Susceptibility of primary human airway epithelial cells to Bordetella pertussis adenylate cyclase toxin in two- and three-dimensional culture conditions

The human pathogen Bordetella pertussis targets the respiratory epithelium and causes whooping cough. Its virulence factor adenylate cyclase toxin (CyaA) plays an important role in the course of infection. Previous studies on the impact of CyaA on human epithelial cells have been carried out using cell lines derived from the airways or the intestinal tract. Here, we investigated the interaction of CyaA and its enzymatically inactive but fully pore-forming toxoid CyaA-AC^– with primary human airway epithelial cells (hAEC) derived from different anatomical sites (nose and tracheo-bronchial region) in two-dimensional culture conditions. To assess possible differences between the response of primary hAEC and respiratory cell lines directly, we included HBEC3-KT in our studies. In comparative analyses, we studied the impact of both the toxin and the toxoid on cell viability, intracellular cAMP concentration and IL-6 secretion. We found that the selected hAEC, which lack CD11b, were differentially susceptible to both CyaA and CyaA-AC^–. HBEC3-KT appeared not to be suitable for subsequent analyses. Since the nasal epithelium first gets in contact with airborne pathogens, we further studied the effect of CyaA and its toxoid on the innate immunity of three-dimensional tissue models of the human nasal mucosa. The present study reveals first insights in toxin–cell interaction using primary hAEC.     

Bordetella Adenylate Cyclase Toxin Differentially Modulates Toll-Like Receptor-Stimulated Activation,Migration and T Cell Stimulatory Capacity of Dendritic Cells

Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC) enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR)-activated murine and human dendritic cells (DCs). cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 in vitro. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4⁺ and CD8⁺ T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4⁺ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4⁺CD25⁺Foxp3⁺ T regulatory cells in vitro. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8⁺ T cell proliferation and limited the induction of IFN-γ producing CD8⁺ T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during B. pertussis infection.     

Almost half of the RTX domain is dispensable for complement receptor 3 binding and cell-invasive activity of the Bordetella adenylate cyclase toxin

The whooping cough agent Bordetella pertussis secretes an adenylate cyclase toxin (CyaA) that through its large carboxy-proximal Repeat-in-ToXin (RTX) domain binds the complement receptor 3 (CR3). The RTX domain consists of five blocks (I–V) of characteristic glycine and aspartate-rich nonapeptides that fold into five Ca²⁺-loaded parallel β-rolls. Previous work indicated that the CR3-binding structure comprises the interface of β-rolls II and III. To test if further portions of the RTX domain contribute to CR3 binding, we generated a construct with the RTX block II/III interface (CyaA residues 1132–1294) linked directly to the C-terminal block V fragment bearing the folding scaffold (CyaA residues 1562–1681). Despite deletion of 267 internal residues of the RTX domain, the Ca²⁺-driven folding of the hybrid block III/V β-roll still supported formation of the CR3-binding structure at the interface of β-rolls II and III. Moreover, upon stabilization by N- and C-terminal flanking segments, the block III/V hybrid-comprising constructs competed with CyaA for CR3 binding and induced formation of CyaA toxin-neutralizing antibodies in mice. Finally, a truncated CyaAΔ_1295-1561 toxin bound and penetrated erythrocytes and CR3-expressing cells, showing that the deleted portions of RTX blocks III, IV, and V (residues 1295–1561) were dispensable for CR3 binding and for toxin translocation across the target cell membrane. This suggests that almost a half of the RTX domain of CyaA is not involved in target cell interaction and rather serves the purpose of toxin secretion.     

Bi-Allelic TCRα or β Recombination Enhances T Cell Development but Is Dispensable for Antigen Responses and Experimental Autoimmune Encephalomyelitis

Dual TCRα-expressing T cells outnumber dual TCRβ-expressing cells by ~10:1. As a result, efforts to understand how dual TCR T cells impact immunity have focused on dual TCRα expression; dual TCRβ expression remains understudied. We recently demonstrated, however, that dual TCRβ expression accelerated disease in a TCR transgenic model of autoimmune arthritis through enhanced positive selection efficiency, indicating that dual TCRβ expression, though rare, can impact thymic selection. Here we generated mice hemizygous for TCRα, TCRβ, or both on the C57BL/6 background to investigate the impact bi-allelic TCR chain recombination has on T cell development, repertoire diversity, and autoimmunity. Lack of bi-allelic TCRα or TCRβ recombination reduced αβ thymocyte development efficiency, and the absence of bi-allelic TCRβ recombination promoted γδ T cell development. However, we observed no differences in the numbers of naïve and expanded antigen-specific T cells between TCRα^+/-β^+/- and wildtype mice, and TCR repertoire analysis revealed only subtle differences in Vβ gene usage. Finally, the absence of dual TCR T cells did not impact induced experimental autoimmune encephalomyelitis pathogenesis. Thus, despite more stringent allelic exclusion of TCRβ relative to TCRα, bi-allelic TCRβ expression can measurably impact thymocyte development and is necessary for maintaining normal αβ/γδ T cell proportions.     

Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin

Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, secretes several virulence factors, among which is the adenylate cyclase toxin (CyaA) that plays a crucial role in the early stages of human respiratory tract colonization. CyaA invades target cells by translocating its catalytic domain directly across the plasma membrane and overproduces cAMP, leading to cell death. The molecular process leading to the translocation of the catalytic domain remains largely unknown. We have previously shown that the catalytic domain per se, AC384, encompassing residues 1–384 of CyaA, did not interact with lipid bilayer, whereas a longer polypeptide, AC489, spanning residues 1–489, binds to membranes and permeabilizes vesicles. Moreover, deletion of residues 375–485 within CyaA abrogated the translocation of the catalytic domain into target cells. Here, we further identified within this region a peptidic segment that exhibits membrane interaction properties. A synthetic peptide, P454, corresponding to this sequence (residues 454–485 of CyaA) was characterized by various biophysical approaches. We found that P454 (i) binds to membranes containing anionic lipids, (ii) adopts an α-helical structure oriented in plane with respect to the lipid bilayer, and (iii) permeabilizes vesicles. We propose that the region encompassing the helix 454–485 of CyaA may insert into target cell membrane and induce a local destabilization of the lipid bilayer, thus favoring the translocation of the catalytic domain across the plasma membrane.     

Bordetella pertussis adenylate cyclase toxin modulates innate and adaptive immune responses: distinct roles for acylation and enzymatic activity in immunomodulation and cell death

Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the repeat in toxin family of pore-forming toxins, which require posttranslational acylation to lyse eukaryotic cells. CyaA modulates dendritic cell (DC) and macrophage function upon stimulation with LPS. In this study, we examined the roles of acylation and enzymatic activity in the immunomodulatory and lytic effects of CyaA. The adenylate cyclase activity of CyaA was necessary for its modulatory effects on murine innate immune cells. In contrast, acylation was not essential for the immunomodulatory function of CyaA, but was required for maximal caspase-3 activation and cytotoxic activity. The wild-type acylated toxin (A-CyaA) and nonacylated CyaA (NA-CyaA), but not CyaA with an inactive adenylate cyclase domain (iAC-CyaA), enhanced TLR-ligand-induced IL-10 and inhibited IL-12, TNF-alpha, and CCL3 production by macrophages and DC. In addition, both A-CyaA and NA-CyaA, but not iAC-CyaA, enhanced surface expression of CD80 and decreased CpG-stimulated CD40 and ICAM-1 expression on immature DC. Furthermore, both A-CyaA and NA-CyaA promoted the induction of murine IgG1 Abs, Th2, and regulatory T cells against coadministered Ags in vivo, whereas iAC-CyaA had more limited adjuvant activity. In contrast, A-CyaA and iAC-CyaA induced caspase-3 activation and cell death in macrophages, but these effects were considerably reduced or absent with NA-CyaA. Our findings demonstrate that the enzymatic activity plays a critical role in the immunomodulatory effects of CyaA, whereas acylation facilitates the induction of apoptosis and cell lysis, and as such, NA-CyaA has considerable potential as a nontoxic therapeutic molecule with potent anti-inflammatory properties.     

Adenylate cyclase toxin translocates across target cell membrane without forming a pore

The adenylate cyclase toxin‐haemolysin of Bordetella (CyaA) targets CD11b⁺ myeloid phagocytes and translocates across their cytoplasmic membrane an adenylate cyclase (AC) enzyme that catalyses conversion of cytosolic ATP into cAMP. In parallel, CyaA acts as a cytolysin forming cation‐selective pores, which permeabilize cell membrane and eventually provoke cell lysis. Using cytolytic activity, potassium efflux and patch‐clamp assays, we show that a combination of substitutions within the pore‐forming (E570Q) and acylation‐bearing domain (K860R) ablates selectively the cell‐permeabilizing activity of CyaA. At the same time, however, the capacity of such mutant CyaA to translocate the AC domain across cytoplasmic membrane into cytosol of macrophage cells and to elevate cellular cAMP concentrations remained intact. Moreover, the combination of E570Q+K860R substitutions suppressed the residual cytolytic activity of the enzymatically inactive CyaA/OVA/AC^‐ toxoid on CD11b‐expressing monocytes, while leaving unaffected the capacity of the mutant toxoid to deliver in vitro a reporter CD8⁺ T cell epitope from ovalbumin (OVA) to the cytosolic pathway of dendritic cells for MHC class I‐restricted presentation and induce in vivo an OVA‐specific cytotoxic T cell response. CyaA, hence, employs a mechanism of AC enzyme domain translocation across cellular membrane that avoids passage across the cytolytic pore formed by toxin oligomers.     

Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.     

Bordetella pertussis Commits Human Dendritic Cells to Promote a Th1/Th17 Response through the Activity of Adenylate Cyclase Toxin and MAPK-Pathways

The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types. We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played by adenylate cyclase toxin (CyaA), an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendritic cells (MDDC), an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The work compared MDDC functions after encounter with wild-type B. pertussis (BpWT) or a mutant lacking CyaA (BpCyaA−), or the BpCyaA− strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As a first step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step, engagement of Toll-like receptors (TLR) 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed. These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, by reducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B. pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance, enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in the absence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion. The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17 responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury.     

Structure-function studies of the adenylate cyclase toxin of Bordetella pertussis and the leukotoxin of Pasteurella haemolytica by heterologous C protein activation and construction of hybrid proteins.

The adenylate cyclase toxin (CyaA) from Bordetella pertussis and the leukotoxin (LktA) from Pasteurella haemolytica are members of the RTX (stands for repeats in toxin) family of cytolytic toxins. They have pore-forming activity and share significant amino acid homology but show marked differences in biological activity. CyaA is an invasive adenylate cyclase and a weak hemolysin which is active on a wide range of mammalian cells. LktA is a cytolytic protein with a high target cell specificity and is able to lyse only leukocytes and platelets from ruminants. Each toxin is synthesized as an inactive protoxin encoded by the A gene, and the product of the accessory C gene is required for posttranslational activation. Heterologous activation of LktA by CyaC did not result in a change in its specificity for nucleated cells, although the toxin showed a greater hemolytic-to-cytotoxic ratio. LktC was unable to activate CyaA. A hybrid toxin (Hyb1), which contained the N-terminal enzymic domain and the pore-forming domain from CyaA (amino acids [aa] 1 to 687), with the remainder of the protein derived from the C-terminal end of LktA (aa 379 to 953), showed no toxic activity. Replacement of part of the LktA C-terminal domain of Hyb1 by the CyaA C-terminal domain (aa 919 to 1706) to create hybrid toxin 2 (Hyb2) partially restored toxic activity. In contrast to CyaA, Hyb2 was activated more efficiently by LktC than by CyaC, showing the importance of the region between aa 379 and 616 of LktA for activation by LktC. LktC-activated Hyb2 was more active against ruminant than murine nucleated cells, whereas CyaC-activated Hyb2 displayed a similar, but lower, activity against both cell types. These data indicate that LktC and the region with which it interacts have an influence on the target cell specificity of the mature toxin.     

CFTR-Adenylyl Cyclase I Association Responsible for UTP Activation of CFTR in Well-Differentiated Primary Human Bronchial Cell Cultures

Chloride secretion by airway epithelial cells is defective in cystic fibrosis (CF). The conventional paradigm is that CFTR is activated through cAMP and protein kinase A (PKA), whereas the Ca²⁺-activated chloride channel (CaCC) is activated by Ca²⁺ agonists like UTP. We found that most chloride current elicited by Ca²⁺ agonists in primary cultures of human bronchial epithelial cells is mediated by CFTR by a mechanism involving Ca²⁺ activation of adenylyl cyclase I (AC1) and cAMP/PKA signaling. Use of selective inhibitors showed that Ca²⁺ agonists produced more chloride secretion from CFTR than from CaCC. CFTR-dependent chloride secretion was reduced by PKA inhibition and was absent in CF cell cultures. Ca²⁺ agonists produced cAMP elevation, which was blocked by adenylyl cyclase inhibition. AC1, a Ca²⁺/calmodulin-stimulated adenylyl cyclase, colocalized with CFTR in the cell apical membrane. RNAi knockdown of AC1 selectively reduced UTP-induced cAMP elevation and chloride secretion. These results, together with correlations between cAMP and chloride current, suggest that compartmentalized AC1–CFTR association is responsible for Ca²⁺/cAMP cross-talk. We further conclude that CFTR is the principal chloride secretory pathway in non-CF airways for both cAMP and Ca²⁺ agonists, providing a novel mechanism to link CFTR dysfunction to CF lung disease.     

Reduction of T Cell Receptor Diversity in NOD Mice Prevents Development of Type 1 Diabetes but Not Sj?gren’s Syndrome

Non-obese diabetic (NOD) mice are well-established models of independently developing spontaneous autoimmune diseases, Sjögren’s syndrome (SS) and type 1 diabetes (T1D). The key determining factor for T1D is the strong association with particular MHCII molecule and recognition by diabetogenic T cell receptor (TCR) of an insulin peptide presented in the context of I-A^g7 molecule. For SS the association with MHCII polymorphism is weaker and TCR diversity involved in the onset of the autoimmune phase of SS remains poorly understood. To compare the impact of TCR diversity reduction on the development of both diseases we generated two lines of TCR transgenic NOD mice. One line expresses transgenic TCRβ chain originated from a pathogenically irrelevant TCR, and the second line additionally expresses transgenic TCRα^mini locus. Analysis of TCR sequences on NOD background reveals lower TCR diversity on Treg cells not only in the thymus, but also in the periphery. This reduction in diversity does not affect conventional CD4⁺ T cells, as compared to the TCR^mini repertoire on B6 background. Interestingly, neither transgenic TCRβ nor TCR^mini mice develop diabetes, which we show is due to lack of insulin B:9–23 specific T cells in the periphery. Conversely SS develops in both lines, with full glandular infiltration, production of autoantibodies and hyposalivation. It shows that SS development is not as sensitive to limited availability of TCR specificities as T1D, which suggests wider range of possible TCR/peptide/MHC interactions driving autoimmunity in SS.     

Adenylate cyclase toxin subverts phagocyte function by RhoA inhibition and unproductive ruffling

Adenylate cyclase toxin (CyaA or ACT) is a key virulence factor of pathogenic Bordetellae. It penetrates phagocytes expressing the alpha(M)beta(2) integrin (CD11b/CD18, Mac-1 or CR3) and paralyzes their bactericidal capacities by uncontrolled conversion of ATP into a key signaling molecule, cAMP. Using pull-down activity assays and transfections with mutant Rho family GTPases, we show that cAMP signaling of CyaA causes transient and selective inactivation of RhoA in mouse macrophages in the absence of detectable activation of Rac1, Rac2, or RhoG. This CyaA/cAMP-induced drop of RhoA activity yielded dephosphorylation of the actin filament severing protein cofilin and massive actin cytoskeleton rearrangements, which were paralleled by rapidly manifested macrophage ruffling and a rapid and unexpected loss of macropinocytic fluid phase uptake. As shown in this study for the first time, CyaA/cAMP signaling further caused a rapid and near-complete block of complement-mediated phagocytosis. Induction of unproductive membrane ruffling, hence, represents a novel sophisticated mechanism of down-modulation of bactericidal activities of macrophages and a new paradigm for action of bacterial toxins that hijack host cell signaling by manipulating cellular cAMP levels.     

Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin. Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.     

cAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin

The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.     

Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) Activity and Sinoatrial Nodal Pacemaker Cell Energetics

Ca²⁺-activated basal adenylate cyclase (AC) in rabbit sinoatrial node cells (SANC) guarantees, via basal cAMP/PKA-calmodulin/CaMKII-dependent protein phosphorylation, the occurrence of rhythmic, sarcoplasmic-reticulum generated, sub-membrane Ca²⁺ releases that prompt rhythmic, spontaneous action potentials (APs). This high-throughput signaling consumes ATP.

Aims

We have previously demonstrated that basal AC-cAMP/PKA signaling directly, and Ca²⁺ indirectly, regulate mitochondrial ATP production. While, clearly, Ca²⁺-calmodulin-CaMKII activity regulates ATP consumption, whether it has a role in the control of ATP production is unknown.

Methods and Results

We superfused single, isolated rabbit SANC at 37°C with physiological saline containing CaMKII inhibitors, (KN-93 or autocamtide-2 Related Inhibitory Peptide (AIP)), or a calmodulin inhibitor (W-7) and measured cytosolic Ca²⁺, flavoprotein fluorescence and spontaneous AP firing rate. We measured cAMP, ATP and O₂ consumption in cell suspensions. Graded reductions in basal CaMKII activity by KN-93 (0.5–3 µmol/L) or AIP (2–10 µmol/L) markedly slow the kinetics of intracellular Ca²⁺ cycling, decrease the spontaneous AP firing rate, decrease cAMP, and reduce O₂ consumption and flavoprotein fluorescence. In this context of graded reductions in ATP demand, however, ATP also becomes depleted, indicating reduced ATP production.

Conclusions

CaMKII signaling, a crucial element of normal automaticity in rabbit SANC, is also involved in SANC bioenergetics.     

Cell cycle arrest induced by the bacterial adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis

Bacillus anthracis oedema toxin (ET) and Bordetella pertussis adenylate cyclase toxin (ACT) enter host cells and produce cAMP. To understand the cellular consequences, we exposed J774 cells to these toxins at ng ml(-1) (pM) concentrations, then followed cell number and changes in cell signalling pathways. Under these conditions, both toxins produce a concentration-dependent inhibition of cell proliferation without cytotoxicity. ET and ACT increase the proportion of cells in G(1) /G(0) and reduce S phase, such that a single addition of ET or ACT inhibits cell division for 3-6 days. Treatment with ET or ACT produces striking changes in proteins controlling cell cycle, including virtual elimination of phosphorylated ERK 1/2 and Cyclin D1 and increases in phospho-CREB and p27(Kip1) . Importantly, PD98059, a MEK inhibitor, elicits a comparable reduction in Cyclin D1 to that produced by the toxins and blocks proliferation. These data show that non-lethal concentrations of ET and ACT impose a prolonged block on the proliferation of J774 cells by impairment of the progression from G(1) /G(0) to S phase in a process involving cAMP-mediated increases in phospho-CREB and p27(Kip1) and reductions in phospho-ERK 1/2 and Cyclin D1. This phenomenon represents a new mechanism by which these toxins affect host cells.     

Conventional and Regulatory CD4+ T Cells That Share Identical TCRs Are Derived from Common Clones

Results from studies comparing the diversity and specificity of the TCR repertoires expressed by conventional (Tconv) and regulatory (Treg) CD4⁺ T cell have varied depending on the experimental system employed. We developed a new model in which T cells express a single fixed TCRα chain, randomly rearranged endogenous TCRβ chains, and a Foxp3-GFP reporter. We purified CD4⁺Foxp3^- and CD4⁺Foxp3⁺ cells, then performed biased controlled multiplex PCR and high throughput sequencing of endogenous TCRβ chains. We identified >7,000 different TCRβ sequences in the periphery of 5 individual mice. On average, ~12% of TCR sequences were expressed by both conventional and regulatory populations within individual mice. The CD4⁺ T cells that expressed shared TCR sequences were present at higher frequencies compared to T cells expressing non-shared TCRs. Furthermore, nearly all (>90%) of the TCR sequences that were shared within mice were identical at the DNA sequence level, indicating that conventional and regulatory T cells that express shared TCRs are derived from common clones. Analysis of TCR repertoire overlap in the thymus reveals that a large proportion of Tconv and Treg sharing observed in the periphery is due to clonal expansion in the thymus. Together these data show that there are a limited number of TCR sequences shared between Tconv and Tregs. Also, Tconv and Tregs sharing identical TCRs are found at relatively high frequencies and are derived from common progenitors, of which a large portion are generated in the thymus.     

Neurochemical and morphological studies on differentiation of NG108-15 cells by phorbol ester and forskolin

12-O-tetradecanoylphorbol 13-acetate (TPA), forskolin or dibutyryl cAMP induced neurite outgrowth and inhibition of cell growth in NG108-15 cells. TPA, forskolin and dibutyryl cAMP significantly increased specific activity of choline acetyltransferase. Forskolin markedly stimulated cAMP accumulation, but not TPA, suggesting that forskolin could induce differentiation by increasing the cAMP content via adenylate cyclase activation, but TPA-induced differentiation seems not to be due to the raise of the cAMP level. Incubation of the cells with TPA, forskolin or dibutyryl cAMP for 24 h resulted in enhancement of 50 mM K⁺-evoked Ca²⁺ influx and neurite elongation, although incubation with these agents for 1 h didn't affect these events. From these results, it is suggested that TPA and forskolin induce differentiation of NG108-15 cells to acetylcholine neurons via different mechanisms: protein kinase C activation by TPA and cAMP-dependent protein kinase activation by forskolin. In addition, it is likely that Ca²⁺ channels in cells differentiated by TPA, forskolin or dibutyryl cAMP become sensitive to depolarization.