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Background: Amphiregulin, a member of the epidermal growth factor family, is expressed by activated mouse T(H)2 cells. Amphiregulin produced by mouse hematopoietic cells contributes to the elimination of a nematode infection by a type 2 effector response., Objective: To identify the human peripheral blood cell population expressing amphiregulin., Methods: Amphiregulin-expressing cells were identified by flow cytometry of cell surface markers and histologic staining. Histamine and amphiregulin in supernatants were measured by enzyme immunoassay. Quantitative real-time PCR was used to measure mRNA expression., Results: Stimulation of human PBMCs by anti-CD3 + anti-CD28 antibodies induced expression of amphiregulin mRNA and protein by a non-T-cell population. The amphiregulin-producing cells were basophils, as judged by morphology and expression of CD203c and CD123 (IL-3 receptor α chain). Activated mouse basophils also produced amphiregulin. Amphiregulin expression by basophils in response to anti-TCR stimulation required IL-3 produced by T cells, and IL-3 alone induced high levels of amphiregulin expression by purified basophils. Amphiregulin was expressed at much higher levels when human basophils were stimulated by IL-3 than by IgE cross-linking, whereas the opposite was true for IL-4 expression and histamine release. Heparin-binding epidermal growth factor-like growth factor was also expressed by IL-3-stimulated human basophils. PBMCs from human subjects with asthma contained significantly higher numbers of basophils able to produce amphiregulin compared with controls with or without allergy., Conclusion: IL-3 can induce basophils to express high levels of amphiregulin, which may contribute to tissue remodeling during type 2 immune responses such as asthma., (Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Aims: Expression of ectoenzymes responsible for nucleotide phosphohydrolysis to form adenosine may represent a mechanism that facilitates the proliferation and spread of malignancy. In this study, we have identified and characterized the ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) family members expressed during the subcutaneous tumor growth and in the ascitic form of Walker 256 mammary tumor cells., Main Methods: The biochemical characteristics in ascitic forms and expression of NPP 1, 2, and 3 in both solid and ascitic forms of Walker 256 tumor were investigated using RT-PCR and real-time PCR., Key Findings: Walker 256 tumor cells demonstrate E-NPP activities that are associated with extracellular hydrolysis of p-Nph-5'-TMP, and define the biochemical characteristics. The K(m) and maximal velocity for the hydrolysis of p-Nph-5'-TMP in the ascitic tumor cells were in accordance with the NPP reaction. The mRNA expression in the cells of the ascitic form of Walker 256 tumor revealed transcripts for NPP2 and NPP3, whereas elevated expression of NPP3 was observed in solid tumor, after 6, 10, and 15days of inoculation. The dominant gene expressed in both forms of the tumor was the NPP3 enzyme. However, this enzyme was expressed more during tumor development in vivo, when compared with the ascitic cells., Significance: We have previously demonstrated that Walker 256 tumor cells express mRNA for ecto-5'-nucleotidase and E-NTPDases. Thus, coexistence with NPP3 suggests an ectonucleotidase "enzyme chain" that is responsible for the sequential hydrolysis of ATP to adenosine, which may be an important therapeutic target in anticancer therapy., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Background: CD203c is a basophil cell surface marker used to diagnose and monitor various allergic diseases, but its relationship to asthma is not clear., Objective: We determined whether CD203c expression levels are associated with stable and exacerbated asthma., Methods: We used flow cytometry to compare spontaneous expression levels of surface markers on basophils from patients with stable or exacerbated asthma and from healthy subjects. Longitudinal changes in these expression levels were measured after basophil stimulation by IgE-dependent or IgE-independent mechanisms and compared with patients' asthma status., Results: Spontaneous expression levels of CD203c were significantly higher on basophils from patients with asthma exacerbation than patients with stable asthma or healthy subjects. In contrast, no differences in spontaneous expression levels of CD63 or CD69 were observed among the 3 groups. Anti-IgE-induced expression of CD203c significantly increased in basophils during asthma exacerbation (P = .005). Low concentrations of Dermatophagoides pteronyssinus or IL-3 induced higher expression levels of CD203c during asthma exacerbation than during clinical improvement; induction of CD203c expression by these antigens therefore correlates with asthma control. In the patients with clinical improvement, there was a correlation between spontaneous CD203c expression levels and the percent predicted values of FEV(1) (r = -0.761; P = .022)., Conclusion: Asthma exacerbation was accompanied by increased expression of CD203c on basophils that decreased significantly during remission. Basophil expression levels of CD203c might therefore be used to monitor asthma in patients., (Copyright 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Importance of the Field: For many cancers, there has been a shift from management with traditional, nonspecific cytotoxic chemotherapies to treatment with molecule-specific targeted therapies that are used either alone or in combination with traditional chemotherapy and radiation therapy. Accumulating data suggest that multi-targeted agents may produce greater benefits than those observed with single-targeted therapies, may have acceptable tolerability profiles, and may be active against a broader range of tumour types. Thus, regulation of cyclic nucleotide signalling is properly regarded as a composite of multiple component pathways involved in diverse aspects of tumour cell function. The impairment of cAMP and/or cGMP generation by overexpression of PDE isoforms that has been described in various cancer pathologies, and the effects of PDE inhibitors in tumour models in vitro and in vivo, may offer promising insight into future cancer treatments because of the numerous advantages of PDE inhibitors., Areas Covered in This Review: In this review, we focus on the expression and regulation of cyclic nucleotide phosphodiesterases (PDEs) in tumour progression and provide evidence that PDE inhibitors may be effective agents for treating cancer; the review covers literature from the past several years., What the Reader Will Gain: PDEs have been studied in a variety of tumours; data have suggested that the levels of PDE activity are elevated and, therefore, the ratio of cGMP to cAMP is affected. In addition, PDE inhibitors may be potential targets for tumour cell growth inhibition and induction of apoptosis. This review explores the prospects of targeting PDEs with therapeutic agents for cancer, as well as the shortcomings of this approach such as dose-limiting side effects, toxicity/efficacy ratio and selectivity towards tumour tissue. In addition, it includes opinions and suggestion for developing PDE inhibition for cancer treatment from initial concept to potential therapeutic application and final relevance in clinical use., Take Home Message: Impaired cAMP and/or cGMP generation upon overexpression of PDE isoforms has been described in various cancer pathologies. Inhibition of selective PDE isoforms, which raises the levels of intracellular cAMP and/or cGMP, induces apoptosis and cell cycle arrest in a broad spectrum of tumour cells and regulates the tumour microenvironment. Therefore, the development and clinical application of inhibitors specific for individual PDE isoenzymes may selectively restore normal intracellular signalling, providing antitumour therapy with reduced adverse effects.

Cyclic adenosine monophosphpate (cAMP) signaling is thought to be involved in the pathophysiology of major depressive disorder and antidepressant action; however, relatively little is known about the possible role of cyclic guanosine monophosphate (cGMP) signaling. Accumulating evidence suggests that crosstalk occurs between cAMP and cGMP pathways. There is a need to clarify the trajectory of cAMP and cGMP concentrations, their synthesis by cyclases, and degradation by phosphodiesterases (PDEs) to understand the role of cyclic mononucleotide signaling in the effect of chronic antidepressant therapy. We used quantitative real-time PCR and enzyme immunoassay to systematically investigate the expression of intracellular signaling cascade elements in the hippocampus of rats chronically treated with the antidepressants fluoxetine and amitriptyline. We found increased cGMP levels, which were consistent with our findings of decreased PDE gene expression. Immunoassay results showed unchanged cAMP levels. We conclude that increased cGMP signaling might underlie the efficacy of chronic antidepressant treatment.

Background: KIT tyrosine kinase inhibitors (TKI), such as nilotinib or midostaurin (PKC412), are increasingly used in clinical trials to counteract neoplastic cell growth in patients with aggressive mast cell (MC) disorders. However, these patients suffer not only from MC infiltration and consecutive organ damage but also from MC mediator-related symptoms., Methods: We examined the effects of three KIT TKI, imatinib, nilotinib, and midostaurin, on IgE-dependent mediator release in normal human blood basophils and cultured cord blood cell-derived MC, and on spontaneous histamine secretion in the MC leukaemia cell line HMC-1 and the basophil cell line KU812., Results: The multi-kinase inhibitor midostaurin that interacts with KIT and protein kinase C was found to counteract anti-IgE-induced mediator release in blood basophils and cultured cord blood cell-derived MC in all samples examined. By contrast, no effects of imatinib or nilotinib on histamine secretion in basophils or MC were found. The effects of midostaurin on histamine release were dose-dependent and occurred at pharmacologic concentrations (IC(50) 10-100 nm). Midostaurin was also found to inhibit the IgE-dependent up-regulation of CD63 on cultured cord blood cell-derived human MC, but did not inhibit IgE-dependent up-regulation of CD63 or CD203c in human blood basophils., Conclusion: Midostaurin may be a beneficial drug in aggressive systemic mastocytosis not only because of its growth-inhibitory effects but also because of its additional effects on activation and mediator release in MC and basophils.

Glycerophosphodiesters are formed by deacylation of phospholipids. Streptomyces coelicolor and other soil-dwelling actinomycetes utilize glycerophosphodiesters as phosphate and carbon sources by the action of glycerophosphodiester phosphodiesterases (GDPDs). Seven genes encoding putative GDPDs occur in the S. coelicolor genome. Two of these genes, glpQ1 and glpQ2, encoding extracellular GDPDs, showed a PhoP-dependent upregulated profile in response to phosphate shiftdown. Expression studies using the luxAB genes as reporter confirmed the PhoP dependence of both glpQ1 and glpQ2. Footprinting analyses with pure GST-PhoP of the glpQ1 promoter revealed four protected direct repeat units (DRu). PhoP binding affinity to the glpQ2 promoter was lower and revealed a protected region containing five DRu. As expected for pho regulon genes, inorganic phosphate, and also glycerol 3-phosphate, inhibited the expression from both glpQ1 and glpQ2. The expression of glpQ1 was also repressed by serine and inositol but expression of glpQ2 was not. In contrast, glucose, fructose and glycerol increased expression of glpQ2 but not that of glpQ1. In summary, our results suggest an interaction of phosphate control mediated by PhoP and carbon source regulation of the glpQ1 and glpQ2 genes involving complex operator structures.

During development, differentiating oligodendrocytes progress in distinct maturation steps from premyelinating to myelinating cells. Such maturing oligodendrocytes express both the receptors mediating signaling via extracellular lysophosphatidic acid (LPA) and the major enzyme generating extracellular LPA, namely phosphodiesterase-Ialpha/autotaxin (PD-Ialpha/ATX). However, the biological role of extracellular LPA during the maturation of differentiating oligodendrocytes is currently unclear. Here, we demonstrate that application of exogenous LPA induced an increase in the area occupied by the oligodendrocytes' process network, but only when PD-Ialpha/ATX expression was down-regulated. This increase in network area was caused primarily by the formation of membranous structures. In addition, LPA increased the number of cells positive for myelin basic protein (MBP). This effect was associated by an increase in the mRNA levels coding for MBP but not myelin oligodendrocyte glycoprotein (MOG). Taken together, these data suggest that LPA may play a crucial role in regulating the later stages of oligodendrocyte maturation.

Vibrio cholerae is a facultative human pathogen. The ability of V. cholerae to form biofilms is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. Formation of mature biofilms requires the production of extracellular matrix components, including Vibrio polysaccharide (VPS) and matrix proteins. Biofilm formation is positively controlled by the transcriptional regulators VpsR and VpsT and is negatively regulated by the quorum-sensing transcriptional regulator HapR, as well as the cyclic AMP (cAMP)-cAMP receptor protein (CRP) regulatory complex. Transcriptome analysis of cyaA (encoding adenylate cyclase) and crp (encoding cAMP receptor protein) deletion mutants revealed that cAMP-CRP negatively regulates transcription of both VPS biosynthesis genes and genes encoding biofilm matrix proteins. Further mutational and expression analysis revealed that cAMP-CRP negatively regulates transcription of vps genes indirectly through its action on vpsR transcription. However, negative regulation of the genes encoding biofilm matrix proteins by cAMP-CRP can also occur independent of VpsR. Transcriptome analysis also revealed that cAMP-CRP regulates the expression of a set of genes encoding diguanylate cyclases (DGCs) and phosphodiesterases. Mutational and phenotypic analysis of the differentially regulated DGCs revealed that a DGC, CdgA, is responsible for the increase in biofilm formation in the Deltacrp mutant, showing the connection between of cyclic di-GMP and cAMP signaling in V. cholerae.

A variety of adrenal tumors and bilateral adrenocortical hyperplasias (BAH) leading to Cushing syndrome (CS) may be caused by aberrant cAMP signaling. We recently identified patients with a micronodular form of BAH that we have called "isolated micronodular adrenocortical disease" (iMAD) in whom CS was associated with inactivating mutations in phosphodiesterase (PDE) 11A ( PDE11A). In the present study, we examined PDE11A expression in normal adrenocortical tissue, sporadic tumors, and hyperplasias without PDE11A mutations, and primary pigmented nodular adrenocortical disease (PPNAD) and adenomas from patients with PRKAR1A and a single tumor with a GNAS mutation. The total number of the tumor samples that we studied was 22. Normal human tissues showed consistent PDE11A expression. There was variable expression of PDE11A in sporadic adrenocortical hyperplasia or adenomas; PPNAD tissues from patients with PRKAR1A mutations expressed consistently high levels of PDE11A in contrast to adenomas caused by GNAS mutations. Phosphorylated CREB was the highest in tissues from patients with iMAD compared to all other forms of BAH and normal adrenal tissue. We conclude that PDE11A is expressed widely in adrenal cortex. Its expression appears to be increased in PPNAD but varies widely among other adrenocortical tumors. PRKAR1A expression appears to be higher in tissues with PDE11A defects. Finally, sequencing defects in PDE11A are associated with a high state of CREB phosphorylation, just like PRKAR1A mutations. These preliminary data suggest that these two molecules are perhaps regulated in a reverse manner in their control of cAMP signaling in adrenocortical tissues.

Autotaxin is a type II ectonucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that it also has a lysophospholipase D activity. This enzyme probably provides most of the extracellular lysophosphatidic acid from lysophosphatidylcholine. The cloning and tissue distribution of the three isoforms (imaginatively called alpha, beta, and gamma) from human and mouse are reported in this study, as well as their tissue distribution by PCR in the human and mouse. The fate of the alpha isoform from human was also studied after purification and using mass spectrometry. Indeed, this particular isoform expresses the intron 12 in which a cleavage site is present, leading to a rapid catabolism of the isoform. For the human isoform gamma and the total autotaxin mRNA expression, quantitative PCR is presented in 21 tissues. The isoforms were expressed in two different hosts, insect cells and Chinese hamster ovary cells, and were highly purified. The characteristics of the six purified isoforms (pH and temperature dependence, K(m) and V(max) values, and their dependence on metal ions) are presented in this study. Their sensitivity to a small molecule inhibitor, hypericin, is also shown. Finally, the specificity of the isoforms toward a large family of lysophosphatidylcholines is reported. This study is the first complete description of the reported autotaxin isoforms.

Human chorionic gonadotropin (hCG) was administered to mares in estrus with large, dominant ovarian follicles to initiate follicular and oocyte maturation. Follicular contents were collected at 0, 2, 4 and 6 h after hCG. Epiregulin, amphiregulin and phosphodiesterase (PDE) mRNA contents of granulosa cells (PDE 4D) were determined by reverse transcription and real-time PCR; PDE 3A mRNA content of single oocytes was determined similarly. Copy numbers of mRNA did not increase for PDE 3A or 4D over the time interval studied. Amounts of epiregulin and amphiregulin mRNA were correlated (r=0.98) when log transformed. Epiregulin and amphiregulin mRNA increased (P<0.01) from controls by 4 h after hCG administration, with amphiregulin increasing (P<0.01) by 2 h after hCG administration. Epiregulin and amphiregulin mRNA levels remained elevated (P<0.01) at 6h after hCG. These results indicate that EGF-like growth factors are likely paracrine mediators of the LH signal in the horse.

Background: Rush immunotherapy (RIT) can confer rapid clinical benefit on patients with allergic rhinitis or asthma. However, biomarkers representing mechanisms for the efficacy of RIT are still to be established. CD203c is a basophil activation marker known to be upregulated by cross-linking of the FcepsilonRIalpha receptor and may serve as a useful marker., Objective: We sought to investigate the changes in allergen-induced CD203c expression in patients with Japanese cedar pollen (JCP) pollinosis who received RIT., Methods: Nine patients treated with RIT were enrolled in the study. Whole blood was incubated with various concentrations of JCP extract. CD203c expression on basophils was quantitated by means of flow cytometry. JCP-specific IgG4 levels in sera were measured with ELISA. Basophil histamine release, CAP-RAST to JCP (JCP-IgE) and total IgE were also examined. The biomarkers listed above were evaluated before and sequentially after RIT. Symptom and quality of life scores were obtained during pre- and posttreatment pollen seasons., Results: All patients showed significant improvement in symptom and quality of life scores after RIT. Serum JCP-specific IgG4 titers were significantly elevated at 1 month and remained at high levels 12 months after the treatment. Stimulation with JCP extract induced enhancement of basophil CD203c expression in a concentration-dependent manner except for 2 subjects in whom no increase in CD203c by an anti-IgE antibody was observed (nonresponders). Significant reductions in the responses were observed in 4 subjects after RIT (reduction in CD203c expression, RCE) whereas no changes were seen in 3 subjects (non-RCE). RCE subjects were older than non-RCE counterparts, with mean ages of 20 and 12 years, respectively. No significant changes in JCP-specific IgE and total IgE levels were seen before and after RIT., Conclusion: Allergen-induced CD203c expression in basophils may represent, at least in part, the cellular mechanism for the therapeutic responses to RIT for JCP pollinosis. However, further larger-scale studies to confirm the utility of the test are necessary., (Copyright 2008 S. Karger AG, Basel.)

Endothelial barrier dysfunction leading to increased permeability and vascular leakage is an underlying cause of several pathological conditions, including edema and sepsis. Whereas cAMP has been shown to decrease endothelial permeability, the role of cGMP is controversial. Endothelial cells express cGMP-inhibited phosphodiesterase (PDE)3A and cGMP-stimulated PDE2A. Thus we hypothesized that the effect of cGMP on endothelial permeability is dependent on the concentration of cGMP present and on the relative expression levels of PDE2A and PDE3A. When cAMP synthesis was slightly elevated with a submaximal concentration of 7-deacetyl-7-(O-[N-methylpiperazino]-gamma-butyryl)-dihydrochloride-forskolin (MPB-forskolin), we found that low doses of either atrial natriuretic peptide (ANP) or NO donors potentiated the inhibitory effects of MPB-forskolin on thrombin-induced permeability. However, this inhibitory effect of forskolin was reversed at higher doses of ANP or NO. These data suggest that cGMP at lower concentrations inhibits PDE3A and thereby increases a local pool of cAMP, whereas higher concentrations cGMP activates PDE2A, reversing the effect. Inhibitors of PDE3A mimicked the effect of low-dose ANP on thrombin-induced permeability, and inhibition of PDE2A reversed the stimulation of permeability seen with higher doses of ANP. Finally, increasing PDE2A expression with tumor necrosis factor-alpha reversed the inhibition of permeability caused by low doses of ANP. As predicted, the effect of tumor necrosis factor-alpha on permeability was reversed by a PDE2A inhibitor. These findings suggest that the effect of increasing concentrations of cGMP on endothelial permeability is biphasic, which, in large part, is attributable to the relative amounts of PDE2A and PDE3A in endothelial cells.

PC-1/PrLZ gene overexpression has been identified to be associated with prostate cancer progression. Previous studies have revealed that PC-1 possesses transforming activity and confers malignant phenotypes to mouse NIH3T3 cells. However, the functional relevance of PC-1 expression changes during prostate cancer development and progression remains to be evaluated. In this study, gain-of-function and loss-of-function analyses in LNCaP and C4-2 cells, respectively, were implemented. Experimental data showed that PC-1 expression was in positive correlation with prostate cancer cell growth and anchor-independent colony formation in vitro, as well as tumorigenicity in athymic BALB/c mice. Moreover, PC-1 expression was also found to promote androgen-independent progression and androgen antagonist Casodex resistance in prostate cancer cells. These results indicate that PC-1 contributes to androgen-independent progression and malignant phenotypes in prostate cancer cells. Furthermore, molecular evidence revealed that PC-1 expression stimulated Akt/protein kinase B signaling pathway, which has been implicated to play important roles in promoting androgen refractory progression in prostate cancer. Increased PC-1 levels in C4-2 cells may represent an adaptive response in prostate cancer, mediating androgen-independent growth and malignant progression. Inhibiting PC-1 expression may represent a novel therapeutic strategy to delay prostate cancer progression.

Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a nonspecific diesterase that enables Escherichia coli to utilize alkyl phosphodiesters, such as diethyl phosphate, as the sole phosphorus source. The catalytic properties of GpdQ were determined, and the best substrate found was bis(p-nitrophenyl) phosphate with a kcat/Km value of 6.7 x 10(3) M-1 s-1. In addition, the E. aerogenes diesterase was tested as a catalyst for the hydrolysis of a series of phosphonate monoesters which are the hydrolysis products of the highly toxic organophosphonate nerve agents sarin, soman, GF, VX, and rVX. Among the phosphonate monoesters tested, the hydrolysis product of rVX, isobutyl methyl phosphonate, was the best substrate with a kcat/Km value of 33 M-1 s-1. The ability of GpdQ to hydrolyze the phosphonate monoesters provides an alternative selection strategy in the search of enhanced variants of the bacterial phosphotriesterase (PTE) for the hydrolysis of organophosphonate nerve agents. This investigation demonstrated that the previously reported activity of GpdQ toward the hydrolysis of methyl demeton-S is due to the presence of a diester contaminant in the commercial material. Furthermore, it was shown that GpdQ is capable of hydrolyzing a close analogue of EA 2192, the most toxic and persistent degradation product of the nerve agent VX.

Background: Fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling is associated with the aberrant mineralization phenotype of the craniosynostosis syndromes. One critical aspect of mineralization involves the elaboration and transport of pyrophosphate into the extracellular matrix with subsequent enzymatic hydrolysis into phosphate. We have previously shown that FGF2 up-regulates expression of the pyrophosphate generating enzyme, PC-1, and the pyrophosphate channel, ANK, while down-regulating expression of the pyrophosphate hydrolyzing enzyme, tissue non-specific alkaline phosphatase in pre-osteoblastic, MC3T3E1(C4) cells. These results suggest that FGF/FGFR signaling may affect mineralization via changes in the elaboration and metabolism of pyrophosphate., Objectives: We are currently conducting experiments towards a more systematic analysis of PC-1 expression in osteoblastic cells, in order to more clearly elucidate the significance of pyrophosphate elaboration in the process of normal bone mineralization and in the molecular etiology of the FGFR-associated craniosynostosis syndromes., Design: Towards this goal we have constructed a PC-1 gene promoter/firefly luciferase reporter construct, in order to more directly investigate the regulation of PC-1 by FGF/FGFR signaling in osteoblastic and non-osteoblastic cells., Results and Conclusions: Preliminary results confirm that FGF/FGFR signaling, either via treatment with FGF2 or via expression of a Crouzon syndrome-associated mutant FGFR2, induces PC-1 promoter activity in osteoblastic cells in culture. This appears to be a cell type specific phenomenon. These results suggest that the expression of PC-1 downstream of FGF signaling is an integral aspect of osteoblastic function, and that pyrophosphate elaboration may play a significant role in the pathology of craniosynostosis.

Background: Anaphylaxis has variable clinical presentations and lacks reliable biomarkers. Expression of activation markers on basophils has been useful in assessing sensitization in IgE-mediated diseases but has not been examined in vivo in anaphylaxis., Objective: The study's goals were to assess the baseline expression of activation markers on basophils in individuals with a sting reaction history, the degree of change in expression of these markers after intentional sting challenge, and the relationship between in vitro and in vivo activation marker expression., Methods: Patients allergic to insect venom were enrolled and grouped by clinical category defined by a history of a systemic or large local reaction and use of venom immunotherapy. Blood was collected before and after sting challenge. Enriched basophils were analyzed for activation marker expression. In select subjects, basophils were examined after in vitro stimulation with insect venom for activation marker expression and histamine release., Results: Of 35 sting-challenge participants, 21 provided adequate samples for analysis. Pre-sting basophil CD63 expression was significantly higher in systemic reactors on immunotherapy. Following sting challenge, the rise in basophil CD69 expression and CD203c was significantly higher in systemic reactors on immunotherapy. Levels of activation markers on basophils were greater after in vitro venom stimulation than after in vivo challenge., Conclusion: Broader shifts in expression of basophil activation markers after in vivo challenge occurred among subjects with a history of in vivo systemic anaphylaxis despite venom immunotherapy., Clinical Implications: Basophil activation markers may be potential biomarkers for anaphylaxis.

We recently identified the ectoenzyme CD203c as a novel basophil activation antigen that is upregulated in response to FcepsilonRI cross-linkage. We investigated the effects of various interleukins (ILs) on expression of CD203c on blood basophils using an antibody against CD203c and flow cytometry. Of all cytokines tested, only IL-3 was found to upregulate expression of CD203c on basophils above baseline levels. The effects of IL-3 were dose- and time-dependent (EC(50): 0.1-1 ng/ml) without differences observed between healthy and allergic donors. Whereas anti-IgE induced maximum upregulation of CD203c within 15 minutes, the IL-3-induced upregulation showed a maximum after 180 minutes. IgE-receptor cross-linking resulted in enhanced expression of both CD63 and CD203c, whereas IL-3 enhanced the levels of CD203c without promoting expression of CD63. The IL-3-induced upregulation of CD203c was also observed in highly enriched basophils and was counteracted by a blocking antibody against the alpha chain of the IL-3 receptor (CD123). The IL-3-induced upregulation of CD203c was also found to depend on the presence of calcium. To analyze signaling pathways involved in IL-3-induced upregulation of CD203c, pharmacologic inhibitors were applied. The PI3-kinase inhibitors, wortmannin and LY294002 counteracted the IL-3-induced expression of CD203c, whereas MEK- and PKC inhibitors showed no effects. In conclusion, IL-3 upregulates expression of CD203c on basophils through a specific receptor and via a PI3-kinase-dependent signaling-pathway. Compared to FcepsilonRI-mediated cell activation, IL-3-induced upregulation of CD203c is a late(r) event and is not accompanied by upregulation of CD63.

Dental preparation sometimes causes transient congestion, edema, and necrosis of the pulp. We hypothesized that nitric oxide (NO) is involved in the pathophysiological changes in pulp after preparation. The mRNA and protein expression of the inducible isoform of NO synthase (iNOS) was examined in murine pulp after dental preparation. The effects of NO on the proliferation, mineralization, and apoptosis of pulp cells were also studied in vitro. We found that not only iNOS, but also mRNAs for alkaline phosphatase and plasma membrane glycoprotein-1, were expressed in the pulp after preparation. NOC-18, an NO donor, suppressed the proliferation of pulp cells without inducing cell death, whereas it promoted the mineralization of cells cultured in the presence of beta-glycerophosphate, ascorbic acid, dexamethasone, and KH(2)PO(4). Under these conditions, NOC-18 induced the apoptosis of pulp cells. These results suggest that NO regulates the growth, apoptosis, and mineralization of pulp cells.

Protein expression and secretion in insect cells have been widely studied in the baculovirus-infected insect cell system. In directly transfected insect cells only intracellular expression and purification of recombinant proteins have been studied in detail. To examine multiple recombinant protein variants, easy and fast expression and a purification screening system are required. The aim of this study was to establish an effective and rapid secretion system for human azurocidin using directly transfected insect cells. We also constructed and tested expression vectors possessing heterologous signal peptides derived from human azurocidin, yellow lupin diphosphonucleotide phosphatase/phosphodiesterase (PPD1), and papaya papain IV to secrete yellow lupin and red kidney bean purple acid phosphatases, PPD1, and papain IV. Our results demonstrate that the secretion vectors used here can direct recombinant proteins to the culture medium very effectively, allowing their simple purification on a small/medium scale. Based on secretion and activity analyses it seems that the azurocidin signal peptide is one of the most potent secretion signals.

On the basis of docking studies carried out using the recently published cannabinoid receptor models,35 new 1,8-naphthyridin-4(1H)-on-3-carboxamide and quinolin-4(1H)-on-3-carboxamide derivatives were designed, synthesized, and tested for their affinities toward the cannabinoid CB1 and CB2 receptors. Compound 10, which presented p-fluorobenzyl and carboxycycloheptylamide substituents bound in the 1 and 3 positions of the 1,8-naphthyiridine-4-one nucleus, showed a high CB2 affinity with a Ki of 1.0 nM. The substitution of the naphthyridine-4-one nucleus with the quinoline-4-one system determined a general increase in CB2 affinity. In particular, the N-cyclohexyl-7-chloro-1-(2-morpholin-4-ylethyl)quinolin-4(1H)-on-3-carboxamide (40) possessed a remarkable affinity, with Ki of 3.3 nM, which was also accompanied by a high selectivity for the CB2 receptor (Ki(CB1)/Ki(CB2) ratio greater than 303). Moreover, the [35S]GTPgamma binding assay and functional studies on human basophils indicated that the 1,8-naphthyridin-4(1H)-on-3-carboxamide derivatives behaved as CB1 and CB2 receptor agonists.

Background: Basophils are inflammatory cells associated with allergy and parasite infections. Investigation of their true biological function has long been hampered by the difficulty in obtaining sufficient amounts of pure basophils and by the lack of phenotypic markers. Moreover, it has been very difficult to clone and identify basophil-specific granule proteins, partially because of an almost complete lack of mRNA in mature circulating basophils., Methods: To obtain transcriptionally active immature basophils, umbilical cord blood cells were cultured in the presence of interleukin (IL)-3. The cells were analysed by flow cytometry and by histological staining., Results: The continuous presence of IL-3 in cord blood cultures resulted in the expansion of basophil precursors co-expressing FcepsilonRI and the recently described mast cell/basophil marker, 97A6 (CD203c). Several nonbasophil markers (i.e. CD3, CD14, CD15, CD16, CD19 and CD21) were absent on the cultured basophils. However, we show that in early cultures, almost 60% of the CD203c+ cells co-express human leukocyte antigen (HLA)-DR, a marker that is absent on mature circulating basophils. The presence of HLA-DR on basophil precursors may explain the low recovery (24+/-5.2%) obtained after isolation of cultured basophils, when using a conventional basophil isolation kit that removes HLA-DR+ cells. A novel purification method was developed, including a two-step cocktail of antibodies against selected markers, which resulted in both high purity (95+/-0.5%) and recovery (59+/-1.5%) of cultured basophils., Conclusions: We here establish cord blood cultures as a source from which transcriptionally active basophil precursors can be isolated in reasonable quantities for thorough biochemical characterization.

Background: Flow cytometric basophil activation tests have been developed as cellular tests for in vitro diagnosis of IgE-mediated reactions. Different activation markers (CD63 or CD203c) with distinct ways of regulation have been used after stimulation with various allergens., Objective: It was the aim of the present study to compare basophil activation tests by measuring both CD63 and CD203c upregulation in patients with insect venom allergy., Materials and Methods: 43 patients with a history of insect venom anaphylaxis were examined. A careful allergy history was taken, and skin tests and determination of specific IgE-antibodies were performed. Basophil activation tests (BAT) using CD63 or CD203c expression were done after stimulation with different concentrations of bee and wasp venom extracts. 25 healthy subjects with negative history of insect venom allergy were studied as controls., Results: The CD203c protocol showed a slightly higher sensitivity than the CD63 protocol (97% vs. 89%) with regard to patients' history. The magnitude of basophil response was higher with CD203c in comparison to CD63 for both insect venoms. Specificity was 100% for the CD63 protocol and 89% for the CD203c protocol with regard to controls with negative history and negative RAST., Conclusion: These results support the reliability of basophil activation tests using either CD63 or CD203c as cellular tests in the in vitro diagnosis of patients with bee or wasp venom allergy with a slightly higher sensitivity for the CD203c protocol.

Background: Approximately 40% of patients with chronic idiopathic urticaria have antibodies to the alpha subunit of the high-affinity IgE receptor. CD203c is a basophil activation marker known to be upregulated by cross-linking of the FcepsilonRIalpha receptor and may serve as a useful marker to identify these patients., Objective: The primary objective was to assess the affect of sera from patients with chronic idiopathic urticaria on basophil CD203c expression. Secondary objectives were to correlate CD203c expression with basophil histamine release and size of the autologous serum skin test and to determine whether the mechanism is mediated by an IgG antibody., Methods: Sera were obtained from patients with chronic idiopathic urticaria and positive autologous serum skin test or negative autologous serum skin test and normal controls. Sera were incubated with donor whole blood. Activated basophils from whole blood were identified by flow cytometry on the basis of the presence of CD203c on high-expressing IgE positive cells., Results: Incubation of donor basophils with sera from patients with chronic idiopathic urticaria and positive autologous serum skin test demonstrated significant upregulation of CD203c. IgG depletion of representative sera from patients with chronic idiopathic urticaria resulted in significant decrease in CD203c expression on donor basophils. CD203c expression correlated with basophil histamine release and the size of the autologous serum skin test., Conclusion: Sera from patients with chronic idiopathic urticaria and positive autologous serum skin test significantly upregulate basophil CD203c and correlate with basophil histamine release., Clinical Implications: This article describes an activation marker on basophils whose expression is increased by sera from patients with chronic idiopathic urticaria.

Objectives: Only a little research has focused on the evaluation of female sexual function. With sexual stimulation, the clitoris becomes engorged with blood and tumescent. Nevertheless, only little is known about the significance of the cyclic nucleotide-mediated signal transduction in the control of this process. We sought to elucidate the presence of the phosphodiesterase (PDE) isoenzymes 3, 4, and 5 in the human clitoris using immunohistochemical and molecular biology methods., Methods: Thin sections of clitoral specimens were incubated with primary antibodies directed against PDE isoenzymes 3, 4, and 5. Next, the sections were incubated with either Texas red or fluorescein isothiocyanate-labeled secondary antibodies, and visualization was done using laser microscopy. The expression of mRNA encoding for various PDE isoenzymes was evaluated using reverse transcriptase polymerase chain reaction., Results: Immunofluorescence indicating the presence of PDE4 (cyclic adenosine monophosphate-PDE) was observed in the nonvascular smooth musculature of the corpus cavernosum clitoris, sinusoidal endothelial and subendothelial layers, and nerve fibers innervating the tissue. Immunoreactivity specific for PDE5 (cyclic guanosine monophosphate-PDE) was limited to the smooth muscle of the clitoral erectile tissue. The fluorescein isothiocyanate reaction indicating the expression of PDE3 (cyclic adenosine monophosphate-PDE) was registered to a certain degree only in the clitoral epidermis. In the reverse transcriptase polymerase chain reaction studies, a predominant expression of mRNA encoding for PDE1A was registered, but only small amounts of mRNA encoding for PDE4 and PDE5 were detected., Conclusions: Our results have demonstrated the presence of cyclic adenosine monophosphate-PDE and cyclic guanosine monophosphate-PDE in the human clitoris and may indicate a regulatory function of these enzymes in the cyclic nucleotide-mediated control of smooth muscle tone.

The ectoenzyme PC-1 is an insulin receptor inhibitor that is elevated in cells and tissues of humans with type 2 diabetes (T2D). We have recently shown that acute PC-1 overexpression in liver causes insulin resistance and glucose intolerance in mice (3), but the chronic effects of PC-1 overexpression on these functions are unknown. Herein we produced transgenic mice overexpressing the potent q allele of human PC-1 in muscle and liver. Compared with controls, these mice had 2- to 3-fold elevations of PC-1 content in liver and 5- to 10-fold elevations in muscle. In the fed state, the PC-1 animals had 100 mg/dl higher glucose levels and sixfold higher insulin levels compared with controls. During glucose tolerance tests, these PC-1 animals had peak glucose levels that were >150 mg/dl higher than controls. In vivo uptake of 2-deoxy-d-glucose in muscle during insulin infusion was decreased in the PC-1 animals. These in vivo data support the concept, therefore, that PC-1 plays a role in insulin resistance and hyperglycemia and suggest that animals with overexpression of human PC-1 in insulin-sensitive tissues may be important models to investigate insulin resistance.

Objective: To compare the differences of expressions of adenylyl cyclase (AC) and phosphodiesterase (PDE) in ejaculated spermatozoa between healthy volunteers and the patients with asthenospermia., Methods: Ejaculated spermatozoa were collected from healthy volunteers and the patients with asthenospermia. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of AC and PDE subtypes in human spermatozoa. The concentrations of cAMP and cGMP in the samples were detected by enzyme-linked immunosorbent assay (ELISA)., Results: Compared with healthy volunteers, expression of sAC mRNA and concentration of cAMP were significantly decreased in the patients with asthenospermia (P < 0.01) , while the expression of PDE4C mRNA was significantly increased at the same time (P <0.01). There were no marked differences in the expression of ACIII mRNA and concentration of cGMP between the two groups., Conclusion: The sAC down-regulation and PDE4C up-regulation are possible reasons for asthenospermia.

Microarray and other studies have reported oligodendrocyte and myelin-related (OMR) deficits in schizophrenia. Here, we employed a quantitative approach to determine the magnitude of OMR gene expression deficits and their brain-region specificity. In addition, we examined how expression levels among the studied OMR genes are interrelated. mRNA of MAG, CNP, SOX10, CLDN11, and PMP22, but not MBP and MOBP, was reduced in the hippocampus and anterior cingulate cortex but not in the putamen of patients with schizophrenia. Expression of the only protein examined (CNP) was decreased in the hippocampus but not in the putamen. Correlation and factor analyses revealed that mRNA levels for genes that did exhibit differential expression in schizophrenia (MAG, CNP, SOX10, CLDN11, and PMP2), as opposed to those that did not (MOBP and MBP), loaded on separate factors. Thus, OMR gene and protein expression deficits in schizophrenia are brain-region specific, and the affected components may share regulatory elements.

The biosynthetic pathway for l-mycarose, a 2,6-dideoxy-3-methylhexose found in the antibiotic tylosin produced by Streptomyces fradiae, has been studied with only two genes in the gene cluster, tylK and tylC2, remaining to be characterized. To verify the postulated functions of these two genes and to establish the sequence of events in the pathway, functional analyses of the proteins encoded by tylK and tylC2 were carried out. The results clearly show that TylK functions as the 5-epimerase and TylC2 functions as the 4-ketoreductase. However, both enzymes exhibit relaxed substrate specificity such that the production of TDP-l-mycarose as the sole product of the pathway depends on the absolute specificity of TylC3, a methyltransferase involved in an early step in the pathway. Exploitation of the promiscuous substrate specificity in the late steps of the mycarose pathway could produce new analogues of tylosin as well as other antibiotics containing this sugar.

Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels.

A promising attempt in the field of tumour therapy is the modulation of intracellular, proliferation-associated signalling pathways. The role of cyclic nucleotide phosphodiesterases (PDEs), key enzymes in cAMP/cGMP signal transduction, was investigated in two human CNS tumour cell lines as well as in the rat glioblastoma cell line C6 in comparison with rat cerebellar astrocytes with the emphasis on target evaluation. We found differential PDE expression patterns in human CNS tumour cell lines as well as in CNS cells of rat origin. In human glioblastoma cells, intracellular cAMP and Ca(2+) levels correlated well with the PDE expression pattern. There were, however, marked differences in PDE expression and Ca(2+) kinetics between the human glioblastoma cell lines. In contrast to human epithelial tumour cells, shown earlier by us to express significantly enhanced cAMP-specific PDE activity, this was not the case in rat glioblastoma cells compared with non-malignant rat astrocytes. Despite different levels of PDE1 and PDE4 expression and activity, cyclic nucleotide and Ca(2+) levels in non-malignant and malignant rat CNS cells were similar. These in vitro data do not support the concept of PDE1C representing a target exploitable for drug treatment of malignant CNS tumours.

Objective: To study the effect of PC-1 gene knockdown on the biological action of prostate cancer cell line C4-2., Methods: Recombinant plasmids of expressing short hairpin RNA targeting PC-1 mRNA were constructed using DNA recombinant technology and transfected into C4-2 cells via liposome. The positive cell clones were selected by G418. The expression of PC-1 gene was analyzed by RT-PCR and Western blotting technology. MTT and soft agar cloning formation were applied to observe the changes of the growth rate and independent anchor ability of C4-2 cells., Results: PC-1 RNA interference severely affected the expression of PC-1 gene and reduced the growth and colony formation ability of C4-2 cells., Conclusion: RNA interference-mediated PC-1 gene knockdown can decrease the growth and cloning formation ability of C4-2 cells.

Basophils are important in allergic diseases such as asthma because they produce a variety of inflammatory mediators. Activation of these cells with IgE and N-formyl-methionyl-leucyl-phenylalanine results in a variety of responses, including increased surface expression of CD203c and CD11b and release of histamine. Although considerable information is available on the effects of eicosanoids on neutrophils, eosinophils, and monocytes, less is known about their effects on basophils. In the present study, we examined the effects of various eicosanoids on the above basophil responses. Of the naturally occurring eicosanoids tested, prostaglandin D(2) (PGD(2); EC(50), 10 nM) was by far the most potent activator of CD203c expression, with other prostanoids having little effect. This response was mediated by the DP(2) receptor/chemoattractant receptor-homologous molecule expressed on Th2 cells because it was shared by the selective agonist 15R-methyl-PGD(2) (EC(50), 3 nM). The 5-lipoxygenase products leuko-triene B(4) and 5-oxo-6,8,11,14-eicosatetraenoic acid also stimulated CD203c expression but to a lesser extent than PGD(2), whereas leukotriene D(4) was inactive. Neither PGD(2) nor 5-oxo-6,8,11,14-eicosatetraenoic acid stimulated histamine release or CD63 expression on basophils. Both PGE(2) and the DP(1) receptor agonist BW245C [(4S)-(3-[(3R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid] strongly inhibited DP(2) receptor-mediated CD203c expression. The DP(1) receptor antagonist BWA868C [3-[(2-cyclohexyl-2-hydroxyethyl)amino]-2,5-dioxo-1-(phenylmethyl)-4-imidazolidine-heptanoic acid] enhanced PGD(2)-induced CD203c expression, suggesting that interaction of PGD(2) with DP(1) receptors can limit activation of basophils by this prostaglandin. In conclusion, PGD(2) is the most potent inducer of basophil CD203c expression among eicosanoids and may be a key mediator in asthma and other allergic diseases. The balance between DP(1) and DP(2) receptors may be important in determining the magnitude of basophil responses to this prostaglandin.

A complex interaction of neural, vascular, and biomolecular activity results in normal erectile function. Two chambers in the penis, the corpus cavernosa, contain sinusoids, which fill with blood and expand to become rigid when smooth muscle is relaxed. Blood flow out of the penis is temporarily stopped (veno-occlusion) to maintain the erection. Neural control is complex but, in general, the sympathetic nervous system inhibits, and the parasympathetic system excites erectile function. The central nervous system can induce erections without peripheral stimuli, e.g. in nocturnal erections. In general, there are 5 recognized phases in erectile response: latent, tumescent, full erection, rigid erection, and detumescent. The key biochemical event in erectile function is an increase in levels of cyclic guanosine monophosphate, which causes smooth-muscle relaxation and allows an erection to occur. This process is mediated by nitric oxide, produced by neurons and elsewhere, which acts as a gaseous messenger molecule.

We have previously purified and characterized a diphosphonucleotide phosphatase/phosphodiesterase (PPD1) from yellow lupin seeds. This report describes an efficient strategy for overexpression in baculovirus-infected Spodoptera frugiperda Sf9 cells and purification of a functional PPD1 enzyme. We tested six variants of recombinant PPD1, differing in secretion peptides, fusion tags, and promoters. The highest expression level of the active PPD1 was achieved when the native signal peptide and the C-terminal V5-6His tag were attached. This recombinant protein was secreted at very high level (18.4 mg/L) to serum-free medium. Single-step purification procedure using metal affinity chromatography resulted in the homogeneous PPD1. The overexpressed protein showed enzymatic activity comparable to the native enzyme isolated previously from plant material. We showed that PPD1, which belongs to purple acid phosphatase family, formed tetrameric structure, which is non-typical for this group of enzymes.

Background: The phosphodiesterase type-5 (PDE-5) inhibitor sildenafil has been reported to improve pulmonary arterial hypertension (PAH), but the mechanisms that account for this effect are incompletely understood. Severe pulmonary hypertension has been characterized by defects in a signaling pathway involving angiopoietin-1 and the bone morphogenetic receptor-2 (BMPR-2). We investigated the effects of sildenafil on hemodynamics and signaling molecules in a piglet overcirculation-induced model of early PAH., Methods and Results: Thirty 3-week-old piglets were randomized to placebo or sildenafil therapy 0.75 mg/kg TID after anastomosis of the left subclavian artery to the pulmonary arterial trunk or after a sham operation. Three months later, the animals underwent a hemodynamic evaluation followed by pulmonary tissue sampling for morphometry, immunohistochemistry or radioimmunoassay, and real-time quantitative-polymerase chain reaction. Chronic systemic-to-pulmonary shunting increased pulmonary mRNA for angiopoietin-1, endothelin-1 (ET-1), angiotensin II, inducible nitric oxide synthase, vascular endothelial growth factor, and PDE-5. Pulmonary messenger RNA for BMPR-1A and BMPR-2 decreased. Pulmonary angiotensin II, ET-1, and vascular endothelial growth factor proteins increased. Pulmonary artery pressure increased from 20+/-2 to 33+/-1 mm Hg, and arteriolar medial thickness increased by 91%. The expressions of angiopoietin-1, ET-1, and angiotensin II were tightly correlated to pulmonary hypertension. Sildenafil prevented the increase in pulmonary artery pressure, limited the increase in medial thickness to 41%, and corrected associated biological perturbations except for the angiopoietin-1/BMPR-2 pathway, PDE-5, and angiotensin II., Conclusions: Sildenafil partially prevents overcirculation-induced PAH and associated changes in signaling molecules. Angiotensin II, PDE-5, and angiopoietin-1/BMPR-2 signaling may play a dominant role in the early stages of the disease.

Dopamine and NO are physiological stimulators of synthesis of cAMP and cGMP, respectively, and NO synthase-containing interneurons in the striatum are physiologically activated by dopamine-containing neurons in the substantia nigra. This study investigated whether lesioning dopamine neurons has multiple consequences in the striatum consistent with the reported sensitization of cAMP synthesis, including alteration of the NO-cGMP pathway and phosphodiesterase-dependent metabolism of cyclic nucleotides. The substantia nigra of adult Sprague-Dawley rats was unilaterally lesioned with 6-hydroxydopamine. Two months later, we determined expression of NO synthase and evaluated cGMP and cAMP levels of intact and deafferented striatum. Moreover, we evaluated cAMP- and cGMP-phosphodiesterase activities in basal conditions and after Ca2+-calmodulin stimulation and determined the expression of the phosphodiesterase-1B isoform and the levels of phosphodiesterase-1B mRNA. Using immunocytochemistry we characterized the distribution of NO synthase and phosphodiesterase-1B within striatal neurons. In the dopamine-deafferented striatum, NO synthase levels were decreased by 42% while NO synthase-immunopositive intrastriatal fibres but not NO synthase neuronal bodies were reduced in number. In the deafferented striatum basal cGMP levels were reduced, and cAMP levels were increased, but cGMP-phosphodiesterase and cAMP-phosphodiesterase activities were both increased in basal and Ca2+-calmodulin-stimulated conditions. Accordingly, phosphodiesterase-1B expression and phosphodiesterase-1B mRNA were upregulated while a large population of medium-sized striatal neurons showed increased phosphodiesterase-1B immunoreactivity. Dopamine deafferentation led to a complex down-regulation of the NO-cGMP pathway in the striatum and to an up-regulation of phosphodiesterase-1B-dependent cyclic nucleotide metabolism, showing new aspects of neuronal plasticity in experimental hemiparkinsonism.

Basophils are effector cells of allergic reactions. These cells produce and store a number of vasoactive and immunomodulatory mediators. During an allergic reaction, basophils can release their mediator substances into the extracellular space and thus contribute to the clinical picture and symptoms in allergy. The phenotypic hallmark of basophils is expression of high-affinity IgE receptors (Fc epsilon RI) on their cell surface together with expression of the activation-linked molecule CD203c. This ectoenzyme is located both on the plasma membrane and in the cytoplasmic compartment of basophils. Cross-linking of the Fc epsilon RI by an allergen or anti-IgE antibody results in a rapid upregulation of intracellular CD203c molecules to the cell surface and is accompanied by mediator release. CD203c is therefore a promising target molecule for a flow cytometry-based test to analyze sensitized individuals and patients with type I allergy. In the present article, we review the current knowledge of CD203c with special regard to its tissue distribution and regulation in basophil activation. In addition, we discuss the application of CD203c in allergy diagnosis., (Copyright 2004 S. Karger AG, Basel)

The aim of the study was to compare the localization of the nitric oxide (NO)-cGMP pathway in hippocampus of mice and rats using cGMP- and soluble guanylyl cyclase (GC) immunocytochemistry and in situ hybridization of the cGMP-hydrolysing phosphodiesterase types 2, 5 and 9. In vitro incubation of hippocampus slices in the absence of a guanylyl cyclase stimulator or a phosphodiesterase inhibitor resulted in cGMP-positive astrocytes mainly in the CA1 area in mouse slices. In contrast, no cGMP immunoreactivity was observed under these conditions in the rat hippocampus. Treatment with an NO synthase inhibitor or inhibitors of soluble or particulate GC did not abolish cGMP immunoreactivity in astrocytes. Incubation with the NO donors sodium nitroprusside or diethylamino NONOate, or with the NO-independent activators of soluble GC, YC-1 and BAY 41-2272, in combination with phosphodiesterase inhibitors, resulted in an increase in cGMP immunoreactivity in numerous astrocytes throughout the mouse hippocampus. In contrast, under these conditions cGMP immunoreactivity was primarily observed in varicose fibers in rat hippocampus. Comparison of the cellular localization of the beta1 subunit of soluble GC and the mRNAs of PDE2, PDE5 and PDE9 revealed that in both species the beta1 subunit was observed in pyramidal and granule cells, which also expressed the mRNAs of the three phosphodiesterase families. Although the beta1 subunit was observed in astrocytes, none of the phosphodiesterases were detected in these cells. We conclude that, although the expression profiles of the soluble GC beta1 subunit and cGMP-hydrolysing phosphodiesterase mRNAs were identical, the cellular patterns of cGMP immunoreactivity differ between rat and mouse hippocampus.

Purpose: Gene transfer of cDNA sequences that confer drug resistance can be used (1) to protect hematopoietic cells against the toxic effects of chemotherapy, (2) for in vivo enrichment of genetically engineered cells and (3) to protect cytotoxic T lymphocytes in drug-resistant immunotherapy approaches for the treatment of cancer. We have previously developed strategies to confer resistance to agents targeting thymidylate synthase (TS) and have now expanded our drug resistance strategies to include retroviral expression of tyrosyl-DNA phosphodiesterase (TDP-I), an enzyme recently implicated in the repair of topoisomerase-I (Top-I)/DNA lesions induced by camptothecin (CPT). The combination of TS and Top-I inhibition has been shown to be an effective treatment for several types of cancer., Materials and Methods: Retroviral vectors were generated that individually encoded TS and TDP-I or that coexpressed both enzymes. Murine fibroblast and Chinese hamster lung transfectants were generated with the vectors and resistance to TS- and Top-I-directed inhibitors was tested. Murine bone marrow progenitor cells were also transduced using recombinant retroviruses encoding TS and TDP-I and the degree of drug resistance conferred to gene-modified cells was tested., Results: Enforced expression of TDP-I increased TDP-I activity in gene-modified cells and conferred up to threefold resistance to CPT. The degree of resistance was dependent on the duration of drug treatment. Simultaneous expression of the TS gene encoding E. coli TS optimized for expression in mammalian cells (optecTS) and TDP-I conferred extremely high-level resistance to concurrent treatment with the TS-inhibitor BW1843U89 and CPT. Furthermore, by direct analysis of DNA fragmentation using the comet assay, substantial protection was conferred (fourfold) against DNA fragmentation associated with combination drug treatments by dual enzyme expression compared to non-modified cells. Hematopoietic progenitor assays of murine bone marrow cells transduced with retroviral vectors encoding TS and TDP-I showed that bone marrow cells could be protected from the cytotoxic effects of TS and Top-I inhibition., Conclusions: Enforced expression of optecTS and TDP-I conferred antifolate and CPT resistance to genetically modified cells. Additionally, this work further illustrated a role for TDP-I in the repair of dead-end Top-I complexes and implied that TDP-I expression analysis may aid in predicting the therapeutic effectiveness of the CPT class of compounds.

An in vivo expression technology (IVET) system was previously developed and used to identify Pasteurella multocida genes, which are upregulated during infection of the host. Of the many genes identified, two encoded products which showed similarity to the Haemophilus influenzae lipoproteins, protein D and PCP, which have been shown to stimulate heterologous immunity against infection with H. influenzae. Therefore, the lipoprotein homologues in P. multocida, designated GlpQ and PCP, were investigated. GlpQ and PCP were shown to be lipoproteins by demonstrating that post-translational processing of the proteins was inhibited by globomycin. The P. multocida GlpQ homologue showed glycerophosphodiester phosphodiesterase enzyme activity, indicating that it is a functional homologue of other characterized GlpQ enzymes. Using surface immunoprecipitation, PCP was found to be surface exposed, but GlpQ was not. Non-lipidated forms of GlpQ and PCP were expressed and purified from Escherichia coli and used to vaccinate mice. However, mice were not protected from challenge with live P. multocida. The lipoproteins were then expressed in E. coli in the lipidated form and used to vaccinate mice and chickens. Protection against challenge with live P. multocida was not observed.

Glycerophosphodiester phosphodiesterase (GDPD) is a glycerol metabolizing enzyme that has been previously identified in Escherichia coli, Haemophilus influenzae, Bacillus subtilis and Borrelia hermsii. It has been also reported that there is putative protein containing GDPD domain in Mus musculus, but not in Homo sapiens. SMART analysis showed that our predicted protein and three others of the same gene also comprise this domain. Because the gene has not been named, we termed it GDPD1 and accordingly termed our splice variant GDPD1&#95;v1 after acquiring the agreement of HUGO. RT-PCR experiment revealed that our splice variant of the gene is mainly expressed in human ovary and small intestine, basically conforming to the result of serial analysis of gene expression.

Osteoblast maturation is a multistep series of events characterized by an integrated cascade of gene expression that are accompanied by specific phenotypic alterations. To find new osteoblast-related genes we cloned differentially expressed cDNAs characteristic of specific differentiation stages in the mouse osteoblast-like MC3T3-E1 cells by a differential display method. We identified a novel cDNA encoding a putative glycerophosphodiester phosphodiesterase, GDE3, which specifically was expressed at the stage of matrix maturation. Interestingly, the deduced amino acid sequence contains 539 amino acids including seven putative transmembrane domains and a glycerophosphodiester phosphodiesterase region in one of the extracellular loops. Northern blot analysis revealed that GDE3 was also expressed in spleen as well as primary calvarial osteoblasts and femur. We next transfected HEK293T cells with GDE3 with green fluorescent protein fused to the C terminus. The green fluorescent protein-fused protein accumulated at the cell periphery, and the transfected cells overexpressing the protein changed from a spread form to rounded form with disappearance of actin filaments. Immunofluorescence staining with GDE3 antibody and phalloidin in MC3T3-E1 cells indicated that endogenous GDE3 might be co-localized with the actin cytoskeleton. To identify a role for GDE3 in osteoblast differentiation, MC3T3-E1 cells stably expressing the full-length protein were constructed. Expression of GDE3 showed morphological changes, resulting in dramatic increases in alkaline phosphatase activity and calcium deposit. These results suggest that GDE3 might be a novel seven-transmembrane protein with a GP-PDE-like extracellular motif expressed during the osteoblast differentiation that dramatically accelerates the program of osteoblast differentiation and is involved in the morphological change of cells.

Background: The prognosis and functional capacity of patients with pulmonary arterial hypertension (PAH) is poor, and there is a need for safe, effective, inexpensive oral treatments. A single dose of sildenafil, an oral phosphodiesterase type-5 (PD-5) inhibitor, is an effective and selective pulmonary vasodilator in PAH. However, the long-term effects of PD-5 inhibition and its mechanism of action in human pulmonary arteries (PAs) are unknown., Methods and Results: We hypothesized that 3 months of sildenafil (50 mg orally every 8 hours) added to standard treatment would be safe and improve functional capacity and hemodynamics in PAH patients. We studied 5 consecutive patients (4 with primary pulmonary hypertension, 1 with Eisenmenger's syndrome; New York Heart Association class II to III). Functional class improved by > or =1 class in all patients. Pretreatment versus posttreatment values (mean+/-SEM) were as follows: 6-minute walk, 376+/-30 versus 504+/-27 m, P<0.0001; mean PA pressure, 70+/-3 versus 52+/-3 mm Hg, P<0.007; pulmonary vascular resistance index 1702+/-151 versus 996+/-92 dyne x s x cm(-5) x m(-2), P<0.006. The systemic arterial pressure was unchanged, and no adverse effects occurred. Sildenafil also reduced right ventricular mass measured by MRI. In 7 human PAs (6 cardiac transplant donors and 1 patient with PAH on autopsy), we showed that PD-5 is present in PA smooth muscle cells and that sildenafil causes relaxation by activating large-conductance, calcium-activated potassium channels., Conclusions: This small pilot study suggests that long-term sildenafil therapy might be a safe and effective treatment for PAH. At a monthly cost of 492 dollars Canadian, sildenafil is more affordable than most approved PAH therapies. A large multicenter trial is indicated to directly compare sildenafil with existing PAH treatments.

Background: Phosphodiesterase type 5 (PDE5) is a novel therapeutic target for the treatment of pulmonary hypertension. This study examined the distribution of PDE5 in normal and hypoxic lung and the effect of chronic PDE5 inhibition with sildenafil, initiated before and during exposure to hypoxia, on pulmonary artery pressure (PAP) and structure., Methods and Results: Sprague-Dawley rats were exposed to hypoxia (10% O2) for up to 42 days. PAP, measured continuously by telemetry, increased gradually by 20 to 40 mm Hg, reaching a plateau between 10 and 14 days, and declined to normal levels on return to normoxia. PDE5 immunoreactivity was localized to smooth muscle cells in the medial layer of pulmonary arteries and veins in the normal lung and in distal muscularized arteries (<25 microm diameter) after hypoxia-induced pulmonary hypertension. Sildenafil (25 or 75 mg x kg(-1) x d(-1)) given before hypoxia produced marked dose-dependent inhibition in the rise of PAP (60% to 90% reduction; P<0.0001) and vascular muscularization (28.4+/-5.0% reduction; P<0.001). When begun after 14 days of hypoxia, sildenafil significantly reduced PAP (30% reduction; P<0.0001) and partially reversed pulmonary artery muscularization (39.9+/-4.9% reduction; P<0.001)., Conclusions: PDE5 is found throughout the muscularized pulmonary vascular tree, including in newly muscularized distal pulmonary arteries exposed to hypoxia. PDE5 inhibition attenuates the rise in PAP and vascular remodeling when given before chronic exposure to hypoxia and when administered as a treatment during ongoing hypoxia-induced pulmonary hypertension.

Recent data suggest that the statins, apart from their lipid-lowering activity, exhibit profound anti-inflammatory effects. Basophils are major proinflammatory effector cells in diverse pathologic reactions. We have examined the in vitro effects of five different statins on primary human basophils, their progenitors, and the basophil cell line KU-812. Preincubation of blood basophils with cerivastatin or atorvastatin (0.1-100 microM) for 24 h reduced their capacity to release histamine on immunoglobulin E (IgE)-dependent stimulation in a dose-dependent manner. These statins also inhibited IgE-dependent up-regulation of the basophil-activation antigen CD203c. Moreover, both statins suppressed interleukin-3-induced differentiation of basophils from their progenitors as well as (3)H-thymidine uptake in KU-812 cells. All inhibitory effects of cerivastatin and atorvastatin were reversed by mevalonic acid (200 microM). The other statins tested (lovastatin, simvastatin, pravastatin) did not show significant inhibitory effects on basophils. Together, these data identify cerivastatin and atorvastatin as novel inhibitors of growth and activation of human basophils.

To understand changes in cyclic nucleotide metabolism in muscle disease states, the expression of phosphodiesterase (PDE) isozymes in normal mouse leg muscle was examined. Four subcellular fractions were generated by differential centrifugation at 10,000 x g and 100,000 x g. cAMP PDE activity was found predominately in the soluble fractions, while cGMP PDE activity was more evenly distributed amongst soluble and particulate fractions. Pharmacological inhibitors demonstrate that PDE4 represents the major cAMP hydrolyzing activity and PDE2 represents the major cGMP hydrolyzing activity in mouse leg muscle. PDE1 is expressed at low levels, while PDE3 and PDE5 are intermediate. Between 20 and 40% of total PDE activity remained in the presence of inhibitors for PDE1-PDE5, indicating that other PDE families contribute to the total PDE pool. Reverse-transcription PCR with family-specific primers showed expression of mRNA for PDE7-PDE9, supporting this conclusion. Total PDE activity was found to be elevated in tissue extracts from a mouse model of Duchenne's muscular dystrophy.

Background: Bee and wasp venom extracts contain potent allergens capable of inducing severe clinical reactions. To analyze immediate-type hypersensitivity to defined hymenoptera venom components, a recently developed in vitro test was applied that is based on the upregulation of CD203c expression on basophils., Methods: CD203c expression on blood basophils of 9 healthy donors and 39 patients allergic to bee and/or wasp venom was analyzed by flow cytometry before and after activation with the purified bee venom allergens phospholipase A2 (Api m 1), hyaluronidase (Api m 2) and melittin (Api m 4), or the purified wasp venom allergens phospholipase A1 (Ves v 1), hyaluronidase (Ves v 2) and the recombinant antigen 5 (Ves v 5). Venom-induced CD203c upregulation on basophils was compared with skin tests and assessment of specific IgE. Basophils of nonresponders were preincubated with 10 ng/ml interleukin-3 (IL-3) prior to allergen stimulation., Results: CD203c upregulation on basophils was induced by defined hymenoptera venom components in 35/39 patients with a diagnosed allergy to wasp and/or bee venom. Twenty-seven of the 34 tested patients with wasp allergy showed CD203c upregulation in response to Ves v 5, 26 of these patients also reacted with Ves v 2 and 17 with Ves v 1. Nine of 13 patients with bee allergy reacted with Api m 1, 13 individuals with Api m 2 and none of these patients with the minor allergen Api m 4. A diagnosed wasp allergy could also be confirmed in the prestimulated basophils (IL-3) of 2 nonresponder individuals who failed to upregulate CD203c in response to IgE receptor cross-linking prior to culture with IL-3., Conclusions: Flow-cytometric determination of CD203c upregulation on basophils activated by molecularly defined allergens is a powerful method to identify the precise allergen reactivity in sensitized individuals., (Copyright 2002 S. Karger AG, Basel)