Monophosphate Signaling Research Articles

Soluble guanylate cyclase agonist, isoliquiritigenin attenuates renal damage and aortic calcification in a rat model of chronic kidney failure

AimsChronic kidney disease (CKD) is a growing fatal health problem worldwide associated with vascular calcification. Therapeutic approaches are limited with higher costs and poor outcomes. Adenine supplementation is one of the most relevant CKD models to human. Insufficient Nitric Oxide (NO)/ cyclic Guanosine Monophosphate (cGMP) signaling plays a key role in rapid development of renal fibrosis. Natural products display proven protection against CKD. Current study therefore explored isoliquiritigenin, a bioflavonoid extracted from licorice roots, potential as a natural activator for soluble Guanylate Cyclase (sGC) in a CKD rat model. Materials and methods60 male Wistar rats were grouped into Control group (n = 10) and the remaining rats received adenine (200 mg/kg, p.o) for 2 wk to induce CKD. They were equally sub-grouped into: Adenine untreated group and 4 groups orally treated by isoliquiritigenin low or high dose (20 or 40 mg/kg) with/without a selective sGC inhibitor, ODQ (1-H(1,2,4)oxadiazolo(4,3-a)-quinoxalin-1-one, 2 mg/kg, i.p) for 8 wk. Key findingsLong-term treatment with isoliquiritigenin dose-dependently and effectively amended adenine-induced chronic renal and endothelial dysfunction. It not only alleviated renal fibrosis and apoptosis markers but also aortic calcification. Additionally, this chalcone neutralized renal inflammatory response and oxidative stress. Isoliquiritigenin beneficial effects were associated with up-regulation of serum NO, renal and aortic sGC, cGMP and its dependent protein kinase (PKG). However, co-treatment with ODQ antagonized isoliquiritigenin therapeutic impact. SignificanceIsoliquiritigenin seems to exert protective effects against CKD and vascular calcification by activating sGC, increasing cGMP and its downstream PKG.

Salivary microbiome and metabolome analysis of severe early childhood caries

BackgroundSevere early childhood caries (SECC) is an inflammatory disease with complex pathology. Although changes in the oral microbiota and metabolic profile of patients with SECC have been identified, the salivary metabolites and the relationship between oral bacteria and biochemical metabolism remains unclear. We aimed to analyse alterations in the salivary microbiome and metabolome of children with SECC as well as their correlations. Accordingly, we aimed to explore potential salivary biomarkers in order to gain further insight into the pathophysiology of dental caries.MethodsWe collected 120 saliva samples from 30 children with SECC and 30 children without caries. The microbial community was identified through 16S ribosomal RNA (rRNA) gene high-throughput sequencing. Additionally, we conducted non-targeted metabolomic analysis through ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry to determine the relative metabolite levels and their correlation with the clinical caries status.ResultsThere was a significant between-group difference in 8 phyla and 32 genera in the microbiome. Further, metabolomic and enrichment analyses revealed significantly altered 32 salivary metabolites in children with dental caries, which involved pathways such as amino acid metabolism, pyrimidine metabolism, purine metabolism, ATP-binding cassette transporters, and cyclic adenosine monophosphate signalling pathway. Moreover, four in vivo differential metabolites (2-benzylmalate, epinephrine, 2-formaminobenzoylacetate, and 3-Indoleacrylic acid) might be jointly applied as biomarkers (area under the curve = 0.734). Furthermore, the caries status was correlated with microorganisms and metabolites. Additionally, Spearman's correlation analysis of differential microorganisms and metabolites revealed that Veillonella, Staphylococcus, Neisseria, and Porphyromonas were closely associated with differential metabolites.ConclusionThis study identified different microbial communities and metabolic profiles in saliva, which may be closely related to caries status. Our findings could inform future strategies for personalized caries prevention, detection, and treatment.

The Effects of Different Doses of Sildenafil on Coronary Blood Flow and Oxidative Stress in Isolated Rat Hearts.

The dose-response relationship of sildenafil effects on cardiac function is not completely elucidated. The aim of this study was to assess the effects of different doses of sildenafil on coronary flow and oxidative stress in isolated rat hearts. Coronary flow and markers of oxidative stress, including nitrite outflow, and superoxide anion production in coronary effluent, were determined for isolated rat hearts. The experiments were performed during control conditions and in the presence of sildenafil (10, 20, 50, 200 nM) alone or with Nω-nitro-L-arginine monomethyl ester (L-NAME) (30 μM). Sildenafil was shown to result in a significant increase in coronary flow at lower coronary perfusion pressure (CPP) values at all administered doses, whereas, with an increase in CPP, a reduction in coronary flow was observed. An increase in nitric oxide (NO) was most pronounced in the group treated with the lowest dose of sildenafil at the highest CPP value. After the inhibition of the NO-cyclic guanosine monophosphate (cGMP) signaling (NOS) system by L-NAME, only a dose of 200 nM sildenafil was high enough to overcome the inhibition and to boost release of O2-. That effect was CPP-dependent, with statistical significance reached at 80, 100 and 120 mmHg. Our findings indicate that sildenafil causes changes in heart vasculature in a dose-dependent manner, with a shift from a vasodilatation effect to vasoconstriction with a pressure increase. The highest dose administered is capable of producing superoxide anion radicals in terms of NOS system inhibition.

Targeting the nitric oxide/cGMP signaling pathway to treat chronic pain.

Nitric oxide (NO)/cyclic guanosine 3',5'-monophosphate (cGMP) signaling has been shown to act as a mediator involved in pain transmission and processing. In this review, we summarize and discuss the mechanisms of the NO/cGMP signaling pathway involved in chronic pain, including neuropathic pain, bone cancer pain, inflammatory pain, and morphine tolerance. The main process in the NO/cGMP signaling pathway in cells involves NO activating soluble guanylate cyclase, which leads to subsequent production of cGMP. cGMP then activates cGMP-dependent protein kinase (PKG), resulting in the activation of multiple targets such as the opening of ATP-sensitive K+ channels. The activation of NO/cGMP signaling in the spinal cord evidently induces upregulation of downstream molecules, as well as reactive astrogliosis and microglial polarization which participate in the process of chronic pain. In dorsal root ganglion neurons, natriuretic peptide binds to particulate guanylyl cyclase, generating and further activating the cGMP/PKG pathway, and it also contributes to the development of chronic pain. Upregulation of multiple receptors is involved in activation of the NO/cGMP signaling pathway in various pain models. Notably the NO/cGMP signaling pathway induces expression of downstream effectors, exerting both algesic and analgesic effects in neuropathic pain and inflammatory pain. These findings suggest that activation of NO/cGMP signaling plays a constituent role in the development of chronic pain, and this signaling pathway with dual effects is an interesting and promising target for chronic pain therapy.

CGMP signaling: a potential therapeutic target for neurodegeneration in glaucoma?

cGMP signaling: a potential therapeutic target for neurodegeneration in glaucoma?

Intestinal Bile Acids Induce Behavioral and Olfactory Electrophysiological Responses in Large Yellow Croaker (Larimichthys crocea)

Chemical cues and pheromones mediate fish reproduction, aggregation, risk assessment, and kin recognition. To better understand the chemical communication of conspecific fish, the behavioral responses to bile acids (BAs), their source, and reception investigated in large yellow croaker (Larimichthys crocea). Behavioral experimental results indicated that juvenile fish were attracted to intestinal contents (ICs) emanating from conspecifics, regardless of whether the fish were feeding. IC BA-targeted metabolomics revealed that cholic acid (CA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), chenodeoxycholic acid (CDCA), and taurodeoxycholic acid (TDCA) were the top five categories. Tests with and without fasting yielded similar categories and proportions of BAs, indicating that the intestinal BA profiles were generally stable. At the nanomolar level, CA led to significant preference behavior (p < 0.01). The electrophysiological results supported the hypothesis that the top five BAs were potent odorants in L. crocea. Moreover, inhibition of adenylate cyclase–cyclic adenosine monophosphate (AC–cAMP) signaling and phospholipase C (PLC) signaling reduced the electro-olfactogram (EOG) responses to CA and CDCA. Collectively, the findings of this study indicate that conspecific individuals could be attracted by ICs unrelated to feeding. As a key intestinal BA, CA led to fish preference behaviors and olfactory responses relying on cAMP and PLC transduction cascades.

Hippocampal Changes Elicited by Metabolic and Inflammatory Stressors following Prenatal Maternal Infection.

The hippocampus participates in spatial navigation and behavioral processes, displays molecular plasticity in response to environmental challenges, and can play a role in neuropsychiatric diseases. The combined effects of inflammatory prenatal and postnatal challenges can disrupt the hippocampal gene networks and regulatory mechanisms. Using a proven pig model of viral maternal immune activation (MIA) matched to controls and an RNA-sequencing approach, the hippocampal transcriptome was profiled on two-month-old female and male offspring assigned to fasting, mimetic viral, or saline treatments. More than 2600 genes presented single or combined effects (FDR-adjusted p-value < 0.05) of MIA, postnatal stress, or sex. Biological processes and pathways encompassing messenger cyclic adenosine 3',5'-monophosphate (cAMP) signaling were enriched with genes including gastric inhibitory polypeptide receptor (GIPR) predominantly over-expressed in the MIA-exposed fasting males relative to groups that differed in sex, prenatal or postnatal challenge. While this pattern was amplified in fasting offspring, the postnatal inflammatory challenge appeared to cancel out the effects of the prenatal challenge. The transcription factors C-terminal binding protein 2 (CTBP2), RE1 silencing transcription factor (REST), signal transducer and activator of transcription 1 (STAT1), and SUZ12 polycomb repressive complex 2 subunit were over-represented among the genes impacted by the prenatal and postnatal factors studied. Our results indicate that one environmental challenge can influence the effect of another challenge on the hippocampal transcriptome. These findings can assist in the identification of molecular targets to ameliorate the effects of pre-and post-natal stressors on hippocampal-associated physiology and behavior.

Calcium-dependent cAMP mediates the mechanoresponsive behaviour of endothelial cells to high-frequency nanomechanostimulation

The endothelial junction plays a central role in regulating intravascular and interstitial tissue permeability. The ability to manipulate its integrity therefore not only facilitates an improved understanding of its underlying molecular mechanisms but also provides insight into potential therapeutic solutions. Herein, we explore the effects of short-duration nanometer-amplitude MHz-order mechanostimulation on interendothelial junction stability and hence the barrier capacity of endothelial monolayers. Following an initial transient in which the endothelial barrier is permeabilised due to Rho–ROCK-activated actin stress fibre formation and junction disruption typical of a cell's response to insults, we observe, quite uniquely, the integrity of the endothelial barrier to not only spontaneously recover but also to be enhanced considerably—without the need for additional stimuli or intervention. Central to this peculiar biphasic response, which has not been observed with other stimuli to date, is the role of second messenger calcium and cyclic adenosine monophosphate (cAMP) signalling. We show that intracellular Ca2+, modulated by the high frequency excitation, is responsible for activating reorganisation of the actin cytoskeleton in the barrier recovery phase, in which circumferential actin bundles are formed to stabilise the adherens junctions via a cAMP-mediated Epac1–Rap1 pathway. Despite the short-duration stimulation (8 min), the approximate 4-fold enhancement in the transendothelial electrical resistance (TEER) of endothelial cells from different tissue sources, and the corresponding reduction in paracellular permeability, was found to persist over hours. The effect can further be extended through multiple treatments without resulting in hyperpermeabilisation of the barrier, as found with prolonged use of chemical stimuli, through which only 1.1- to 1.2-fold improvement in TEER has been reported. Such an ability to regulate and enhance endothelial barrier capacity is particularly useful in the development of in vitro barrier models that more closely resemble their in vivo counterparts.

Identification of Novel, Replicable Genetic Risk Loci for Suicidal Thoughts and Behaviors Among US Military Veterans

Suicide is a leading cause of death; however, the molecular genetic basis of suicidal thoughts and behaviors (SITB) remains unknown. To identify novel, replicable genomic risk loci for SITB. This genome-wide association study included 633 778 US military veterans with and without SITB, as identified through electronic health records. GWAS was performed separately by ancestry, controlling for sex, age, and genetic substructure. Cross-ancestry risk loci were identified through meta-analysis. Study enrollment began in 2011 and is ongoing. Data were analyzed from November 2021 to August 2022. SITB. A total of 633 778 US military veterans were included in the analysis (57 152 [9%] female; 121 118 [19.1%] African ancestry, 8285 [1.3%] Asian ancestry, 452 767 [71.4%] European ancestry, and 51 608 [8.1%] Hispanic ancestry), including 121 211 individuals with SITB (19.1%). Meta-analysis identified more than 200 GWS (P < 5 × 10-8) cross-ancestry risk single-nucleotide variants for SITB concentrated in 7 regions on chromosomes 2, 6, 9, 11, 14, 16, and 18. Top single-nucleotide variants were largely intronic in nature; 5 were independently replicated in ISGC, including rs6557168 in ESR1, rs12808482 in DRD2, rs77641763 in EXD3, rs10671545 in DCC, and rs36006172 in TRAF3. Associations for FBXL19 and AC018880.2 were not replicated. Gene-based analyses implicated 24 additional GWS cross-ancestry risk genes, including FURIN, TSNARE1, and the NCAM1-TTC12-ANKK1-DRD2 gene cluster. Cross-ancestry enrichment analyses revealed significant enrichment for expression in brain and pituitary tissue, synapse and ubiquitination processes, amphetamine addiction, parathyroid hormone synthesis, axon guidance, and dopaminergic pathways. Seven other unique European ancestry-specific GWS loci were identified, 2 of which (POM121L2 and METTL15/LINC02758) were replicated. Two additional GWS ancestry-specific loci were identified within the African ancestry (PET112/GATB) and Hispanic ancestry (intergenic locus on chromosome 4) subsets, both of which were replicated. No GWS loci were identified within the Asian ancestry subset; however, significant enrichment was observed for axon guidance, cyclic adenosine monophosphate signaling, focal adhesion, glutamatergic synapse, and oxytocin signaling pathways across all ancestries. Within the European ancestry subset, genetic correlations (r > 0.75) were observed between the SITB phenotype and a suicide attempt-only phenotype, depression, and posttraumatic stress disorder. Additionally, polygenic risk score analyses revealed that the Million Veteran Program polygenic risk score had nominally significant main effects in 2 independent samples of veterans of European and African ancestry. The findings of this analysis may advance understanding of the molecular genetic basis of SITB and provide evidence for ESR1, DRD2, TRAF3, and DCC as cross-ancestry candidate risk genes. More work is needed to replicate these findings and to determine if and how these genes might impact clinical care.

Molecular Mechanisms of PTH/PTHrP Class B GPCR Signaling and Pharmacological Implications.

The classical paradigm of G protein-coupled receptor (GPCR) signaling via G proteins is grounded in a view that downstream responses are relatively transient and confined to the cell surface, but this notion has been revised in recent years following the identification of several receptors that engage in sustained signaling responses from subcellular compartments following internalization of the ligand-receptor complex. This phenomenon was initially discovered for the parathyroid hormone (PTH) type 1 receptor (PTH1R), a vital GPCR for maintaining normal calcium and phosphate levels in the body with the paradoxical ability to build or break down bone in response to PTH binding. The diverse biological processes regulated by this receptor are thought to depend on its capacity to mediate diverse modes of cyclic adenosine monophosphate (cAMP) signaling. These include transient signaling at the plasma membrane and sustained signaling from internalized PTH1R within early endosomes mediated by PTH. Here we discuss recent structural, cell signaling, and in vivo studies that unveil potential pharmacological outputs of the spatial versus temporal dimension of PTH1R signaling via cAMP. Notably, the combination of molecular dynamics simulations and elastic network model-based methods revealed how precise modulation of PTH signaling responses is achieved through structure-encoded allosteric coupling within the receptor and between the peptide hormone binding site and the G protein coupling interface. The implications of recent findings are now being explored for addressing key questions on how location bias in receptor signaling contributes to pharmacological functions, and how to drug a difficult target such as the PTH1R toward discovering nonpeptidic small molecule candidates for the treatment of metabolic bone and mineral diseases.

Increased protein phosphatase 5 expression in inflammation-induced left ventricular dysfunction in rats

Titin phosphorylation contributes to left ventricular (LV) diastolic dysfunction. The independent effects of inflammation on the molecular pathways that regulate titin phosphorylation are unclear. We investigated the effects of collagen-induced inflammation and subsequent tumor necrosis factor-α (TNF-α) inhibition on mRNA expression of genes involved in regulating titin phosphorylation in 70 Sprague-Dawley rats. LV diastolic function was assessed with echocardiography. Circulating inflammatory markers were quantified by enzyme-linked immunosorbent assay and relative LV gene expression was assessed by Taqman® polymerase chain reaction. Differences in normally distributed variables between the groups were determined by two-way analysis of variance (ANOVA), followed by Tukey post-hoc tests. For non-normally distributed variables, group differences were determined by Kruskal-Wallis tests. Collagen inoculation increased LV relative mRNA expression of vascular cell adhesion molecule 1 (VCAM1), pentraxin 3 (PTX3), and inducible nitric oxide synthase (iNOS) compared to controls, indicating local microvascular inflammation. Collagen inoculation decreased soluble guanylate cyclase alpha-2 (sGCα2) and soluble guanylate cyclase beta-2 (sGCβ2) expression, suggesting downregulation of nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling. Inhibiting TNF-α prevented collagen-induced changes in VCAM1, iNOS, sGCα2 and sGCβ2 expression. Collagen inoculation increased protein phosphatase 5 (PP5) expression. Like LV diastolic dysfunction, increased PP5 expression was not prevented by TNF-α inhibition. Inflammation-induced LV diastolic dysfunction may be mediated by a TNF-α-independent increase in PP5 expression and dephosphorylation of the N2-Bus stretch element of titin, rather than by TNF-α-induced downregulation of NO-sGC-cGMP pathway-dependent titin phosphorylation. The steady rise in number of patients with inflammation-induced diastolic dysfunction, coupled with low success rates of current therapies warrants a better understanding of the systemic signals and molecular pathways responsible for decreased titin phosphorylation in development of LV diastolic dysfunction. The therapeutic potential of inhibiting PP5 upregulation in LV diastolic dysfunction requires investigation.

Cyclic adenosine 3′-5′ monophosphate (cAMP) signaling and the major actors of the cardiac excitation contraction coupling (ECC) are differentially altered in diabetic cardiomyopathy

Cyclic adenosine 3′-5′ monophosphate (cAMP) signaling and the major actors of the cardiac excitation contraction coupling (ECC) are differentially altered in diabetic cardiomyopathy

Gαs-Coupled CGRP Receptor Signaling Axis from the Trigeminal Ganglion Neuron to Odontoblast Negatively Regulates Dentin Mineralization.

An inflammatory response following dental pulp injury and/or infection often leads to neurogenic inflammation via the axon reflex. However, the detailed mechanism underlying the occurrence of the axon reflex in the dental pulp remains unclear. We sought to examine the intracellular cyclic adenosine monophosphate (cAMP) signaling pathway in odontoblasts via the activation of Gs protein-coupled receptors and intercellular trigeminal ganglion (TG) neuron-odontoblast communication following direct mechanical stimulation of TG neurons. Odontoblasts express heterotrimeric G-protein α-subunit Gαs and calcitonin receptor-like receptors. The application of an adenylyl cyclase (AC) activator and a calcitonin gene-related peptide (CGRP) receptor agonist increased the intracellular cAMP levels ([cAMP]i) in odontoblasts, which were significantly inhibited by the selective CGRP receptor antagonist and AC inhibitor. Mechanical stimulation of the small-sized CGRP-positive but neurofilament heavy chain-negative TG neurons increased [cAMP]i in odontoblasts localized near the stimulated neuron. This increase was inhibited by the CGRP receptor antagonist. In the mineralization assay, CGRP impaired the mineralization ability of the odontoblasts, which was reversed by treatment with a CGRP receptor antagonist and AC inhibitor. CGRP establishes an axon reflex in the dental pulp via intercellular communication between TG neurons and odontoblasts. Overall, CGRP and cAMP signaling negatively regulate dentinogenesis as defensive mechanisms.

CGMP Signaling in the Neurovascular Unit-Implications for Retinal Ganglion Cell Survival in Glaucoma.

Glaucoma is a progressive age-related disease of the visual system and the leading cause of irreversible blindness worldwide. Currently, intraocular pressure (IOP) is the only modifiable risk factor for the disease, but even as IOP is lowered, the pathology of the disease often progresses. Hence, effective clinical targets for the treatment of glaucoma remain elusive. Glaucoma shares comorbidities with a multitude of vascular diseases, and evidence in humans and animal models demonstrates an association between vascular dysfunction of the retina and glaucoma pathology. Integral to the survival of retinal ganglion cells (RGCs) is functional neurovascular coupling (NVC), providing RGCs with metabolic support in response to neuronal activity. NVC is mediated by cells of the neurovascular unit (NVU), which include vascular cells, glial cells, and neurons. Nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling is a prime mediator of NVC between endothelial cells and neurons, but emerging evidence suggests that cGMP signaling is also important in the physiology of other cells of the NVU. NO-cGMP signaling has been implicated in glaucomatous neurodegeneration in humans and mice. In this review, we explore the role of cGMP signaling in the different cell types of the NVU and investigate the potential links between cGMP signaling, breakdown of neurovascular function, and glaucoma pathology.

Sensitive genetically encoded sensors for population and subcellular imaging of cAMP in vivo.

Cyclic adenosine monophosphate (cAMP) signaling integrates information from diverse G-protein-coupled receptors, such as neuromodulator receptors, to regulate pivotal biological processes in a cellular-specific and subcellular-specific manner. However, in vivo cellular-resolution imaging of cAMP dynamics remains challenging. Here, we screen existing genetically encoded cAMP sensors and further develop the best performer to derive three improved variants, called cAMPFIREs. Compared with their parental sensor, these sensors exhibit up to 10-fold increased sensitivity to cAMP and a cytosolic distribution. cAMPFIREs are compatible with both ratiometric and fluorescence lifetime imaging and can detect cAMP dynamics elicited by norepinephrine at physiologically relevant, nanomolar concentrations. Imaging of cAMPFIREs in awake mice reveals tonic levels of cAMP in cortical neurons that are associated with wakefulness, modulated by opioids, and differentially regulated across subcellular compartments. Furthermore, enforced locomotion elicits neuron-specific, bidirectional cAMP dynamics. cAMPFIREs also function in Drosophila. Overall, cAMPFIREs may have broad applicability for studying intracellular signaling in vivo.

Design, synthesis, and biological evaluation of novel NO-releasing 4-chromanone derivatives as potential vasodilator agents.

The nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling pathway is an effective mechanism involved in the treatment of hypertension. In our search for potential antihypertensive agents, a series of novel NO-donor derivatives of the 4-chromanone skeleton were designed and synthesized by coupling furoxans or nitrooxy NO-donor moieties. All derivatives showed enhanced nitric oxide releasing capacity and vasodilator activity with EC50 values ranging from 0.0215 μM to 1.46 μM, obviously superior to those of precursor 3. These biological evaluations indicated that all compounds displayed an important vasorelaxant effect, and several compounds (9c, 14b, 14c, 14d) presented good vasodilator activity, with 14c being the best. Furthermore, molecular modeling studies revealed that compound 14c occupied the pocket well with the phosphodiesterase 5 domain in a favorable conformation. In conclusion, we observed that these novel compounds can act as structural templates for the design and subsequent development of new vasodilators and antihypertensive drugs.

The Primary Cilium and Neuronal Migration.

The primary cilium (PC) is a microtubule-based tiny sensory organelle emanating from the centrosome and protruding from the surface of most eukaryotic cells, including neurons. The extremely severe phenotypes of ciliopathies have suggested their paramount importance for multiple developmental events, including brain formation. Neuronal migration is an essential step of neural development, with all neurons traveling from their site of birth to their site of integration. Neurons perform a unique type of cellular migration called cyclic saltatory migration, where their soma periodically jumps along with the stereotyped movement of their centrosome. We will review here how the role of the PC on cell motility was first described in non-neuronal cells as a guide pointing to the direction of migration. We will see then how these findings are extended to neuronal migration. In neurons, the PC appears to regulate the rhythm of cyclic saltatory neuronal migration in multiple systems. Finally, we will review recent findings starting to elucidate how extracellular cues sensed by the PC could be intracellularly transduced to regulate the machinery of neuronal migration. The PC of migrating neurons was unexpectedly discovered to display a rhythmic extracellular emergence during each cycle of migration, with this transient exposure to the external environment associated with periodic transduction of cyclic adenosine monophosphate (cAMP) signaling at the centrosome. The PC in migrating neurons thus uniquely appears as a beat maker, regulating the tempo of cyclic saltatory migration.

The N-Linked Glycosylation Site N191 Is Necessary for PKA Signal Transduction in Eel Follicle-Stimulating Hormone Receptor.

The follicle-stimulating hormone receptor (FSHR) contains several N-linked glycosylation sites in its extracellular region. We conducted the present study to determine whether conserved glycosylated sites in eel FSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction. We used site-directed mutagenesis to induce four mutations (N120Q, N191Q, N272Q, and N288Q) in the N-linked glycosylation sites of eel FSHR. In the eel FSHR wild-type (wt), the cAMP response was gradually increased in a dose-dependent manner (0.01-1500 ng/mL), displaying a high response (approximately 57.5 nM/104 cells) at the Rmax level. Three mutants (N120Q, N272Q, and N288Q) showed a considerably decreased signal transduction as a result of high-ligand treatment, whereas one mutant (N191Q) exhibited a completely impaired signal transduction. The expression level of the N191Q mutant was only 9.2% relative to that of the eel FSHR-wt, indicating a negligible expression level. The expression levels of the N120Q and N272Q mutants were approximately 35.9% and 24% of the FSHG-wt, respectively. The N288Q mutant had an expression level similar to that of the eel FSHR-wt, despite the mostly impaired cAMP responsiveness. The loss of the cell surface agonist-receptor complexes was very rapid in the cells expressing eel FSHR-wt and FSHR-N288Q mutants. Specifically, the N191Q mutant was completely impaired by the loss of cell surface receptors, despite treatment with a high concentration of the agonist. Therefore, we suggest that the N191 site is necessary for cAMP signal transduction. This finding implies that the cAMP response, mediated by G proteins, is directly related to the loss of cell surface receptors as a result of high-agonist treatment.

What makes another life possible in bacteria? Global regulators as architects of bacterial biofilms.

Biofilm structures are the main mode of evolutionary reproductive adaptation of bacteria, and even these features alone, are sufficient to make them the focus of genetic and physiological studies. As this life form is a multicellular-like life form coordinated by genetic and physiological programming, it is quite different from the planktonic form. In bacterial biofilms, which are often composed of more than one species in nature, there is a clear division of labor, nutrient channels, and a language (signaling) established between the cells forming the biofilm. On the other hand, biofilms, especially formed by pathogens, cause important industrial and clinical problems due to their high resistance to environmental stress conditions. Obtaining new data on the molecular basis of bacterial evolution and understanding the intra- and inter-species ecosystem relations in this context, as well as finding permanent solutions to the serious problems they create, are directly related to a detailed understanding of the genetic regulation of bacterial biofilm structures. Today, it is becoming increasingly certain that environmental signals effective in the transition from planktonic form to biofilm form and their receptor/response molecules are generally managed by similar systems and global regulator molecules in bacteria. In this sense; Besides the quorum sensing (QS) systems, cyclic adenosine monophosphate-catabolite suppressor protein (cAMP-CRP) and bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP) signaling molecules are of critical importance. In this review article, current information on bacterial biofilms is summarized and interpreted based on this framework.

A Robust and Sensitive Spectrophotometric Assay for the Enzymatic Activity of Bacterial Adenylate Cyclase Toxins.

Various bacterial pathogens are producing toxins that target the cyclic Nucleotide Monophosphate (cNMPs) signaling pathways in order to facilitate host colonization. Among them, several are exhibiting potent nucleotidyl cyclase activities that are activated by eukaryotic factors, such as the adenylate cyclase (AC) toxin, CyaA, from Bordetella pertussis or the edema factor, EF, from Bacillus anthracis. The characterization of these toxins frequently requires accurate measurements of their enzymatic activity in vitro, in particular for deciphering their structure-to-function relationships by protein engineering and site-directed mutagenesis. Here we describe a simple and robust in vitro assay for AC activity based on the spectrophotometric detection of cyclic AMP (cAMP) after chromatographic separation on aluminum oxide. This assay can accurately detect down to fmol amounts of B. pertussis CyaA and can even be used in complex media, such as cell extracts. The relative advantages and disadvantages of this assay in comparison with other currently available methods are briefly discussed.