Novel multiparameter correlates of Coxiella burnetii infection and vaccination identified by longitudinal deep immune profiling.

Q-fever is a flu-like illness caused by Coxiella burnetii (Cb), a highly infectious intracellular bacterium. There is an unmet need for a safe and effective vaccine for Q-fever. Correlates of immune protection to Cb infection are limited. We proposed that analysis by longitudinal high dimensional immune (HDI) profiling using mass cytometry combined with other measures of vaccination and protection could be used to identify novel correlates of effective vaccination and control of Cb infection. Using a vaccine-challenge model in HLA-DR transgenic mice, we demonstrated significant alterations in circulating T-cell and innate immune populations that distinguished vaccinated from naïve mice within 10 days, and persisted until at least 35 days post-vaccination. Following challenge, vaccinated mice exhibited reduced bacterial burden and splenomegaly, along with distinct effector T-cell and monocyte profiles. Correlation of HDI data to serological and pathological measurements was performed. Our data indicate a Th1-biased response to Cb, consistent with previous reports, and identify Ly6C, CD73, and T-bet expression in T-cell, NK-cell, and monocytic populations as distinguishing features between vaccinated and naïve mice. This study refines the understanding of the integrated immune response to Cb vaccine and challenge, which can inform the assessment of candidate vaccines for Cb.

Sphingosine-1-phosphate acts as a developmental stage specific inhibitor of platelet-derived growth factor-induced chemotaxis of osteoblasts.

The development and maintenance of a healthy skeleton depends on the migration of cells to areas of new bone formation. Osteoblasts, the bone forming cells of the body, mature from mesenchymal stem cells under the influence of bone morphogenetic protein. It is unclear at what developmental stage the osteoblasts start to migrate to their functional location. We have studied migration of immature pre-osteoblasts and of mature osteoblasts in response to Platelet-derived growth factor (PDGF) and sphingosine-1-phosphate (S1P). PDGF is a growth factor involved in bone remodeling and fracture healing whereas S1P is a circulating sphingolipid known to control cell trafficking. Our data indicate that PDGF acts as a chemotactic cue for pre-osteoblasts. In contrast, S1P is a chemorepellent to these cells. Upon Bone Morphogenetic Protein 2-induced conversion to the osteoblast phenotype, the chemotaxis response to PDGF is retained whereas the sensitivity to S1P is lost. By RNA interference and overexpression experiments we showed that the expression level of the S1P2 receptor is the sole determinant controlling responsiveness to S1P. The combined data indicate that migration of osteoblasts is controlled by the balance between PDGF, S1P and the differentiation state of the cells. We propose that this mechanism preserves the osteoprogenitor pool in the bone marrow, only allowing the more differentiated cell to travel to sites of bone formation.

Fluorescence assays for high-throughput screening of protein kinases.

Protein kinases comprise one of the most important group of targets for drug discovery research today. Methods to identify novel kinase inhibitors by high-throughput screening have evolved rapidly in recent years. An important aspect is the availability of fluorescent probes that can be applied in a homogeneous, or mix-and-measure, assay format. Here, we illustrate the application of fluorescence read-out technologies for kinase targets in light of our own experiences in assay development and high-throughput screening.

The coiled-coil region of the G protein beta subunit. Mutational analysis of Ggamma and effector interactions.

The beta and gamma subunits of the heterotrimeric G proteins remain tightly associated throughout the signaling cycle as the betagamma dimer interacts with Galpha, receptors, and effectors. A coiled-coil structure involving alpha-helical segments at the N termini of the beta and gamma subunits contributes to the dimerization interface and has been implicated in effector signaling in yeast. Scanning mutagenesis of the coiled-coil region of the mammalian beta1 subunit was performed to examine the effect of point mutations on betagamma assembly and effector signaling in COS cell cotransfection assays. In addition to the E10K mutation described previously, mutations A11E, L14E, and I18E in beta1 were found to block betagamma association, as evidenced by the failure of the Gbeta mutants to undergo cytosolic translocation with cotransfected nonisoprenylated Ggamma. Although none of 14 beta1 point mutations prevented the betagamma-dependent activation of the c-Jun N-terminal kinase (JNK) effector pathway, the D20K point mutation enhanced JNK but not phospholipase C-beta2 activation. These findings implicate the coiled-coil region of Gbeta in JNK signaling, provide further evidence that the structural features of the betagamma complex mediating effector regulation may differ among effectors, and identify single codons in the mammalian beta subunit where mutation might yield a phenotype of defective signal transduction.

Human serotonin 5-HT7 receptor: cloning and pharmacological characterisation of two receptor variants.

Two splice variants of the 5-HT7 receptor were identified in human brain that differ in the lengths of their intracellular carboxy terminal tail. Identification of the variants of this receptor is of particular interest since the 5-HT7 receptor is known to have a high affinity for a number of antidepressants and is localized in brain regions thought to be implicated in depression. The two isoforms are expressed in roughly equal amounts in various regions of the human brain. When expressed in NIH-3T3 cells, both variants encode functional 5-HT7 receptors, positively coupled to adenylyl cyclase. We suggest that both variants are derived from a single gene by alternative mRNA splicing. Furthermore, our results from Southern blot analysis studies suggest that additional 5-HT7 receptor genes may exist in human.

Multiple domains of G protein beta confer subunit specificity in beta gamma interaction.

The expression and assembly of particular combinations of beta and gamma subunit isoforms into beta gamma heterodimers may contribute to the specificity of signal transduction mediated by heterotrimeric guanine nucleotide binding regulatory proteins. Using a transient transfection paradigm to examine selectivity in beta gamma heterodimer formation, we find that gamma 1 interacts with beta 1 but not with beta 2, while both beta subunits interact with gamma 2 and that a beta 2/beta 1 chimera containing the N-terminal 41 residues of beta 2 retained a beta 1 phenotype. These results confirm previous reports and imply that a structure or structures between residues 42 and 340 in the C-terminal region of the beta subunit imparts the ability to distinguish between gamma chains. Extending this chimeric approach to further localize the regions of beta important for selectivity between gamma subunits, we find that the region of beta 1 or beta 2 between residue 215 and the C terminus is sufficient to confer the parental phenotype. Additional mutants implicate multiple regions of beta, including discrete residues in the variable connecting segments joining conserved elements of the fifth and sixth GH-WD repeats toward the C terminus of beta. These findings suggest a multidomain interaction between beta and gamma.

The N-terminal coiled-coil domain of beta is essential for gamma association: a model for G-protein beta gamma subunit interaction.

We have identified the N terminus of the beta subunit as an essential domain for G-protein beta gamma assembly. A C-terminal fragment, beta 1-(130-340), fails to bind gamma unless coexpressed with the complementary N-terminal fragment, beta 1-(1-129). Deletion of the N-terminal 33 residues of beta 1, a region identified by computer algorithm to favor coiled-coil formation, abolishes gamma 2 association. On the basis of these findings, we propose a coiled-coil model of beta gamma interaction and refine this by computer-assisted molecular modeling. The model is tested by further mutagenesis: reversing the charge of residues in beta 1 that are hypothesized to be involved in interhelical salt bridges precludes gamma association. Insertions in the coiled-coil region, which disrupt the proposed hydrophobic interface, prevent gamma association. This structural basis for beta gamma dimerization provides a starting point for the design of beta and gamma mutants that can be used to map regions in beta gamma critical for interactions with the alpha subunit, receptors, and effectors.

Inhibition of cyclic AMP accumulation in intact NCB-20 cells as a direct result of elevation of cytosolic Ca2+.

Earlier studies established that adenylyl cyclase in NCB-20 cell plasma membranes is inhibited by concentrations of Ca2+ that are achieved in intact cells. The present studies were undertaken to prove that agents such as bradykinin and ATP, which elevate the cytosolic Ca2+ concentration ([Ca2+]i) from internal stores in NCB-20 cells, could inhibit cyclic AMP (cAMP) accumulation as a result of their mobilization of [Ca2+]i and not by other mechanisms. Both bradykinin and ATP transiently inhibited [3H]cAMP accumulation in parallel with their transient mobilization of [Ca2+]i. The [Ca2+]i rise stimulated by bradykinin could be blocked by treatment with thapsigargin; this thapsigargin treatment precluded the inhibition of cAMP accumulation mediated by bradykinin (and ATP). A rapid rise in [Ca2+]i, as elicited by bradykinin, rather than the slow rise evoked by thapsigargin was required for inhibition of [3H]cAMP accumulation. Desensitization of protein kinase C did not modify the inhibitory action of bradykinin on [3H]cAMP. Effects of Ca2+ on phosphodiesterase were also excluded in the present studies. The accumulated data are consistent with the hypothesis that hormonal mobilization of [Ca2+]i leads directly to the inhibition of cAMP accumulation in these cells and presumably in other cells that express the Ca(2+)-inhibitable form of adenylyl cyclase.

Manipulation of intracellular calcium in NCB-20 cells.

A number of lines of evidence indicate that the Ca2+ and cyclic AMP signalling systems interact in NCB-20 cells. However, to date, the regulation of [Ca2+]i homeostasis has not been studied in this cell line. The present study aimed to clarify our understanding of [Ca2+]i homeostasis in these cells and to evaluate tools that manipulate [Ca2+]i, independently of protein kinase C effects. Bradykinin, by a B2-receptor, elevated [Ca2+]i by a pertussis-toxin-insensitive mechanism. The BK-stimulated [Ca2+]i rise originated from intracellular sources, without a contribution from Ca2+ entry mechanisms. The effect of BK was precluded by pretreatment with thapsigargin and ionomycin--compounds that elevated [Ca2+]i independent of phospholipase C activation. Both compounds, however, exerted effects in addition to stimulating release of Ca2+ from BK-sensitive stores; the BK-sensitive Ca2+ pool was a subset of the thapsigargin-sensitive pool; ionomycin strongly stimulates Ca2+ entry. Activation of protein kinases A and C attenuated the duration of the BK-induced rise in [Ca2+]i, without affecting the peak [Ca2+]i, suggesting interference with the BK response at a step downstream of the activation of phospholipase C. Application of these approaches should enhance the delineation of the consequences of Ca2+ mobilization on cyclic AMP accumulation.

Purinergic receptor regulation of signal transduction in NCB-20 cells.

In the present paper, P1 and P2 purinergic receptors and their control of signal transduction pathways were investigated in NCB-20 cells. ATP elicited an increase in [Ca2+]i. The purinergic receptor subtype involved was identified by comparing the actions of a range of nucleotides. UTP was the most potent agonist in elevating [Ca2+]i, with an EC50 value of 6.2 +/- 0.5 microM. UTP, ATP (EC50, 17.3 +/- 1.5 microM), adenosine-5'-O-(3-thio)triphosphate (23 +/- 3 microM), and ITP (55 +/- 4 microM) exerted similar maximal effects. Other nucleotides tested, including beta, gamma-methylene-ATP and 2-methylthio-ATP, which are considered prototypic agonists for P2x and P2y receptors, respectively, were ineffective; in general, modifications in the ribose-triphosphate chain and substitution on the 2-position of the purines reduced the efficacy of nucleotides. This pharmacological characterization indicated that a putative P2u receptor mediates the [Ca2+]i elevation elicited by nucleotides in NCB-20 cells. The increase in [Ca2+]i originates from intracellular Ca2+ stores; blockade of Ca2+ entry does not affect the rise in [Ca2+]i. In contrast, pretreatment with the Ca(2+)-ATPase inhibitor thapsigargin or with bradykinin, a hormone that releases Ca2+ from inositol trisphosphate-sensitive stores, does preclude the increase in [Ca2+]i induced by ATP. ATP and UTP also transiently inhibit cAMP accumulation in the intact cell, presumably via a Ca(2+)-mediated mechanism. The finding of a P2u receptor in NCB-20 cells adds to a growing perception that P2 receptors are widely distributed. Besides the P2u receptor, NCB-20 cells express adenosine A2 receptors, coupled to stimulation of cAMP accumulation. The presence of both P1 and P2 purinergic receptors permits a sequential modulation of distinct second messenger levels associated with a common stimulus, ATP.

The mode of interaction of amiloride and some of its analogues with the adenosine A1 receptor.

Amiloride, a potassium sparing diuretic, inhibits adenosine A1 receptor-radioligand binding in calf and rat brain membranes in the low micromolar range. The drug interacted with the A1 receptor in a manner different from classical A1 ligands, but structure-activity relationship studies indicated that this inhibitory effect is not related to the ion transport inhibiting properties of amiloride (Garritsen et al., 1990a,b) In the present study, the question is addressed how amiloride interacts with the adenosine A1 receptor. Amiloride and two of its analogues, in concentrations equivalent to their Ki values in displacement studies, decrease the affinity of the A1 antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine, but not the maximal binding capacity of the radioligand. Furthermore, the dissociation rate of the receptor-ligand complex is unaltered in the presence of amiloride or its analogues in a concentration exceeding the Ki value 10-fold. These characteristics argue for a purely competitive mode of interaction. The functional consequences of the interaction between amiloride analogues and the A1 receptor were investigated at the level of cyclic adenosine 3',5'-monophosphate (cAMP) formation. The amiloride analogue 5-(N-butyl-N-methyl) amiloride (MBA) reversed A1-receptor mediated inhibition of forskolin-stimulated cAMP formation in rat fat cell membranes. In this model, the antagonist potency of MBA is ca 5 microM. This value is in fair agreement with a Ki value of 3.5 microM in binding assays under similar conditions. In conclusion, amiloride inhibits A1 receptor binding in an apparently competitive manner. This suggests that the binding sites of amiloride and the classic A1 receptor ligands may at least partially overlap.(ABSTRACT TRUNCATED AT 250 WORDS)

Bradykinin stimulates Ca2+ mobilization in NCB-20 cells leading to direct inhibition of adenylylcyclase. A novel mechanism for inhibition of cAMP production.

The modulation of neuronal adenylylcyclase by Ca2+, acting via calmodulin, is a long-established example of a positive interaction between the Ca2(+)-mobilizing and cAMP-generating systems. In the present study, concentrations of Ca2+ that stimulate brain adenylylcyclase inhibit the adenylylcyclase of NCB-20 plasma membranes. These inhibitory effects of Ca2+ have been characterized and seem to be exerted at the catalytic unit of the enzyme; they are independent of calmodulin, Gi, and phosphodiesterase. To determine whether this inhibition of adenylylcyclase by Ca2+ could occur in the intact cell, cAMP accumulation was measured in response to bradykinin. Bradykinin, which mobilizes Ca2+ in NCB-20 cells, as a consequence of stimulating inositol phosphate production, causes a transient inhibition of prostaglandin E1 stimulation of cAMP accumulation. The inhibitory action of bradykinin is attenuated significantly by treatment of cells with the cell-permeant Ca2+ chelator, 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid. It seems likely that the inhibition of adenylylcyclase by low concentrations of Ca2+ represents a novel means for a negative interaction between Ca2(+)-mobilizing and cAMP-generating systems.

Evaluation of the role of GTP hydrolysis in the interaction between Mg2+ and GTP in regulating agonist binding to the adenosine A1 receptor.

Binding of agonists to adenosine receptors is reduced by GTP, whereas it is enhanced by Mg2+. The effect of GTP can be completely reversed by divalent cations, in contrast to the effect of the nonhydrolyzable analogue 5'-guanylylimidodiphosphate (GPPNHP). The present study addresses the role of divalent cation-stimulated specific and nonspecific GTP-ases in this reversal process. Under the conditions commonly employed in binding assays, almost all GTP is rapidly converted to GMP and Pi, indicating that maintenance of GTP levels is essential for the proper interpretation of results. A combination of a GTP-generating system and a competing substrate for high Km GTP-ases minimizes GTP breakdown. In the presence of these additions, the reversal of GTP effects is almost eliminated, and the inhibitory effects of both GTP and GPPNHP on agonist binding are reduced by divalent cations to a similar extent. Besides enhancing nonspecific GTP hydrolysis, Mg2+, but not Mn2+ or Ca2+, also stimulates specific agonist-dependent GTP-ase activity. Thus, it is evident that specific regulatory effects of Mg2+ and other divalent cations can only be identified when other, nonspecific, effects have been evaluated and controlled.

Receptor binding profiles of amiloride analogues provide no evidence for a link between receptors and the Na+/H+ exchanger, but indicate a common structure on receptor proteins.

Amiloride and its analogues affect radioligand binding to the adenosine-A1 receptor. In this paper, the specificity of this effect is investigated by generating receptor binding profiles for amiloride and two of its analogues. A limited structure-activity relationships study is performed to probe the relationship between inhibition of receptor binding by amiloride analogues and the effects of these compounds on Na+ transport, in particular Na+/H+ exchange. The receptor binding profiles of amiloride, benzamil and 5'-(N,N-hexamethylene)amiloride (HMA) indicate that the compounds affect a variety of receptors and that none of the compounds is highly selective for any of these. The SAR study indicates that it is very unlikely that a direct coupling between receptors and Na+/H+ exchange or another amiloride-sensitive ion transport system is responsible for the inhibition of receptor binding. A correlation between the signal transduction systems coupled to the receptors involved and the potency of the amiloride analogues is also absent. The varying nature of the receptors, affected by amiloride or its analogues, suggests a wide-spread presence of an amiloride binding site on receptors and other membrane proteins.

Interaction of amiloride and its analogues with adenosine A1 receptors in calf brain.

Amiloride, a potassium sparing diuretic, is known to interact with a number of ion transport systems, receptors and enzymes. Here, we report on the interaction between this drug and the adenosine A1 receptor as present in calf brain membranes. Adenosine A1 receptors are characterized by a subnanomolar affinity for the antagonists [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and the agonist [3H]N6-R-1-phenyl-2-propyladenosine ([3H]PIA). Amiloride displaces both agonist and antagonist binding with a Ki value in the low micromolar range. This inhibition is counteracted by NaCl and protons, in contrast to the binding of [3H]PIA and [3H]DPCPX. The results suggest that amiloride interacts with the adenosine A1 receptor at a site distinct from the ligand binding site. In order to elucidate the role of one of the ion transport systems known to be inhibited by amiloride, eight amiloride analogues with different sensitivities for these systems were tested. The potency and order of potency of these compounds towards adenosine A1 receptors excludes the involvement of the epithelial Na+ channel, Na+/H+ exchanger or Na+/Ca2+ exchanger.

Chemical modification of adenosine A1 receptors. Implications for the interaction with R-PIA, DPCPX and amiloride.

Amiloride, a potassium sparing diuretic, inhibits the specific binding of [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and [3H]N6-R-1-phenyl-2-propyladenosine (PIA) to adenosine A1 receptors in calf brain. This interaction is different from the agonist-receptor or the antagonist-receptor interaction as Na+ and H+ counteract the inhibitory effect of amiloride whereas these ions hardly affect the binding of the classic A1 receptor ligands. In the present study, the effects of protein modifiers on the equilibrium inhibition constant of amiloride are compared with effects of these reagents on the affinities of DPCPX and PIA. It is demonstrated that the affinities of amiloride and [3H]DPCPX are changed after treatment with a carboxyl-modifying reagent but unaffected by modification of histidyl, arginyl and cystein residues. The maximal binding capacity of [3H]DPCPX is enhanced by sulfhydryl modification, whereas the number of [3H]DPCPX binding sites is reduced by treatment with a histidine-modifying reagent. The histidyl residues of the [3H]DPCPX binding site can be partially protected against modification by 300 microM amiloride, present during treatment of the membranes. An equivalent concentration of 8-phenyltheophylline results in complete protection. The apparent affinity of PIA is altered by modification of histidyl, carboxyl, arginyl and cystein residues. In the latter two cases, uncoupling of the G protein seems to be the major reason for the decrease in affinity of PIA. The results suggest that amiloride is an A1 antagonist with binding characteristics that differ from the classic A1 antagonists such as DPCPX.

[3H]batrachotoxinin-A 20-alpha-benzoate binding to sodium channels in rat brain: sensitivity to tetrodotoxin and divalent cations.

The sodium channel blocker tetrodotoxin partially inhibited [3H]batrachotoxinin-A 20-alpha-benzoate binding to rat brain synaptosomes. This inhibition results from a decrease in the apparent affinity of the radioligand, which indicates that sites 1 and 2 of the sodium channel are not independent as thought previously. Computer-assisted data analysis allowed two binding sites for BTX-B to be distinguished. These sites could be differentiated by means of the divalent cations Mg2+ and Ca2+, that inhibit BTX-B binding completely. Tetrodotoxin diminished the inhibition by Mg2+ and vice versa, suggesting a common mechanism of action for the inhibition.

The membrane stabilizing activity of beta-adrenoceptor ligands. Quantitative evaluation of the interaction of phenoxypropanolamines with the [3H]batrachotoxinin A 20-alpha-benzoate binding site on voltage-sensitive sodium channels in rat brain.

The interaction of 12 phenoxypropanolamines, all ligands with high affinity towards beta-adrenoceptors, with the [3H]batrachotoxinin A 20-alpha-benzoate ([3H]BTX-B) binding site on voltage-sensitive sodium channels in a rat brain synaptosomal preparation, was studied as a measure for local anaesthetic activity. All derivatives are capable of displacing [3H]BTX-B from its specific binding site, penbutolol displaying the highest affinity. Multiple regression analyses were performed, correlating biological activity (pKi values) with physicochemical parameters. Lipophilicity is of prime importance in the established regression equations, but steric factors are relevant as well. Comparisons were made with analogous equations relating beta 1- and beta 2-adrenoceptor affinity with physicochemical parameters, leading to the conclusion that "cardioselective" beta-adrenoceptor ligands appear to have fewer membrane stabilizing properties.