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1.
Eur J Med Chem ; 181: 111560, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31382118

RESUMO

A new series of raloxifene sulfonate/sulfamate derivatives were designed and synthesized. The target compounds were tested for inhibitory effect against nucleotide pyrophosphatase/phosphodiesterase-1 and -3 (NPP1 and NPP3) enzymes. Furthermore, all the ten target compounds were subjected to cytotoxic studies on various cancer cell lines, and the most potent derivatives were explored for their potency against these cancer cell lines as well as F180 fibroblasts to investigate the selectivity indexes. Compound 1f exerted the highest potency against HT-29 colon cancer cell line (IC50 = 1.4 µM) with 8.43-fold selectivity towards HT-29 than F180 fibroblasts. Compound 1f exerted sub-micromolar IC50 values against NPP1 and NPP3 (IC50 = 0.29 µM and 0.71 µM, respectively). The most potent inhibitors were docked in developed homology model of NPP1 and crystal structure of NPP3. All the docked analogues manifested remarkable interactions within the active pocket of NPP1 and NPP3.

3.
Biochim Biophys Acta Mol Basis Dis ; 1865(10): 2595-2605, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31271845

RESUMO

Extracellular nucleotides are released as constitutive danger signals by various cell types and activate nucleotide (P2) receptors such as P2Y6 receptor. P2Y6 activation on monocytes induces the secretion of the chemokine CXCL8 which may propagate intestinal inflammation. Also, P2Y6 expression is increased in infiltrating T cells of Crohn's disease patients. As inflammatory bowel disease (IBD) is associated with immune cell recruitment, we hypothesised that P2Y6 would participate to the establishment of inflammation in this disease. To address this, we used P2Y6 deficient (P2ry6--/-) mice in the dextran sodium sulfate (DSS) murine model of IBD. In disagreement with our hypothesis, P2Y6 deficient mice were more susceptible to inflammation induced by DSS than WT mice. DSS treated-P2ry6-/- mice showed increased histological damage and increased neutrophil and macrophage infiltration that correlated with increased mRNA levels of the chemokines KC and MCP-1. DSS treated-P2ry6-/- mice exhibited also higher levels of Th17/Th1 lymphocytes in their colon which correlated with increased levels of IFN-γ and IL-17A in the sera as well as increased mRNA levels of IFN-γ, IL-17A, IL-6, IL-23 and IL-1ß in P2ry6-/- colons. This inflammation was also accompanied by a decreased cell proliferation and goblet cell number. Importantly, injection of anti-IL-17 intraperitoneally partially protected P2ry6-/- mice from DSS-induced colitis. Taken together, in the absence of P2Y6, an exacerbated intestinal inflammation to DSS was observed which correlated with increased recruitment of Th17/Th1 lymphocytes. These data suggest a protective role of P2Y6 expressed on leukocytes in intestinal inflammation.

4.
Resuscitation ; 139: 241-252, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31029714

RESUMO

OBJECTIVE: To determine the initial defibrillation energy dose that is associated with sustained return of spontaneous circulation (ROSC) during paediatric cardiac arrest with ventricular fibrillation or pulseless ventricular tachycardia. METHODS: A systematic review was performed using four databases (PROSPERO: CRD42016036734). Human studies and animal model studies of pediatric cardiac arrest involving assessment of external defibrillation energy dosing were considered. The primary outcome was sustained ROSC. Survival and defibrillation-induced complications were also evaluated. RESULTS: The search strategy identified 14,471 citations of which 232 manuscripts were reviewed. Ten human and 10 animal model studies met the inclusion criteria. Human studies were prospective (n = 6) or retrospective (n = 4) cohort studies and included between 11 and 266 patients (median = 46 patients). Sustained ROSC rates ranged from 0 to 61% (n = 7). No studies reported a statistically significant association between the initial defibrillation energy dose and the rate of sustained ROSC (n = 7) or survival (n = 6). Meta-analysis was not considered appropriate due to clinical heterogeneity. Risk of bias was moderate. All animal studies were randomized controlled trials with 8 and 52 (median = 27) piglets. ROSC was frequently achieved (≥85%) with energy dose ranging from 2 to 7 J/kg (n = 7). The defibrillation threshold varied according to the body weight and appears to be higher in infant. CONCLUSION: Defibrillation energy doses and thresholds varied according to the body weight and trended higher for infants. No definitive association between initial defibrillation doses and the sustained ROSC or survival could be demonstrated. Clinicians should follow local consensus-based guidelines.

5.
Purinergic Signal ; 15(2): 247-263, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31025169

RESUMO

Overproduction of extracellular diphosphate due to hydrolysis of ATP by NPP1 leads to pathological calcium diphosphate (pyrophosphate) dihydrate deposition (CPPD) in cartilage, resulting in a degenerative joint disease that today lacks a cure. Here, we targeted the identification of novel NPP1 inhibitors as potential therapeutic agents for CPPD deposition disease. Specifically, we synthesized novel analogs of AMP (NPP1 reaction product) and ADP (NPP1 inhibitor). These derivatives incorporate several chemical modifications of the natural nucleotides including (1) a methylene group replacing the Pα,ß-bridging oxygen atom to provide metabolic resistance, (2) sulfonate group(s) replacing phosphonate(s) to improve binding to NPP1's catalytic zinc ions, (3) an acyclic nucleotide analog to allow flexible binding in the NPP1 catalytic site, and (4) a benzimidazole base replacing adenine. Among the investigated compounds, adenine-N9-(methoxy)ethyl-ß-bisphosphonate, 10, was identified as an NPP1 inhibitor (Ki 16.3 µM vs. the artificial substrate p-nitrophenyl thymidine-5'-monophosphate (p-Nph-5'-TMP), and 9.60 µM vs. the natural substrate, ATP). Compound 10 was selective for NPP1 vs. human NPP3, human CD39, and tissue non-specific alkaline phosphatase (TNAP), but also inhibited human CD73 (Ki 12.6 µM). Thus, 10 is a dual NPP1/CD73 inhibitor, which could not only be of interest for treating CPPD deposition disease and calcific aortic valve disease but may also be considered for the immunotherapy of cancer. Compound 10 proved to be a promising inhibitor, which almost completely reduces NPPase activity in human osteoarthritic chondrocytes at a concentration of 100 µM.

6.
J Integr Plant Biol ; 61(5): 564-580, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30916433

RESUMO

Seed development is a complex period of the flowering plant life cycle. After fertilization, the three main regions of the seed, embryo, endosperm and seed coat, undergo a series of developmental processes that result in the production of a mature seed that is developmentally arrested, desiccated, and metabolically quiescent. These processes are highly coordinated, both temporally and spatially, to ensure the proper growth and development of the seed. The transcription factor, LEAFY COTYLEDON1 (LEC1), is a central regulator that controls several aspects of embryo and endosperm development, including embryo morphogenesis, photosynthesis, and storage reserve accumulation. Thus, LEC1 regulates distinct sets of genes at different stages of seed development. Despite its critical importance for seed development, an understanding of the mechanisms underlying LEC1's multifunctionality is only beginning to be obtained. Recent studies describe the roles of specific transcription factors and the hormones, gibberellic acid and abscisic acid, in controlling the activity and transcriptional specificity of LEC1 across seed development. Moreover, studies indicate that LEC1 acts as a pioneer transcription factor to promote epigenetic reprogramming during embryogenesis. In this review, we discuss the mechanisms that enable LEC1 to serve as a central regulator of seed development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Sementes/metabolismo , Sementes/fisiologia , Fatores de Transcrição/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Giberelinas/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Sementes/genética , Fatores de Transcrição/genética
7.
Cell Metab ; 2018 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-30449685

RESUMO

Identification of cell-surface markers specific to human pancreatic ß cells would allow in vivo analysis and imaging. Here we introduce a biomarker, ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), that is expressed on the cell surface of essentially all adult human ß cells, including those from individuals with type 1 or type 2 diabetes. NTPDase3 is expressed dynamically during postnatal human pancreas development, appearing first in acinar cells at birth, but several months later its expression declines in acinar cells while concurrently emerging in islet ß cells. Given its specificity and membrane localization, we utilized an NTPDase3 antibody for purification of live human ß cells as confirmed by transcriptional profiling, and, in addition, for in vivo imaging of transplanted human ß cells. Thus, NTPDase3 is a cell-surface biomarker of adult human ß cells, and the antibody directed to this protein should be a useful new reagent for ß cell sorting, in vivo imaging, and targeting.

8.
Analyst ; 143(22): 5417-5430, 2018 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-30303204

RESUMO

Ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) is a major ectonucleotidase that hydrolyzes proinflammatory ATP via ADP to AMP, which is subsequently converted by ecto-5'-nucleotidase (CD73) to immunosuppressive adenosine. Activation of CD39 has potential for treating inflammatory diseases, while inhibition was suggested as a novel strategy for the immunotherapy of cancer. In the present study, we developed a selective and highly sensitive capillary electrophoresis (CE) assay using a novel fluorescent CD39 substrate, a fluorescein-labelled ATP (PSB-170621A) that is converted to its AMP derivative. To accelerate the assays, a two-directional (forward and reverse) CE system was implemented using 96-well plates, which is suitable for the screening of compound libraries (Z'-factor: 0.7). The detection limits for the forward and reverse operation were 11.7 and 2.00 pM, respectively, indicating a large enhancement in sensitivity as compared to previous methods (e.g. malachite-green assay: 1 000 000-fold, CE-UV assay: 500 000-fold, fluorescence polarization immunoassay: 12 500-fold). Enzyme kinetic studies at human CD39 revealed a Km value of 19.6 µM, and a kcat value of 119 × 10-3 s-1 for PSB-170621A, which shows similar substrate properties as ATP (11.4 µM and 82.5 × 10-3 s-1). The compound displayed similar properties at rat and mouse CD39. Subsequent docking studies into a homology model of human CD39 revealed a hydrophobic pocket that accommodates the fluorescein tag. PSB-170621A was found to be preferably hydrolyzed by CD39 as compared to other ectonucleotidases. The new assay was validated by performing inhibition assays with several standard CD39 inhibitors yielding results that were consonant with data using the natural substrates.

9.
Proc Natl Acad Sci U S A ; 115(35): E8315-E8322, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30104383

RESUMO

The precise mechanisms that control gene activity during seed development remain largely unknown. Previously, we showed that several genes essential for seed development, including those encoding storage proteins, fatty acid biosynthesis enzymes, and transcriptional regulators (e.g., ABI3, FUS3) are located within hypomethylated regions of the soybean genome. These hypomethylated regions are similar to the DNA methylation valleys (DMVs), or canyons, found in mammalian cells. Here, we address the question of the extent to which DMVs are present within seed genomes and what role they might play in seed development. We scanned soybean and Arabidopsis seed genomes from postfertilization through dormancy and germination for regions that contain <5% or <0.4% bulk methylation in CG, CHG, and CHH contexts over all developmental stages. We found that DMVs represent extensive portions of seed genomes, range in size from 5-76 kb, are scattered throughout all chromosomes, and are hypomethylated throughout the plant life cycle. Significantly, DMVs are enriched greatly in transcription factor (TF) genes and other developmental genes that play critical roles in seed formation. Many DMV genes are regulated with respect to seed stage, region, and tissue, and contain H3K4me3, H3K27me3, or bivalent marks that fluctuate during development. Our results indicate that DMVs are a unique regulatory feature of both plant and animal genomes, and that a large number of seed genes are regulated in the absence of methylation changes during development, probably by the action of specific TFs and epigenetic events at the chromatin level.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Metilação de DNA/fisiologia , DNA de Plantas , Genoma de Planta/fisiologia , Sementes , Soja , Fatores de Transcrição , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA de Plantas/genética , DNA de Plantas/metabolismo , Epigênese Genética/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Sementes/genética , Sementes/metabolismo , Soja/genética , Soja/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
J Med Chem ; 61(9): 3939-3951, 2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29681152

RESUMO

Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) hydrolyzes phosphodiester bonds of nucleotides such as ATP, resulting mainly in the formation of AMP and pyrophosphate. NPP1 activity plays a deleterious function in calcified aortic valve disease and calcium pyrophosphate deposition disease. Thus, inhibitors of NPP1 represent a medical need. We developed novel NPP1 inhibitors based on uridine 5'-Pα,α-dithiophosphate analogues, 9-12. All these analogues potently inhibited hNPP1 (80-100% inhibition) at 100 µM, with no, or minimal, inhibition of NPP3 and other ectonucleotidases (NTPDase1,2,3,8). These compounds showed nearly no activity at uracil-nucleotide sensitive P2Y2,4,6-receptors and thus represent highly selective NPP1 inhibitors. The most promising inhibitor was diuridine 5'-Pα,α,5″-Pα,α-tetrathiotetraphosphate, 12, exhibiting Ki of 27 nM. Analogues 9-12 proved to be highly stable to air oxidation and to acidic and basic pH. Docking simulations suggested that the enhanced NPP1 inhibitory activity and selectivity of analogue 12 could be attributed to the simultaneous occupancy of two sites (the AMP site and an alternative site) of NPP1 by this compound.

11.
Front Pharmacol ; 9: 149, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29541027

RESUMO

In this study, we investigated the role of extracellular nucleotides in chemokine (KC, MIP-2, MCP-1, and CXCL10) expression and secretion by murine primary intestinal epithelial cells (IECs) with a focus on P2Y6 receptors. qRT-PCR experiments showed that P2Y6 was the dominant nucleotide receptor expressed in mouse IEC. In addition, the P2Y6 ligand UDP induced expression and secretion of CXCL10. For the other studies, we took advantage of mice deficient in P2Y6 (P2ry6-/-). Similar expression levels of P2Y1, P2Y2, P2X2, P2X4, and A2A were detected in P2ry6-/- and WT IEC. Agonists of TLR3 (poly(I:C)), TLR4 (LPS), P2Y1, and P2Y2 increased the expression and secretion of CXCL10 more prominently in P2ry6-/- IEC than in WT IEC. CXCL10 expression and secretion induced by poly(I:C) in both P2ry6-/- and WT IEC were inhibited by general P2 antagonists (suramin and Reactive-Blue-2), by apyrase, and by specific antagonists of P2Y1, P2Y2, P2Y6 (only in WT), and P2X4. Neither adenosine nor an A2A antagonist had an effect on CXCL10 expression and secretion. Macrophage chemotaxis was induced by the supernatant of poly(I:C)-treated IEC which was consistent with the level of CXCL10 secreted. Finally, the non-nucleotide agonist FGF2 induced MMP9 mRNA expression also at a higher level in P2ry6-/- IEC than in WT IEC. In conclusion, extracellular nucleotides regulate CXCL10 expression and secretion by IEC. In the absence of P2Y6, these effects are modulated by other P2 receptors also present on IEC. These data suggest that the presence of P2Y6 regulates chemokine secretion and may also regulate IEC homeostasis.

12.
Addict Behav ; 80: 154-160, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29407687

RESUMO

BACKGROUND: Methamphetamine has long been considered as a neurotoxic substance causing cognitive deficits. Recently, however, the magnitude and the clinical significance of the cognitive effects associated with methamphetamine use disorder (MUD) have been debated. To help clarify this controversy, we performed a meta-analysis of the cognitive deficits associated with MUD. METHODS: A literature search yielded 44 studies that assessed cognitive dysfunction in 1592 subjects with MUD and 1820 healthy controls. Effect size estimates were calculated using the Comprehensive Meta-Analysis, for the following 12 cognitive domains: attention, executive functions, impulsivity/reward processing, social cognition, speed of processing, verbal fluency/language, verbal learning and memory, visual learning and memory, visuo-spatial abilities and working memory. RESULTS: Findings revealed moderate impairment across most cognitive domains, including attention, executive functions, language/verbal fluency, verbal learning and memory, visual memory and working memory. Deficits in impulsivity/reward processing and social cognition were more prominent, whereas visual learning and visuo-spatial abilities were relatively spared cognitive domains. A publication bias was observed. DISCUSSION: These results show that MUD is associated with broad cognitive deficits that are in the same range as those associated with alcohol and cocaine use disorder, as recently shown by way of meta-analysis. The prominent effects of MUD on social cognition and impulsivity/reward processing are based on a small number of studies, and as such, these results will need to be replicated. The functional consequences (social and occupational) of the cognitive deficits of methamphetamine will also need to be determined.

13.
Proc Natl Acad Sci U S A ; 114(45): E9730-E9739, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29078418

RESUMO

We profiled soybean and Arabidopsis methylomes from the globular stage through dormancy and germination to understand the role of methylation in seed formation. CHH methylation increases significantly during development throughout the entire seed, targets primarily transposable elements (TEs), is maintained during endoreduplication, and drops precipitously within the germinating seedling. By contrast, no significant global changes in CG- and CHG-context methylation occur during the same developmental period. An Arabidopsis ddcc mutant lacking CHH and CHG methylation does not affect seed development, germination, or major patterns of gene expression, implying that CHH and CHG methylation does not play a significant role in seed development or in regulating seed gene activity. By contrast, over 100 TEs are transcriptionally de-repressed in ddcc seeds, suggesting that the increase in CHH-context methylation may be a failsafe mechanism to reinforce transposon silencing. Many genes encoding important classes of seed proteins, such as storage proteins, oil biosynthesis enzymes, and transcription factors, reside in genomic regions devoid of methylation at any stage of seed development. Many other genes in these classes have similar methylation patterns, whether the genes are active or repressed. Our results suggest that methylation does not play a significant role in regulating large numbers of genes important for programming seed development in both soybean and Arabidopsis. We conclude that understanding the mechanisms controlling seed development will require determining how cis-regulatory elements and their cognate transcription factors are organized in genetic regulatory networks.


Assuntos
Arabidopsis/genética , Metilação de DNA/fisiologia , DNA de Plantas/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/genética , Soja/genética , Sequência de Bases , Metilação de DNA/genética , Elementos de DNA Transponíveis/genética , Elementos de DNA Transponíveis/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Redes Reguladoras de Genes , Inativação Gênica , Genes de Plantas/genética , Genoma de Planta/genética , Germinação/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Plântula/genética , Plântula/metabolismo , Sementes/citologia
14.
Proc Natl Acad Sci U S A ; 114(32): E6710-E6719, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28739919

RESUMO

LEAFY COTYLEDON1 (LEC1), an atypical subunit of the nuclear transcription factor Y (NF-Y) CCAAT-binding transcription factor, is a central regulator that controls many aspects of seed development including the maturation phase during which seeds accumulate storage macromolecules and embryos acquire the ability to withstand desiccation. To define the gene networks and developmental processes controlled by LEC1, genes regulated directly by and downstream of LEC1 were identified. We compared the mRNA profiles of wild-type and lec1-null mutant seeds at several stages of development to define genes that are down-regulated or up-regulated by the lec1 mutation. We used ChIP and differential gene-expression analyses in Arabidopsis seedlings overexpressing LEC1 and in developing Arabidopsis and soybean seeds to identify globally the target genes that are transcriptionally regulated by LEC1 in planta Collectively, our results show that LEC1 controls distinct gene sets at different developmental stages, including those that mediate the temporal transition between photosynthesis and chloroplast biogenesis early in seed development and seed maturation late in development. Analyses of enriched DNA sequence motifs that may act as cis-regulatory elements in the promoters of LEC1 target genes suggest that LEC1 may interact with other transcription factors to regulate distinct gene sets at different stages of seed development. Moreover, our results demonstrate strong conservation in the developmental processes and gene networks regulated by LEC1 in two dicotyledonous plants that diverged ∼92 Mya.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Sementes/metabolismo , Soja/metabolismo , Transcrição Genética/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , Motivos de Nucleotídeos/fisiologia , Sementes/genética , Soja/genética
15.
Purinergic Signal ; 13(3): 293-304, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28409324

RESUMO

Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) is an ectonucleotidase that modulates P2 receptor activation by hydrolyzing ATP to ADP. In rodents, NTPDase2 is expressed by several specialized cell types such as vascular adventitial cells, neuroglial cells, hepatic portal fibroblasts, gustatory type I cells, and cells within the connective tissues of reproductive and gastrointestinal organs. Much less is known regarding the expression and function of NTPDase2 in humans. Here, we developed specific research tools to study human NTPDase2. We generated mouse monoclonal antibodies and rabbit polyclonal antibodies specific to human NTPDase2 and validated their specificity by western blot, immunocytochemistry, immunohistochemistry, and flow cytometry. In addition, one monoclonal antibody named hN2-D5 s specifically inhibits human NTPDase2 enzymatic activity but not mouse nor rat NTPDase2. Using these antibodies, NTPDase2 immunoreactivity was detected on glial cells of the human enteric nervous system suggesting a function of the enzyme in intestinal motility. In conclusion, the new antibodies described in our work are novel tools that will enhance future studies of NTPDase2 expression and function in humans.


Assuntos
Adenosina Trifosfatases/imunologia , Anticorpos/imunologia , Apirase/imunologia , Adenosina Trifosfatases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Apirase/metabolismo , Humanos , Imuno-Histoquímica/métodos , Camundongos Endogâmicos BALB C , Neuroglia/metabolismo , Transdução de Sinais/fisiologia
16.
Front Pharmacol ; 8: 115, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28337144

RESUMO

The ectonucleotidase nucleoside triphosphate diphosphohydrolase-8 (NTPDase8) is the last member of the Ecto-NTPDase family to be discovered and characterized. It is a transmembrane protein which regulates the concentration of the agonists of P1 and P2 receptors at the cell surface. The functions of the enzyme are still not known partly due to the lack of specific tools such as antibodies. In this work, guinea pig polyclonal antibodies against mouse NTPDase8 and mouse monoclonal antibodies against human NTPDase8 have been generated and characterized. For the production of antibodies against mouse NTPDase8 several techniques have been tried. Several peptide antigens in several hosts (rabbit, rat, hamster, and guinea pig) failed to give a positive reaction suggesting that NTPDase8 is poorly immunogenic. In this study, we describe the successful process that led to anti-mouse NTPDase8, namely the cDNA immunization technique. Monoclonal antibodies to human NTPDase8 were also obtained by cDNA immunization followed by a final injection with transfected human embryonic kidney (HEK 293T) cells expressing human NTPDase8. The specificity of these antibodies was evaluated by Western blot, immunocytochemistry, immunohistochemistry and flow cytometry. In contrast, all commercial antibodies to NTPDase8 peptides that we have tested failed to give a specific positive signal against the expressed NTPDase8 protein when used to probe Western blots. In addition, immunohistochemistry experiments confirmed the presence of NTPDase8 in mouse liver canaliculi. The tools generated in this work will help characterize NTPDase8 localization and function in future studies and its contribution to the modulation of P1 and P2 receptor activation.

17.
J Appl Res Intellect Disabil ; 30(5): 830-846, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27461950

RESUMO

BACKGROUND: The benefits of autonomy support with the general population have been demonstrated numerous times. However, little research has been conducted to verify if these benefits apply to people with special needs. METHODS: The goal of the study was to examine whether autonomy support (AS) can foster the sense of autonomy of people with a mild intellectual disabilities (MIDs) and improve their experience while engaging in an important but unpleasant learning activity. This experiment compares the effects of two contexts: with and without AS. All participants (N = 51) had a mild intellectual disability and were recruited from rehabilitation centres. RESULTS: Compared to participants in the control group, participants in the AS group tended to experience greater autonomy satisfaction and tended to perceive more value to the activity. They were also significantly more engaged in it, and they experienced a steeper decrease in anxiety over time. CONCLUSIONS: This study suggests that the benefits of AS extend to individuals with mild intellectual disability.


Assuntos
Deficiência Intelectual/reabilitação , Pessoas com Deficiência Mental/reabilitação , Autonomia Pessoal , Satisfação Pessoal , Resolução de Problemas/fisiologia , Adolescente , Adulto , Feminino , Humanos , Deficiência Intelectual/psicologia , Masculino , Pessoas com Deficiência Mental/psicologia , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Adulto Jovem
18.
CJEM ; 18(6): 437-442, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27780500

RESUMO

OBJECTIVES: The purpose of this study was to assess adverse events associated with diagnostic urethral catheterization (UC) in young children and to determine their impact on the patient and their family. METHODS: This was a prospective cohort study conducted in the emergency department of a tertiary-care pediatric hospital. All 3- to 24-month-old children with fever who had a diagnostic UC were eligible. Parents who consented to participate were contacted by phone within 7 to 10 days after the UC to answer a standardized questionnaire inquiring about complications. The primary outcome was the occurrence of an unfavourable event in the seven days following UC, defined as painful urination, genital pain, urinary retention, hematuria or secondary urinary tract infection. Secondary outcomes included the need for further medical care and the need for parents to miss school or work. RESULTS: Of the 199 patients who completed the study, 41 (21%) reported a complication: painful urination in 19 (10%) children, genital pain in 16 (8%), urinary retention in 11 (6%), gross hematuria in 9 (5%), and secondary urinary tract infection in 1 (0.5%). Three (1%) parents reported the need for further medical care and three (1%) missed work. Two independent variables (male sex and age 12-23 months) were associated with a higher risk of adverse events. CONCLUSIONS: Urethral catheterization is associated with adverse events in 21% of young children in the week following the procedure. Accordingly, this procedure should be used judiciously in children, considering its potential to cause unfavourable events.


Assuntos
Serviço Hospitalar de Emergência , Cateterismo Urinário/efeitos adversos , Cateterismo Urinário/métodos , Infecções Urinárias/epidemiologia , Fatores Etários , Pré-Escolar , Estudos de Coortes , Feminino , Seguimentos , Humanos , Incidência , Lactente , Masculino , Pediatria , Estudos Prospectivos , Quebeque , Medição de Risco , Fatores Sexuais , Centros de Atenção Terciária , Infecções Urinárias/diagnóstico , Doenças Urológicas/diagnóstico
19.
Purinergic Signal ; 12(4): 719-734, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27650530

RESUMO

Refractoriness to existing medications of up to 80 % of the patients with mesial temporal lobe epilepsy (MTLE) prompts for finding new antiepileptic drug targets. The adenosine A2A receptor emerges as an interesting pharmacological target since its excitatory nature partially counteracts the dominant antiepileptic role of endogenous adenosine acting via inhibitory A1 receptors. Gain of function of the excitatory A2A receptor has been implicated in a significant number of brain pathologies commonly characterized by neuronal excitotoxicity. Here, we investigated changes in the expression and cellular localization of the A2A receptor and of the adenosine-generating enzyme, ecto-5'-nucleotidase/CD73, in the hippocampus of control individuals and MTLE human patients. Western blot analysis indicates that the A2A receptor is more abundant in the hippocampus of MTLE patients compared to control individuals. Immunoreactivity against the A2A receptor predominates in astrocytes staining positively for the glial fibrillary acidic protein (GFAP). No co-localization was observed between the A2A receptor and neuronal cell markers, like synaptotagmin 1/2 (nerve terminals) and neurofilament 200 (axon fibers). Hippocampal astrogliosis observed in MTLE patients was accompanied by a proportionate increase in A2A receptor and ecto-5'-nucleotidase/CD73 immunoreactivities. Given our data, we hypothesize that selective blockade of excessive activation of astrocytic A2A receptors and/or inhibition of surplus adenosine formation by membrane-bound ecto-5'-nucleotidase/CD73 may reduce neuronal excitability, thus providing a novel therapeutic target for drug-refractory seizures in MTLE patients.


Assuntos
5'-Nucleotidase/metabolismo , Astrócitos/metabolismo , Epilepsia do Lobo Temporal/metabolismo , Hipocampo/metabolismo , Receptor A2A de Adenosina/metabolismo , Regulação para Cima , 5'-Nucleotidase/genética , Adulto , Idoso , Epilepsia do Lobo Temporal/genética , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Receptor A2A de Adenosina/genética
20.
Plant Physiol ; 169(3): 2030-47, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26381315

RESUMO

Plants sense the foliar shade of competitors and alter their developmental programs through the shade-avoidance response. Internode and petiole elongation, and changes in overall leaf area and leaf mass per area, are the stereotypical architectural responses to foliar shade in the shoot. However, changes in leaf shape and complexity in response to shade remain incompletely, and qualitatively, described. Using a meta-analysis of more than 18,000 previously published leaflet outlines, we demonstrate that shade avoidance alters leaf shape in domesticated tomato (Solanum lycopersicum) and wild relatives. The effects of shade avoidance on leaf shape are subtle with respect to individual traits but are combinatorially strong. We then seek to describe the developmental origins of shade-induced changes in leaf shape by swapping plants between light treatments. Leaf size is light responsive late into development, but patterning events, such as stomatal index, are irrevocably specified earlier. Observing that shade induces increases in shoot apical meristem size, we then describe gene expression changes in early leaf primordia and the meristem using laser microdissection. We find that in leaf primordia, shade avoidance is not mediated through canonical pathways described in mature organs but rather through the expression of KNOTTED1-LIKE HOMEOBOX and other indeterminacy genes, altering known developmental pathways responsible for patterning leaf shape. We also demonstrate that shade-induced changes in leaf primordium gene expression largely do not overlap with those found in successively initiated leaf primordia, providing evidence against classic hypotheses that shaded leaf morphology results from the prolonged production of juvenile leaf types.


Assuntos
Regulação da Expressão Gênica de Plantas/efeitos da radiação , Proteínas de Homeodomínio/metabolismo , Lycopersicon esculentum/efeitos da radiação , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Proteínas de Homeodomínio/genética , Luz , Lycopersicon esculentum/anatomia & histologia , Lycopersicon esculentum/genética , Lycopersicon esculentum/fisiologia , Meristema/anatomia & histologia , Meristema/genética , Meristema/fisiologia , Meristema/efeitos da radiação , Modelos Biológicos , Fenótipo , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética
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