Analysis of core circadian feedback loop in suprachiasmatic nucleus of mCry1-luc transgenic reporter mouse

The suprachiasmatic nucleus (SCN) coordinates circadian rhythms that adapt the individual to solar time. SCN pacemaking revolves around feedback loops in which expression of Period (Per) and Cryptochrome (Cry) genes is periodically suppressed by their protein products. Specifically, PER/CRY complexes act at E-box sequences in Per and Cry to inhibit their transactivation by CLOCK/BMAL1 heterodimers. To function effectively, these closed intracellular loops need to be synchronized between SCN cells and to the light/dark cycle. For Per expression, this is mediated by neuropeptidergic and glutamatergic extracellular cues acting via cAMP/calcium-responsive elements (CREs) in Per genes. Cry genes, however, carry no CREs, and how CRY-dependent SCN pacemaking is synchronized remains unclear. Furthermore, whereas reporter lines are available to explore Per circadian expression in real time, no Cry equivalent exists. We therefore created a mouse, B6.Cg-Tg(Cry1-luc)01Ld, carrying a transgene (mCry1-luc) consisting of mCry1 elements containing an E-box and E′-box driving firefly luciferase. mCry1-luc organotypic SCN slices exhibited stable circadian bioluminescence rhythms with appropriate phase, period, profile, and spatial organization. In SCN lacking vasoactive intestinal peptide or its receptor, mCry1 expression was damped and desynchronized between cells. Despite the absence of CREs, mCry1-luc expression was nevertheless (indirectly) sensitive to manipulation of cAMP-dependent signaling. In mPer1/2-null SCN, mCry1-luc bioluminescence was arrhythmic and no longer suppressed by elevation of cAMP. Finally, an SCN graft procedure showed that PER-independent as well as PER-dependent mechanisms could sustain circadian expression of mCry1. The mCry1-luc mouse therefore reports circadian mCry1 expression and its interactions with vasoactive intestinal peptide, cAMP, and PER at the heart of the SCN pacemaker.     

Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca²⁺-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.     

C-di-GMP turnover influences motility and biofilm formation in Bacillus amyloliquefaciens PG12

Bis-(3′→5′) cyclic dimeric guanosine monophosphate (c-di-GMP) is defined as a highly versatile secondary messenger in bacteria, coordinating diverse aspects of bacterial growth and behavior, including motility and biofilm formation. Bacillus amyloliquefaciens PG12 is an effective biocontrol agent against apple ring rot caused by Botryosphaeria dothidea. In this study, we characterized the core regulators of c-di-GMP turnover in B. amyloliquefaciens PG12. Using bioinformatic analysis, heterologous expression and biochemical characterization of knockout and overexpression derivatives, we identified and characterized two active diguanylate cyclases (which catalyze c-di-GMP biosynthesis), YhcK and YtrP and one active c-di-GMP phosphodiesterase (which degrades c-di-GMP), YuxH. Furthermore, we showed that elevating c-di-GMP levels up to a certain threshold inhibited the swimming motility of B. amyloliquefaciens PG12. Although yhcK, ytrP and yuxH knockout mutants did not display defects in biofilm formation, significant increases in c-di-GMP levels induced by YtrP or YuxH overexpression stimulated biofilm formation in B. amyloliquefaciens PG12. Our results indicate that B. amyloliquefaciens possesses a functional c-di-GMP signaling system that influences the bacterium's motility and ability to form biofilms. Since motility and biofilm formation influence the efficacy of biological control agent, our work provides a basis for engineering a more effective strain of B. amyloliquefaciens PG12.     

Defective Responsiveness of Adenylate Cyclase to Forskolin in the Drosophila Memory Mutant rutabaga

The Drosophila memory mutant rutabaga (rut) has been previously shown to have a defective subpopu-lation (or functional state) of the enzyme adenylate cyclase. We report here that the reduced adenylate cyclase activity is also associated with a defective responsiveness of the enzyme to forskolin. Forskolin activation isotherms of the enzyme in normal membranes reveal low- and high-affinity forskolin-interacting components; the residual enzyme in the mutant shows a smaller proportion of the high-affinity response. In addition, in mutant membrane preparations, forskolin fails to shift the K_m of the enzyme for free Mg²⁺ and for MgATP, in contrast to the situation in the normal tissue. The defect in the responsiveness to forskolin in rut is even more pronounced in a Lubrol-solubilized enzyme preparation, and is due to intrinsic properties of the cyclase system rather than to the absence (or presence) of a soluble, or detergent solubilized, factor in rut. The reduced forskolin responsiveness maps to the X chromosomal segment 12F5-6 to 13A1-5, within the region previously reported to span the locus that controls both the abortive memory and the lack of Ca^{2 +}-stimulation of adenylate cyclase in rut¹⁷. The possible relevance of the findings to postulated molecular mechanisms of short-term memory formation is discussed.     

Free radicals generated by xanthine oxidase-hypoxanthine damage adenylate cyclase and ATPase in gerbil cerebral cortex

The generation of superoxide radicals from xanthine oxidase-hypoxanthine in a particulate fraction of gerbil cerebral cortex influenced the activity of the synaptic enzyme adenylate cyclase, as well as Mn²⁺- and Na⁺,K⁺-sensitive forms of ATPase. Low concentrations of xanthine oxidase actually elevated the sensitivity of adenylate cyclase to GTP, GTP + norepinephrine (NE), and forskolin but not significantly to Mn²⁺. Higher levels of xanthine oxidase elicited a marked inhibition of these responses. The stimulation of adenylate cyclase mechanisms requiring GTP (GTP, forskolin, and NE) was more susceptible than was Mn²⁺, suggesting that the guanine nucleotide stimulatory protein was more vulnerable to free radical attack than the catalytic site of adenylate cyclase. superoxide dismutase (SOD), but not catalase, partially protected the forskolin-sensitive enzyme from the action of xanthine oxidase-hypoxanthine. A combination of SOD plus catalase preserved enzyme responses to forskolin. In comparison, additions of SOD plus mannitol or catalase plus flunarizine were less effective. The sensitivity of the particulate ATPase to Mn²⁺ was more labile to the consequence of superoxide formation than Na⁺, K⁺-ATPase. In this regard the Ca²⁺, Mg²⁺ sensitivity of the enzyme was reduced only to a marginal extent. The findings might be analogous toin vivo data in which cerebral adenylate cyclase and Na⁺, K⁺-ATPase are damaged following postischemic reperfusion in gerbils, a process thought to be mediated by free radicals.     

Overexpression of adenylate cyclase‐associated protein 2 is a novel prognostic marker in malignant melanoma

Malignant melanoma is one of the lethal malignant tumors worldwide. Previously we reported that adenylate cyclase‐associated protein 2 (CAP2), which is a well‐conserved actin regulator, was overexpressed in hepatocellular carcinoma; however, CAP2 expression in other clinical cancers remains unclear. The aim of the current study was to clarify the clinicopathological significance of CAP2 overexpression in malignant melanoma. Immunohistochemical analyses revealed that many melanoma cells exhibited diffuse cytoplasmic expression of CAP2, whereas no normal melanocytes showed detectable immunostaining for CAP2. A high level of CAP2 expression was seen in 14 of 50 melanomas and was significantly correlated with greater tumor thickness and nodular melanoma subtypes. In addition, a high level of CAP2 expression was associated with poor overall survival in univariate and multivariate analyses. For 13 patients, samples of primary and metastatic melanoma tissue were available: four patients exhibited higher levels of CAP2 expression in metastatic tumor compared to the primary site, whereas no patient showed lower levels of CAP2 expression in metastatic melanomas. Our findings show that CAP2 overexpression is a novel prognostic marker in malignant melanoma and that CAP2 expression seems to increase stepwise during tumor progression, suggesting the involvement of CAP2 in the aggressive behavior of malignant melanoma.     

Mechanisms underlying the effects of estrogen on nocturnal melatonin synthesis in peripubertal female rats: relation to norepinephrine and adenylate cyclase

We previously reported that estrogen modulates the nocturnal synthesis of melatonin in the pineal gland of peripubertal female rats. These effects appeared to be mediated by the modulation of N-acetyltransferase (NAT) activity. The present study assessed the mechanism underlying the effects of estrogen deficiency and stimulation on pineal melatonin synthesis in peripubertal female rats. We measured the norepinephrine levels and adenylate cyclase activity in pineal gland homogenates obtained from 4-10-wk-of-age female Sprague Dawley rats at mid-dark during the daily light/dark cycle. The animals were ovariectomized and daily s.c. administration of estradiol benzoate (E2B, 1.0 microgram/d) was initiated at 4 wk of age. Pineal norepinephrine levels increased significantly from Week 3 to 4 (P < 0.0001), and remained unchanged thereafter. Neither ovariectomy nor E2B administration significantly affected norepinephrine levels. Adenylate cyclase activity in the pineal gland peaked at 4 wk in untreated (control) rats. Ovariectomy at Week 4 led to a significant increase in adenylate cyclase activity at Week 8. At Week 10, adenylate cyclase activity returned to control levels. S.c. injection of E2B suppressed the ovariectomy-induced increase in adenylate cyclase activity to the level seen in control rats. These changes in mid-dark adenylate cyclase activity resembled those previously observed with NAT activity. The results suggest that estrogen modulates adenylate cyclase activity in the pineal gland of peripubertal female rats. The inhibitory effect of estrogen on melatonin synthesis appeared to be mediated in part, by changes in the norepinephrine-induced stimulation of pineal adenylate cyclase activity.     

Effect of replacement of extracellular sodium ions and of D-600 on the activation by adrenalin of adenylate cyclase in intact pigeon erythrocytes

The effect of replacement of extracellular Na⁺ by Li⁺, choline, K⁺ or sucrose on cyclic AMP formation in pigeon erythrocytes has been investigated. Replacement of extracellular Na⁺ by Li⁺, choline or sucrose but not by K⁺ inhibited the stimulation by adrenalin of cyclic AMP formation, but had no detectable effect on cyclic AMP content in the absence of adrenalin. This inhibition was observed in the presence or absence of extracellular Ca²⁺. The relative inhibition caused by Na⁺ removal decreased with increasing adrenalin concentration. It was concluded that extracellular Na⁺ or K⁺ ions were required for maximal activation of adenylate cyclase by low concentrations of adrenalin, and that this effect of monovalent cations may have been due to an effect on the affinity of the receptor for adrenalin.The verapamil derivative D-600 also inhibited the stimulation by adrenalin of cyclic AMP formation. This effect occurred in the absence of extracellular Ca²⁺ and hence seemed to be unrelated to the inhibition by D-600 of the slow Ca²⁺ channel in electrically excitable tissues.     

Prostacyclin analogs stimulate receptor-mediated cAMP synthesis and ATP release from rabbit and human erythrocytes

Objectives: The purpose of this study was to establish that the prostacyclin (PGI₂) receptor (IP receptor) is present on rabbit and human erythrocytes and that its activation stimulates cyclic adenosine monophosphate (cAMP) synthesis and adenosine triphosphate (ATP) release. Methods: The effect of incubation of erythrocytes with the active PGI₂ analogs, iloprost or UT‐15C, on cAMP levels and ATP release was determined in the absence and presence of the IP receptor antagonist, CAY10441. Western analysis was used to determine the presence of the IP receptor on isolated membranes. To establish that effects of PGI₂ analogs were not due to prostaglandin E₂(PGE₂) receptor activation, the effect of PGE₂ on cAMP levels and ATP release was determined. Results: Rabbit and human erythrocytes possess IP receptors. Iloprost and UT‐15C stimulated increases in cAMP and ATP release that were prevented by the IP receptor antagonist, CAY10441. PGE₂ did not stimulate cAMP accumulation or ATP release and did not inhibit iloprost‐induced increases in cAMP. Conclusions: This study establishes that the IP receptor is present on rabbit and human erythrocytes and that its activation results in increases in cAMP and ATP release. These results suggest a novel mechanism by which PGI₂ and its active analogs, when administered pharmacologically, could produce vasodilation.