFP fluorescence recovered with halftimes of 1.11 s and 6.27 s . While t2+ elevation. In order to test this hypothesis, we conducted parallel bleaching experiments on cells transfected to express the NCS-1 constructs in the presence or absence of 1 µM thapsigargin. We used this reagent to raise intracellular Ca2+ since we had already demonstrated that it produces comparable, long-lasting calcium elevations in cells expressing NCS-1-EGFP or NCS-1(R102Q)-EGFP -EGFP . As comp2Q)-EGFP . Further2Q)-EGFP . We conc1H/15N HSQC spectra of recombinant NCS-1 and NCS-1(R102Q). The 1H/15N HSQC spectrum of the mutant protein showed a number of resonances from residues towards the C-terminus which are either absent or significantly reduced in intensities when compared to the spectrum of the wild-type protein. The mutation of this single surface residue appeared to have produced pronounced, long-range as well as local changes. It is interesting to note that while R102 is not conserved in lower organisms such as C. elegans and Drosophila, it is conserved in all species from fish to man. The AGADIR analysis predicts a reduction in helical content in helix F when R102 is mutated to a glutamine. Therefore, it is most likely that the destabilisation of helix F affects the hydrogen-bond network which in the wild-type protein restraints the conformational dynamics of the C-terminus region. A result of this hydrogen bond perturbation is that the C-terminus is able to adopt several conformations which are in chemical exchange. Despite these structural changes in NCS-1(R102Q), the protein shows a Ca2+-dependent conformation change as monitored by tryptophan fluorescence at the same Ca2+ concentration as wild type protein, localised essentially identically to wild-type NCS-1 when expressed in N2A cells and had a similar effect on thapsigargin-induced Ca2+ signals.The data presented in this paper provide insights into the cellular localisation and dynamics of NCS-1 and describe structural and functional deficits in the R102Q mutation/polymorphism of NCS-1 that had been discovered in an individual with ASD 2+ elevation 2+ elevation stabilises a fraction of cellular NCS-1 at the plasma membrane. Presumably, this stabilisation follows Ca2+ -binding by NCS-1 and possibly Ca2+-dependent interaction with a binding partner. It has been previously shown that bradykinin stimulation, which raises intracellular Ca2+ via IP3 -induced calcium release, can shift a proportion of cellular NCS-1 into detergent resistant plasma membrane microdomains The localisation of exogenous NCS-1 in N2A cells that we identified in this study is similar to that seen in other cell types in previous studies 2+-dependent component of cytosol/plasma membrane exchange dynamics. Thus, one possibility is that in cells, wild type NCS-1 binds to an endogenous plasma membrane target protein effectively only when Ca2+ is elevated, but NCS-1(R102Q) is able to bind to the target protein via its C-terminus at resting Ca2+ concentrations. Assay of in vitro binding of NCS-1 and NCS-1(R102Q) to GST-IL1RAPL1(569–644) did not reveal any differences, but this may not be surprising, as NCS-1 does not need to be fully folded to bind to IL1RAPL1. Indeed, the C-terminus alone, NCS-1(174–190), is sufficient for an interaction While the major structural changes in NCS-1(R102Q) were in the domain required for binding to IL1RAPL1, the main functional effect of the mutation was loss of a Ca2+-dependent stabilisation at the plasma membrane are not yet clear, but it is tempting to speculate that the dynamic interaction between NCS-1 and membranes might allow regulation of its local levels on neuronal membranes. Impairment of this regulation could have subtle effects on neuronal signalling and physiology in developing and adult brain. It is noteworthy that NCS-1 is highly conserved having an identical amino acid sequence in birds and all mammals. There are, for example, only four amino acid differences between NCS-1 in humans and the most similar NCS-1 in the teleost fish Danio rerio suggesting strong evolutionary pressure against any changes in its sequence. There is currently little evidence available for non-synonomous polymorphisms in NCS-1 in human populations. A direct linkage of the R102Q mutation/polymorphism to ASD has not yet been established as it is based on data from a single subject. Further genetic and functional analysis may now be warranted, however, to determine the relevance of the R102Q mutation/polymorphism,in NCS-1 to the pathology of ASD.The functional significance of NCS-1 dynamics and its CaBamHI/XhoI and were ligated using standard methods. The vector used for the bacterial expression of GST-NCS-1 was as described previously nd codon, AGG, for CAG, using the primer pair CAGTGGGCCTTC-3′5′-GGACCCTGGATGAGAAGTTG (sense) and 5′-CAACTTCTCATCCAGGGTCCCCCTTGAGG-3′ (antisense). Mutants were generated using the GeneTailor system  according to manufacturer's instructions. All constructs were verified by automated sequencing . The Golgi-YFP fusion construct was obtained from Clontech, and the pIL1RAPL1-GFP The NCS-1 sequence was excised from an existing vector 15N-labeled GST-NCS-1 and GST-NCS-1(R102Q) were produced by similar inoculation of M9 minimal media, supplemented with 100 mg/l ampicillin and containing (15NH4)SO4 as a sole nitrogen source with growth at 37°C to a density of 0.8 OD units and induction with 1 mM IPTG overnight at 30°C. Following cell disruption by use of a French press, recombinant proteins were purified on glutathione-conjugated Sepharose. GST tags were removed from NCS proteins using PreScission protease (Invitrogen) according to the manufacturer's instructions prior to use in all experiments.GST, GST-IL1RAPL1(569–644), GST-NCS-1 and GST-NCS-1(R102Q) were expressed in BL21 E.coli. Unlabeled protein was produced by inoculating SB media, supplemented with 100 mg/l ampicillin, with single transformed clones, allowing cell growth at 37°C for 15–17 h, and inducing protein expression with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 3–4 h. 2, 10 mm CaCl2, 1 mM NaN3 in 90% H2O/10% D2O at pH 6.5. Spectra were recorded at 30°C using a Bruker Avance 800 MHz spectrometer fitted with a triple resonance, single z-axis gradient cryogenic probehead. 15N-1H spectra were acquired using water flip-back pulses. Spectra were processed using Bruker Topspin software.The NMR samples of NCS-1 and NCS-1(R102Q) were approximately 0.5 mM in 20 mM Tris, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, pH 7.4) containing CaCl2 to give free calcium concentrations of 0, 0.1, 0.3 or 1 µM, before incubation with 1 µM recombinant NCS-1 or NCS-1(R102Q) for 1 h at 4°C. The beads were washed three times in the appropriate buffer, and then bound proteins were eluted by boiling in SDS/PAGE sample buffer. Samples were separated by SDS PAGE and proteins were stained using Coomassie Brilliant Blue. To allow quantification of binding to IL1RAPL1, multiple binding assays were carried out at 1 µM free Ca2+ concentration and samples of bound proteins and input proteins run on the same gels. Quantification of bound NCS-1 protein was carried out using ImageJ  and values corrected for concentration of the input proteins.GST or GST-IL1RAPL1(569–644) was bound to 15 µl glutathione-Sepharose beads for 1 h at 4°C. Beads were washed with binding buffer ,1 mM DTT, pH 7.4) were excited at room temperature with 280 nm light, and their emission spectra from 290–410 nm were measured, using a Jasco FP-6300 spectrofluorometer . The concentration of free calcium was increased by incremental addition of CaCl2 to give calculated free Ca2+ concentrations, and additional emission spectra were measured as before.To monitor intrinsic tryptophan fluorescence of NCS proteins 2 culture flasks at 37°C, 5% CO2 in Dulbecco's Modified Eagle Medium  supplemented with 10% foetal calf serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were passaged twice weekly. Transfection of the cells was carried out as follows. Coverslips in live cell dishes  or in 6- or 24-well plates were seeded with ∼3×105 cells/ml media, and cells were allowed to adhere overnight. Transfection mixes were made up with 3 µl of the Gene Juice reagent  per µg plasmid in 100 µl Optimem 1 (Gibco), incubated for 30 minutes, and then applied to cells. Cells were incubated for a further 18 hours prior to experiments. Cells expressing VSVG-GFP were incubated for 18 h at 20°C and then washed in PBS and fixed in 4% (v/v) paraformaldehyde for 30 min prior to imaging. All other imaging was carried out on live cells.Adherent N2A cells  were grown in 75 cmCell lysates were made by direct addition of 200 µl/well Laemmlli buffer (Sigma) to cells in 6-well plates following washing with PBS. Samples were resolved using SDS-PAGE (12.5% gels) and transferred to nitrocellulose filters for Western blotting by transverse electrophoresis. Anti-NCS-1 , anti-IL1RAPL1  and anti-GFP (JL-8) (Clontech) were used at 1∶800, 1∶200 and 1∶1000 dilutions respectively. HRP-conjugated secondary antibodies (Sigma) were used at a 1∶400 dilution, and blots were analysed by detection of chemiluminescence using a Biorad Chemidoc XRS system .2, 1.2 mM NaH2PO4).Confocal microscopy following fixation, in live cell single-frame imaging and in calcium indicator experiments, was carried out on a Leica TCS-SP2 microscope  using a 63x oil-immersion objective with a 1.4 numerical aperture. The pinhole was set to airy2 for calcium indicator experiments and to airy1 for all other experiments. Fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP) experiments were carried out on a Leica TCS-SP2-MP microscope (Leica Microsystems). A 63x water-immersion objective with a 1.2 numerical aperture was used and the pinhole set to airy 2.06. Where variant GFP-containing constructs were used, wells/dishes were transfected with 0.5 µg of each plasmid. ECFP-, EGFP-, and mCherry-containing constructs were excited using 405 nm, 488 nm and 594 nm lasers respectively, and emitted light was collected between 450–500 nm, 500–550 nm and 625–675 nm. Fluo-4 was excited using a 488 nm laser, and emitted light was collected between 495–545 nm. Golgi-YFP was excited using a 514 nm laser, and emitted light was collected between 530–600 nm. All imaging of live cells and was carried out on cells at room temperature in Krebs Ringer buffer , Fluo-4 fluorescence measurements for each cell were normalised with respect to fluorescence at the first time-point.In calcium indicator experiments, cells were loaded with 5 µM Fluo-4-AM (Invitrogen) for 30 minutes at room temperature to allow measurement of changes in [CaIn FLIP experiments, rectangular regions of interest (ROIs), 4 µm by 2 µm, in the cytosol of cells, were repetitively bleached using 75% of laser power. To discriminate fluorescence loss in different cellular regions, background fluorescence was subtracted from images and ROIs drawn around the outside perimeter, inside perimeter, nucleus, and Golgi of cells. Values for average plasma membrane pixel intensity were calculated after subtracting fluorescence totals and pixel number contained by the inner perimeter from those contained by the outer perimeter. Values for average cytosol pixel intensity were calculated after subtracting fluorescence totals and pixel numbers contained by the nucleus, Golgi and bleached region from those contained by the inner perimeter. Data was normalised with respect to fluorescence of adjacent cells in each experiment in order to correct for bleaching during low power laser excitation.In FRAP experiments, circular regions of interest of diameter 2 µm, were bleached using 75% of laser power following prior capture of 10 frames. 100 post-bleach frames were recorded per cell. To determine the rate of fluorescence recovery, fluorescence in these regions was measured over time, and then normalised with respect to corresponding total cellular fluorescence at each individual time point to correct for bleaching during low power laser excitation. Bleaching experiments with NCS-1-EGFP and NCS-1(R102Q)-EGFP, and experiments in the presence and absence of thapsigargin, were conducted in parallel. Non-linear regression analysis was used to calculate the rate and extent of fluorescence recovery of each construct and was carried out using Graphpad Prism software .Figure S12+ concentration, and four lanes of the input NCS-1 protein on the right.Coomassie Blue-stained SDS-gel used for quantification of NCS-1 and NCS-1(R102Q) binding to GST-IL1RAPL1(569-644). Each gel has 4 independent binding assays on the left, carried out at 1 µM free Ca(5.82 MB TIF)Click here for additional data file.Figure S2IL1RAPL1 and NCS-1 are endogenously expressed by N2A cells. A. Samples of N2A cells were separated by SDS-PAGE, prepared for Western blotting and probed with antisera specific for IL1RAPL1 or NCS-1 as indicated. B-D N2A cells were fixed and processed for immunufluorescence staining with anti-NCS-1 and anti-IL1RAPL1. The overlay shows anti-IL1RAPL1 in green and anti-NCS-1 in red with co-localisation in yellow in the overlay image.(3.37 MB TIF)Click here for additional data file.Table S1Propensity of helix formation predicted by use of Agadir (reference 48).(0.09 MB TIF)Click here for additional data file.


O- derivative of betulinic acid, blocks the last step of protease-mediated processing of HIV-1 Gag precursor (Pr55Gag), which leads to immature, noninfectious virions. When administered to Pr55Gag-expressing insect cells (Sf9), DSB inhibits the assembly and budding of membrane-enveloped virus-like particles (VLP). In order to explore the possibility that viral factors could modulate the susceptibility to DSB of the VLP assembly process, several viral proteins were coexpressed individually with Pr55Gag in DSB-treated cells, and VLP yields assayed in the extracellular medium.DSB, the 3-wt) restored the VLP production in DSB-treated cells to levels observed in control, untreated cells. DSB-counteracting effect was also observed with Vif mutants defective in encapsidation into VLP, suggesting that packaging and anti-DSB effect were separate functions in Vif. The anti-DSB effect was abolished for VifC133S and VifS116V, two mutants which lacked the zinc binding domain (ZBD) formed by the four H108C114C133H139 coordinates with a Zn atom. Electron microscopic analysis of cells coexpressing Pr55Gag and Vifwt showed that a large proportion of VLP budded into cytoplasmic vesicles and were released from Sf9 cells by exocytosis. However, in the presence of mutant VifC133S or VifS116V, most of the VLP assembled and budded at the plasma membrane, as in control cells expressing Pr55Gag alone.Wild-type Vif (Vifwt, but not with ZBD mutants VifC133S and VifS116V, VLP were redirected to a vesicular compartment and egressed via the exocytic pathway.The function of HIV-1 Vif protein which negated the DSB inhibition of VLP assembly was independent of its packaging capability, but depended on the integrity of ZBD. In the presence of Vif O--betulinic acid (or YK-FH312 [in vitro experiments [in vivo [The 3-YK-FH312 ), has beYK-FH312 -8. DSB dYK-FH312 . DSB bloYK-FH312 ,6,10. SeYK-FH312 ,8,11. Al[in vivo , suggestSIV), although at significantly higher DSB concentrations, suggesting that the DSB inhibitory activity on Gag assembly was not as strictly sequence-dependent as the negative effect on Gag processing at the CA-SP1 junction [The latter observation incited us to study the possible effect of DSB on assembly of recombinant HIV-1 Gag precursor (Pr55Gag) expressed in heterologous, eukaryotic system. We observed a dose-dependent negative effect of DSB on the process of assembly and release of HIV-1 VLP from recombinant baculovirus AcMNPV-Pr55Gag-infected cells . This efjunction . In addijunction . Using Gjunction .8 per cell [6 cells corresponded to 12 × 109 DSB molecules per cell, i.e. a DSB to Gag stoichiometric ratio of 24: 1 at this DSB concentration. A 24-fold excess of DSB over Gag was therefore compatible with a mechanism of Gag assembly inhibition due to a stoichiometric interaction between the drug and its protein target.The DSB concentration at which we observed an inhibitory activity on Gag assembly in insect cells (IC50 ~8–10 μM) was apparently disproportionate compared to the usual doses required for blocking the CAp25 cleavage in HIV-1-infected mammalian cells. However, a wide range of IC-50 values have been reported for the DSB inhibition of virus maturation, varying from nanomolar  with Pr55Gag restored the VLP assembly in DSB-treated Sf9 cells at levels observed in the absence of the drug, suggesting an antagonistic effect of Vif towards DSB. Data obtained with Vif mutants indicated that the anti-DSB function of Vif required the integrity of the zinc binding domain (ZBD) recently identified in the Vif protein [wt, in the presence or absence of DSB, resulted in a major change in the VLP egress pathway: the majority of VLP budded in intracytoplasmic vesicles and were released by exocytosis, instead of budding at the plasma membrane as in cells expressing Pr55Gag alone. With ZBD mutants of Vif however, the VLP budding pathway was similar to that observed in cells expressing Pr55Gag alone. Our data suggested that the anti-DSB effect of Vif, a novel function associated with its ZBD, was the indirect consequence of its effect on the cellular pathway of VLP assembly and budding.We found that coexpression of wild-type Vif protein  possibly interfered with DSB inhibitory activity and accounted for the difference in DSB response between Sf9 and 5BD.1 cells, as well as other mammalian cells.The best candidates to act as viral modulators of the Gag assembly response to DSB were the HIV-1 proteins coencapsidated with Gag, in particular those which are active participants in the virus assembly pathway . ThiIn order to test this possibility, Sf9 were coinfected with AcMNPV-Pr55Gag and AcMNPV-Gp160, and subjected to increasing doses of DSB for 30 h, at 18 h pi. Culture medium samples were collected at 48 h pi and assayed for production of extracellular VLP. Results were compared with VLP yields from Sf9 cells infected with AcMNPV-Pr55Gag alone and treated in parallel with DSB at the same doses. No significant difference in the DSB effect on VLP assembly was detectable with or without coexpression of EnvGp160 (data not shown). This excluded the direct or indirect participation of HIV-1 envelope glycoproteins in the level of susceptibility to DSB of assembly and extracellular release of VLP by Sf9 cells.bona fide component of the virion inner core [Nef in its processed form, called Nef core, has been shown to be a ner core -40. In 5ner core ,30, we oVpr is coencapsidated with Gag via interaction of the N-terminal alpha-helical domain encompassing residues 17–33 in Vpr -44 with in vitro and in vivo [wt showed a pattern of DSB effect different from that observed in cells expressing Pr55Gag alone: there was no significant decrease in the VLP yields from DSB-treated Sf9 cells, up to drug concentrations as high as 20 μg/ml, implying that expression of Vifwt protein negated the DSB inhibition of VLP assembly process in Pr55Gag-expressing insect cells , VifsubB (89WR90-Y94 to 89FE90-F94), double mutant VifsubC (subA+subB), and triple mutant VifsubCΔ170 carrying the double mutation subA+subB and a deletion of the C-terminal twenty-three residues, were found to be defective to various degrees in the encapsidation of Vif into VLP: VifsubA, VifsubB and VifsubC were partially defective in Vif packaging (40–50% the levels of Vifwt), whereas this function was totally abolished in VifsubCΔ170 [wt [In a previous study, we have constructed and characterized Vif mutants which differed from Vifed cells . The twosubCΔ170 , was pacsubC, the production of extracellular VLP remained virtually unchanged throughout the DSB concentration range, with less than 15% decrease in VLP production at high DSB doses . Likewise, the packaging-efficient mutant VifKRA8 showed the same phenotype as Vifwt and the packaging-defecting mutants in terms of anti-DSB activity (not shown). These results suggested that the DSB-counteracting function of Vif was independent from the packaging function of Vif.We then tested the anti-DSB activity of Vif mutants with different encapsidation phenotypes. With Vif108, C114, C133 and H139 coordinates (HCCH) with a Zn atom [A conserved region of the Vif protein, within residues 108 to 140, has been recently characterized as a non-canonical zinc-coordinating structure, generated by the H Zn atom ,28. This Zn atom ,28,58-60 Zn atom ,28  and immunoelectron microscopy (immuno-EM). Cells were infected with AcMNPV-Pr55Gag and AcMNPV-Vif, untreated or treated with DSB at 10 μg/ml at 18 h pi, harvested at 48 h pi and processed for EM or immuno-EM using anti-Vif antibody. In control Sf9 cells expressing Pr55Gag alone, the vast majority of VLP assembled at and budded from the plasma membrane  domain [The proportion of VLP following the intravesicular budding and exocytosis pathway compared to the ones using the plasma membrane pathway was estimated under the EM, by counting several hundreds of VLP in subcellular compartments of more than 20 different cells. In control Sf9 cells expressing Pr55Gag alone, less than 5% VLP were found within the vesicular compartment, whereas in Gag+Vifway Fig. . Interesway Fig. , resembl) domain .wt, the VLP assembly and budding process was redirected to the vesicular compartment, and that the VLP egress via exocytosis represented a salvage pathway through which HIV-1 VLP escaped the negative effect of DSB.We next examined cells coexpressing Pr55Gag and ZBD mutants of Vif under the EM, and found that, in the presence of Vif116V and VifC133S, the VLP budding pathway was similar to the one observed in Sf9 cells expressing Pr55Gag alone, i.e. a majority of VLP budding at the plasma membrane and rare intravesicular VLP  which involves the bypass of a drug-sensitive assembly and budding pathway by the virus or virus-like particle progeny.It has been shown that the assembly and release of HIV-1 virions proceeds via two pathways, depending upon the cell type : (i) in The results of our study suggested that DSB and other betulinic acid derivatives could be considered not only as antivirals for patients treatment in vivo, but also as chemical probes to analyse the molecular and cellular mechanisms of retroviral Gag assembly in vitro. In the latter context, considering Vif as a determinant of the budding pathway usage in Sf9 cells, and as a modulator of the DSB response in terms of VLP assembly, any evaluation of potential HIV-1 assembly inhibitors using the baculovirus-insect cell system should be carried out in the presence of the Vif protein.O--betulinic acid  was obtained as originally described [The title compound 3-escribed .Spodoptera frugiperda Sf9 cells were maintained as monolayers, and infected with recombinant baculovirus at a multiplicity of infection (MOI) ranging from 2.5 to 20 PFU/cell, as previously described [Simian 5BD.1 packaging cells  were CMT3-COS-derived cells that stably express HIV-1 Gag-Pol and Env proteins but no Nef ,30. Theyescribed ,65,70,71vpr, were inserted into the genome of Autographa californica MultiCapsid NucleoPolyhedrosis Virus (AcMNPV) under the control of a chimeric AcMNPV-GmNPV polyhedrin promoter [(i) Gag. AcMNPV-Pr55Gag, expressing the full-length wild type (WT) HIV-1 Gag polyprotein (Pr55Gag), has been described in detail in previous studies [(ii) Envelope glycoprotein Gp160. AcMNPV-Gp160 expressed the CCR5-tropic YU2 envelope glycoprotein. (iii) Vpr. The baculovirus clone expressing the oligohistidine-tagged Vpr protein (AcMNPV-Vpr) was obtained from Eric Cohen [(iv) Vif clones  and VifsubB (WRxxxY-to-FExxxF substitution) were mutated in two tryptophan-containing motifs, at position 76–80 and 89–94, respectively; the double mutant VifsubC carried both subA and subB mutations; mutant VifKRA8 had 8 basic residues in the C-terminal domain (residues 156–192) replaced by alanine residues. VifsubCΔ170 carried the VifsubC multiple substitutions and an additional deletion of the 23 C-terminal residues of Vif. VifsubA, VifsubB, VifsubC, VifKRA8 and VifsubCΔ170 have been characterized in previous studies [All the different HIV-1 genes used in the present study, except for promoter ,65,70.  Vi studies ,50. Subs6) were infected with recombinant AcMNPV at MOI 10. At 18 h postinfection (pi), increasing quantities of DSB in DMSO were added. To avoid possible interference with DMSO effect, DMSO was kept constant in volume in the different samples. A stock solution of DSB (10 mg/ml DMSO) was diluted with DMSO to obtain a range of DSB concentrations from 0.5 to 30 μg DSB per 3 μl-aliquot of DMSO, and each 3 μl-aliquot was added to 1 ml of culture medium overlaying the cell monolayers. The cells were harvested at 48 h pi, and extracellular VLP quantitatively assayed in the culture medium.Aliquots of Sf9 cells (102EDTA). The pellets were gently resuspended in PBS (0.20–0.25 ml), and VLP further purified by isopycnic ultracentrifugation in linear sucrose-D2O gradients [2O buffered to pH 7.2 with NaOH, and a 30% sucrose solution made in 10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5.7 mM Na2EDTA. The gradients were centrifuged for 18 h at 28 krpm in a Beckman SW41 rotor. Aliquots of 0.5 ml were collected from the top, and proteins analyzed by SDS-PAGE, immunoblot analysis with or without autoradiography.Sf9 cell culture supernatants were clarified by low-speed centrifugation, then VLP recovered using sucrose-step gradient centrifugation, by pellradients . GradienPolyacrylamide gel electrophoresis of SDS-denatured protein samples (SDS-PAGE), and immunoblotting analysis have been described in detail in previous studies ,71,74. BPst I site; gagLai sequence). Mouse monoclonal antibody (mAb) anti-CAp24 (Epiclone #5001) and mAb anti-MAp17 (Epiclone #5003) were obtained from Cylex Inc. . MAb 41A9, directed against the Gp41 domain of the EnvGp160, was obtained from Hybridolab . Mouse anti-Hisx6-tag antibody (Tag-100 antibody) was purchased from Qiagen SA . Anti-Vif antibody was raised in rabbit by injection of bacterially-expressed His-tagged Vif protein purified by guanidine denaturation and progressive renaturation of insoluble protein inclusion, followed by affinity chromatography on Ni-column , and exposed to radiographic films . Autoradiograms were scanned and quantitated by densitometric analysis, using the VersaDoc image analyzer and the Quantity One program (BioRad). Alternatively, protein bands were excised from blots and radioactivity measured in a scintillation counter (Beckman LS-6500), as previously described [Anti-HIV-1 Gag polyclonal antibody  was raiDecroly; ). Phosphescribed ,50.2O) and treated with 0.5% tannic acid solution in H2O. The specimens were dehydrated and embedded in Epon . Ultrathin sections were stained with 2.6% alkaline lead citrate and 0.5% uranyl acetate in 50% ethanol, and post-stained with 0.5% uranyl acetate solution in H2O [2O [Baculovirus-infected Sf9 cells were harvested at 48 h pi, pelleted, fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.5, post-fixed with osmium tetroxide (2% in Hn in H2O . For imm H2O [2O ,74,76. GThe authors declare that they have no competing interests.SDF performed the bench work, and BG and PB performed the EM analyses. PC synthesized the DSB. SSH and PB conceived the strategies and designed the experiments. SB contributed to data analysis. PB wrote the manuscript. All the authors read and approved the final manuscript.


Rheumatoid arthritis (RA) is characterized by bone and cartilage invasion by fibroblast-like synoviocytes (FLSs). Adrenomedullin, a peptide with anabolic and antiapoptotic properties, is secreted by rheumatoid FLSs. Adrenomedullin also increases the expression of adhesion molecules in endothelial cells and keratinocytes. Here, we investigated whether adrenomedullin mediated FLS adhesion to extracellular matrix (ECM) proteins.2 and β1 integrins was evaluated by fluorescent microscopy; integrin inhibition, by RGD peptides; and the talin-integrin interaction, by immunoprecipitation (IP).FLSs were isolated from synovial tissues from RA and osteoarthritis (OA) patients. Plates were coated overnight with the ECM proteins vitronectin, fibronectin, and type I collagen (Coll.I). Adrenomedullin was used as a soluble FLS ligand before plating. We tested interactions with the adrenomedullin receptor antagonist (22-52)adrenomedullin and with the protein kinase A (PKA) inhibitor H-89, and inhibition of co-receptor RAMP-2 by siRNA. Cell adhesion was measured by using color densitometry. Activation of α2 and β1 integrin activation. This activation was shown with IP to be related to an integrin-talin interaction and was significantly decreased by (22-52)adrenomedullin.Adrenomedullin specifically increased RA-FLS adhesion to vitronectin, fibronectin, and Coll.I; no such effect was found for OA-FLS adhesion. Basal or adrenomedullin-stimulated RA-FLS adhesion was inhibited by (22-52)adrenomedullin, H-89, and RAMP-2 siRNA. Adrenomedullin-stimulated adhesion was inhibited by RGD peptides, and associated with α2 and β1 integrins. This effect of adrenomedullin was dependent on adrenomedullin receptors. These results support a new role for adrenomedullin in rheumatoid synovial fibroblast pathobiology.Adrenomedullin-stimulated RA-FLS adhesion was specific for ECM proteins and mediated by α Rheumatoid arthritis (RA) is a chronic inflammatory joint disease in which fibroblast-like synoviocytes (FLSs) invade the extracellular matrix (ECM) of cartilage and bone, causing destruction of both tissues. FLSs in patients with RA (RA-FLSs) differ from normal FLSs regarding both morphologic and biologic characteristics . More sp1β1, α2β1, α3β1, and αvβ3 , was used. Cells were transfected by using the INTERFERIN transfection reagent (Ozyme) according to the manufacturer's instructions. siRNA was diluted in transfection reagent and culture medium, and cells were incubated with siRNA  for 2 hours at 37°C. After incubation, nonadherent cells were removed along with the culture medium. The cells were fixed with 4% PFA in PBS. Nonspecific sites were blocked with 3% BSA in PBS for 1 hour at RT. For immunostaining, cells were incubated for 1 hour with both monoclonal AK-7 α2- FITC and HUTS-21 β1-PE antibodies (Mc Ab) diluted 1/40 in 3% BSA. As a negative control, cells were incubated with γ2-FITC and γ1-PE isotype control. The nuclei were then stained with DAPI for 5 minutes. Cells were mounted in fluorescent mounting medium (Dako) and viewed by using a fluorescence microscope (Nikon) interfaced with the software package Microvision Instrument.FLSs were incubated for 1 hour with 10-6 M (22-52)AM for 30 minutes before being treated with adrenomedullin or left untreated for 1 hour and then processed for fluorescence microscopy, as described.Where indicated, FLSs were preincubated with 105 cells were seeded in triplicate for 2 hours. After incubation, cell proteins were extracted in lysis buffer. After centrifugation, the protein content of the supernatants was determined by using the Pierce protein assay. For immunoprecipitation (IP), 1 μg of specific anti-talin antibody (Santa-Cruz) was incubated overnight at 4°C in a rotating device with Dynabeads Protein G  and 100-μg aliquots of protein lysates. The magnetic beads were placed on a magnet, washed 3 times, suspended in lysis buffer and 4× running buffer , and 0.8 mg bromophenol blue), and denatured for 5 minutes at 95°C. The magnetic beads were placed on a magnet, and the denatured proteins were retained in 100 μl lysis buffer, whereas the beads were discarded. Aliquots (30 μl) were then subjected to electrophoresis resolved on 4-20% SDS-PAGE gel (Bio-Rad) and transferred onto Hybond polyvinylidene difluoride membrane (Amersham Biosciences). The membranes were incubated for 1 hour with 1× blocking buffer and then reacted overnight with anti-talin antibody diluted 1:500 in blocking solution. The membranes were washed and incubated with the appropriate peroxidase-coupled secondary antibody. The signal was visualized by using a chemiluminescence detection system (Bio-Rad).At confluence, FLSs were harvested with trypsin-EDTA, pelleted, and resuspended in HAM-F12 without FCS. FLSs were incubated with adrenomedullin for 1 hour, and 10post hoc tests (Fisher test). All values are normalized for the control value. P values less than 0.05 were considered significant.The data are reported as mean ± SEM for adhesion experiments (at least three separate experiments with different donors). Groups were compared by using ANOVA and -7 M adrenomedullin for 1 hour and studied in a 2-hour adhesion assay, as described earlier. The 10-7 M adrenomedullin significantly enhanced RA-FLS adhesion to all three ECM proteins . No effect was seen with the negative controls (BSA and polylysin). These results suggest that adrenomedullin may specifically affect adhesion to FLS integrins, which are receptors of ECM proteins.RA-FLSs were treated with 10s Figure , with a -8 M (P = 0.04); twofold with 10-7 M (P < 0.0001) of adrenomedullin; 1.6-fold at 10-8 M (P = 0.0001); and 1.7-fold at 10-7 M (P < 0.0001) on vitronectin and fibronectin, respectively . Nevertheless, this effect remained significant  after 2 hours  with vitronectin only. Spontaneous RA-FLSs adhesion increased gradually with time from 0.43 OD at 15 minutes to 1.52 OD at 2 hours Figure , i. By cs Figure , ii. FurP < 0.0001 for all comparisons) but had no effect on OA-FLSs adhesion  than did OA-FLS , we compn Figure . This co-6 M)   significantly increased RA-FLSs adhesion . This increase was significantly and dose-dependently inhibited by (22-52)adrenomedullin  adrenomedullin, a specific antagonist of adrenomedullin receptors . Without) Figure . Adrenom) Figure .P = 0.005)  was significantly decreased by H-89 .To confirm further the role for the adrenomedullin receptor and downstream signaling molecules, we used the pharmacologic PKA inhibitor H-89. Basal RA-FLS adhesion was significantly and dose-dependently decreased by H-89  Figure . MoreoveP < 0.0001) with 5, 10, and 20 nM RAMP-2 siRNA, respectively  inhibited basal and AM-stimulated RA-FLSs adhesion in a dose-dependent manner, whereas the scrambled peptide had no effect Figure .1 [2 integrin subunits, respectively. Adrenomedullin significantly increased the expression of both activated α2  and β1 subunits  . We then used HUTS-21 and AK-7, which specifically recognize the activated β1  and α2 i) Figure . Co-loca1 subunit, thereby causing integrin activation. The β1 subunit co-immunoprecipitated with talin, confirming that the two proteins are associated in a complex. Immunoblotting showed that adrenomedullin increased the talin-β1 interaction adrenomedullin, for 30 minutes, before adding 10-7 M adrenomedullin adrenomedullin significantly decreased AM-stimulated α2  and α2β1 (the Coll.I receptor). Coll.I accounts for 90% of the organic matrix of mineralized bone [Adrenomedullin increased RA-FLSs adhesion to the ECM proteins vitronectin, fibronectin, and Coll.I, which are abundant in the rheumatoid pannus. Increased levels of fibronectin have been found in rheumatoid synovial tissues -25. Vitr subunit ,27,28. Tzed bone , and RA zed bone , a procezed bone . For theet al. [et al. [Few studies have compared OA-FLSs and RA-FLSs adhesion to cartilage and bone ECM proteins . Schedelet al.  showed t [et al.  showed tin vitro receptor binding kinetics compared (22-52)adrenomedullin with adrenomedullin.We found that adrenomedullin specifically increased RA-FLS adhesion to ECM proteins but had no effect on OA-FLS adhesion. This difference may be ascribable to the stronger expression of adrenomedullin receptor (CLR/RAMP2) mRNA and proteins by RA-FLSs than by OA-FLSs . MoreoveOur findings suggest that the effect of adrenomedullin may be specific for adhesion molecules, such as integrins. The effect of adrenomedullin on RA-FLSs adhesion was already at its peak after 15 minutes, when adhesion was loose, whereas after 2 hours, the RA-FLSs exhibited the typical spreadout fibroblast-like appearance. In our attempt to demonstrate early adhesion-molecule expression, we selected integrins, because these are presynthesized in an inactive form and expressed at the membrane cell surface, and they are natural receptors for ECM proteins. With the specific antibodies AK7 and HUTS-21, we found that adrenomedullin induced integrin activation through a mechanism involving the adrenomedullin receptors. Integrin-mediated cell adhesion has been shown to depend on cAMP/PKA .2 and β1 integrin subunits, which are involved in cell adhesion, not only to Coll.I and fibronectin, but also to other ECM molecules of interest, such as type II collagen [1 immunoblotting to investigate the interaction of the two proteins with adrenomedullin. We found that adrenomedullin stimulated integrin activation by increasing this talin-β1 interaction. Moreover, by using the adrenomedullin-receptor antagonist (22-52)AM, we were able to confirm the role for adrenomedullin in α2 and β1 integrin activation.We studied only the αcollagen . The sigPrevious studies have established that adrenomedullin activates adhesion molecules in other cell types. With HUVECs, adrenomedullin induces cell-surface expression of E-selectin, vascular cell adhesion molecule (VCAM-1), and ICAM-1 , throughin vivo, respectively. Cell adhesion is the first event in a multistep process and is followed by migration and invasion in angiogenesis, tumor growth, and invasiveness. We have obtained preliminary evidence supporting a role for adrenomedullin in increasing RA-FLSs migration in a two-dimensional system . Overall, adrenomedullin may contribute to cell adhesion, migration, proliferation, and reduced apoptosis. These events are involved not only in pannus progression but also in tumor development, indicating a new role for adrenomedullin.Adrenomedullin also increased capillary cell activation and neoangiogenesis in various animal models of tissue repair or tumor growth ,35. Angi1 interaction followed by integrin activation (Figure In this work, we showed that soluble adrenomedullin increases RA-FLSs adhesion to various ECM proteins expressed in synovial tissue, cartilage, or bone. This effect is mediated by the adrenomedullin receptor CLR, and it does not occur with OA-FLSs, which express fewer adrenomedullin receptors . This adn Figure . Thus, aAC: adenylate cyclase; BSA: bovine serum albumin; CLR: calcitonin receptor-like receptor; Coll.I: type I collagen; ECM: extracellular matrix; FCS: fetal calf serum; FITC: fluorescein isothiocyanate; FLS: fibroblast-like synoviocyte; HUVEC: human umbilical vein endothelial cell; ICAM: intercellular adhesion molecule; IP: immunoprecipitation; OA: osteoarthritis; PE: phycoerythrin; PFA: paraformaldehyde; PKA: protein kinase A; RA: rheumatoid arthritis; RAMP: receptor activity-modifying proteins; RT: room temperature; VCAM-1: vascular cell adhesion molecule.The authors declare that they have no competing interests.MDAK was responsible for the conception and design, acquisition of data, analysis and interpretation of data, and drafting of the manuscript; CA, for acquisition of data and drafting of the manuscript; HKE, for conception and design, analysis and interpretation of data, and drafting of the manuscript; BU, for acquisition of data, analysis and interpretation of data, and drafting of the manuscript; MCS, for analysis and interpretation of data and drafting of the manuscript; FL, for conception and design, analysis and interpretation of data, drafting of the manuscript, and supervision. All authors read and approved the final manuscript.