cAMP Treatment Research Articles

Rapid degradation of ABCA1 protein following cAMP withdrawal and treatment with PKA inhibitor suggests ABCA1 is a short-lived protein primarily regulated at the transcriptional level.

ATP-binding cassette transporter A1 (ABCA1) is a key player in the reverse cholesterol transport (RCT) and HDL biogenesis. Since RCT is compromised as a result of ABCA1 dysfunction in diabetic state, the objective of this study was to investigate the regulation of ABCA1 in a stably transfected 293 cells expressing ABCA1 under the control of cAMP response element. To delineate transcriptional and posttranscriptional regulation of ABCA1, 293 cells were stably transfected with the full length ABCA1 cDNA under the control of CMV promoter harboring cAMP response element. cAMP-mediated regulation of ABCA1 and cholesterol efflux were studied in the presence of 8-Br-cAMP and after withdrawal of 8-Br-cAMP. The mechanism of cAMP-mediated transcriptional induction of the ABCA1 gene was studied in protein kinase A (PKA) inhibitors-treated cells. The transfected 293 cells expressed high levels of ABCA1, while non-transfected wild-type 293 cells showed very low levels of ABCA1. Treatments of transfected cells with 8-Br-cAMP increased ABCA1 protein by 10-fold and mRNA by 20-fold. Cholesterol efflux also increased in parallel. Withdrawal of 8-Br-cAMP caused time-dependent rapid diminution of ABCA1 protein and mRNA, suggesting ABCA1 regulation at the transcriptional level. Treatment with PKA inhibitors abolished the cAMP-mediated induction of the ABCA1 mRNA and protein, resulting dampening of ABCA1-dependent cholesterol efflux. These results demonstrate that transfected cell line mimics cAMP response similar to normal cells with natural ABCA1 promoter and suggest that ABCA1 is a short-lived protein primarily regulated at the transcriptional level to maintain cellular cholesterol homeostasis.

Genome-wide identification of CNGC genes in Chinese jujube (Ziziphus jujuba Mill.) and ZjCNGC2 mediated signalling cascades in response to cold stress

BackgroundsCyclic nucleotide gated channels (CNGCs) play multifaceted roles in plant physiological processes, especially with respect to signalling processes, plant development, and responses to environmental stresses. However, little information is known about the CNGC family in the large cosmopolitan family Rhamnaceae, which has strong tolerance to biotic and abiotic stresses.ResultsIn the current study, a total of 15 ZjCNGCs which located on 7 chromosomes were firstly identified in Chinese jujube (Ziziphus jujuba Mill.), the most important species of Rhamnaceae in terms of economic and ecological values. Phylogenetic analysis showed that these ZjCNGCs could be classified into four groups, ZjCNGC12 belonged to group IVA, and ZjCNGC13, 14, 15 belonged to group IVB. In addition, the paralogous and orthologous homology duplication of ZjCNGC15 occurred during the evolutionary process. The characteristics of ZjCNGCs regarding to exon-intron numbers and post-translational modifications showed diversified structures and functions. Motif composition and protein sequence analysis revealed that the phosphate-binding cassette and hinge regions were conserved among ZjCNGCs. Prediction of the cis-acting regulatory elements and expression profiles by real-time quantitative PCR analysis showed that some of the ZjCNGCs responded to environmental changes, especially ZjCNGC2, which was significantly downregulated in response to cold stress, and ZjCNGC4 was highly induced in response to cold, salt and alkaline stresses. ZjCNGC13 and 14 were highly induced in the phytoplasma-resistant cultivar and downregulated in the susceptible cultivar. Furthermore, ZjCNGC2 could be regulated by cAMP treatment, microtubule changes and interact with ZjMAPKK4, which suggested that cAMP and microtubule might play important roles in ZjCNGC2 mediated ZjMAPKK4 signalling transduction involved in cold stress.ConclusionsThe identification and classification analysis of ZjCNGCs were firstly reported, and some key individual ZjCNGCs might play essential roles in the response to biotic and abiotic stresses, especially ZjCNGC2 mediated ZjMAPKK4 signalling transduction involved in cold stress. This systematic analysis could provide important information for further functional characterization of ZjCNGCs with the aim of breeding stress-resistant cultivars.

Inhibitory effects of glycopyrronium, formoterol, and budesonide on coronavirus HCoV-229E replication and cytokine production by primary cultures of human nasal and tracheal epithelial cells.

BackgroundCoronavirus 229E (HCoV-229E), one of the causes of the common cold, exacerbates chronic obstructive pulmonary disease (COPD) and bronchial asthma. Long-acting muscarinic antagonists and β2-agonists and inhaled corticosteroids inhibit the exacerbation of COPD and bronchial asthma caused by infection with viruses, including HCoV-229E. However, the effects of these drugs on HCoV-229E replication and infection-induced inflammation in the human airway are unknown.MethodsPrimary human nasal (HNE) and tracheal (HTE) epithelial cell cultures were infected with HCoV-229E.ResultsPretreatment of HNE and HTE cells with glycopyrronium or formoterol decreased viral RNA levels and/or titers, the expression of the HCoV-229E receptor CD13, the number and fluorescence intensity of acidic endosomes where HCoV-229E RNA enters the cytoplasm, and the infection-induced production of cytokines, including IL-6, IL-8, and IFN-β. Treatment of the cells with the CD13 inhibitor 2′2′-dipyridyl decreased viral titers. Pretreatment of the cells with a combination of three drugs (glycopyrronium, formoterol, and budesonide) exerted additive inhibitory effects on viral titers and cytokine production. Pretreatment of HNE cells with glycopyrronium or formoterol reduced the susceptibility to infection, and pretreatment with the three drugs inhibited activation of nuclear factor-kappa B p50 and p65 proteins. Pretreatment with formoterol increased cAMP levels and treatment with cAMP decreased viral titers, CD13 expression, and the fluorescence intensity of acidic endosomes.ConclusionsThese findings suggest that glycopyrronium, formoterol, and a combination of glycopyrronium, formoterol, and budesonide inhibit HCoV-229E replication partly by inhibiting receptor expression and/or endosomal function and that these drugs modulate infection-induced inflammation in the airway.

Quantitatively relating brain endothelial cell\u2013cell junction phenotype to global and local barrier properties under varied culture conditions via the Junction Analyzer Program

BackgroundThe endothelial cell–cell junctions of the blood–brain barrier (BBB) play a pivotal role in the barrier’s function. Altered cell–cell junctions can lead to barrier dysfunction and have been implicated in several diseases. Despite this, the driving forces regulating junctional protein presentation remain relatively understudied, largely due to the lack of efficient techniques to quantify their presentation at sites of cell–cell adhesion. Here, we used our novel Junction Analyzer Program (JAnaP) to quantify junction phenotype (i.e., continuous, punctate, or perpendicular) in response to various substrate compositions, cell culture times, and cAMP treatments in human brain microvascular endothelial cells (HBMECs). We then quantitatively correlated junction presentation with barrier permeability on both a “global” and “local” scale.MethodsWe cultured HBMECs on collagen I, fibronectin, collagen IV, laminin, fibronectin/collagen IV/laminin, or hyaluronic acid/gelatin for 2, 4, and 7 days with varying cAMP treatment schedules. Images of immunostained ZO-1, VE-cadherin, and claudin-5 were analyzed using the JAnaP to calculate the percent of the cell perimeter presenting continuous, punctate, or perpendicular junctions. Transwell permeability assays and resistance measurements were used to measure bulk (“global”) barrier properties, and a “local” permeability assay was used to correlate junction presentation proximal to permeable monolayer regions.ResultsSubstrate composition was found to play little role in junction presentation, while cAMP supplements significantly increased the continuous junction architecture. Increased culture time required increased cAMP treatment time to reach similar ZO-1 and VE-cadherin coverage observed with shorter culture, though longer cultures were required for claudin-5 presentation. Prolonged cAMP treatment (6 days) disrupted junction integrity for all three junction proteins. Transwell permeability and TEER assays showed no correlation with junction phenotype, but a local permeability assay revealed a correlation between the number of discontinuous and no junction regions with barrier penetration.ConclusionsThese results suggest that cAMP signaling influences HBMEC junction architecture more than matrix composition. Our studies emphasized the need for local barrier measurement to mechanistically understand the role of junction phenotype and supported previous results that continuous junctions are indicative of a more mature/stable endothelial barrier. Understanding what conditions influence junction presentations, and how they, in turn, affect barrier integrity, could lead to the development of therapeutics for diseases associated with BBB dysfunction.

CATARACT & OTHER CO MORBIDITIES – STUDY FROM NORTH INDIA.

Background: Rural areas are away from medical facilities & these areas catters more than 3/4th of Indian populations. Thus, eye care services need to be planned & executed in these areas.
 Aim: To determine the prevalence of other ocular and systemic co-morbidities among the camp selected cataract patients in rural areas.
 Methodology: The present observational study was conducted at postgraduate department of Ophthalmology of GMC Jammu & involved patients from various eye camps selected for ECCE (SICS with PC-IOL implantation surgery). Total of 84 patients were participated in the study. Detailed history was taken from the patient & relevant ocular & systemic examination was done.Routine investigations were also done.
 Results: The most frequently occuring systemic co-morbidities was hypertension i.e.36.9% followed by back pain in 32.1% in the present study. The ocular co-morbities other than cataract were diabetic retinopathy in 4.8%, glaucoma in 3.6% etc. Among other ocular co morbidity, optic atrophy, amblyopia, chorioretinal atrophy, macular scar etc were noted.
 Conclusion: From present study it has been concluded that large number of patients who came to seek ocular treatment in eye camps has systemic co morbidities also. Thus, other specialities should also be part of team of eye camp so that along with eye other comorbidities should also be treated.
 Key words: Cataract, Ocular co-morbidities, Systemic co-morbidities.

Characterization and identification of human immortalized granulosa cells derived from ovarian follicular fluid.

Follicular fluid serves a crucial role in follicular development and oocyte maturation. Increasing evidence indicates that follicular fluid is rich in proteins and functional cells. In addition to oocyte cells, follicular fluid contains granulosa, thecal and ovarian surface epithelial cells. Granulosa cells (GCs) represent the predominant somatic cell type of the ovarian follicle and are involved in steroidogenesis and folliculogenesis. However, the long-term culture of GCs in vitro remains challenging. The present study aimed to extend the culture of GCs in vitro. Human GCs were collected from the follicular fluid of patients included in an in vitro fertilization program and cultured in the presence of conditioned medium obtained from mouse embryonic fibroblasts. GCs were cultured for over a year and 130 passages, and the population doubling time was ~22 h. Cells presented epithelial-like morphology and a cobblestone-like appearance when they reached confluence. Flow cytometric analysis demonstrated that cells expressed CD29, CD166 and CD49f but not CD31, CD34, CD45, CD90, CD105 or CD13. Immunofluorescence staining revealed that cells expressed follicle stimulating hormone receptor, luteinizing hormone receptor and cytochrome P450 aromatase, which was confirmed by reverse transcription-quantitative polymerase chain reaction. In the presence of androstenedione, cells secreted estradiol. In addition, estradiol level was further stimulated by dibutyryl cAMP treatment. In addition, intracellular cAMP and progesterone expression levels were upregulated by follicle stimulating hormone and/or human chorionic gonadotropin. Furthermore, cells survived in severe combined immunodeficiency mice following intra-ovarian injection. Histological analysis revealed that certain cells formed follicle-like structures. The results from the present study suggested that immortalized GCs may be a useful tool for further research on GC and improve the clinical application of drugs such as follicle-stimulating hormone or human chorionic gonadotropin.

Cyclic adenosine phosphate improves functional recovery after spinal cord injury via activating unfolded protein response.

The current study aimed to evaluate the role and underlying mechanism of cyclic adenosine phosphate (cAMP) on the functional recovery of spinal cord injury (SCI). Basso, Beattie and Bresnahan (BBB) scoring and inclined plane test indicated that cAMP treatment improved the functional recovery of SCI rats. Real time PCR and western blot analysis showed the mRNA and protein levels of IRE1, PERK, and ATF6 were increased in the SCI rats than those of sham control. However, higher levels of IRE1, PERK, and ATF6 were indicated after cAMP treatment. Meanwhile, more apoptotic cells were observed in the SCI rats, as evidenced by TUNEL staining and increased expression of GRP78, CHOP, and caspase12. In contrast, the expression of GRP78, CHOP, and caspase12 was decreased in SCI rats after cAMP treatment. In summary, we showed novel data that cAMP reduced cell apoptosis and functional recover after SCI mainly via activating UPR.

A Small GTPase RHO2 Plays an Important Role in Pre-infection Development in the Rice Blast Pathogen Magnaporthe oryzae.

The rice blast pathogen Magnaporthe oryzae is a global threat to rice production. Here we characterized RHO2 gene (MGG_02457) that belongs to the Rho GTPase family, using a deletion mutant. This mutant ΔMorho2 exhibited no defects in conidiation and germination but developed only 6% of appressoria in response to a hydrophobic surface when compared to the wild-type progenitor. This result indicates that MoRHO2 plays a role in appressorium development. Furthermore, exogenous cAMP treatment on the mutant led to appressoria that exhibited abnormal morphology on both hydrophobic and hydrophilic surfaces. These outcomes suggested the involvement of MoRHO2 in cAMP-mediated appressorium development. ΔMorho2 mutation also delayed the development of appressorium-like structures (ALS) at hyphal tips on hydrophobic surface, which were also abnormally shaped. These results suggested that MoRHO2 is involved in morphological development of appressoria and ALS from conidia and hyphae, respectively. As expected, ΔMorho2 mutant was defective in plant penetration, but was still able to cause lesions, albeit at a reduced rate on wounded plants. These results implied that MoRHO2 plays a role in M. oryzae virulence as well.

Choroby i ich leczenie w stalinowskich więzieniach i obozach w Polsce w latach 1944–1956

Artykul traktuje o opiece medycznej, w tym o rozwoju medycyny ludowej w warunkach wieziennych w latach 1944–1956. Jej rozwoj byl spowodowany kilkoma problemami: niskim stanem higieny wieźniow i powszechnie panującym brudem w zakladach karnych, brakiem szpitali i ambulatoriow oraz lekarstw i personelu medycznego, w koncu polityką komunistow wobec wieźniow, w tym tzw. antypanstwowych (czyli wieźniow politycznych, ktorzy stanowili okolo 30–35% wszystkich skazanych). Komunistom nie zalezalo na ich leczeniu. Choroby i dolegliwości, nieleczone, byly jedną z kolejnych represji wobec uwiezionych w zakladach karnych, obozach pracy przymusowej i ośrodkach pracy wieźniow. Diseases and their treatment in Stalinist prisons and camps in Poland in the years 1944–1956 In post-war Stalinist prisons and labour camps in Poland (to 1956) several diseases and ailments were treated with the help of the so-called folk or traditional medicine. Inmates, with all means at their disposal, tried to treat themselves as efficiently as possible, for example curing scabies with their own urine, a toothache and scurvy with garlic, or hyperacidity with chalk and lime scratched off walls. They could also soothe the itching in infestation with lice and ailments in furunculosis. But they could do nothing in the case of epidemic of typhus, tuberculosis, and venereal diseases. Only in 1945 ca. five thousand inmates went down with typhus. In the labour camp of Świetochlowice– Zgodna at least 1855 inmates died of diseases. Surgeons, feldshers, and other medical personnel were helpless in the face of diseases and epidemics in prisons and camps. Medical treatment, when it was possible, was hampered by dirt, insects, chronic shortages of food and medicines. This was further enhanced by a small number of hospital beds, but first of all by insufficient number of medical staff. Only a handful of surgeons decided to treat inmates.

CAMP-induced activation of protein kinase A and p190B RhoGAP mediates down-regulation of TC10 activity at the plasma membrane and neurite outgrowth.

Cyclic AMP plays a pivotal role in neurite growth. During outgrowth, a trafficking system supplies membrane at growth cones. However, the cAMP-induced signaling leading to the regulation of membrane trafficking remains unknown. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking. Recent studies have shown a role of TC10 in neurite growth in NGF-treated PC12 cells. Here, we investigated a mechanical linkage between cAMP and TC10 in neuritogenesis. Plasmalemmal TC10 activity decreased abruptly after cAMP addition in neuronal cells. TC10 was locally inactivated at extending neurite tips in cAMP-treated PC12 cells. TC10 depletion led to a decrease in cAMP-induced neurite outgrowth. Constitutively active TC10 could not rescue this growth reduction, supporting our model for a role of GTP hydrolysis of TC10 in neuritogenesis by accelerating vesicle fusion. The cAMP-induced TC10 inactivation was mediated by PKA. Considering cAMP-induced RhoA inactivation, we found that p190B, but not p190A, mediated inactivation of TC10 and RhoA. Upon cAMP treatment, p190B was recruited to the plasma membrane. STEF depletion and Rac1-N17 expression reduced cAMP-induced TC10 inactivation. Together, the PKA-STEF-Rac1-p190B pathway leading to inactivation of TC10 and RhoA at the plasma membrane plays an important role in cAMP-induced neurite outgrowth.

Icaritin inhibits decidualization of endometrial stromal cells.

The aim of the present study was to investigate the effects of Icaritin on the proliferation and decidualization of endometrial stromal cells (ESCs). A total of 20 specimens of endometrium were collected during hysterectomy at the Gynecology Department of Shenzhen Nanshan People's Hospital (Shenzhen, China) between August 2014 and December 2015. The endometrium was digested with high concentrations of collagenase and DNase and filtered with meshes, and then the glandular epithelial and stromal cells were separated by the adhesion purification method. The purity of stromal cells was identified by vimetin and cytokeratin 7 immunostaining. The estradiol + progesterone (E2+P4) and/or cyclic adenosine monophosphate (cAMP) were added to induce an in vitro decidualization model, which was used to analyze the effect of Icaritin on the decidualization ability of the human ESCs. The decidualization markers of human ESCs, prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP-1), was analyzed by reverse-transcription quantitative polymerase chain reaction measurements of the mRNA levels, PRL immunostaining and ELISA analysis of the IGFBP-1 protein levels in the cells or cell culture supernatant separately. The results demonstrated that treatment with E2+P4 and/or cAMP for 96 h was able to induce decidualization in ESCs, and that the cells demonstrated polygon-shaped epithelioid changes. The cell nuclei revealed multinuclear changes, and the cells were also observed to be large and round in shape. The PRL expression and upregulated IGFBP-1 mRNA and protein levels in the E2+P4+cAMP treatment group indicated successful decidualization of the in vitro model. However, the addition of Icaritin inhibited the expression of PRL and IGFBP-1 mRNA, as well as IGFBP-1 protein in the induced ESCs compared with groups without Icaritin. These results suggest that Icaritin was able to inhibit the expression of decidualization-related genes in ESCs in vitro. However, the exact mechanisms require further investigation.

An intensive boot camp approach to the remediation of speech sound disorders: a case report

ABSTRACTBackground: Understanding treatment dose is essential to optimizing therapeutic frequency and intensity for speech sound disorders. Intensive therapies, known as boot camp approaches, have proven efficacious in certain speech therapy contexts such as voice disorder treatment. However, few data exist regarding intervention intensity for developmental speech sound disorders.Method: This investigation examined the effects of an intensive boot camp speech therapy approach to remediate interdental /s/ production over two consecutive days. Treatment occurred in morning and afternoon blocks for a total of 5.5 h per day. Auditory-perceptual ratings were used to evaluate /s/ productions pre-treatment, immediately post-treatment, and one week following treatment.Results: Interdental /s/ production improved significantly immediately following the boot camp treatment; improvements were sustained at one week post-treatment (p < .05). Improvement was more pronounced for /s/ productions in medial and final word position versus initial word position.Conclusion: The findings of this study indicate that an intensive boot camp speech therapy approach might be useful to stimulate or accelerate treatment of speech sound disorders in certain populations. Future research should explore dose–response relationships among treatment frequency, intensity, and duration in children with a variety of speech sound disorders.

PKA activity is essential for relieving the suppression of hyphal growth and appressorium formation by MoSfl1 in Magnaporthe oryzae.

In the rice blast fungus Magnaporthe oryzae, the cAMP-PKA pathway regulates surface recognition, appressorium turgor generation, and invasive growth. However, deletion of CPKA failed to block appressorium formation and responses to exogenous cAMP. In this study, we generated and characterized the cpk2 and cpkA cpk2 mutants and spontaneous suppressors of cpkA cpk2 in M. oryzae. Our results demonstrate that CPKA and CPK2 have specific and overlapping functions, and PKA activity is essential for appressorium formation and plant infection. Unlike the single mutants, the cpkA cpk2 mutant was significantly reduced in growth and rarely produced conidia. It failed to form appressoria although the intracellular cAMP level and phosphorylation of Pmk1 MAP kinase were increased. The double mutant also was defective in plant penetration and Mps1 activation. Interestingly, it often produced fast-growing spontaneous suppressors that formed appressoria but were still non-pathogenic. Two suppressor strains of cpkA cpk2 had deletion and insertion mutations in the MoSFL1 transcription factor gene. Deletion of MoSFL1 or its C-terminal 93-aa (MoSFL1ΔCT) was confirmed to suppress the defects of cpkA cpk2 in hyphal growth but not appressorium formation or pathogenesis. We also isolated 30 spontaneous suppressors of the cpkA cpk2 mutant in Fusarium graminearum and identified mutations in 29 of them in FgSFL1. Affinity purification and co-IP assays showed that this C-terminal region of MoSfl1 was essential for its interaction with the conserved Cyc8-Tup1 transcriptional co-repressor, which was reduced by cAMP treatment. Furthermore, the S211D mutation at the conserved PKA-phosphorylation site in MoSFL1 partially suppressed the defects of cpkA cpk2. Overall, our results indicate that PKA activity is essential for appressorium formation and proper activation of Pmk1 or Mps1 in M. oryzae, and phosphorylation of MoSfl1 by PKA relieves its interaction with the Cyc8-Tup1 co-repressor and suppression of genes important for hyphal growth.

Infected T98G glioblastoma cells support human cytomegalovirus reactivation from latency

T98G cells have been shown to support long-term human cytomegalovirus (HCMV) genome maintenance without infectious virus release. However, it remains unclear whether these viral genomes could be reactivated. To address this question, a recombinant HCMV (rHCMV) containing a GFP gene was used to infect T98G cells, and the infected cells absent of infectious virus production were designated T98G-LrV. Upon dibutyryl cAMP plus IBMX (cAMP/IBMX) treatment, a serial of phenomena were observed, including GFP signal increase, viral genome replication, lytic genes expression and infectious viruses release, indicating the reactivation of HCMV in T98G-LrV cells from a latent status. Mechanistically, HCMV reactivation in the T98G-LrV cells induced by cAMP/IBMX was associated with the PKA-CREB signaling pathway. These results demonstrate that HCMV was latent in T98G-LrV cells and could be reactivated. The T98G-LrV cells represent an effective model for investigating the mechanisms of HCMV reactivation from latency in the context of neural cells.

A survey of hypertension prevalence, awareness, treatment, and control in health screening camps of rural central Punjab, Pakistan

Hypertension is a global public health problem with increasing prevalence. There is limited updated information on the prevalence of hypertension in the Pakistani population. This is a cross-sectional study based on data collected during multiple health screening camps held at multiple locations in rural central Punjab, Pakistan in the period between 2008 and 2015. A total of 13,722 patients were included in this study. Crude prevalence of hypertension was 35.1% and age-standardized prevalence was 34.4%. Among patients with hypertension, 62.3% were aware of having high blood pressure; among these patients, 75.3% were already on treatment for hypertension. Blood pressure was controlled in 22.3% of all patients with hypertension. Among those on treatment for hypertension, blood pressure was controlled in 32.3%. Nearly one-third of patients in health screening camps of rural central Punjab had hypertension. Blood pressure control rate was poor among these patients.

H2O2 is involved in cAMP-induced inhibition of sclerotia initiation and maturation in the sunflower pathogen Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a destructive pathogen and has a wide range of hosts, including sunflower. The fungus produces sclerotia, which is a dormant structure and plays a vital role in the infection cycle of S. sclerotiorum . The initiation and maturation of sclerotia can be influenced by many factors. In this study, S. sclerotiorum isolate X-8, was collected from a diseased sunflower plant, and used to determine the role of H 2 O 2 and cyclic adenosine 3', 5'-monophosphate (cAMP) in the sclerotia initiation and maturation. Results showed that the transition of S. sclerotiorum from vegetative growth to the initial stage of sclerotia is accompanied by a decrease of H2O2 and exogenously supplying H2O2 or diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase, in the culture medium promoted or inhibited the initiation of sclerotia, respectively. In addition, the cAMP-induced inhibition of sclerotia initiation and maturation were also accompanied by significantly decrease of H2O2. This was achieved by down- regulating the transcripts of Nox1 and Nox2 genes, the isoform of NADPH oxidase. Additionally, a decreased enzymatic activity of both superoxide dismutase (SOD) and catalase (CAT) were also detected in S. sclerotiorum after cAMP treatment. We concluded that H2O2 not only regulates the formation of sclerotia, but also cross-talks with cAMP in regulating the inhibition of sclerotial initiation and maturation.

Role of CREB on heme oxygenase-1 induction in adrenal cells: involvement of the PI3K pathway.

In addition to the well-known function of ACTH as the main regulator of adrenal steroidogenesis, we have previously demonstrated its effect on the transcriptional stimulation of HO-1 expression, a component of the cellular antioxidant defense system. In agreement, we hereby demonstrate that, in adrenocortical Y1 cells, HO-1 induction correlates with a significant prevention of the generation of reactive oxygen species induced by H2O2/Fe(2+) ACTH/cAMP-dependent activation of redox-imbalanced related factors such as NRF2 or NFκB and the participation of MAPKs in this mechanism was, however, discarded based on results with specific inhibitors and reporter plasmids. We suggest the involvement of CREB in HO-1 induction by ACTH/cAMP, as transfection of cells with a dominant-negative isoform of CREB (DN-CREB-M1) decreased, while overexpression of CREB increased HO-1 protein levels. Sequence screening of the murine HO-1 promoter revealed CRE-like sites located at -146 and -37 of the transcription start site and ChIP studies indicated that this region recruits phosphorylated CREB (pCREB) upon cAMP stimulation in Y1 cells. In agreement, H89 (PKA inhibitor) or cotransfection with DN-CREB-M1 prevented the 8Br-cAMP-dependent increase in luciferase activity in cells transfected with pHO-1[-295/+74].LUC. ACTH and cAMP treatment induced the activation of the PI3K/Akt signaling pathway in a PKA-independent mechanism. Inhibition of this pathway prevented the cAMP-dependent increase in HO-1 protein levels and luciferase activity in cells transfected with pHO-1[-295/+74].LUC. Finally, here we show a crosstalk between the cAMP/PKA and PI3K pathways that affects the binding of p-CREB to its cognate element in the murine promoter of the Hmox1 gene.

CREB regulates TNF-α-induced GM-CSF secretion via p38 MAPK in human lung fibroblasts

BackgroundGranulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that mediates eosinophilic differentiation, migration and survival, causing respiratory tract inflammation. GM-CSF is also known to be secreted from respiratory tract structural cells. However, the mechanisms of GM-CSF secretion have not been well established. MethodsHuman fetal lung fibroblasts and human primary asthmatic lung fibroblasts were used for the study of tumor necrosis factor alpha (TNF-α)-induced GM-CSF secretion. GM-CSF secretion and mRNA expression were measured by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction, respectively. Knockdown of cAMP response element-binding protein (CREB) in fibroblasts was carried out by using specific small interfering RNAs of CREB. ResultsAmong respiratory tract structural cells, pulmonary fibroblasts exhibited increased GM-CSF secretion and mRNA expression after stimulation with TNF-α in a concentration-dependent manner. Moreover, a p38 mitogen-activated protein kinase (MAPK) inhibitor controlled TNF-α-induced GM-CSF secretion, and roflumilast and rolipram, inhibitors of phosphodiesterase-4, suppressed TNF-α-induced GM-CSF secretion. Consistent with this, forskolin also completely blocked GM-CSF secretion, and similar results were observed in response to cAMP treatment, suggesting that cAMP signaling suppressed TNF-α-induced GM-CSF secretion in human lung fibroblasts. Furthermore, CREB was phosphorylated through p38 MAPK but not cAMP signaling after TNF-α stimulation, and GM-CSF secretion was inhibited by CREB knockdown. Finally, these effects were also demonstrated in human primary lung fibroblasts in a patient with asthma. ConclusionsCREB signaled independent of cAMP signaling and was phosphorylated by p38 MAPK following TNF-α stimulation, playing a critical role in GM-CSF secretion in human lung fibroblasts.

TRI6 and TRI10 play different roles in the regulation of deoxynivalenol (DON) production by cAMP signalling in Fusarium graminearum.

The biosynthesis of mycotoxin deoxynivalenol (DON) in Fusarium graminearum is regulated by two pathway-specific transcription factors Tri6 and Tri10 and affected by various host and environmental factors. In this study, we showed that cyclic adenosine monophosphate (cAMP) treatment induced DON production by stimulating TRI gene expression and DON-associated cellular differentiation in F. graminearum. Interestingly, exogenous cAMP had no effects on the tri6 mutant but partially recovered the defect of tri10 mutant in DON biosynthesis. Although the two cAMP phosphodiesterase genes PDE1 and PDE2 had overlapping functions in vegetative growth, conidiation, sexual reproduction and plant infection, deletion of PDE2 but not PDE1 activated intracellular PKA activities and increased DON production. Whereas the tri6 pde2 mutant failed to produce DON, the tri10 pde2 double mutant produced a significantly higher level of DON than the tri10 mutant. Cellular differentiation associated with DON production was stimulated by exogenous cAMP or deletion of PDE2 in both tri10 and tri6 mutants. These data indicate that TRI6 is essential for the regulation of DON biosynthesis by cAMP signalling but elevated PKA activities could partially bypass the requirement of TRI10 for TRI gene-expression and DON production, and Pde2 is the major cAMP phosphodiesterase to negatively regulate DON biosynthesis in F. graminearum.

Tumor necrosis factor reduces Plasmodium falciparum growth and activates calcium signaling in human malaria parasites

BackgroundPlasmodium has a complex biology including the ability to interact with host signals modulating their function through cellular machinery. Tumor necrosis factor (TNF) elicits diverse cellular responses including effects in malarial pathology and increased infected erythrocyte cytoadherence. As TNF levels are raised during Plasmodium falciparum infection we have investigated whether it has an effect on the parasite asexual stage. MethodsFlow cytometry, spectrofluorimetric determinations, confocal microscopy and PCR real time quantifications were employed for characterizing TNF induced effects and membrane integrity verified by wheat germ agglutinin staining. ResultsTNF is able to decrease intracellular parasitemia, involving calcium as a second messenger of the pathway. Parasites incubated for 48h with TNF showed reduced erythrocyte invasion. Thus, TNF induced rises in intracellular calcium concentration, which were blocked by prior addition of the purinergic receptor agonists KN62 and A438079, or interfering with intra- or extracellular calcium release by thapsigargin or EGTA (ethylene glycol tetraacetic acid). Importantly, expression of PfPCNA1 which encodes the Plasmodium falciparum Proliferating-Cell Nuclear Antigen 1, decreased after P. falciparum treatment of TNF (tumor necrosis factor) or 6-Bnz cAMP (N6-benzoyladenosine-3′,5′-cyclic monophosphate sodium salt). ConclusionsThis is potentially interesting data showing the relevance of calcium in downregulating a gene involved in cellular proliferation, triggered by TNF. General significanceThe data show that Plasmodium may subvert the immunological system and use TNF for the control of its proliferation within the vertebrate host.