Obligaciones tributarias y la recaudación tributaria de las MYPES comercializadoras de Lima Metropolitana, período 2017

La investigación realizada nos permitió conocer una realidad tangible de las mypes comerciales en Lima Metropolitana y está relacionado con las obligaciones en el aspecto tributario que tienen las empresas y la forma como se recaudan impuestos; este grupo de empresas así como otras tienen muchos problemas por poder cumplir puntualmente con el pago de sus impuestos bien sea por deficiencias en el manejo interno de impuestos, por las altas tasas de impuestos, por la presión tributaria, por el desconocimiento del manejo tributario o cualquier otro factor que incide en esta problemática. El objetivo de la investigación planteada fue determinar si el cumplimiento de las obligaciones tributarias contribuye con la recaudación tributaria en las Mypes comerciales de Lima Metropolitana, período 2017 El tipo de investigación fue aplicada, en cuanto al nivel de Investigación fue de tipo descriptiva y explicativa; en cuanto al método fue mixto (cuantitativo y cualitativo). Se aplicó un instrumento (encuesta) a un total de 91 empleados de las mypes comerciales en Lima Metropolitana. La principal conclusión a la que se llegó, permitió demostrar que con el cumplimiento de las obligaciones tributarias se puede influir significativamente con la recaudación tributaria en las Mypes comerciales de Lima Metropolitana, período 2017

Rat Adrenal Chromaffin Cells Are Neonatal CO2 Sensors

We studied the participation of adrenal medulla (AM) chromaffin cells in hypercapnic chemotransduction. Using amperometric recordings, we measured catecholamine (CAT) secretion from cells inAMslices of neonatal and adult rats perfused with solutions bubbled with different concentrations of CO2. The secretory activity augmented from 1.74_0.19 pC/min at 5% CO2 to 6.36_0.77 pC/min at 10% CO2. This response to CO2 was dose dependent and appeared without changes in extracellular pH, although it was paralleled by a drop in intracellular pH. Responsiveness to hypercapnia was higher in neonatal than in adult slices. The secretory response to hypercapnia required extracellular Ca2_ influx. Both the CO2-induced internal pH drop and increase in CAT secretion were markedly diminished by methazolamide (2_M), a membrane-permeant carbonic anhydrase (CA) inhibitor.Wedetected the presence of twoCAisoforms (CAI and CAII) in neonatalAMslices by in situ hybridization and real-time PCR. The expression of these enzymes decreased in adultAMtogether with the disappearance of responsiveness to CO2. In patch-clamped chromaffin cells, hypercapnia elicited a depolarizing receptor potential, which led to action potential firing, extracellular Ca2_ influx, and CAT secretion. This receptor potential (inhibited by methazolamide) was primarily attributable to activation of a resting cationic conductance. In addition, voltage-gated K_ current amplitude was also decreased by high CO2. The CO2-sensing properties of chromaffin cells may be of physiologic relevance, particularly for the adaptation of neonates to extrauterine life, before complete maturation of peripheral and central chemoreceptors

Trophic restoration of the nigrostriatal dopaminergic pathway in long-term carotid body-grafted parkinsonian rats

We studied the mechanisms underlying long-term functional recovery of hemiparkinsonian rats grafted intrastriatally with carotid body (CB) cell aggregates. Amelioration of their motor syndrome is a result of the trophic actions of these grafts on the remaining ipsilateral substantia nigra neurons rather than of the release of dopamine from the CB grafts. The grafts maintain a stable morphological appearance and differentiated cell phenotype for the duration of the life of the host. Adult CB expresses high levels of glial cell line-derived neurotrophic factor (GDNF) and the multicomponent GDNF receptor complex. These properties may contribute to the trophic actions of the CB transplants on nigrostriatal neurons and to their extraordinary longevity. We show that CB glomus cells, although highly dopaminergic, are protected from dopamine-mediated oxidative damage because of the absence of the high-affinity dopamine transporter. Thus, intrastriatal CB grafts are uniquely suited for long-term delivery of trophic factors capable of promoting restoration of the nigrostriatal pathway

Aquaporin-4 facilitates cell proliferation in retinal müller cells: implications in neuromyelitis optica

Müller cells are involved in controlling extracellular homeostasis in the retina, regulating cell swelling by a regulatory volume decrease (RVD) mechanism that depends on the efflux of solutes and water through Aquaporin-4 (AQP4). Müller cells are also important for retinal integrity, as they respond to injury by re-entering the cell cycle for tissue repair. Since AQP4 was reported to modulate cell volume during cell cycle progression and facilitate proliferation in astrocytes, the aim of this study was to evaluate, using the novel inhibitor TGN-020, if AQP4 was involved in human Müller cells? proliferation in physiological conditions. Considering that AQP4 is the target of autoantibody IgG-NMO present in the sera of patients with Neuromyelitis Optica (NMO), we also evaluated if cell proliferation was altered in the presence of IgG-NMO. MIO-M1 human Müller cells were exposed to 100 nM TNG-020 or vehicle or to 1/50 dilution of IgG-NMO positive or control sera. Cell volume (videomicroscopy) and cell proliferation (cell count, cell cycle analysis by flow cytometry and BrdU incorporation by immunofluorescence) were measured. AQP4 inhibition with TGN-020 reduced osmotic water permeability (Pf, µm/s) from 20.3±1.2 to 12.2±0.4 (n=5, p<0.001) and %RVD 15min from 54±4 to 17±3 (n=5, p<0.001). MIO-M1 cell proliferation was decreased by TGN-020 (doubling time in hours, control vs. TGN-020: 31±1 vs. 40±3, n=4, p<0.05) without affecting cell viability. TGN-020 also increased the % of cells in G1/G0 phase, decreased the S phase of cell cycle and reduced BrdU incorporation by 20%. IgG-NMO positive sera decreased AQP4 plasma membrane expression in MIO-M1 cells, reducing Pf from 22.4±1.5 to 15.9±0.6 µm/s (n=6, p<0.001) and %RVD 15min from 66±5 to 48±4 (n=6, p<0.005), as well as cell proliferation (doubling time in hours, control vs. IgG-NMO: 59±5 vs. 86±4, n=3, p<0.05) in comparison to control sera. We propose that inhibition or removal of AQP4 from the plasma membrane reduces AQP4-mediated water permeability altering cell proliferation. This is of particular importance in NMO, as the decreased ability of Müller cells to proliferate may affect retinal tissue repair.Fil: Netti, Vanina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: White, Alan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Di Giusto, Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Fernández, Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Ford, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Echevarría, Miriam. Universidad de Sevilla; EspañaFil: Capurro, Claudia Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaReunión Anual de la Sociedad Argentina de FisiologíaRosarioArgentinaSociedad Argentina de Fisiologi

The Expression of AQP1 IS Modified in Lung of Patients With Idiopathic Pulmonary Fibrosis: Addressing a Possible New Target

Activation of the epithelial-mesenchymal transition process (EMT) by which alveolar cells in human lung tissue undergo differentiation giving rise to a mesenchymal phenotype (fibroblast/miofibroblasts) has been well recognized as a key element in the origin of idiopathic pulmonary fibrosis (IPF). Here we analyzed expression of AQP1 in lung biopsies of patients diagnosed with IPF, and compared it to biopsies derived from patients with diverse lung pneumonies, such as hypersensitivity pneumonitis, sarcoidosis or normal lungs. Immunostaining for AQP1 showed a clear increment of AQP1 localized in the alveolar epithelium in biopsies from IPF patients alone. Moreover, to examine the possible participation of AQP1 in the pathophysiology of IPF, we evaluated its role in the pro-fibrotic transformation induced by transforming growth factor (TGF-β) in vitro. Human alveolar epithelial cells (A549), and fibroblasts derived from an IPF patient (LL29), or fibroblasts from healthy normal lung tissue (MRC-5), were treated with TGF-β, and levels of expression of AQP1, as well as those of E-cadherin, vimentin, α-SMA and collagen were analyzed by RT-qPCR, western blot and immunohistochemistry. An increase of AQP1 mRNA and protein after TGF-β treatment (4-72h) was observed either in A549 or IPF fibroblast-LL29 but not in MRC-5 fibroblasts. A gradual reduction of E-cadherin, and increased expression of vimentin, with no changes in α-SMA levels were observed in A549. Whereas in LL29 and MRC-5, TGF-β1 elicited a large production of collagen and α-SMA that was significantly greater in IPF fibroblast-LL29. Changes observed are consistent with activation of EMT by TGF-β, but whether modifications in AQP1 expression are responsible or independent events occurring at the same time is still unknown. Our results suggest that AQP1 plays a role in the pro-fibrotic TGF-β action and contributes to the etiology and pathophysiology of IPF. Understanding AQP1's role will help us comprehend the fate of this disease.Fondos FEDER (UE)Fundación NeumosurFundación SEPARISCIII Instituto de Salud Carlos II

Predictive Value of Serum Antibodies and Point Mutations of AQP4, AQP1 and MOG in A Cohort of Spanish Patients with Neuromyelitis Optica Spectrum Disorders

The detection of IgG aquaporin-4 antibodies in the serum of patients with Neuromyelitis optica (NMO) has dramatically improved the diagnosis of this disease and its distinction from multiple sclerosis. Recently, a group of patients have been described who have an NMO spectrum disorder (NMOsd) and who are seronegative for AQP4 antibodies but positive for IgG aquaporin-1 (AQP1) or myelin oligodendrocyte glycoprotein (MOG) antibodies. The purpose of this study was to determine whether AQP1 and MOG could be considered new biomarkers of this disease; and if point mutations in the gDNA of AQP4, AQP1 and MOG genes could be associated with the etiology of NMOsd. We evaluated the diagnostic capability of ELISA and cell-based assays (CBA), and analyzed their reliability, specificity, and sensitivity in detecting antibodies against these three proteins. The results showed that both assays can recognize these antigen proteins under appropriate conditions, but only anti-AQP4 antibodies, and not AQP1 or MOG, appears to be a clear biomarker for NMOsd. CBA is the best method for detecting these antibodies; and serum levels of AQP4 antibodies do not correlate with the progression of this disease. So far, the sequencing analysis has not revealed a genetic basis for the etiology of NMOsd, but a more extensive analysis is required before definitive conclusions can be drawn.Ministerio de Economía y CompetitividadFEDER (Grants PI16/01249 y PI16/00493

Phosphate-activated glutaminase activity is enhanced in brain, intestine and kidneys of rats following portacaval anastomosis

AIM: To assess whether portacaval anastomosis (PCA) in rats affects the protein expression and/or activity of glutaminase in kidneys, intestines and in three brain areas of cortex, basal ganglia and cerebellum and to explain the neurological alterations found in hepatic encephalopathy (HE). METHODS: Sixteen male Wistar rats weighing 250-350 gwere grouped into sham-operation control (n = 8) or portacaval shunt (n = 8). Twenty-eight days after the procedure, the animals were sacrificed. The duodenum, kidney and brain were removed, homogenised and mitochondria were isolated. Ammonia was measured in brain and blood. Phosphate-activated glutaminase (PAG) activity was determined by measuring ammonia production following incubation for one hour at 37 with O-phthalaldehyde (OPA) and specific activity expressed in units per gram of protein (μkat/g of protein). Protein expression was measured by immunoblotting. RESULTS: Duodenal and kidney PAG activities together with protein content were significantly higher in PCA group than in control or sham-operated rats (duodenum PAG activity was 976.95±268.87 μkat/g of protein in PCA rats vs 429.19±126.92 μkat/g of protein in shamoperated rats; kidneys PAG activity was 1259.18 ± 228.79 μkat/g protein in PCA rats vs 669.67± 400.8 μkat/g of protein in controls, P < 0.05; duodenal protein content: 173% in PCA vs sham-operated rats; in kidneys the content of protein was 152% in PCA vs sham-operated rats). PAG activity and protein expression in PCA rats were higher in cortex and basal ganglia than those in shamoperated rats (cortex: 6646.6 ± 1870.4 μkat/g of protein vs 3573.8 ± 2037.4 μkat/g of protein in control rats, P < 0.01; basal ganglia, PAG activity was 3657.3 ± 1469.6 μkat/g of protein in PCA rats vs 2271.2 ± 384 μkat/g of protein in sham operated rats, P < 0.05; In the cerebellum, the PAG activity was 2471.6 ± 701.4 μkat/g of protein vs 1452.9 ± 567.8 μkat/g of protein in the PCA and sham rats, respectively, P < 0.05; content of protein:cerebral cortex: 162% ± 40% vs 100% ± 26%, P < 0.009;and basal gangl ia: 140% ± 39% vs 100% ± 14%,P < 0.05; but not in cerebel lum: 100% ± 25% vs 100% ± 16%, P = ns). CONCLUSION: Increased PAG activity in kidney and duodenum could contribute significantly to the hyperammonaemia in PCA rats, animal model of encephalopathy. PAG is increased in non-synaptic mitochondria from the cortex and basal ganglia and could be implicated in the pathogenesis of hepatic encephalopathy. Therefore, PAG could be a possible target for the treatment of HE orliver dysfunction

An immunoassay that distinguishes real neuromyelitis optica signals from a labeling detected in patients receiving natalizumab

BACKGROUND: Cell-based assays for neuromyelitis optica (NMO) diagnosis are the most sensitive and specific methods to detect anti-aquaporin 4 (AQP4) antibodies in serum, but some improvements in their quantitative and specificity capacities would be desirable. Thus the aim of the present work was to develop a sensitive quantitative method for detection of anti-AQP4 antibodies that allows clear diagnosis of NMO and distinction of false labeling produced by natalizumab treatment. METHODS: Sera from 167 individuals, patients diagnosed with NMO (16), multiple sclerosis (85), optic neuritis (24), idiopathic myelitis (21), or other neurological disorders (13) and healthy controls (8), were used as the primary antibody in an immunofluorescence assay on HEK cells transfected with the M23 isoform of human AQP4 fused with enhanced green fluorescent protein. Cells used were freshly transfected or stored frozen and then thawed just before adding the serum. RESULTS: Microscopic observation and fluorescence quantification produced similar results in fresh and frozen samples. Serum samples from patients diagnosed with NMO were 100% positive for anti-AQP4 antibodies, while all the other sera were negative. Using serum from patients treated with natalizumab, a small and unspecific fluorescent signal was produced from all HEK cells, regardless of AQP4 expression. CONCLUSIONS: Our cell-based double-label fluorescence immunoassay protocol significantly increases the signal specificity and reduces false diagnosis of NMO patients, especially in those receiving natalizumab treatment. Frozen pretreated cells allow faster detection of anti-AQP4 antibodies

Comparative Analysis for the Presence of IgG Anti-Aquaporin-1 in Patients with NMO-Spectrum Disorders

Detection of IgG anti-Aquaporin-4 (AQP4) in serum of patients with Neuromyelitis optica syndrome disorders (NMOSD) has improved diagnosis of these processes and differentiation from Multiple sclerosis (MS). Recent findings also claim that a subgroup of patients with NMOSD, serum negative for IgG-anti-AQP4, present antibodies anti-AQP1 instead. Explore the presence of IgG-anti-AQP1 using a previously developed cell-based assay (CBA) highly sensitive to IgG-anti-AQP4. Serum of 205 patients diagnosed as NMOSD (8), multiple sclerosis (94), optic neuritis (39), idiopathic myelitis (29), other idiopathic demyelinating disorders of the central nervous system (9), other neurological diseases (18) and healthy controls (8), were used in a CBA over fixed HEK cells transfected with hAQP1-EGFP or hM23-AQP4-EGFP, treated with Triton X-100 and untreated. ELISA was also performed. Analysis of serum with our CBA indicated absence of anti-AQP1 antibodies, whereas in cells pretreated with detergent, noisy signal made reliable detection impossible. ELISA showed positive results in few serums. The low number of NMOSD serums included in our study reduces its power to conclude the specificity of AQP1 antibodies as new biomarkers of NMOSD. Our study does not sustain detection of anti-AQP1 in serum of NMOSD patients but further experiments are expected.Grants from “La Junta de Andalucía, Consejería de Innovación Ciencia y Empresa” (P08-CTS-03574) and Consejería de Salud (PI0298-2010), and from the “Instituto de Salud Carlos III” (Exp. PI12/01882) to Miriam Echevarría funded this work. We thank Genzyme Foundation in multiple sclerosis for giving to Miriam Echevarría one of their 2012 fellowships. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI

Comparative analysis for the presence of IgG anti-aquaporin-1 in patients with NMO-Spectrum disorders

Detection of IgG anti-Aquaporin-4 (AQP4) in serum of patients with Neuromyelitis optica syndrome disorders (NMOSD) has improved diagnosis of these processes and differentiation from Multiple sclerosis (MS). Recent findings also claim that a subgroup of patients with NMOSD, serum negative for IgG-anti-AQP4, present antibodies anti-AQP1 instead. Explore the presence of IgG-anti-AQP1 using a previously developed cell-based assay (CBA) highly sensitive to IgG-anti-AQP4. Serum of 205 patients diagnosed as NMOSD (8), multiple sclerosis (94), optic neuritis (39), idiopathic myelitis (29), other idiopathic demyelinating disorders of the central nervous system (9), other neurological diseases (18) and healthy controls (8), were used in a CBA over fixed HEK cells transfected with hAQP1-EGFP or hM23-AQP4-EGFP, treated with Triton X-100 and untreated. ELISA was also performed. Analysis of serum with our CBA indicated absence of anti-AQP1 antibodies, whereas in cells pretreated with detergent, noisy signal made reliable detection impossible. ELISA showed positive results in few serums. The low number of NMOSD serums included in our study reduces its power to conclude the specificity of AQP1 antibodies as new biomarkers of NMOSD. Our study does not sustain detection of anti-AQP1 in serum of NMOSD patients but further experiments are expected