Adjuvant and protective properties of native and recombinant Bordetella pertussis adenylate cyclase toxin preparations in mice

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin which is synthesised from the cyaA gene as an inactive protoxin that is post-translationally activated by the product of the cyaC gene. Purified active and inactive CyaA proteins were prepared from B. pertussis or from recombinant Escherichia coli expressing both cyaA and cyaC genes or the cyaA gene alone. respectively. In addition, a hybrid toxin (Hyb2) in which an internal region of CyaA had been replaced with the analogous region from the leukotoxin (LktA) of Pasteurella haemolytica, and which had low cell-invasive activity, was also prepared from E. coli expressing the cyaC gene. The CyaA preparations showed no evidence of toxicity in a mouse weight-gain test. Active toxin preparations were protective in mice against intranasal challenge with wild-type B. pertussis, as evidenced by lung:body weight ratios and bacterial numbers in the lungs, which were comparable to those in mice given whole-cell DPT vaccine. Hyb2 was not as protective as active CyaA and inactive CyaA preparations were not protective. Active CyaA, when co-administered with ovalbumin (OA), had a marked adjuvant effect on the anti-OA IgG antibody response which was not as apparent with inactive CyaA preparations. Similarly, active CyaA stimulated a greater anti-CyaA response than the inactive form.     

1-Methyladenine inhibits adenylate cyclase in starfish oocytes

Summary

The effect of 1-methyladenine (1-MeA) on adenylate cyclase (AC) basal activity and on preliminary stimulated AC activity was investigated in oocyte membrane preparations of the starfish Aphelasterias japonica. 1-MeA inhibited the membrane-bound AC activity both after its addition to intact oocytes and in cell-free experiments. GTP did not affect AC activity but it intensified the inhibitory effect of 1-MeA on AC activity. Sodium fluoride (F″) stimulated the oocyte AC (8 fold), while 1-MeA significantly reduced F″-stimulated activity. Manganese (MnCl₂, 5mM) stimulated AC (150 fold), but 1-MeA did not reduce Mn²⁺-stimulated activity. However, Mn²⁺-stimulated AC activity was inhibited by 1-MeA in the presence of MgCl₂. Forskolin stimulated AC activity (7 fold) and 1-MeA had no effect on AC. Thus, the inhibitory effect of 1-MeA on stimulated AC activity is displayed only after stimulation of the regulatory AC subunit. We suggest that 1-MeA inhibits the oocyte AC acting via inhibitory regulatory Gi protein of AC.     

Oligomeric behavior of Bordetella pertussis adenylate cyclase toxin in solution

Adenylate cyclase (AC) toxin from Bordetella pertussis inserts into eukaryotic cells, producing intracellular cAMP, as well as hemolysis and cytotoxicity. Concentration dependence of hemolysis suggests oligomers as the functional unit and inactive deletion mutants permit partial restoration of intoxication and/or hemolysis, when added in pairs [M. Iwaki, A. Ullmann, P. Sebo, Mol. Microbiol. 17 (1995) 1015-1024], suggesting dimerization/oligomerization. Using affinity co-precipitation and fluorescence resonance energy transfer (FRET), we demonstrate specific self-association of AC toxin molecules in solution. Flag-tagged AC toxin mixed with biotinylated-AC toxin, followed by streptavidin beads, yields both forms of the toxin. FRET measurements of toxin, labeled with different fluorophores, demonstrate association in solution, requiring post-translational acylation, but not calcium. AC toxin mixed with DeltaR, an inactive mutant, results in enhancement of hemolysis over that with wild type alone, suggesting that oligomers are functional. Dimers and perhaps higher molecular mass forms of AC toxin occur in solution in a manner that is relevant to toxin action.     

Determination of adenylate cyclase activity in a variety of organisms: Evidence against the occurrence of the enzyme in higher plants

Adenylate cyclase activity was examined in a variety of organisms using a highly sensitive assay. Activity was found in a blue-green alga, four green algae, two cellular slime molds, a fungus and moss protonemata. Fern prothalli and fronds gave variable results. No activity was detected in any of the higher plant tissues tested. The results throw further doubt on the existence of adenosine 3:5-cyclic monophosphate in higher plants.Abbreviation cAMP adenosine 3:5-cyclic monophosphate     

东菱克栓酶治疗对脑梗死患者腺苷酸环化酶影响的研究

目的研究东菱克栓酶治疗对脑梗死患者腺苷酸环化酶（AC）的影响。方法选取于2012年1月至2014年1月在本溪市中心医院治疗的脑梗死患者56例作为研究对象,采用抽签法将患者分为两组,对照组采用常规治疗,观察组在常规治疗的基础上采取东菱克栓酶治疗,利用RT-PCR技术检测两组患者治疗前后AC表达情况（AC/GPR-DH）,并观察临床疗效。结果治疗前对比分析两组患者AC/GPRDH比值,差异无统计学意义（P〉0.05）;经针对性治疗后,观察组患者的AC/GPR-DH比值上升幅度为（0.861±0.030）,较对照组（0.443±0.024）显著上升,经比较差异有统计学意义（P〈0.05）;观察组治疗总有效率（89.29%）较对照组（60.71%）高,差异有统计学意义（P〈0.05）;治疗后,观察组血脂指标浓度（TC：4.74±1.20;TG：1.06±1.04;LDL-C：3.19±1.22）下降量明显多于对照组（TC：5.25±1.15;TG：1.51±1.12;LDL-C：3.87±1.25）,差异均有统计学意义（均P〈0.05）。结论研究表明观察组治疗可明显缓解病患的并发症,具有积极意义。采用中医体质辨识理论结合解郁合欢汤治疗失眠患者具有较好的临床疗效,值得推广使用。     

Decreased adenylate cyclase responsiveness of transformed cells correlates with the presence of a viral transforming protein

The adenylate cyclase responsiveness of transformed fibroblastic and epithelial cell lines to forskolin, fluoride, guanine nucleotides and cholera toxin was reduced compared to their parental counterparts. This phenomenon was observed in lines transformed by either RNA or DNA tumor viruses, and in the case of polyoma virus, coincided with the expression of middle T antigen. The data suggest that decreased responsiveness of adenylate cyclase to non-hormone activators is a general consequence of viral transformation and may be related to viral regulation of protein kinase activity.     

Supra-molecular organization of adenylate cyclase in a neuroblastoma × glioma hybrid

Studies on the thermal inactivation of adenylate cyclase from neuroblastoma x glioma hybrid cells have been carried out. Inactivation curves show marked deviation from first-order kinetics, and as a first approximation can be adequately described as a sum of two negative exponentials. Half-lives of the rapidly decaying component have been estimated to be 5, 3.4,1.2 and 0.5 min at 37, 40, 44 and 48°C, respectively. The corresponding values for the slow-decaying component are found to be 90, 30, 11 and 5 min. Plausible inactivation pathways responsible for multi-exponential decay curves are discussed. Kinetic curves describing fractional loss of stimulatory response of adenylate cyclase to prostaglandin E₁ are shifted downwards with reference to basal activity. In contrast, an upward shift is observed for the inhibitory response of the enzyme to etorphine. A quantitative analysis of the inactivation curves for prostaglandin and etorphine-responsiveness has led to definitive predictions regarding the heat-sensitivity of the ‘hypothetical’ temperature-labile component responsible for the observed shifts.     

Ultrastructural cytochemical localization of adenylate cyclase in the early chick embryo

Summary Adenylate cyclase activity was localized in various tissues of the early chick embryo using an ultrastructural histochemical technique. Reaction product was deposited on the lateral plasma membrane of all cells, but with a preferential localization at the apical terminal complex in the epiblast. There was no activity associated with the free surfaces of these or other cells in the embryo. Intracellular deposits were found in all cells associated with the endoplasmic reticulum, nuclear envelope and Golgi bodies. In the last organelle, the deposit was sometimes observed to be distributed through the stack in a non-uniform way, with the heaviest deposits occurring at the forming face. No clear difference could be detected between the cytochemical activity associated with cells in various regions of the embryo, or with embryos at different stages of early development.     

Multiple effects of guanine nucleotides on human platelet adenylate cyclase

We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E₁. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E₁-stimulated adenylate cyclase activity when the enzyme was assayed in the presence of Mg²⁺. The basal and prostaglandin E₁-stimulated adenylate cyclase activities were also increased by washing, which presumably removes endogenous GTP. The analog, guanyl-5′-yl-imidodiphosphate mimics the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase activity but it stimulates basal enzyme activity. The onset of the inhibitory effect of GTP on the adenylate cyclase system is rapid (1 min) and is maintained at a constant rate during incubation for 10 min. GTP and guanyl-5′-yl-imidodiphosphate were noncompetitive inhibitors of prostaglandin E₁. An increase in the concentration of Mg²⁺ gradually reduces the effect of GTP while having little influence on the effect of guanyl-5′-yl-imidodiphosphate. Neither the substrate concentration nor the pH (7.2–8.5) is related to the inhibitory effect of guanine nucleotides. The inhibition by nucleotides was found to show a specificity for purine nucleotides with the order of potency being guanyl-5′-yl-imidodiphosphate > dGTP > GTP > ITP > XTP > CTP > TTP. The inhibitory effect of GTP is reversible while the effect of guanyl-5′-yl-imidodiphosphate is irreversible. The GTP inhibitory effect was abolished by preparing the lysates in the presence of Ca²⁺. However, the inhibitory effect of guanyl-5′-yl-imidodiphosphate persisted. Substitution of Mn²⁺ for Mg²⁺ in the assay medium resulted in a diminution of the inhibitory effect of GTP on basal activity and converted the inhibitory effect of GTP on prostaglandin E₁-stimulated activity to a stimulatory effect. At a lower concentration of Mn²⁺ (less than 2 mM) guanyl-5′-yl-imidodiphosphate inhibited prostaglandin E₁-stimulated adenylate cyclase activity, but at a higher concentration of Mn²⁺, it caused an increase in enzyme activity exceeding that occuring in the presence of prostaglandin E₁. In the presence of Mn²⁺, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E₁ receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase.     

Escherichia coli cells with mutations in the gene for adenylate cyclase (cya) exhibit a heat shock response

Abstract Adenylate cyclase mutants of Escherichia coli showed the heat-shock response. The heat-shock response was studied in two different mutants and in different growth media, including rich and minimal media. These results are in disagreement with the proposal that the cya gene regulates the expression of the heat-shock genes.     

Somatostatin acts through G-proteins on dopaminergic adenylate cyclase in the caudate-putamen of the rat

Somatostatin was incubated in an adenylate cyclase assay of a particulate fraction of caudateputamen tissue of the rat in order to examine the effect of the peptide on D-1 receptor coupled adenylate cyclase in vitro. Somatostatin was able to enhance cyclic AMP formation in the presence of guanylylimidodiphosphate and guanosine-triphosphate. In contrast to this, somatostatin inhibited both dopamine and forskolin-stimulated cyclic AMP accumulation. Pertussis toxin and cholera toxin also depressed forskolin-induced stimulation. Somatostatin was found to antagonize these inhibitory effects of pertussis toxin and cholera toxin. The results suggest that somatostatin acts through a stimulatory as well as an inhibitory guanine nucleotide regulatory protein subtype to affect dopaminergic adenylate cyclase activity.     

Mechanism of glucagon activation of adenylate cyclase in the presence of Mn2+

For a variety of ligand states, adenylate cyclase activity in the presence of Mn2+ was greater than with Mg2+. Trypsin treatment of intact hepatocytes, under conditions which destroy cell surface glucagon receptors, led to a first order loss of glucagon-stimulated adenylate cyclase activity in isolated membranes assayed in the presence of Mn2+ whether or not GTP (100 microM) was present in the assays. Arrhenius plots of basal activity exhibited a break at around 22 degrees C, those with NaF were linear and those with glucagon +/- GTP (100 microM) were biphasic with a break at around 28 degrees C. It is suggested that Mn2+ perturbs the coupling interaction between the glucagon receptor and catalytic unit of adenylate cyclase at the level of the guanine nucleotide regulatory protein. This appears to take the form of Mn2+ preventing GTP from initiating glucagon's activation of adenylate cyclase through a collision coupling mechanism.     

Cytochemical localization of adenylate cyclase activity in the rat anterior pituitary

Summary The cytochemical localization of adenylate cyclase was studied in relation to the secretory function of the anterior pituitary glands of male rats. The reaction product of adenylate cyclase was localized on the outside of plasma membranes, but was not detected intracellularly. High activity of adenylate cyclase was detected on somatotrophs and microvilli of follicular cells, whereas no activity was found on thyrotrophs or corticotrophs. Although most of the gonadotrophs showed little or no adenylate-cyclase activity, some was detected in a small number of gonadotrophs in the central portion of the gland. In somatotrophs, activity was not detected on the plasma membranes facing perivascular spaces where exocytotic extrusion of secretory granules was frequently observed, although the remaining areas of plasma membranes of the same somatotrophs were associated with high levels of adenylate-cyclase activity. These findings indicate that the association of a high level of adenylate-cyclase activity is not directly related to the ability of the plasma membranes to fuse with secretory granule membranes.     

Role of calmodulin in the effect of guanyl nucleotides on rat hippocampal adenylate cyclase: involvement of adenosine and opiates

Abstract: The adenylate cyclase activity of rat hippocampal plasma membranes can be stimulated by vaso-active intestinal polypeptide (VIP). Low concentrations (10⁻⁹ to 10⁻⁷M) of 5'-guanylyl-imido diphosphate (GppNHp) evoke a transient inhibition of the enzyme, which is followed by stimulation with increasing GppNHp concentrations (10⁻⁶ to 10⁻⁴M). Inclusion of ethyleneglycol - bis - (β - aminoethylether) - N,N' - tetraacetic acid (EGTA) during incubation abolishes the GppNHp inhibition while preserving GppNHp activation. The stimulation induced by GppNHp is amplified by VIP, but the inhibition is unaffected. Adenosine analogs and opiates are inhibitory ligands in the presence of GTP, and their effects can be reversed by the appropiate receptor antagonists, 3-isobutyl-1-methylxanthine and naloxone. Treatment of membranes with trypsin abolishes the GppNHp-induced inhibition without affecting the GppNHp stimulation. The inhibition induced by GppNHp is also abolished by EGTA treatment followed by washing, which coincides wtih a reduction in the adenosine- and opiate-mediated, GTP-dependent inhibition. The GppNHp inhibition can be restored in EGTA-treated but not in trypsin-treated membranes by addition of calcium-calmodulin but not by Ca²⁺ or Mg²⁺. Calcium-calmodulindepleted membranes lack calcium stimulation as well as GppNHp-induced inhibition, whereas untreated membranes and calcium-calmodulin-depleted membranes plus exogenous calcium-calmodulin showed calcium stimulation and GppNHp inhibition. These results suggest that calmodulin is involved in both Ca²⁺ stimulation and guanine nucleotide-mediated inhibition of rat hippocampal adenylate cyclase.     

Effects of proteolysis on the actions of monovalent cations and 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine on platelet adenylate cyclase

NaCl stimulated the adenylate cyclase activities of human and rabbit platelet particulate fractions prepared in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetate, but inhibited the activities of particulate fractions proteolysed by endogenous Ca2+-activated protease or treatment with alpha-chymotrypsin. Studies with other monovalent cations showed that LiCl had weak effects similar to those of NaCl, whereas KCl inhibited the enzyme in both proteolysed and non-proteolysed preparations. The results suggest that NaCl exerts stimulatory and inhibitory effects through different sites. NaCl potentiated and proteolysis greatly reduced the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (platelet-activating factor).     

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.