RESUMEN
Mitochondria are dynamic organelles essential for cell survival whose structural and functional integrity rely on selective and regulated transport of lipids from/to the endoplasmic reticulum (ER) and across the mitochondrial intermembrane space. As they are not connected by vesicular transport, the exchange of lipids between ER and mitochondria occurs at membrane contact sites. However, the mechanisms and proteins involved in these processes are only beginning to emerge. Here, we show that the main physiological localization of the lipid transfer proteins ORP5 and ORP8 is at mitochondria-associated ER membrane (MAM) subdomains, physically linked to the mitochondrial intermembrane space bridging (MIB)/mitochondrial contact sites and cristae junction organizing system (MICOS) complexes that bridge the two mitochondrial membranes. We also show that ORP5/ORP8 mediate non-vesicular transport of phosphatidylserine (PS) lipids from the ER to mitochondria by cooperating with the MIB/MICOS complexes. Overall our study reveals a physical and functional link between ER-mitochondria contacts involved in lipid transfer and intra-mitochondrial membrane contacts maintained by the MIB/MICOS complexes.
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Proteínas Mitocondriales , Fosfatidilserinas , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Fosfatidilserinas/metabolismoRESUMEN
Bradycardia, renal failure, atrioventricular (AV) nodal blockade, shock, and hyperkalemia (BRASH) syndrome commonly occurs in the elderly population with compromised renal function and a history of taking AV nodal blocking agents on a regular basis. Hypovolemia and worsening of renal function are considered to be the major risk factors. BRASH syndrome should be differentiated from pure intoxication with AV nodal blocking agents, as the therapeutic goals of these conditions are different from each other. It encompasses a vicious cycle of bradycardia and decreased cardiac output leading to organ dysfunction including renal failure with hyperkalemia, further augmenting bradycardia. It is usually associated with high morbidity and mortality. Typically, the treatment involves increasing renal blood flow by augmenting cardiac output using catecholamine infusion. Very rarely, interventions such as intralipid emulsion and continuous renal replacement therapy (CRRT) may be required on a case-to-case basis. Promptly recognizing the symptoms of BRASH syndrome can help to avoid diagnostic delays and reduce mortality rates.
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Atrial fibrillation (AF) is one of the most common arrhythmia exhibiting a dramatic rise in prevalence with associated increased risk of stroke, heart failure, and death. No standard symptoms have been categorized yet to set a gold standard in diagnosing this clinical attribute. A highly variable symptoms array has increased the challenges of management in terms of AF. An obvious relationship has not been established between symptoms and the onset or recurrence of arrhythmia. We present a case of a 43-year-old male patient who complained of chronic fatigue as a primary symptom and was diagnosed with AF with myocardial infarction.
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Acute pyelonephritis (APN) is considered a rare cause of acute kidney injury (AKI), especially when no anatomical abnormalities or predisposing factors are identified. Additionally, non-obstructive pyelonephritis is a very infrequent cause of rapidly progressive acute kidney injury. Herein, we present a rare case of a 55-year-old female patient who was diagnosed with acute non-obstructive pyelonephritis leading to AKI eventually requiring hemodialysis. The patient eventually recovered with the administration of intravenous antibiotics with a significant recovery of renal function.
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Bronchospasm is acute narrowing of the airways of lungs, which gives rise to wheezing and shortness of breath. Commonly seen in obstructive lung disease, but a rare finding in patients with hypocalcemia. This is a case that outlines a rare presentation of hypocalcemia induced bronchospasm in a patient with no known history of asthma or chronic obstructive pulmonary disease (COPD). In this report, we present a case of a 57 years old male with no history of asthma or COPD who presented with intractable bronchospasm. Initial work-up for common entities was negative. Patient was found to be profoundly hypocalcemic and treatment provided resolution of symptoms. Early recognition of hypocalcemia induced bronchospasm is important in clinical practice to optimize management and provide improvement in symptoms.
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During angiogenesis, endothelial cells form protrusive sprouts and migrate towards the angiogenic stimulus. In this study, we investigate the role of the endoplasmic reticulum (ER)-anchored protein, Protrudin, in endothelial cell protrusion, migration and angiogenesis. Our results demonstrate that Protrudin regulates angiogenic tube formation in primary endothelial cells, Human umbilical vein endothelial cells (HUVECs). Analysis of RNA sequencing data and its experimental validation revealed cell migration as a prominent cellular function affected in HUVECs subjected to Protrudin knockdown. Further, our results demonstrate that knockdown of Protrudin inhibits focal adhesion kinase (FAK) activation in HUVECs and human aortic endothelial cells (HAECs). This is associated with a loss of polarized phospho-FAK distribution upon Protrudin knockdown as compared to Protrudin expressing HUVECs. Reduction of Protrudin also results in a perinuclear accumulation of mTOR and a decrease in VEGF-mediated S6K activation. However, further experiments suggest that the observed inhibition of angiogenesis in Protrudin knockdown cells is not affected by mTOR disturbance. Therefore, our findings suggest that defects in FAK activation and its abnormal subcellular distribution upon Protrudin knockdown are associated with a detrimental effect on endothelial cell migration and angiogenesis. Furthermore, mice with global Protrudin deletion demonstrate reduced retinal vascular progression. To conclude, our results provide evidence for a novel key role of Protrudin in endothelial cell migration and angiogenesis.
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Neovascularización Patológica , Neovascularización Fisiológica , Animales , Movimiento Celular/genética , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Neovascularización Patológica/genética , Neovascularización Fisiológica/genética , Proteínas de Transporte VesicularRESUMEN
Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute one of the largest families of lipid-binding/transfer proteins (LTPs) in eukaryotes. The current view is that many of them mediate inter-organelle lipid transfer over membrane contact sites (MCS). The transfer occurs in several cases in a 'counter-current' fashion: A lipid such as cholesterol or phosphatidylserine (PS) is transferred against its concentration gradient driven by transport of a phosphoinositide in the opposite direction. In this way ORPs are envisioned to maintain the distinct organelle lipid compositions, with impacts on multiple organelle functions. However, the functions of ORPs extend beyond lipid homeostasis to regulation of processes such as cell survival, proliferation and migration. Important expanding areas of mammalian ORP research include their roles in viral and bacterial infections, cancers, and neuronal function. The yeast OSBP homologue (Osh) proteins execute multifaceted functions in sterol and glycerophospholipid homeostasis, post-Golgi vesicle transport, phosphatidylinositol-4-phosphate, sphingolipid and target of rapamycin (TOR) signalling, and cell cycle control. These observations identify ORPs as lipid transporters and coordinators of signals with an unforeseen variety of cellular processes. Understanding their activities not only enlightens the biology of the living cell but also allows their employment as targets of new therapeutic approaches for disease.
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Receptores de Esteroides , Animales , Transporte Biológico , Colesterol/metabolismo , Glicerofosfolípidos/metabolismo , Mamíferos/metabolismo , Orgánulos/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Esteroles/metabolismoRESUMEN
Oxysterol-binding protein-related protein 2 (ORP2), a cholesterol-PI(4,5)P2 countercurrent transporter, was recently identified as a novel regulator of plasma membrane (PM) cholesterol and PI(4,5)P2 content in HeLa cells. Here, we investigate the role of ORP2 in endothelial cell (EC) cholesterol and PI(4,5)P2 distribution, angiogenic signaling, and angiogenesis. We show that ORP2 knock-down modifies the distribution of cholesterol accessible to a D4H probe, between late endosomes and the PM. Depletion of ORP2 from ECs inhibits their angiogenic tube formation capacity, alters the gene expression of angiogenic signaling pathways such as VEGFR2, Akt, mTOR, eNOS, and Notch, and reduces EC migration, proliferation, and cell viability. We show that ORP2 regulates the integrity of VEGFR2 at the PM in a cholesterol-dependent manner, the depletion of ORP2 resulting in proteolytic cleavage by matrix metalloproteinases, and reduced activity of VEGFR2 and its downstream signaling. We demonstrate that ORP2 depletion increases the PM PI(4,5)P2 coincident with altered F-actin morphology, and reduces both VEGFR2 and cholesterol in buoyant raft membranes. Moreover, ORP2 knock-down suppresses the expression of the lipid raft-associated proteins VE-cadherin and caveolin-1. Analysis of the retinal microvasculature in ORP2 knock-out mice generated during this study demonstrates the subtle alterations of morphology characterized by reduced vessel length and increased density of tip cells and perpendicular sprouts. Gene expression changes in the retina suggest disturbance of sterol homeostasis, downregulation of VE-cadherin, and a putative disturbance of Notch signaling. Our data identifies ORP2 as a novel regulator of EC cholesterol and PI(4,5)P2 homeostasis and cholesterol-dependent angiogenic signaling.
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Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Fisiológica , Receptores de Esteroides/metabolismo , Transducción de Señal , Actinas/metabolismo , Antígenos CD/metabolismo , Cadherinas/metabolismo , Caveolinas/metabolismo , Membrana Celular/metabolismo , Movimiento Celular , Endosomas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Metaloproteinasas de la Matriz/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Notch/metabolismo , Receptores de Esteroides/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Invadopodia are dynamic protrusions that harbor matrix metalloproteinases for pericellular matrix degradation. However, the mechanisms underlying their maturation are poorly understood. Pedersen et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.202003063) demonstrate a dual role of Protrudin in invadopodia elongation and matrix degradation, central to cell invasion and cancer metastasis.
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Podosomas , Endosomas , Exocitosis , Matriz Extracelular , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la MatrizRESUMEN
Oxysterol-binding protein (OSBP)-related proteins (ORPs) constitute a family of intracellular lipid-binding/transport proteins (LTPs) in eukaryotes. They typically have a modular structure comprising a lipid-binding domain and membrane targeting determinants, being thus suited for function at membrane contact sites. Among the mammalian ORPs, ORP2/OSBPL2 is the only member that only exists as a 'short' variant lacking a membrane-targeting pleckstrin homology domain. ORP2 is expressed ubiquitously and has been assigned a multitude of functions. Its OSBP-related domain binds cholesterol, oxysterols, and phosphoinositides, and its overexpression enhances cellular cholesterol efflux. Consistently, the latest observations suggest a function of ORP2 in cholesterol transport to the plasma membrane (PM) in exchange for phosphatidylinositol 4,5-bisphosphate (PI4,5P2), with significant impacts on the concentrations of PM cholesterol and PI4,5P2. On the other hand, ORP2 localizes at the surface of cytoplasmic lipid droplets (LDs) and at endoplasmic-reticulum-LD contact sites, and its depletion modifies cellular triglyceride (TG) metabolism. Study in an adrenocortical cell line further suggested a function of ORP2 in the synthesis of steroid hormones. Our recent knock-out of ORP2 in human hepatoma cells revealed its function in hepatocellular PI3K/Akt signaling, glucose and triglyceride metabolism, as well as in actin cytoskeletal regulation, cell adhesion, migration and proliferation. ORP2 was shown to interact physically with F-actin regulators such as DIAPH1, ARHGAP12, SEPT9 and MLC12, as well as with IQGAP1 and the Cdc37-Hsp90 chaperone complex controlling the activity of Akt. Interestingly, mutations in OSBPL2 encoding ORP2 are associated with autosomal dominant non-syndromic hearing loss, and the protein was found to localize in cochlear hair cell stereocilia. The functions assigned to ORP2 suggest that this protein, in concert with other LTPs, controls the subcellular distribution of cholesterol in various cell types and steroid hormone synthesis in adrenocortical cells. However, it also impacts cellular TG and carbohydrate metabolism and F-actin-dependent functions, revealing a bewildering spectrum of activities.
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Actinas/metabolismo , Metabolismo de los Hidratos de Carbono , Metabolismo de los Lípidos , Receptores de Esteroides/metabolismo , Transducción de Señal , Transporte Biológico , HumanosRESUMEN
ORP2 is a sterol-binding protein with documented functions in lipid and glucose metabolism, Akt signaling, steroidogenesis, cell adhesion, migration and proliferation. Here we investigate the interactions of ORP2 with phosphoinositides (PIPs) by surface plasmon resonance (SPR), its affinity for cholesterol with a pull-down assay, and its capacity to transfer sterol in vitro. Moreover, we determine the effects of wild-type (wt) ORP2 and a mutant with attenuated PIP binding, ORP2(mHHK), on the subcellular distribution of cholesterol, and analyze the interaction of ORP2 with the related cholesterol transporter ORP1L. ORP2 showed specific affinity for PI(4,5)P2, PI(3,4,5)P3 and PI(4)P, with suggestive Kd values in the µM range. Also binding of cholesterol by ORP2 was detectable, but a Kd could not be determined. Wt ORP2 was in HeLa cells mainly detected in the cytosol, ER, late endosomes, and occasionally on lipid droplets (LDs), while ORP2(mHHK) displayed an enhanced LD localization. Overexpression of wt ORP2 shifted the D4H cholesterol probe away from endosomes, while ORP2(mHHK) caused endosomal accumulation of the probe. Although ORP2 failed to transfer dehydroergosterol in an in vitro assay where OSBP is active, its knock-down resulted in the accumulation of cholesterol in late endocytic compartments, as detected by both D4H and filipin probes. Interestingly, ORP2 was shown to interact and partially co-localize on late endosomes with ORP1L, a cholesterol transporter/sensor at ER-late endosome junctions. Our data demonstrates that ORP2 binds several phosphoinositides, both PI(4)P and multiply phosphorylated species. ORP2 regulates the subcellular distribution of cholesterol dependent on its PIP-binding capacity. The interaction of ORP2 with ORP1L suggests a concerted action of the two ORPs.
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Colesterol/metabolismo , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Gotas Lipídicas/metabolismo , Fosfatidilinositoles/metabolismo , Receptores de Esteroides/metabolismo , Colesterol/genética , Retículo Endoplásmico/genética , Endosomas/genética , Células HeLa , Humanos , Fosfatidilinositoles/genética , Receptores de Esteroides/genéticaRESUMEN
BACKGROUND: Skin adnexal neoplasms are relatively rare and thus uncommonly encountered in routine pathology practice. AIMS: The present study was conducted with the aim to diagnose various skin adnexal tumors on fine-needle aspiration cytology (FNAC) followed by confirmation on histopathology and immunohistochemistry of the excised tumor. MATERIALS AND METHODS: A total of 11 cases of superficial nodular swellings of the skin were studied over a period of 2 years from 2015 to 2016. FNAC of the swelling was performed and slides were stained with Giemsa stain. Following FNAC, the swelling was excised and subjected to histopathological examination. Immunohistochemistry was performed in some cases for confirmation. RESULTS: It was observed that overall incidence of adnexal tumors (ATs) encountered on FNAC was very low. Typing of ATs on cytology was possible in 82% cases (9/11 cases) with diagnostic accuracy of 88% (8/9 cases). Most common tumor encountered in the study was nodular hidradenoma followed by pilomatrixoma, trichoepithelioma, cylindroma, chondroid syringoma, and trichilemmal cyst. Majority of the cases were benign except one case, which subsequently turned malignant. CONCLUSION: FNAC is a simple, safe, and cost-effective tool in diagnosing skin ATs. Cytological diagnosis of these lesions is depends on clinical correlation and cytomorphological features. FNAC in lesions suspected to be malignant can guide the surgeon regarding extent of excision.
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Insulin resistance in brain is well-associated with pathophysiology of deficits in whole-body energy metabolism, neurodegenerative diseases etc. Among the seven sirtuins, SIRT2 is the major deacetylase expressed in brain. Inhibition of SIRT2 confers neuroprotection in case of Parkinson's disease (PD) and Huntington's disease (HD). However, the role of this sirtuin in neuronal insulin resistance is not known. In this study, we report the role of SIRT2 in regulating insulin-sensitivity in neuronal cells in vitro. Using approaches like pharmacological inhibition of SIRT2, siRNA mediated SIRT2 knockdown and over-expression of wild-type and catalytically-mutated SIRT2, we observed that downregulation of SIRT2 ameliorated the reduced activity of AKT and increased insulin-stimulated glucose uptake in insulin resistant neuro-2a cells. The data was supported by over expression of catalytically-inactive SIRT2 in insulin-resistant human SH-SY5Y neuronal cells. Data highlights a crucial role of SIRT2 in regulation of neuronal insulin sensitivity under insulin resistant condition.
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Resistencia a la Insulina/fisiología , Insulina/metabolismo , Neuronas/metabolismo , Sirtuina 2/metabolismo , Línea Celular , Células Cultivadas , Regulación hacia Abajo , HumanosRESUMEN
SIRT2 is primarily a cytoplasmic protein deacetylase and is abundantly expressed in metabolically active tissues like adipocytes and brain. However, its role, if any, in regulating insulin signaling in skeletal muscle cells, is not known. We have examined the role of SIRT2 in insulin-mediated glucose disposal in normal and insulin resistant C2C12 skeletal muscle cells in vitro. SIRT2 was over expressed in insulin resistant skeletal muscle cells. Pharmacological inhibition of SIRT2 increased insulin-stimulated glucose uptake and improved phosphorylation of Akt and GSK3ß in insulin resistant cells. Knockdown of endogenous SIRT2 and over expression of catalytically-inactive SIRT2 mutant under insulin-resistant condition showed similar amelioration of insulin sensitivity. Our results suggest that down-regulation of SIRT2 improved insulin sensitivity in skeletal muscle cells under insulin-resistant condition. Previously it has been reported that down-regulation of SIRT1 and SIRT3 in C2C12 cells results in impairment of insulin signaling and induces insulin resistance. However, we have observed an altogether different role of SIRT2 in skeletal muscle. This implicates a differential regulation of insulin resistance by sirtuins which otherwise share a conserved catalytic domain. The study significantly directs towards future approaches in targeting inhibition of SIRT2 for therapeutic treatment of insulin resistance which is the major risk factor in Type 2 diabetes.
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Diferenciación Celular , Resistencia a la Insulina , Insulina/farmacología , Músculo Esquelético/patología , Sirtuina 2/metabolismo , Animales , Western Blotting , Proliferación Celular , Células Cultivadas , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Inmunoprecipitación , Ratones , Músculo Esquelético/metabolismo , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Sirtuina 2/antagonistas & inhibidores , Sirtuina 2/genéticaRESUMEN
The alkalitolerant and xylanolytic bacterial strain (RMV1) isolated from red mud pond was identified as Paenibacillus montaniterrae based on both biochemical and 16S rDNA sequence analysis. The RMV1 bacterial isolate produced alkalistable and thermostable endoxylanase active over a broad range of pH (4.0-11.0) and temperature (20-100 °C), with optima at 50 °C and pH 9.0 with a T1/2 of 6.7 hours at 50 °C. This is the first report on the isolation of P. montaniterrae from bauxite residue with quite a high xylanase producing ability.
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Endo-1,4-beta Xilanasas/metabolismo , Residuos Industriales , Paenibacillus/enzimología , Paenibacillus/aislamiento & purificación , Óxido de Aluminio/análisis , Endo-1,4-beta Xilanasas/química , Estabilidad de Enzimas , Paenibacillus/química , Paenibacillus/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
OBJECTIVE: To determine (1) whether long-term treatment with exenatide is associated with reductions in C-reactive protein (CRP), systolic blood pressure (BP), and triglyceride concentrations in addition to reductions in body weight and hemoglobin A(1c) (A1C) levels and (2) whether these beneficial results persist without any loss of effect while exenatide is being used, and whether they reverse after its cessation. METHODS: We conducted a retrospective review of 141 patients with type 2 diabetes mellitus treated with exenatide at a tertiary clinic. RESULTS: Exenatide (mean duration of treatment, 1.4 years) decreased A1C (0.7%), weight (5 kg), systolic BP (8 mm Hg), and triglyceride concentrations (46 mg/dL) (P<.05 for all). Sixty-one patients continued exenatide therapy throughout the study (mean duration of use, 2.4 years). Exenatide treatment reduced their mean weight by 7 kg, systolic BP by 8 mm Hg, triglycerides by 52 mg/dL, A1C by 1.3%, and CRP by 2.4 mg/L (P<.05 for all). Reductions in systolic BP and CRP were not related to weight loss. The reduction in CRP concentration was significantly related to the baseline CRP concentration (r = 0.78; P<.001) and to change in A1C (r = 0.68; P = .02). Patients who stopped taking exenatide had a reversal of the benefits within 6 months after cessation of treatment. CONCLUSION: Exenatide treatment in patients with type 2 diabetes has durable and persistent beneficial effects on A1C, weight, CRP, systolic BP, and triglyceride concentrations. Cessation of treatment reverses all these beneficial effects within 6 months. There was no evidence of loss of its effects while exenatide treatment was continued.