Receptor-Mediated Enhancement of Beta Adrenergic Drug Activity by Ascorbate In Vitro and In Vivo

RATIONALE: Previous in vitro research demonstrated that ascorbate enhances potency and duration of activity of agonists binding to alpha 1 adrenergic and histamine receptors. OBJECTIVES: Extending this work to beta 2 adrenergic systems in vitro and in vivo. METHODS: Ultraviolet spectroscopy was used to study ascorbate binding to adrenergic receptor preparations and peptides. Force transduction studies on acetylcholine-contracted trachealis preparations from pigs and guinea pigs measured the effect of ascorbate on relaxation due to submaximal doses of beta adrenergic agonists. The effect of inhaled albuterol with and without ascorbate was tested on horses with heaves and sheep with carbachol-induced bronchoconstriction. MEASUREMENTS: Binding constants for ascorbate binding to beta adrenergic receptor were derived from concentration-dependent spectral shifts. Dose- dependence curves were obtained for the relaxation of pre-contracted trachealis preparations due to beta agonists in the presence and absence of varied ascorbate. Tachyphylaxis and fade were also measured. Dose response curves were determined for the effect of albuterol plus-and-minus ascorbate on airway resistance in horses and sheep. MAIN RESULTS: Ascorbate binds to the beta 2 adrenergic receptor at physiological concentrations. The receptor recycles dehydroascorbate. Physiological and supra-physiological concentrations of ascorbate enhance submaximal epinephrine and isoproterenol relaxation of trachealis, producing a 3-10-fold increase in sensitivity, preventing tachyphylaxis, and reversing fade. In vivo, ascorbate improves albuterol's effect on heaves and produces a 10-fold enhancement of albuterol activity in "asthmatic" sheep. CONCLUSIONS: Ascorbate enhances beta-adrenergic activity via a novel receptor-mediated mechanism; increases potency and duration of beta adrenergic agonists effective in asthma and COPD; prevents tachyphylaxis; and reverses fade. These novel effects are probably caused by a novel mechanism involving phosphorylation of aminergic receptors and have clinical and drug-development applications

Trypsin-Like Serine Proteases in Lutzomyia longipalpis – Expression, Activity and Possible Modulation by Leishmania infantum chagasi

Background: Midgut enzymatic activity is one of the obstacles that Leishmania must surpass to succeed in establishing infection. Trypsins are abundant digestive enzymes in most insects. We have previously described two trypsin cDNAs of L. longipalpis: one (Lltryp1) with a bloodmeal induced transcription pattern, the other (Lltryp2) with a constitutive transcription pattern. We have now characterized the expression and activity of trypsin-like proteases of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil. Methodology and Principal Findings: In order to study trypsin expression profiles we produced antibodies against peptides specific for Lltryp1 and Lltryp2. The anti-Lltryp1-peptide antibody revealed a band of 28 kDa between 6 and 48 hours. The anti-Lltryp2 peptide antibody did not evidence any band. When proteinaceous substrates (gelatin, hemoglobin, casein or albumin) were co-polymerized in polyacrylamide gels, insect midguts obtained at 12 hours after feeding showed a unique proteolytic pattern for each substrate. All activity bands were strongly inhibited by TLCK, benzamidine and 4-amino-benzamidine, indicating that they are trypsin-like proteases. The trypsin-like activity was also measured in vitro at different time points after ingestion of blood or blood containing Leishmania infantum chagasi, using the chromogenic substrate BArNA. L. longipalpis females fed on blood infected with L. i. chagasi had lower levels of trypsin activity after 12 and 48 hours than non-infected insects, suggesting that the parasite may have a role in this modulation. Conclusions and Significance: Trypsins are important and abundant digestive enzymes in L. longipalpis. Protein production and enzymatic activity followed previously identified gene expression of a blood modulated trypsin gene. A decrease of enzymatic activity upon the parasite infection, previously detected mostly in Old World vectors, was detected for the first time in the natural vector-parasite pair L. longipalpis-L. i. chagasi

Conservation Genetics of a Critically Endangered Limpet Genus and Rediscovery of an Extinct Species

A third of all known freshwater mollusk extinctions worldwide have occurred within a single medium-sized American drainage. The Mobile River Basin (MRB) of Alabama, a global hotspot of temperate freshwater biodiversity, was intensively industrialized during the 20(th) century, driving 47 of its 139 endemic mollusk species to extinction. These include the ancylinid limpet Rhodacmea filosa, currently classified as extinct (IUCN Red List), a member of a critically endangered southeastern North American genus reduced to a single known extant population (of R. elatior) in the MRB.We document here the tripling of known extant populations of this North American limpet genus with the rediscovery of enduring Rhodacmea filosa in a MRB tributary and of R. elatior in its type locality: the Green River, Kentucky, an Ohio River Basin (ORB) tributary. Rhodacmea species are diagnosed using untested conchological traits and we reassessed their systematic and conservation status across both basins using morphometric and genetic characters. Our data corroborated the taxonomic validity of Rhodacmea filosa and we inferred a within-MRB cladogenic origin from a common ancestor bearing the R. elatior shell phenotype. The geographically-isolated MRB and ORB R. elatior populations formed a cryptic species complex: although overlapping morphometrically, they exhibited a pronounced phylogenetic disjunction that greatly exceeded that of within-MRB R. elatior and R. filosa sister species.Rhodacmea filosa, the type species of the genus, is not extinct. It persists in a Coosa River tributary and morphometric and phylogenetic analyses confirm its taxonomic validity. All three surviving populations of the genus Rhodacmea merit specific status. They collectively contain all known survivors of a phylogenetically highly distinctive North American endemic genus and therefore represent a concentrated fraction of continental freshwater gastropod biodiversity. We recommend the establishment of a proactive targeted conservation program that may include their captive propagation and reintroduction

An Atlas for Schistosoma mansoni Organs and Life-Cycle Stages Using Cell Type-Specific Markers and Confocal Microscopy

Schistosomiasis (bilharzia) is a tropical disease caused by trematode parasites (Schistosoma) that affects hundreds of millions of people in the developing world. Currently only a single drug (praziquantel) is available to treat this disease, highlighting the importance of developing new techniques to study Schistosoma. While molecular advances, including RNA interference and the availability of complete genome sequences for two Schistosoma species, will help to revolutionize studies of these animals, an array of tools for visualizing the consequences of experimental perturbations on tissue integrity and development needs to be made widely available. To this end, we screened a battery of commercially available stains, antibodies and fluorescently labeled lectins, many of which have not been described previously for analyzing schistosomes, for their ability to label various cell and tissue types in the cercarial stage of S. mansoni. This analysis uncovered more than 20 new markers that label most cercarial tissues, including the tegument, the musculature, the protonephridia, the secretory system and the nervous system. Using these markers we present a high-resolution visual depiction of cercarial anatomy. Examining the effectiveness of a subset of these markers in S. mansoni adults and miracidia, we demonstrate the value of these tools for labeling tissues in a variety of life-cycle stages. The methodologies described here will facilitate functional analyses aimed at understanding fundamental biological processes in these parasites

Prenylation inhibitors stimulate both estrogen receptor α transcriptional activity through AF-1 and AF-2 and estrogen receptor β transcriptional activity

INTRODUCTION: We showed in a previous study that prenylated proteins play a role in estradiol stimulation of proliferation. However, these proteins antagonize the ability of estrogen receptor (ER) α to stimulate estrogen response element (ERE)-dependent transcriptional activity, potentially through the formation of a co-regulator complex. The present study investigates, in further detail, how prenylated proteins modulate the transcriptional activities mediated by ERα and by ERβ. METHODS: The ERE-β-globin-Luc-SV-Neo plasmid was either stably transfected into MCF-7 cells or HeLa cells (MELN cells and HELN cells, respectively) or transiently transfected into MCF-7 cells using polyethylenimine. Cells deprived of estradiol were analyzed for ERE-dependent luciferase activity 16 hours after estradiol stimulation and treatment with FTI-277 (a farnesyltransferase inhibitor) or with GGTI-298 (a geranylgeranyltransferase I inhibitor). In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ERα, the full-length ERβ, the AF-1-deleted ERα or the AF-2-deleted ERα. The presence of ERα was then detected by immunocytochemistry in either the nuclei or the cytoplasms of MCF-7 cells. Finally, Clostridium botulinum C3 exoenzyme treatment was used to determine the involvement of Rho proteins in ERE-dependent luciferase activity. RESULTS: FTI-277 and GGTI-298 only stimulate ERE-dependent luciferase activity in stably transfected MCF-7 cells. They stimulate both ERα-mediated and ERβ-mediated ERE-dependent luciferase activity in HELN cells, in the presence of and in the absence of estradiol. The roles of both AF-1 and AF-2 are significant in this effect. Nuclear ERα is decreased in the presence of prenyltransferase inhibitors in MCF-7 cells, again in the presence of and in the absence of estradiol. By contrast, cytoplasmic ERα is mainly decreased after treatment with FTI-277, in the presence of and in the absence of estradiol. The involvement of Rho proteins in ERE-dependent luciferase activity in MELN cells is clearly established. CONCLUSIONS: Together, these results demonstrate that prenylated proteins (at least RhoA, RhoB and/or RhoC) antagonize the ability of ERα and ERβ to stimulate ERE-dependent transcriptional activity, potentially acting through both AF-1 and AF-2 transcriptional activities

Schistosoma mansoni Venom Allergen Like Proteins Present Differential Allergic Responses in a Murine Model of Airway Inflammation

The Schistosoma mansoni Venom Allergen Like proteins (SmVALs) have been identified in the Transcriptome and Post-Genomic studies as targets for immune interventions. Two secreted members of the family were obtained as recombinant proteins in the native conformation. Antibodies produced against them showed that SmVAL4 was present mostly in cercarial secretions and SmVAL26 in egg secretions and that only the native SmVAL4 contained carbohydrate moieties. Due to concerns with potential allergic characteristics of this class of molecules, we have explored the mouse model of airway inflammation in order to investigate these properties in a more confined system. Sensitization and challenge with rSmVAL4, but not rSmVAL26, induced extensive migration of cells to the lungs, mostly eosinophils and macrophages; moreover, immunological parameters were also characteristic of an allergic inflammatory response. Our results showed that the allergic potential of this class of proteins can be variable and that the vaccine candidates should be characterized; the mouse model of airway inflammation can be useful to evaluate these properties

Identification of the Schistosoma mansoni TNF-Alpha Receptor Gene and the Effect of Human TNF-Alpha on the Parasite Gene Expression Profile

Schistosoma mansoni is the major causative agent of schistosomiasis in the Americas. This parasite takes advantage of host signaling molecules such as cytokines and hormones to complete its development inside the host. Tumor necrosis factor-alpha (TNF-α) is one of the most important host cytokines involved in the inflammatory response. When cercariae, the infective stage, penetrates the human skin the release of TNF-α is started. In this work the authors describe the complete sequence of a possible TNF-α receptor in S. mansoni and detect that the receptor is most highly expressed in cercariae among all life cycle stages. Aiming to mimic the situation at the site of skin penetration, cercariae were mechanically transformed in vitro into schistosomula and exposed to human TNF-α. Exposure of early-developing schistosomula to the human hormone caused a large-scale change in the expression of parasite genes. Exposure of adult worms to human TNF-α caused gene expression changes as well, and the set of parasite altered genes in the adult parasite was different from that of schistosomula. This work increases the number of known signaling pathways of the parasite, and opens new perspectives into understanding the molecular components of TNF-α response as well as into possibly interfering with parasite–host interaction

Transcriptional Changes in Schistosoma mansoni during Early Schistosomula Development and in the Presence of Erythrocytes

Schistosome blood flukes cause more mortality and morbidity than any other human worm infection, but current control methods primarily rely on a single drug. There is a desperate need for new approaches to control this parasite, including vaccines. People become infected when the free-swimming larva, the cercaria, enters through the skin and becomes the schistosomulum. Schistosomula are susceptible to immune responses during their first few days in the host before they become adult parasites. We characterised the genes that these newly transformed parasites switch on when they enter the host to identify molecules that are critical for survival in the human host. Some of these highly up-regulated genes can be targeted for future development of new vaccines and drugs

MRI compared to conventional diagnostic work-up in the detection and evaluation of invasive lobular carcinoma of the breast: a review of existing literature

Item does not contain fulltextPURPOSE: The clinical diagnosis and management of invasive lobular carcinoma (ILC) of the breast presents difficulties. Magnetic resonance imaging (MRI) has been proposed as the imaging modality of choice for the evaluation of ILC. Small studies addressing different aspects of MRI in ILC have been presented but no large series to date. To address the usefulness of MRI in the work-up of ILC, we performed a review of the currently published literature. MATERIALS AND METHODS: We performed a literature search using the query "lobular AND (MRI OR MR OR MRT OR magnetic)" in the Cochrane library, PubMed and scholar.google.com, to retrieve all articles that dealt with the use of MRI in patients with ILC. We addressed sensitivity, morphologic appearance, correlation with pathology, detection of additional lesions, and impact of MRI on surgery as different endpoints. Whenever possible we performed meta-analysis of the pooled data. RESULTS: Sensitivity is 93.3% and equal to overall sensitivity of MRI for malignancy in the breast. Morphologic appearance is highly heterogeneous and probably heavily influenced by interreader variability. Correlation with pathology ranges from 0.81 to 0.97; overestimation of lesion size occurs but is rare. In 32% of patients, additional ipsilateral lesions are detected and in 7% contralateral lesions are only detected by MRI. Consequently, MRI induces change in surgical management in 28.3% of cases. CONCLUSION: This analysis indicates MRI to be valuable in the work-up of ILC. It provides additional knowledge that cannot be obtained by conventional imaging modalities which can be helpful in patient treatment

Use of Genomic DNA as an Indirect Reference for Identifying Gender-Associated Transcripts in Morphologically Identical, but Chromosomally Distinct, Schistosoma mansoni Cercariae

BACKGROUND: The use of DNA microarray technology to study global Schistosoma gene expression has led to the rapid identification of novel biological processes, pathways or associations. Implementation of standardized DNA microarray protocols across laboratories would assist maximal interpretation of generated datasets and extend productive application of this technology. METHODOLOGY/PRINCIPAL FINDINGS: Utilizing a new Schistosoma mansoni oligonucleotide DNA microarray composed of 37,632 elements, we show that schistosome genomic DNA (gDNA) hybridizes with less variation compared to complex mixed pools of S. mansoni cDNA material (R = 0.993 for gDNA compared to R = 0.956 for cDNA during ‘self versus self’ hybridizations). Furthermore, these effects are species-specific, with S. japonicum or Mus musculus gDNA failing to bind significantly to S. mansoni oligonucleotide DNA microarrays (e.g R = 0.350 when S. mansoni gDNA is co-hybridized with S. japonicum gDNA). Increased median fluorescent intensities (209.9) were also observed for DNA microarray elements hybridized with S. mansoni gDNA compared to complex mixed pools of S. mansoni cDNA (112.2). Exploiting these valuable characteristics, S. mansoni gDNA was used in two-channel DNA microarray hybridization experiments as a common reference for indirect identification of gender-associated transcripts in cercariae, a schistosome life-stage in which there is no overt sexual dimorphism. This led to the identification of 2,648 gender-associated transcripts. When compared to the 780 gender-associated transcripts identified by hybridization experiments utilizing a two-channel direct method (co-hybridization of male and female cercariae cDNA), indirect methods using gDNA were far superior in identifying greater quantities of differentially expressed transcripts. Interestingly, both methods identified a concordant subset of 188 male-associated and 156 female-associated cercarial transcripts, respectively. Gene ontology classification of these differentially expressed transcripts revealed a greater diversity of categories in male cercariae. Quantitative real-time PCR analysis confirmed the DNA microarray results and supported the reliability of this platform for identifying gender-associated transcripts. CONCLUSIONS/SIGNIFICANCE: Schistosome gDNA displays characteristics highly suitable for the comparison of two-channel DNA microarray results obtained from experiments conducted independently across laboratories. The schistosome transcripts identified here demonstrate, for the first time, that gender-associated patterns of expression are already well established in the morphologically identical, but chromosomally distinct, cercariae stage