Nuclear membrane stretch and its role in mechanotransduction

Most research in nuclear mechanotransduction has focused on the nuclear lamina and lamin binding proteins. These structures provide mechanical stability to the nucleus, establish a link between the cytoskeleton and chromatin, and can transmit mechanical signals. At the same time, mechanical perturbations to the nucleus also affect its phospholipid membranes. In this commentary, we discuss how changes in nuclear membrane tension can mediate mechanotransduction. © 2017 Taylor & Franci

TRESK Background K+ Channel Is Inhibited by PAR-1/MARK Microtubule Affinity-Regulating Kinases in Xenopus Oocytes

TRESK (TWIK-related spinal cord K+ channel, KCNK18) is a major background K+ channel of sensory neurons. Dominant-negative mutation of TRESK is linked to familial migraine. This important two-pore domain K+ channel is uniquely activated by calcineurin. The calcium/calmodulin-dependent protein phosphatase directly binds to the channel and activates TRESK current several-fold in Xenopus oocytes and HEK293 cells. We have recently shown that the kinase, which is responsible for the basal inhibition of the K+ current, is sensitive to the adaptor protein 14-3-3. Therefore we have examined the effect of the 14-3-3-inhibited PAR-1/MARK, microtubule-associated-protein/microtubule affinity-regulating kinase on TRESK in the Xenopus oocyte expression system. MARK1, MARK2 and MARK3 accelerated the return of TRESK current to the resting state after the calcium-dependent activation. Several other serine-threonine kinase types, generally involved in the modulation of other ion channels, failed to influence TRESK current recovery. MARK2 phosphorylated the primary determinant of regulation, the cluster of three adjacent serine residues (S274, 276 and 279) in the intracellular loop of mouse TRESK. In contrast, serine 264, the 14-3-3-binding site of TRESK, was not phosphorylated by the kinase. Thus MARK2 selectively inhibits TRESK activity via the S274/276/279 cluster, but does not affect the direct recruitment of 14-3-3 to the channel. TRESK is the first example of an ion channel phosphorylated by the dynamically membrane-localized MARK kinases, also known as general determinants of cellular polarity. These results raise the possibility that microtubule dynamics is coupled to the regulation of excitability in the neurons, which express TRESK background potassium channel

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

The endoplasmic reticulum (ER) is a metabolically active organelle, which has a central role in proteostasis by translating, modifying, folding, and occasionally degrading secretory and membrane proteins. The lumen of the ER represents a separate compartment of the eukaryotic cell, with a characteristic proteome and metabolome. Although the redox metabolome and proteome of the compartment have not been holistically explored, it is evident that proper redox conditions are necessary for the functioning of many luminal pathways. These redox conditions are defined by local oxidoreductases and the membrane transport of electron donors and acceptors. The main electron carriers of the compartment are identical with those of the other organelles: glutathione, pyridine and flavin nucleotides, ascorbate, and others. However, their composition, concentration, and redox state in the ER lumen can be different from those observed in other compartments. The terminal oxidases of oxidative protein folding generate and maintain an "oxidative environment" by oxidizing protein thiols and producing hydrogen peroxide. ER-specific mechanisms reutilize hydrogen peroxide as an electron acceptor of oxidative folding. These mechanisms, together with membrane and kinetic barriers, guarantee that redox systems in the reduced or oxidized state can be present simultaneously in the lumen. The present knowledge on the in vivo conditions of ER redox is rather limited; development of new genetically encoded targetable sensors for the measurement of the luminal state of redox systems other than thiol/disulfide will contribute to a better understanding of ER redox homeostasis

Reaktív oxigén származékok szerepe a fibrózis kialakulásában = Reactive oxygen species in the development of organ fibrosis

Kutatásaink legfontosabb eredményei a következők: 1. Kimutattuk, hogy a peroxidazin fehérje expressziója fokozódik a miofibroblasztok differenciálódása folyamán és a fehérje szekretálódik a sejtek közötti térbe. Azt is kimutattuk, hogy a vese fibrotikus átalakulása során a peroxidazin felszaporodik a tubulus hámsejtek közötti térben. A peroxidazin sejtek közötti térbe történő szekréciója fontos, eddig ismeretlen eleme lehet a szöveti fibrózisnak. 2. Kimutattuk, hogy az emlős peroxidázok családjába tartozó laktoperoxidáz enzim hatékonyan katalizálja tirozin aminosavak összekapcsolását. Az emlős peroxidázok ditirozin-képző aktivitásának szerepe lehat a sejtek közötti állomány módosításában. 3. A NADPH oxidáz enzimcsalád Duox1 tagjáról kimutattuk, hogy szerepet játszhat a húgyhólyag hámsejtjeinek jelátviteli folyamataiban. 4. Elsőként mutattuk ki élő sejtekben, hogy az endoplazmás retikulum lumenében magas a H2O2 szintje, ami elsősorban az Ero-1L enzim aktivitásának köszönhető és független a Nox enzimek aktivitásától. 5. Genetikai modellekkel alátámasztva kimutattuk, hogy a fibroblaszt-miofibroblaszt átalakulás közben megfigyelhető H2O2 termelés a Nox4-p22phox enzimkomplex aktivitásának köszönhető. | The most important results of the research project are the followings: 1. We demonstrated the increased expression and secretion of peroxidasin during myofibroblastic differentiation. We showed that during the course of kidney fibrosis, peroxidasin accumulates in the peritubular space. The secretion of peroxidasin represents a previously unknown mechanism in tissue fibrosis. 2. We showed that lactoperoxidase, a member of the mammalian peroxidase family, efficiently catalyzes the formation of dityrosine residues. Dityrosine formation by mammalian peroxidases may play a role in the modification of the extracellular matrix. 3. We showed that the NADPH oxidase Duox1 has a role in the signaling mechanisms of urothelial cells. 4. We were the first to show in live cells that lumen of the mammalian endoplasmic reticulum is highly oxidative. The high level of H2O2 in the lumen is mainly due to Ero-1L activity and seems to be independent of Nox enzymes. 5. Using genetic models we showed that H2O2 production during myofibroblastic differentiation is due to the activity of the Nox4-p22phox enzyme complex

Későközépkori prédikációirodalmunk latin nyelvű forrásai = Latin sermons of medieval ages

A "Késő középkori prédikációirodalmunk latin nyelvű forrásai" című projekt keretében Temesvári Pelbárt és Laskai Osvát beszédminta-gyűjteményeinek (modell-sermon-collection) internetes szövegkiadása kezdődött meg. 2007 elejére http://sermones.elte.hu című honlapunkon megjelent Temesvári Pelbárt Pomerium de sanctisa (mintegy 80 szerzői ív terjedelemben), a másolt példány hasonmásával, a szövegkiadás magyar és angol nyelvű szabályzatával, névmutatóval és az eredeti kötet tárgymutatóinak adatbázis-szerű, bővített, hiperhivatkozásokkal ellátott megjelenítésével. Munkánk elvi alapjait és eredményeit bővebben az Ars compilandi - A későközépkori prédikációs segédkönyvek forráshasználata című monográfia foglalja össze, röviden és világnyelven pedig a La diversité thématique dans les prédications de Pelbart de Temesvár című tanulmány (Archivum Franciscanum Historicum, 2007). | Within the framework of the project 'Latin source texts of the late medieval Hungarian sermon literature' Internet publication of Pelbartus de Themeswar's and Osualdus de Lasko's model sermon collections has been started. By the beginning of 2007 on our website http://sermones.elte.hu Pebartus de Themeswar's Pomerium de sanctis (approximately 80 author's sheets) has been published along with the facsimile of the copy, the rules of transferring incunabulum into digital media in Hungarian and English, the index of names, and the database-like, extended indexes of the original text supplied by hyperlinks. The principles and results of our work is summarized in detail in the monography Ars compilandi - Source usage of late medieval sermon handbooks, and briefly in a world language in the study La diversité thématique dans les prédications de Pelbart de Temesvár (Archivum Franciscanum Historicum, 2007)

The plasma membrane Ca2+ pump PMCA4b inhibits the migratory and metastatic activity of BRAF mutant melanoma cells

Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca2+ signaling is a well-known regulator of tumor progression, the crosstalk between Ca2+ signaling and the Ras-BRAF-MEK-ERK pathway is much less explored. Here we show that in BRAF mutant melanoma cells the abundance of the plasma membrane Ca2+ ATPase isoform 4b (PMCA4b, ATP2B4) is low at baseline but markedly elevated by treatment with the mutant BRAF specific inhibitor vemurafenib. In line with these findings gene expression microarray data also shows decreased PMCA4b expression in cutaneous melanoma when compared to benign nevi. The MEK inhibitor selumetinib-similarly to that of the BRAF-specific inhibitor-also increases PMCA4b levels in both BRAF and NRAS mutant melanoma cells suggesting that the MAPK pathway is involved in the regulation of PMCA4b expression. The increased abundance of PMCA4b in the plasma membrane enhances [Ca2+ ]i clearance from cells after Ca2+ entry. Moreover we show that both vemurafenib treatment and PMCA4b overexpression induce marked inhibition of migration of BRAF mutant melanoma cells. Importantly, reduced migration of PMCA4b expressing BRAF mutant cells is associated with a marked decrease in their metastatic potential in vivo. Taken together, our data reveal an important crosstalk between Ca2+ signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor

Hyperspectral Microscopy of Near-Infrared Fluorescence Enables 17-Chirality Carbon Nanotube Imaging

The intrinsic near-infrared photoluminescence (fluorescence) of single-walled carbon nanotubes exhibits unique photostability, narrow bandwidth, penetration through biological media, environmental sensitivity, and both chromatic variety and range. Biomedical applications exploiting this large family of fluorophores will require the spectral and spatial resolution of individual (n,m) nanotube species € fluorescence and its modulation within live cells and tissues, which is not possible with current microscopy methods. We present a wide-field hyperspectral approach to spatially delineate and spectroscopically measure single nanotube fluorescence in living systems. This approach resolved up to 17 distinct (n,m) species (chiralities) with single nanotube spatial resolution in live mammalian cells, murine tissues ex vivo, and zebrafish endothelium in vivo. We anticipate that this approach will facilitate multiplexed nanotube imaging in biomedical applications while enabling deep-tissue optical penetration, and single-molecule resolution in vivo

Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress

Introduction: The etiology of radiation-induced erectile dysfunction (ED) is complex and multifactorial, and it appears to be mainly atherogenic. Aim: To focus on vascular aspects of radiation-induced ED and to elucidate whether the protective effects of sildenafil are mediated by attenuation of oxidative stress and apoptosis in the endothelial cells. Methods: Bovine aortic endothelial cells (BAECs), with or without pretreatment of sildenafil (5 μM at 5 minutes before radiation), were used to test endothelial dysfunction in response to external beam radiation at 10–15 Gy. Generation of reactive oxygen species (ROS) was studied. Extracellular hydrogen peroxide (H2O2) was measured using the Amplex Red assay and intracellular H2O2 using a fluorescent sensor. In addition, ROS superoxide (O2•-) was measured using a O2•- chemiluminescence enhancer. Both H2O2 and O2•- are known to reduce the bioavailability of nitric oxide, which is the most significant chemical mediator of penile erection. Generation of cellular peroxynitrite (ONOO−) was measured using a chemiluminescence assay with the PNCL probe. Subsequently, we measured the activation of acid sphingomyelinase (ASMase) enzyme by radioenzymatic assay using [14C-methylcholine] sphingomyelin as substrate, and the generation of the proapoptotic C16-ceramide was assessed using the diacylglycerol kinase assay. Endothelial cells apoptosis was measured as a readout of these cells’ dysfunction. Main Outcome Measures: Single high-dose radiation therapy induced NADPH oxidases (NOXs) activation and ROS generation via the proapoptotic ASMase/ceramide pathway. The radio-protective effect of sildenafil on BAECs was due to inhibition of this pathway. Results: Here, we demonstrate for the first time that radiation activated NOXs and induced generation of ROS in BAECs. In addition, we showed that sildenafil significantly reduced radiation-induced O2•- and as a result there was reduction in the generation of peroxynitrite in these cells. Subsequently, sildenafil protected the endothelial cells from radiation therapy-induced apoptosis. Strengths and Limitations: This is the first study demonstrating that single high-dose radiation therapy induced NOXs activation, resulting in the generation of O2•- and peroxynitrite in endothelial cells. Sildenafil reduced ROS generation by inhibiting the ASMase/ceramide pathway. These studies should be followed in an animal model of ED. Conclusions: This study demonstrated that sildenafil protects BAECs from radiation-induced oxidative stress by reducing NOX-induced ROS generation, thus resulting in decreased endothelial dysfunction. Therefore, it provides a potential mechanism to better understand the atherogenic etiology of postradiation ED. Wortel RC, Mizrachi A, Li H, et al. Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress. J Sex Med 2019;16:1721–1733. © 201