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1.
Braz J Biol ; 84: e281236, 2024.
Article in English | MEDLINE | ID: mdl-38775525

ABSTRACT

Munguba butter has bioactive compounds such as vitamin E and phytosterols, which has valued its application in the development of new products, with advantages in its use in emulsified formulations. Therefore, the objective was to develop and evaluate the stability of a nanoemulsion containing munguba butter as the oily phase. Munguba butter was extracted by the ultrasound assisted method and its HLB (hydrophilic-lipophilic balance) was determined. Next, formulations varying the concentration of butter from 1-40% were developed and classified into liquid or solid emulsion and phase separation. Liquid emulsions were evaluated for hydrodynamic particle diameter, polydispersity index (PDI), Zeta potential (ζ), rheological characterization, and stability assays. The butter had an HLB of 6.98. The NE 1.0% formulation was selected and demonstrated to be unstable at high temperatures (45 ± 2 °C) and remained stable at room temperature, refrigeration and light radiation for 90 days. Munguba butter, because it has high amounts of saturated fatty acids, hinders its application in the development of new products. However, the success in the development of the NE 1.0% formulation is noteworthy, remaining stable when exposed to refrigeration, room temperature and light radiation.


Subject(s)
Emulsions , Emulsions/chemistry , Vigna/chemistry , Butter/analysis , Particle Size , Drug Stability , Rheology
2.
J Mol Biol ; 435(2): 167890, 2023 01 30.
Article in English | MEDLINE | ID: mdl-36402225

ABSTRACT

14-3-3s are abundant proteins that regulate essentially all aspects of cell biology, including cell cycle, motility, metabolism, and cell death. 14-3-3s work by docking to phosphorylated Ser/Thr residues on a large network of client proteins and modulating client protein function in a variety of ways. In recent years, aided by improvements in proteomics, the discovery of 14-3-3 client proteins has far outpaced our ability to understand the biological impact of individual 14-3-3 interactions. The rate-limiting step in this process is often the identification of the individual phospho-serines/threonines that mediate 14-3-3 binding, which are difficult to distinguish from other phospho-sites by sequence alone. Furthermore, trial-and-error molecular approaches to identify these phosphorylations are costly and can take months or years to identify even a single 14-3-3 docking site phosphorylation. To help overcome this challenge, we used machine learning to analyze predictive features of 14-3-3 binding sites. We found that accounting for intrinsic protein disorder and the unbiased mass spectrometry identification rate of a given phosphorylation significantly improves the identification of 14-3-3 docking site phosphorylations across the proteome. We incorporated these features, coupled with consensus sequence prediction, into a publicly available web app, called "14-3-3 site-finder". We demonstrate the strength of this approach through its ability to identify 14-3-3 binding sites that do not conform to the loose consensus sequence of 14-3-3 docking phosphorylations, which we validate with 14-3-3 client proteins, including TNK1, CHEK1, MAPK7, and others. In addition, by using this approach, we identify a phosphorylation on A-kinase anchor protein-13 (AKAP13) at Ser2467 that dominantly controls its interaction with 14-3-3.


Subject(s)
14-3-3 Proteins , Protein Interaction Maps , Humans , 14-3-3 Proteins/metabolism , Binding Sites , Fetal Proteins/metabolism , Machine Learning , Mitogen-Activated Protein Kinase 7/metabolism , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Proteome/metabolism , Serine/metabolism , Threonine/metabolism
3.
Oncogene ; 37(42): 5587-5604, 2018 10.
Article in English | MEDLINE | ID: mdl-29915393

ABSTRACT

14-3-3 proteins are a family of structurally similar phospho-binding proteins that regulate essentially every major cellular function. Decades of research on 14-3-3s have revealed a remarkable network of interacting proteins that demonstrate how 14-3-3s integrate and control multiple signaling pathways. In particular, these interactions place 14-3-3 at the center of the signaling hub that governs critical processes in cancer, including apoptosis, cell cycle progression, autophagy, glucose metabolism, and cell motility. Historically, the majority of 14-3-3 interactions have been identified and studied under nutrient-replete cell culture conditions, which has revealed important nutrient driven interactions. However, this underestimates the reach of 14-3-3s. Indeed, the loss of nutrients, growth factors, or changes in other environmental conditions (e.g., genotoxic stress) will not only lead to the loss of homeostatic 14-3-3 interactions, but also trigger new interactions, many of which are likely stress adaptive. This dynamic nature of the 14-3-3 interactome is beginning to come into focus as advancements in mass spectrometry are helping to probe deeper and identify context-dependent 14-3-3 interactions-providing a window into adaptive phosphorylation-driven cellular mechanisms that orchestrate the tumor cell's response to a variety of environmental conditions including hypoxia and chemotherapy. In this review, we discuss emerging 14-3-3 regulatory mechanisms with a focus on post-translational regulation of 14-3-3 and dynamic protein-protein interactions that illustrate 14-3-3's role as a stress-adaptive signaling hub in cancer.


Subject(s)
14-3-3 Proteins/metabolism , Signal Transduction/physiology , Stress, Physiological/physiology , Animals , Humans
4.
Sci Total Environ ; 586: 1057-1065, 2017 May 15.
Article in English | MEDLINE | ID: mdl-28214114

ABSTRACT

Post-fire management can have an additional impact on the ecosystem; in some cases, even more severe than the fire. Salvage logging (SL) is a common practice in most fire-affected areas. The management of burnt wood can determine microclimatic conditions and seriously affect soil properties. In some cases, the way of doing it, using heavy machinery, and the vulnerability of soils to erosion and degradation can make this management potentially aggressive to soil. Research was done in "Sierra de Mariola Natural Park" (E Spain). A forest fire (>500ha) occurred in July 2012. In February 2013, SL treatment was applied in a part of the affected forest. Plots for monitoring this effect were installed in this area and in a similar nearby area where no treatment was done, used as control (C). Soil samplings were done immediately after treatment and every 6months during two years. Some soil properties were analysed, including organic matter (OM) content, nitrogen (N) available phosphorous (P) basal soil respiration (BSR), microbial biomass carbon (Cmic), bulk density (BD), water repellency (WR), aggregate stability (AS) and field capacity (FC). SL treatment caused an increase in BD, a decrease of AS, FC, OM and N. In the control area, in general the soil properties remained constant across the 2years of monitoring, and the microbial parameters (BSR and Cmic), initially affected by the fire, recovered faster in C than in the SL area. Plant recovery also showed some differences between treatments. No significant differences were observed in the number of plant species recorded (richness) comparing C versus SL plots, but the number of individuals of each species (evenness) was significantly higher in C plots. In conclusion, we can affirm that for the conditions of this study case, SL had a negative effect on the soil-plant system.

5.
Sci Total Environ ; 575: 1203-1210, 2017 Jan 01.
Article in English | MEDLINE | ID: mdl-27697347

ABSTRACT

Knowledge of the arbuscular mycorrhizal fungal assemblages in the Trachypogon savanna ecosystems is very important to a better understanding of the ecological processes mediated by this soil microbial group that affects multiple ecosystem functions. Considering the hypothesis that the biocrusts can be linked to vegetation through the arbuscular fungi mycelial network, the objectives proposed in this study were to determine (i) whether there are arbuscular mycorrhizal fungi (AMF) in the biocrusts (ii) whether arbuscular mycorrhizal fungal assemblages are linked to the Trachypogon patches, and (iii) whether the composition of the assemblages is related to soil properties affected by microbiological activity. The community structure of the AMF was investigated in three habitats: rhizospheric soil and roots of Trachypogon vestitus, biological soil crusts, and bare soil. The canonical correspondence analysis showed that two soil properties related to enzymatic activity (protease and ß-glucosidase) significantly affected the community composition of the AMF. The biocrusts in the Venezuelan savanna are colonized by an AM fungal community linked to that of the bare soil and significantly different from that hosted by the roots of the surrounding T. vestitus, suggesting that assemblages of AMF in biocrusts might be related more closely to those of annual plant species appearing in favorable conditions.


Subject(s)
Grassland , Mycorrhizae , Poaceae , Soil Microbiology , Ecosystem , Plant Roots , Rhizosphere , Soil , Venezuela
6.
Neurologia ; 30(9): 536-44, 2015.
Article in English, Spanish | MEDLINE | ID: mdl-25066492

ABSTRACT

INTRODUCTION: Stroke is the main cause of admission to Neurology departments and cardioembolic stroke (CS) is one of the most common subtypes of stroke. METHODS: A multicentre prospective observational study was performed in 5 Neurology departments in public hospitals in the Region of Madrid (Spain). The objective was to estimate the use of healthcare resources and costs of acute CS management. Patients with acute CS at<48h from onset were recruited. Patients' socio-demographic, clinical, and healthcare resource use data were collected during hospitalisation and at discharge up to 30 days after admission, including data for rehabilitation treatment after discharge. RESULTS: During an 8-month recruitment period, 128 patients were recruited: mean age, 75.3±11.25; 46.9% women; mortality rate, 4.7%. All patients met the CS diagnostic criteria established by GEENCV-SEN, based on medical history or diagnostic tests. Fifty per cent of the patients had a history of atrial fibrillation and 18.8% presented other major cardioembolic sources. Non-valvular atrial fibrillation was the most frequent cause of CS (33.6%). Data for healthcare resource use, given a mean total hospital stay of 10.3±9.3 days, are as follows: rehabilitation therapy during hospital stay (46.9%, mean 4.5 days) and after discharge (56.3%, mean 26.8 days), complications (32%), specific interventions (19.5%), and laboratory and diagnostic tests (100%). Head CT (98.4%), duplex ultrasound of supra-aortic trunks (87.5%), and electrocardiogram (85.9%) were the most frequently performed diagnostic procedures. Average total cost per patient during acute-phase management and rehabilitation was €13,139. Hospital stay (45.0%) and rehabilitation at discharge (29.2%) accounted for the largest part of resources used. CONCLUSIONS: Acute CS management in the Region of Madrid resulted consumes large amounts of resources (€13,139), mainly due to hospital stays and rehabilitation.


Subject(s)
Embolism/complications , Heart Diseases/complications , Stroke/economics , Stroke/therapy , Adult , Aged , Aged, 80 and over , Atrial Fibrillation/complications , Embolism/therapy , Female , Heart Diseases/therapy , Hospital Costs , Humans , Male , Middle Aged , Prospective Studies , Rehabilitation/economics , Spain/epidemiology , Stroke/etiology
7.
Oncogene ; 34(16): 2052-60, 2015 Apr 16.
Article in English | MEDLINE | ID: mdl-24909166

ABSTRACT

Smoking is an established risk factor for pancreatic cancer (PC), but late diagnosis limits the evaluation of its mechanistic role in the progression of PC. We used a well-established genetically engineered mouse model (LSL-K-ras(G12D)) of PC to elucidate the role of smoking during initiation and development of pancreatic intraepithelial neoplasia (PanIN). The 10-week-old floxed mice (K-ras(G12D); Pdx-1cre) and their control unfloxed (LSL-K-ras(G12D)) littermates were exposed to cigarette smoke (total suspended particles: 150 mg/m(3)) for 20 weeks. Smoke exposure significantly accelerated the development of PanIN lesions in the floxed mice, which correlated with tenfold increase in the expression of cytokeratin19. The systemic accumulation of myeloid-derived suppressor cells (MDSCs) decreased significantly in floxed mice compared with unfloxed controls (P<0.01) after the smoke exposure with the concurrent increase in the macrophage (P<0.05) and dendritic cell (DCs) (P<0.01) population. Further, smoking-induced inflammation (IFN-γ, CXCL2; P<0.05) was accompanied by enhanced activation of pancreatic stellate cells and elevated levels of serum retinoic acid-binding protein 4, indicating increased bioavailability of retinoic acid which contributes to differentiation of MDSCs to tumor-associated macrophages (TAMs) and DCs. TAMs predominantly contribute to the increased expression of heparin-binding epidermal growth factor-like growth factor (EGFR ligand) in pre-neoplastic lesions in smoke-exposed floxed mice that facilitate acinar-to-ductal metaplasia (ADM). Further, smoke exposure also resulted in partial suppression of the immune system early during PC progression. Overall, the present study provides a novel mechanism of smoking-induced increase in ADM in the presence of constitutively active K-ras mutation.


Subject(s)
Carcinoma in Situ/pathology , Heparin-binding EGF-like Growth Factor/biosynthesis , Macrophages/cytology , Myeloid Cells/cytology , Pancreatic Neoplasms/pathology , Smoking/adverse effects , Acinar Cells/pathology , Animals , Carcinoma, Pancreatic Ductal/pathology , Cell Differentiation/genetics , Chemokine CXCL2/biosynthesis , Dendritic Cells/cytology , Disease Progression , Genes, ras/genetics , Inflammation/chemically induced , Interferon-gamma/biosynthesis , Keratin-19/biosynthesis , Macrophages/metabolism , Metaplasia/chemically induced , Mice , Mice, Transgenic , Pancreatic Ducts/pathology , Pancreatic Stellate Cells/metabolism , Receptors, Retinoic Acid/metabolism , Signal Transduction/genetics , Smoke/adverse effects , Tretinoin/metabolism
8.
Minerva Med ; 100(5): 385-400, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19910891

ABSTRACT

Ovarian cancer, the most aggressive gynecologic cancer, is the foremost cause of death from gynecologic malignancies in the developed world. Over 90% of ovarian cancers arise from the surface epithelium, which are classified as epithelial ovarian cancer (EOC). EOCs can be categorized as serous, mucinous, endometrioid, clear cell, and transitional cell types. The molecular pathology of ovarian carcinomas is heterogeneous and involves various putative precursor lesions and multiple pathways of development. Furthermore, in another aspect, immune deficiencies that are present in the ovarian tumor environment enhance the progression of the tumor in the host. The presence of regulatory T cells, the inhibition of natural killer cytotoxic responses, the accumulation of myeloid suppressor cells in the tumor, deficiencies on interferon signaling, the secretion of cytokines that enhance tumor growth (i.e., IL-6, IL-10, CSF-1, TGF-b, TNF), and the expression of surface molecules (i.e., HLA-G, B7-H1, B7-H4, CD40, CD80) that have a role on immune suppression, are discussed in detail. The aim of this review is to provide insight of the evidence that supports the role of immunodeficiency in the progression of ovarian cancer and future directions for ovarian cancer therapies. It also discusses the genetic alterations in the subtypes of ovarian cancers.


Subject(s)
Biomarkers, Tumor/immunology , Carcinoma/immunology , Immunocompromised Host , Ovarian Neoplasms/immunology , Antigens, CD/immunology , B7-1 Antigen/immunology , B7-H1 Antigen , CD40 Antigens/immunology , Carcinoma/classification , Carcinoma/genetics , Carcinoma/pathology , Cell Transformation, Neoplastic/immunology , Cytokines/immunology , Disease Progression , Female , HLA Antigens/immunology , HLA-G Antigens , Histocompatibility Antigens Class I/immunology , Humans , Killer Cells, Natural/immunology , Ovarian Neoplasms/classification , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , T-Lymphocytes/immunology , V-Set Domain-Containing T-Cell Activation Inhibitor 1
10.
Environ Microbiol ; 11(10): 2649-59, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19573133

ABSTRACT

In the present study, we used PCR-Single-Stranded Conformation Polymorphism (SSCP) techniques to analyse arbuscular mycorrhizal fungi (AMF) communities in four sites within a 10 km(2) gypsum area in Southern Spain. Four common plant species from these ecosystems were selected. The AM fungal small-subunit (SSU) rRNA genes were subjected to PCR, cloning, SSCP analysis, sequencing and phylogenetic analyses. A total of 1443 SSU rRNA sequences were analysed, for 21 AM fungal types: 19 belonged to the genus Glomus, 1 to the genus Diversispora and 1 to the Scutellospora. Four sequence groups were identified, which showed high similarity to sequences of known glomalean species or isolates: Glo G18 to Glomus constrictum, Glo G1 to Glomus intraradices, Glo G16 to Glomus clarum, Scut to Scutellospora dipurpurescens and Div to one new genus in the family Diversisporaceae identified recently as Otospora bareai. There were three sequence groups that received strong support in the phylogenetic analysis, and did not seem to be related to any sequences of AM fungi in culture or previously found in the database; thus, they could be novel taxa within the genus Glomus: Glo G4, Glo G2 and Glo G14. We have detected the presence of both generalist and potential specialist AMF in gypsum ecosystems. The AMF communities were different in the plant studied suggesting some degree of preference in the interactions between these symbionts.


Subject(s)
Biodiversity , Desert Climate , Glomeromycota/isolation & purification , Magnoliopsida/microbiology , Mycorrhizae/isolation & purification , Apocynaceae/microbiology , Caryophyllaceae/microbiology , Cistaceae/microbiology , DNA, Fungal/analysis , DNA, Fungal/genetics , Genes, Fungal , Genes, rRNA , Glomeromycota/genetics , Glomeromycota/growth & development , Lamiaceae/microbiology , Molecular Sequence Data , Mycorrhizae/genetics , Mycorrhizae/growth & development , Phylogeny , Spain , Species Specificity
11.
Microb Ecol ; 57(4): 718-27, 2009 May.
Article in English | MEDLINE | ID: mdl-18766400

ABSTRACT

The community composition of arbuscular mycorrhizal fungi (AMF) was analyzed in roots of Gypsophila struthium growing in gypsum soils under semiarid conditions. In order to investigate the effect of plant community degradation on the AMF biodiversity at the single species level, on the basis of the plant community complexity level, we selected four areas affected by degradation and shrub species spatial heterogeneity. The AM fungal community colonizing G. struthium was investigated from the morphological and molecular points of view. All plants were well colonized and showed a high level of infective AM propagules. Roots were analyzed by polymerase chain reaction, restriction fragment length polymorphism screening, and sequence analyses of the ribosomal DNA small subunit region. Four AM fungal types were identified and clustered into the AM fungal family: Glomeraceae, Glomus being the only taxon present. One fungal type was present in all the selected areas. Two fungal types are distinct from any previously published sequences and could be specific to gypsum soils. The chemical-physical properties of the soil were not correlated with the AMF diversity in roots. Our data show vegetation cover complexity-dependent differences in the AM fungal community composition.


Subject(s)
Biodiversity , Caryophyllaceae/microbiology , Glomeromycota/genetics , Mycorrhizae/genetics , Soil Microbiology , DNA, Fungal/genetics , DNA, Ribosomal/genetics , Glomeromycota/classification , Mycorrhizae/classification , Phylogeny , Plant Roots/microbiology , Polymorphism, Restriction Fragment Length , Soil/analysis , Species Specificity , Spores, Fungal/classification , Spores, Fungal/genetics
12.
Int J Biochem Cell Biol ; 39(1): 133-45, 2007.
Article in English | MEDLINE | ID: mdl-16978906

ABSTRACT

While arginine-glycine-aspartic acid-based peptidomimetics have been employed for the treatment of cardiovascular disorders and cancer, their use in other contexts remains to be explored. Arginine-glycine-aspartic acid-serine induces Transforming growth factor-beta1 transcription in human mesangial cells, but the molecular mechanisms involved have not been studied extensively. We explored whether this effect could be due to Activator protein-1 activation and studied the potential pathways involved. Addition of arginine-glycine-aspartic acid-serine promoted Activator protein-1 binding to its cognate sequence within the Transforming growth factor-beta1 promoter as well as c-jun and c-fos protein abundance. Moreover, this effect was suppressed by curcumin, a c-Jun N terminal kinase inhibitor, and was absent when the Activator protein-1 cis-regulatory element was deleted. Activator protein-1 binding was dependent on the activity of integrin linked kinase, as transfection with a dominant negative mutant suppressed both Activator protein-1 binding and c-jun and c-fos protein increment. Integrin linked kinase was, in turn, dependent on Phosphoinositol-3 kinase activity. Arginine-glycine-aspartic acid-serine stimulated Phosphoinositol-3 kinase activity, and Transforming growth factor-beta1 promoter activation was abrogated by the use of Phosphoinositol-3 kinase specific inhibitors. In summary, we propose that arginine-glycine-aspartic acid-serine activates Integrin linked kinase via the Phosphoinositol-3 kinase pathway and this leads to activation of c-jun and c-fos and increased Activator protein-1 binding and Transforming growth factor-beta1 promoter activity. These data may contribute to understand the molecular mechanisms involved in the cellular actions of arginine-glycine-aspartic acid-related peptides and enhance their relevance as these products evolve into clinical therapeutic use.


Subject(s)
Mesangial Cells/metabolism , Peptides, Cyclic/pharmacology , Promoter Regions, Genetic , Signal Transduction/drug effects , Transcription Factor AP-1/metabolism , Transforming Growth Factor beta1/biosynthesis , Up-Regulation/drug effects , Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/metabolism , Cells, Cultured , Enzyme Activation/drug effects , Enzyme Activation/genetics , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Mutation , Neoplasms/drug therapy , Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction/genetics , Transforming Growth Factor beta1/genetics
13.
Nefrología (Madr.) ; 25(supl.2): 46-50, jun. 2005. graf
Article in Es | IBECS | ID: ibc-040024

ABSTRACT

Frecuentemente infraestimado, el deterioro de la función renal característico delenvejecimiento tiene consecuencias clínicas muy relevantes. En el presente artículose analizan algunos aspectos de la biología celular y molecular de este proceso,subrayándose el papel crítico del stress oxidativo y del TGF ��, así como tambiénprobablemente de condicionantes genéticos. Estos conocimientos pueden contribuira desarrollar estrategias terapéuticas útiles para prevenir el declinar de la funciónrenal que acontece con el envejecimiento


Frequently underestimated, the deterioration of the renal function characteristicof the aging has very prominent clinical consequences. In the present article someaspects of the cellular and molecular biology of this process are analysed. The criticalrole of the oxidative stress and of TGF �� are underlined. Determinant geneticfactors are also mentioned. Such a knowledge can contribute to develop therapeuticalstrategies to prevent the decline of the renal function that happens withthe aging


Subject(s)
Aged , Animals , Rats , Humans , Aging/metabolism , Aging/psychology , Kidney/metabolism , Kidney Cortex/metabolism , Kidney Cortex/physiology , Kidney/physiology , Age Factors , Antioxidants/administration & dosage , Blotting, Northern , Extracellular Matrix/metabolism , Free Radicals , Glomerular Filtration Rate/physiology , Oxidative Stress/physiology , RNA, Messenger/analysis
14.
Am J Physiol Cell Physiol ; 286(6): C1335-43, 2004 Jun.
Article in English | MEDLINE | ID: mdl-14761892

ABSTRACT

Progressive renal diseases are characterized by an increased synthesis of extracellular matrix (ECM) components. The mechanisms involved in the development of these alterations are not completely known, but a crucial role for TGF-beta 1 has been suggested. Moreover, the ability of the ECM to modulate the phenotypic expression of different cell types has been widely described. In experiments presented here, human mesangial cells (HMC) were grown on collagen type I (COL I) or IV (COL IV). ECM protein and TGF-beta 1 mRNA expression were evaluated by Northern blot analysis, and TGF-beta 1 secretion was evaluated by ELISA. The involvement of tyrosine kinase and serine-threonine kinase pathways was studied by Western blot analysis, immunofluorescence, and in vitro kinase assays. HMC cultured on COL I showed an increased mRNA expression of COL I and COL IV, fibronectin, and TGF-beta 1. Both tyrosine phosphorylation and integrin-linked kinase (ILK) activity increased when HMC were cultured on COL I, and blockade of these pathways inhibited the increased secretion of TGF-beta 1. In conclusion, the present results support a role for extracellular COL I in the regulation of TGF-beta 1 synthesis during progressive renal sclerosis and fibrosis and the subsequent increase in newly synthesized ECM proteins. In addition, ILK, along with the tyrosine kinases, participates in the genesis of this effect.


Subject(s)
Collagen Type I/metabolism , Extracellular Matrix/genetics , Fibroblasts/metabolism , Glomerular Mesangium/metabolism , Transforming Growth Factor beta/metabolism , Cells, Cultured , Collagen Type I/pharmacology , Collagen Type IV/metabolism , Collagen Type IV/pharmacology , Extracellular Matrix Proteins/biosynthesis , Extracellular Matrix Proteins/genetics , Fibroblasts/drug effects , Glomerular Mesangium/cytology , Glomerular Mesangium/drug effects , Humans , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta1 , Tyrosine/metabolism , Up-Regulation/drug effects , Up-Regulation/genetics
16.
FASEB J ; 17(11): 1529-31, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12824296

ABSTRACT

Extracellular matrix (ECM) components, through specific peptide motifs such as Arg-Gly-Asp (RGD), interact with integrins and can modify the behavior of cells. Transforming growth factor-beta1 (TGF-beta1) is the main cytokine involved in the synthesis of ECM proteins. We analyzed the effect of a RGD-containing peptide, as Arg-Gly-Asp-Ser (RGDS), on the regulation of TGF-beta1 secretion in cultured human mesangial cells. We found that RGDS increased mRNA expression and secretion of TGF-beta1 by stimulating the TGF-beta1 gene promoter. This effect was dependent on the interaction of RGDS with integrins. We evaluated the signaling pathways implicated in TGF-beta1 production by analyzing the effect of RGDS on kinase-related integrins. RGDS stimulated tyrosine phosphorylation as well as integrin-linked kinase (ILK) activity. However, tyrosine kinase inhibitors did not prevent the RGDS effect. In contrast, the inhibition of ILK by cell transfection with a kinase dead-ILK completely abolished the increased TGF-beta1 secretion and promoter activity in the presence of RGDS. Thus RGDS modulates the secretion of TGF-beta1, probably through increased synthesis by interacting with integrins and activating ILK. This supports a role for ECM components in the regulation of their own secretion.


Subject(s)
Integrins/metabolism , Oligopeptides/pharmacology , Transforming Growth Factor beta/biosynthesis , Cells, Cultured , Glomerular Mesangium/cytology , Glomerular Mesangium/metabolism , Humans , Models, Biological , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/biosynthesis , Transcriptional Activation , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1 , Tyrosine/metabolism
17.
Antioxid Redox Signal ; 4(6): 869-75, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12573135

ABSTRACT

Vascular smooth muscle cells (VSMC) exhibit a hypertrophic and contractile response after angiotensin II (Ang II) treatment, and the NADH/NADPH oxidase-dependent synthesis of hydrogen peroxide (H(2)O(2)) seems to play a central role in these responses. Present experiments were designed to analyze the mechanisms responsible for the rapid changes induced by Ang II in the intracellular H(2)O(2) concentration in VSMC. Ang II induced a quick and transient increase of dichlorodihydrofluorescein (DCHF) fluorescence in VSMC, an effect that was completely abolished by catalase and by diethyldithiocarbamate, a cell-permeable superoxide dismutase inhibitor. Losartan and pertussis toxin prevented the stimulatory effect of Ang II. Both diphenylene iodonium (NADH/NADPH oxidase blocker) and 3-(4-octadecylbenzoyl)acrylic acid (phospholipase A2 blocker) inhibited the changes in DCHF fluorescence induced by Ang II, in a dose-dependent fashion, and the effects of both inhibitors were additive. These data demonstrate that Ang II induces a very quick and transient increase of H(2)O(2) in VSMC. This effect depends on the receptor type 1, is linked to a G protein, and involves both NADH/NADPH oxidase and phospholipase A2 activation. The mechanism may be related to the previously proposed role of H(2)O(2) in the genesis of the Ang II-induced cell contraction.


Subject(s)
Angiotensin II/pharmacology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Reactive Oxygen Species/metabolism , Acrylates/pharmacology , Angiotensin II/metabolism , Animals , Benzoates , Catalase/metabolism , Catalase/pharmacology , Cells, Cultured , Ditiocarb/pharmacology , Enzyme Inhibitors/pharmacology , Fluoresceins/chemistry , Fluoresceins/metabolism , Fluorescence , Hydrogen Peroxide/metabolism , Indomethacin/pharmacology , Losartan/pharmacology , Muscle, Smooth, Vascular/cytology , NADH, NADPH Oxidoreductases/antagonists & inhibitors , NADH, NADPH Oxidoreductases/metabolism , Onium Compounds/pharmacology , Pertussis Toxin/pharmacology , Phospholipases A/antagonists & inhibitors , Phospholipases A/metabolism , Phospholipases A2 , Rats , Rats, Wistar
18.
Anal Chem ; 73(24): 5777-83, 2001 Dec 15.
Article in English | MEDLINE | ID: mdl-11791544

ABSTRACT

Chemiluminescence (CL) detection is seldom used in two-dimensional solid support microarray platforms because adequate sensitivity and spatial resolution is difficult to achieve. The three-dimensional ordered microchannels of the Flow-thru Chip increase both the sensitivity and spatial resolution required for quantitative CL measurements on microarrays. Enzyme-catalyzed CL reactions for the detection of hybridizations on microchannel glass were imaged using a CCD camera. Signal uniformity, sensitivity, and dynamic range of the detection method were determined. The relative standard deviation of signal intensities across an array of 64 spots was 8.1%. A detection limit of 250 amol of target with a linear dynamic range of 3 orders of magnitude was obtained for a 3-h assay. Similar to two-color fluorescence measurements, multiple enzyme labels were employed to demonstrate two-channel chemiluminescence. A unique method for measuring the relaxation time of a chemiluminescent species is also described.


Subject(s)
Nanotechnology/instrumentation , Oligonucleotide Array Sequence Analysis/instrumentation , Calibration , Fluorescence , Luminescent Measurements
19.
Aten Primaria ; 26(5): 298-301, 2000 Sep 30.
Article in Spanish | MEDLINE | ID: mdl-11100598

ABSTRACT

OBJECTIVE: To evaluate the association between urinary tract infection (UTI), vesical-uretero-renal reflux (VUR) and renal parenchyma damage, without chronic renal failure. PATIENTS AND METHODS: Retrospective study of 85 children with UTI consulting at the ambulatory paediatric unit of a private university centre in Chile, with at least 3 anthropometric evaluations: before, during and after the UTI. The group was divided into two subgroups: with and without VUR. A descriptive and inferential analysis was made. Simple regression modelling and likelihood ratio test was done for the anthropometric measures. RESULTS: Thirty nine children had VUR (46%). Both subgroups were similar. There were no difference in the nutritional status between these. Only girls with VUR have a tendency toward lower height. There was significant association between renal damage, VUR and degree of severity of it. There was no difference in the index weight for height among children with renal damage with or without RVU. In this series, other risk factors as less age, recurrent UTI and bilateral VUR with renal damage, were not cause of malnourish and lower height. CONCLUSIONS: UTI with or without VUR, with or without renal damage, excluding chronic renal failure, might not be a cause of bad physical development in children.


Subject(s)
Growth , Urinary Tract Infections/complications , Vesico-Ureteral Reflux/complications , Age Factors , Body Height , Body Weight , Child, Preschool , Female , Growth Disorders/etiology , Humans , Infant , Infant, Newborn , Male , Retrospective Studies , Sex Factors
20.
Aten. prim. (Barc., Ed. impr.) ; 26(5): 298-301, sept. 2000.
Article in Es | IBECS | ID: ibc-4270

ABSTRACT

Objetivo. Evaluar la asociación entre infección urinaria (ITU), reflujo vesicoureteral (RVU) y daño del parénquima renal cintigráfico, con alteraciones del crecimiento pondoestatural, en ausencia de insuficiencia renal. Pacientes y métodos. Estudio retrospectivo de 85 niños con ITU que consultaron a la unidad de pediatría ambulatoria de un centro privado universitario chileno, con al menos 3 evaluaciones antropométricas: antes, durante y después de ITU. Se dividieron en 2 grupos: con y sin RVU. Se realizó análisis estadístico descriptivo e inferencias para las variables; así mismo se llevó cabo regresión simple y test de verosimilitud para las curvas antropométricas. Resultados. Treinta y nueve niños (46 por ciento) tenían RVU. Las características de la población fueron similares en ambos grupos, sin presentar alteraciones significativas en el estado nutricional. Sólo las niñas con RVU mostraron tendencia a una menor talla. La presencia de lesión renal se asoció a RVU y a grados severos de éste. No hubo diferencia significativa en el IPT en niños con lesión renal cintigráfica con y sin RVU. Otros factores de riesgo como menor edad, ITU recurrente y presencia de RVU bilateral con daño del parénquima renal cintigráfico, no constituyeron una causa de desnutrición o talla baja en esta serie. Conclusiones. La ITU asociada o no a RVU, con o sin lesión del parénquima renal, en ausencia de insuficiencia renal crónica, no constituiría causa de deterioro del crecimiento pondoestatural en los niños (AU)


Subject(s)
Child, Preschool , Male , Infant , Infant, Newborn , Female , Humans , Growth , Sex Factors , Urinary Tract Infections , Vesico-Ureteral Reflux , Retrospective Studies , Body Height , Body Weight , Age Factors , Growth Disorders
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