bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modification of specific amino acid side chains during hydroxyl radical‐dependent bovine serum albumin (BSA) oxidation
Authors: Turrens Julio F , Mott Ashley , Pannell Lewis F , Ruiz Julio C 
Published: June 2021

Exposure of proteins to different oxidants is accompanied by light emission from decaying electronically excited intermediates. One of our goals is to identify what amino acid side chains are responsible for this process. In a previous study we showed that exposure of 1mg/ml BSA to 1 mM H2O2/200 μM FeCl2 (a source of hydroxyl radical) resulted in modifications of H42, M111, P120, F251, F353, P596, and M571. In order to determine whether these modifications are random or specific, we repeated the experiment but changed the concentrations of ferrous iron (0, 100 μM and 1 mM), H2O2 (100 μM and 1 mM). Some experiments were run in the presence of ascorbic acid to maintain iron in the reduced state (0.1 mM or 1 mM). The samples were digested and screened through a Q‐Tof Ultima ™ (MS Waters, Micromass, Beverly, MA) at a low flow state of 1 μl/min. Samples that showed proper sample material were sent to the LTQ Orbitrap (Thermo Electron, San Jose, CA) for a reliable and higher resolution search for sequence coverage. Data was analyzed with an in‐house copy of Mascot ™, an internet‐based search engine. The results showed consistent oxidation of specific side chains (F51, C58, H63, F73, F172, F353, M469 and M571). As the concentration of oxidants increased, we observed an increase in non specific modification.Conclusionalthough hydroxyl radical is a very powerful oxidant, it tends to attack specific amino acids in proteins. Another possibility is that these amino acids become associated iron, and therefore become the nearest targets for hydroxyl radicals formed during the decomposition of H2O2.
PPARγ Ligands Inhibit Hyperglycemia Induced Endothelial Nox‐4 Expression and Oxidative Stress
Authors: Williams Clintoria Richards , DeShazer Garland , Murphy Tammy , Hart C. Michael 
Published: June 2021

Hyperglycemia‐induced oxidative stress is implicated in the pathogenesis of diabetes‐associated vascular diseases. We demonstrated that the expression and activity of NADPH oxidase, was increased in the aortas of diabetic, leptin receptor‐deficient mice and that administration of the PPARγ ligand, rosiglitazone (RSG), reduced the expression of several NADPH oxidase subunits to control levels. In this study, we tested the hypothesis that endothelial NADPH oxidases contribute to hyperglycemia‐induced oxidative stress. Human aortic endothelial cells were treated with normal (5 mmol/L) or high (30 mmol/L) glucose concentrations for 24 – 72 hours. Compared to normal, high glucose increased hydrogen peroxide (H2O2) generation measured with Amplex Red and increased mRNA and protein levels of the H2O2‐generating subunit of NADPH oxidase, Nox‐4. Treatment with either the non‐specific, NADPH oxidase inhibitor, diphenyliodonium (10 μmol/L), or RSG (10 μmol/L) attenuated glucose‐stimulated H2O2 generation. These findings suggest that high glucose concentrations can induce increases in the expression and activity of endothelial Nox‐4 that may contribute to vascular oxidative stress and that ligands for PPARγ may represent a novel target to inhibit NADPH oxidase expression and oxidative stress in the vascular endothelium.Supported by: The Atlanta VA Research Service and NIH RO1 (DK074518).
Retinoic acid‐induced neuroblastoma differentiation is dependent from NOX activation
Authors: Nitti Mariapaola , Furfaro AnnaLisa , Cevasco Claudia , Balbis Emanuela , Traverso Nicola , Marinari Umberto Maria , Pronzato Maria Adelaide , Domenicotti Cinzia 
Published: June 2021

NOX is a multimeric enzyme able to generate superoxide anion as main reaction product. The wide distribution of NOX isoforms in many tissues and cells underlies the role of superoxide anion and reactive oxygen species in cell pathophysiology. In this work, we found that in SH‐SY5Y neuroblastoma cells treated up to 7 days with 10microM retinoic acid (RA) cell differentiation, monitored by cell count, MAP2 expression increase and morphological changes, was accompanied by the up‐regulation of NOX activity, as revealed by regulatory subunit translocation and the overproduction of superoxide anion. Moreover, PKC delta protein level was increased by RA and was involved in NOX activation and in MAP2 induction as shown using siRNA tools.We suppose NOX involvement in cytoskeleton remodelling during cell differentiation, since the enzyme activity inhibition obtained with DPI exerted a strong preventing effect on cell morphological changes induced by RA. NOX silencing strategy will be applied to confirm these findings.Grants form Genoa University and MIUR‐PRIN 20077S9A32_002
WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway
Authors: Zhou Bo , Zhang Yiqian , Wang Dexuan , Zhuang Jieqiu , Zhang Xuemei , Gu Dingying , Cai Hui 
Published: June 2021

WNK ((with no lysine (k)) kinase is a subfamily of serine/threonine kinases. Mutations in two members of this kinase family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia and metabolic acidosis. WNK1 and WNK4 were shown to modulate NCC activity through SPAK and OSR1. Activation of PKC by a phorbol ester inhibits NCC function via activation of ERK1/2 kinase. To investigate whether WNK4 affects NCC via MAPK signaling pathway besides SPAK and OSR1, we first determine whether WNK4 phosphorylates ERK1/2 kinase. We found that WNK4 increases the phosphorylation of ERK1/2 in a dose‐dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) do not increase the ERK1/2 phosphorylation. We next showed that hypertonic stimulation significantly increased ERK1/2 phosphorylation in mDCT cells. The ERK1/2 activation by hypertonic stimulation was significantly attenuated by WNK4 siRNA knock‐down expression (23.19 ± 8.21 % in siWNK4 group vs 100 % in control group, p <0.01, n=3). We further showed that WNK4 knock‐down significantly increases total NCC expression (100 ± 0 % vs 120.7 ± 6.2 %, p <0.05, n=4) as well as NCC surface expression (100 ± 0 % vs 256.8 ± 50.3 %, p <0.05, n=4). We conclude that the inhibitory effect of WNK4 on NCC is mediated by MAPK ERK 1/2 signaling pathway.Note: BZ and YZ contributed equally to this work.
Vasomotion in rat small mesenteric arteries is bestrophin‐3 dependent.
Authors: Aalkjær Christian , Brøgger Torbjørn , Bødtkjer Donna Briggs , Matchkov Vladimir 
Published: June 2021

We have hypothesized the importance of a cGMP‐dependent Ca‐activated Cl current for synchronization of smooth muscle cells (SMCs) in the vascular wall and have shown that cGMP‐dependent Ca‐activated Cl current dependens on bestrophin‐3 (Best‐3) expression.To study the role of Best‐3 for vascular tone, we transfected rat small mesenteric arteries in vivo with siRNA against Best‐3 and with non‐functional mutated Best‐3 siRNA, as a control. The arteries were tested 3 days after in vitro for isometric force development and for [Ca2+]i.Best‐3 protein expression was downregulated with specific siRNA by 65.0±5.8 % versus control arteries transfected with mutated siRNA. The transfection did not affect either maximal contractile response or sensitivity to noradrenalin (NA). Relaxation to acetylcholine was not affected either. The amplitude of NA‐induced vasomotion in arteries downregulated for Best‐3 was significantly lower than in the controls, while frequency was not different. The downregulation of Best‐3 partly desynchronized the global [Ca2+]i oscillations in SMCs and asynchronous Ca waves appeared.Our study demonstrates the importance of Best‐3 for synchronization of SMCs and, thus, generation of vasomotion.
Mineralocorticoids stimulate the activity and expression of renal H,K‐ATPases
Authors: Greenlee Megan Michelle , Lynch I. Jeanette , Gumz Michelle L. , Cain Brian D. , Wingo Charles S. 
Published: June 2021

Mineralocorticoids such as deoxycorticosterone activate Na+ reabsorption and H+ secretion in the renal collecting duct. In this study, the effect of deoxycorticosterone pivalate (DOCP) on renal H+,K+‐ATPase activity and expression was investigated. Eight days after female wild type (WT) mice received DOCP, collecting ducts were perfused in vitro to measure H+,K+‐ATPase‐dependent H+ secretion after an acute intracellular acid load. DOCP stimulated H+,K+‐ATPase mediated H+ secretion in A type intercalated cells of inner cortical collecting ducts by 70% whereas no effect of DOCP was observed in B type intercalated cells compared to control. DOCP dramatically increased HKα2 mRNA expression in outer and inner medulla (246% and 1040%, respectively) and did not affect HKα1 mRNA expression. DOCP treatment caused significant body weight gain in WT mice and had little effect on body weight in mice lacking both α subunits (HKα1,2−/−). DOCP caused a similar degree of hypokalemia in WT and HKα1,2−/− mice. Importantly, WT developed metabolic alkalosis whereas HKα1,2−/− mice did not. In summary, these data clearly demonstrate a physiological role for renal H+,K+‐ATPases in mineralocorticoid stimulated H+ secretion and suggest a role for these enzymes in Na+ reabsorption.Studies were funded by NIH RO1‐DK‐049750 to C.S.W and 5T32DK007518‐22 to M.M.G.
Vasopressin‐mediated signaling in the distal convoluted tubule regulates phosphorylation of the thiazide‐sensitive Na+‐Cl− cotransporter (NCC) at two conserved threonine residues
Authors: Pedersen Nis Borbye , Hofmeister Marlene Vind , Nielsen Jakob , Fenton Robert Andrew 
Published: June 2021

The Na+‐Cl− cotransporter (NCC) is important for renal electrolyte balance. Phosphorylation of NCC has been proposed to play a major role in its transport activity and cellular localization. Using recently generated phospho‐specific antibodies against two conserved N‐terminal phosphorylation sites (Thr53, Thr58 and Thr53/Thr58) we assessed the role of vasopressin (AVP) in regulating NCC in vivo. In AVP‐deficient Brattleboro rats, acute dDAVP exposure for 15 min significantly increased pNCC abundance at the apical plasma membrane approximately 300%. Acute dDAVP did not alter the abundance of the STE20/SPS1‐related proline/alanine‐rich kinase (SPAK) previously implicated in NCC phosphorylation. Early and late DCTs increased their intracellular Ca2+ levels in response to 1 min of superfusion of dDAVP (2 × 10−10 M), confirming that the DCT is an AVP responsive segment. In rats fed a high salt diet alongside AT1‐R blockade by candesartan, similar increases in pNCC abundance were observed following chronic or acute dDAVP. Candesartan treatment reduced the abundances of total SPAK and pSPAK, suggesting that this kinase can be regulated via the AT1‐R in vivo. Taken together our results indicate that AVP is a potent regulator of NCC activity.Funding is provided by the Danish Medical Research Council, the Novo Nordisk Fond and the Carlsberg Foundation.
Sonic Hedgehog signaling mediates bone marrow‐derived mesenchymal stem cell proliferation and recruitment
Authors: Donnelly Jessica M , Chawla Ambreesh , Houghton JeanMarie , Zavros Yana 
Published: June 2021

ObjectiveBone marrow‐derived mesenchymal stem cells (MSCs) are recruited to the site of chronic inflammation during gastric cancer progression. Given that increased interferon‐gamma (IFNγ) and Sonic Hedgehog (Shh) expression contribute to cancer development, the role of the Shh signaling pathway as a mediator of IFNγ induced proliferation and recruitment of MSCs was studied.MethodsMSCs transduced with lentiviral particles expressing shRNA for Shh (MSCsShhKO) or empty vector (MSCsVect) were analyzed for cell cycle changes by flow cytometry. In vivo, MSCs tagged with RFP were transferred (via tail vein injection) into C57BL/6 mice and treated with either PBS or IFNγ (1μg/mouse/day) for 14 days. To identify the mechanism of recruitment, chemokine SDF‐1α was used in a chemotaxis assay with MSCsShhKO and MSCsVect.ResultsIFNγ significantly increased MSC proliferation, a response mediated by the secretion of Shh from cultured cells and blocked by Hedgehog signaling inhibitor cyclopamine. MSCsShhKO were arrested in G0/G1 phase compared to MSCsVect. Mice injected with IFNγ showed significant recruitment of RFP‐tagged MSCs to the gastric mucosa. Compared to MSCsVect, MSCsShhKO failed to migrate toward the SDF‐1α reflected by decreased CXCR4 expression.ConclusionIFNγ induces proliferation and initiates recruitment of MSCs, a mechanism mediated by Shh signaling.(Supported by start‐up funds, Zavros)
MK571‐sensitive extracellular ATP release in intestinal epithelia
Authors: Nakano Takanari , Akiba Yasutada , Engel Eli , Guth Paul H , Kaunitz Jonathan D 
Published: June 2021

Epithelial ATP release is an essential component of purinergic secretory regulation. Although the mechanism of extracellular ATP release from epithelial cells is unknown, the involvement of ATP channels, such as pannexin hemichannels and the ATP‐binding cassette (ABC) transporters has been hypothesized. We measured extracellular ATP release from confluent Caco‐2 cell monolayers in order to further study this mechanism. Ecto‐ATPase activity of alkaline phosphatase, CD39, 5′‐nucleotidase, and other brush border ecto‐enzymes was inhibited by non‐cytotoxic concentrations of EDTA, enabling the measurement of supernatant ATP using the luciferin‐luciferase assay. In the presence of EDTA, [ATP] increased linearly, consistent with constitutive cellular ATP release. Among the pharmacological reagents reported as inhibitors for ATP release, MK571, a specific multidrug resistant‐associated protein (MRP) inhibitor dose‐dependently inhibited the ATP release, with no inhibition due to stilbene anion transport inhibitors, pannexin inhibitors, or multidrug resistance protein (MDR) 1 substrates. These results suggest MRP‐associated ATP release in intestinal epithelia, a novel mechanism for transmembrane ATP transport.NIH‐NIDDK R01 DK54221 & R21 DK081199
The G‐protein coupled receptor GPR40 mediates long chain fatty acid induced cholecystokinin secretion
Authors: Liou Alice Peiyu , Lu Xinping , Zhao Xilin , Pechhold Susanne , Raybould Helen , Wank Steve 
Published: June 2021

INTRODUCTIONLong chain fatty acid receptors (LCFARs) have been implicated in the chemosensation of dietary fats, however direct studies on native “I” cells have been difficult due to their rare and isolated distribution. The objective of this study was to determine whether dietary fat‐induced cholecystokinin (CCK) secretion is directly mediated by GPR40 expressed on I cells isolated from duodenal mucosa of CCK‐eGFP BAC transgenic mice by fluorescence activated cell sorting.METHODSIsolated CCK‐eGFP cells were evaluated for LCFAR gene expression by Taqman RT‐PCR. GPR40−/− mice were crossed with CCK‐eGFP mice to evaluate the functional relevance of GPR40 on linolenic acid‐stimulated CCK secretion, measured by radioimmunoassay, in isolated CCK‐eGFP cells. Also, plasma CCK secretion in response to olive oil gavage was compared between GPR40+/+ and GPR40−/− mice.RESULTSIsolated CCK‐eGFP cells display a 100‐fold greater expression of GPR40 compared to non‐eGFP cells. In vitro, linolenic acid increased CCK secretion by 53% in GPR40+/+ CCK‐eGFP cells compared to no significant secretion in GPR40−/− CCK‐eGFP cells. In vivo, an olive oil gavage significantly increased plasma CCK levels by 5.7‐fold in GPR40+/+ mice compared to an increase of 3.1‐fold in GPR40−/− mice.CONCLUSIONThis study is the first to demonstrate a direct chemosensory role of GPR40 in dietary fat induced CCK secretion.
Therapeutic targeting of mitochondrial superoxide in hypertension
Authors: Dikalov Sergey , Bikineyeva Alfiya , Budzyn Klaudia , Nazarewicz Rafal , Lewis William , Harrison David G , Dikalova Anna 
Published: June 2021

INTRODUCTIONSuperoxide (O2•) has been implicated in the pathogenesis of many human diseases including hypertension, however commonly employed antioxidants have proven ineffective in clinical trials. It is possible that these agents are not adequately delivered to the subcellular sites of O2• production.HYPOTHESISBecause the mitochondria are important source of O2•, we postulated that mitochondrial targeting of O2• scavenging would have therapeutic benefit.RESULTSIn this study, we found that the hormone angiotensin II increased endothelial mitochondrial O2•. Treatment with the mitochondria‐targeted SOD mimetic mitoTEMPO decreased mitochondrial O2•, inhibited the total cellular O2•, reduced cellular NADPH oxidase activity and restored endothelial NO, while a similar dose of non‐targeted TEMPOL was not effective. These effects were mimicked by overexpressing the mitochondrial MnSOD (SOD2), while SOD2 depletion with siRNA increased both basal and angiotensin II‐stimulated cellular O2•. Treatment of mice in vivo with mitoTEMPO after the onset of angiotensin II‐ and DOCA‐salt induced hypertension decreased blood pressure by 30 mm Hg, decreased vascular O2•, increased NO• production and improved endothelium‐dependent relaxation. Transgenic mice overexpressing mitochondrial SOD2 demonstrated attenuated angiotensin II‐induced hypertension and vascular oxidative stress similar to mice treated with mitoTEMPO.CONCLUSIONThese studies show that mitochondrial O2• is important for the development of hypertension and that mitochondria‐targeted SOD mimetic could have therapeutic benefits in this and possibly other diseases.Supported by AHA and NIH grant No 09GRNT2220128, HL058000 and HL075209.
Cardiac autonomic regulation in hyperthyroid subjects during sleep
Authors: Tobaldini Eleonora , Pecis Marica , Bulgheroni Mara , Colombo Silvia , Muratori Milena , Bevilacqua Maurizio , Porta Alberto , Montano Nicola 
Published: June 2021

Hyperthyroidism, a model of reversible hyperadrenergic state, is characterized by an altered autonomic regulation and by a reduced sleep quality, but no data are available on sleep characteristics and autonomic control during sleep in hyperthyroid (HYPERTH) compared to euthyroid (EUTH) state.The aim of our study was to assess autonomic control during sleep in Hyperth and Euth using symbolic analysis (SA) of heart rate variability (HRV). A polysomnographic study was performed in six women with diagnosed HYPERTH and when recovered to EUTH, after suppression therapy. The time series of RR intervals were constructed for each sleep stage and analyzed using SA, a method that is based on the conversion of time series into symbols, the construction of patterns and the identification of four families: 0V (no variation), marker of sympathetic modulation, 1V, 2LV and 2UV (two unlike variations), marker of parasympathetic modulation.Sleep quality parameters were similar in HYPERTH and EUTH. As to HRV, in Hyperth REM sleep was characterized by an increased 0V% and a decreased 2UV%. Comparing to Hypert, Euthyr showed a decrease of heart rate and sympathetic modulation and an increase of vagal modulation in all sleep stages. In conclusion, in HYPERTH patients, REM is characterized by a predominant sympathetic control and the alterations of autonomic cardiac control during sleep seem to be reverted after treatment.
Toward comprehensive quantitative proteomics using subcellular fractionation and LC‐MS/MS
Authors: Luginbuhl Kelli , Pisitkun Trairak , Rinschen Markus , Hoffert Jason , Knepper Mark A 
Published: June 2021

Our previous LC‐MS/MS profiling in cultured renal mpkCCD cells has detected 2999 unique proteins. This compares with 7922 transcripts found by Affymetrix microarrays in these cells (http://dir.nhlbi.nih.gov/papers/lkem/mpkccdtr/Default.aspx).Comprehensive quantification requires, as a pre‐requisite, that we can detect virtually all of the proteins that correspond to the expressed transcripts. With a goal of comprehensive proteomic profiling, we asked if subcellular fractionation prior to LC‐MS/MS could increase the number of proteins detected. Gene ontology analysis of the transcriptomic data revealed that approximately 29% of the detected transcripts code for nuclear proteins, a much larger percentage than previously found by LC‐MS/MS. To increase protein identifications in mpkCCD cells, we used a standard procedure for subcellular fractionation into nuclear extract, nuclear pellet and cytoplasmic fractions followed by 1D SDS‐PAGE and in‐gel trysinization. Using high sensitivity LC‐MS/MS (LTQ‐Orbitrap) and multiple spectral search algorithms, while limiting false positive identifications to <1% (target/decoy), we identified a total of 6188 unique proteins including 2521 that were in the two nuclear fractions but not in the cytoplasmic fraction. With further improvements in mass spectrometry, comprehensive identification (and quantification) of proteins should soon be feasible.
PDGF‐BB signaling in adventitial fibroblasts during hypoxia‐induced pulmonary hypertension
Authors: Panzhinskiy Evgeniy , Shields Kristin , Zawada Michael , Stenmark Kurt , Das Mita 
Published: June 2021

PDGF‐BB contributes to vascular remodeling during the development of hypoxia‐induced pulmonary hypertension (HPH). Adventitial fibroblasts (AdFibs) are the first cells activated in HPH. We tested the hypothesis that PDGF‐BB‐induced activation of AdFibs is a key regulator of HPH development. Newborn calves were exposed to high altitude (4750 m) (HA) for 2 weeks to induce HPH. Phosphorylated PDGFβ receptor (PDGFβ‐R) levels were markedly elevated in the thickened adventitia of the vessels in HA lungs compared to that of control. PDGFβ‐R expression was similarly activated in AdFib isolated from the main pulmonary artery of HA animals as evaluated by immunoblot analysis. PDGF‐BB stimulated greater proliferation rate and reactive oxygen species (ROS) production in HA cells compared to the AdFibs of control animals. ROS scavengers, N‐Acetyl Cysteine (NAC), and superoxide dismutase mimetic, TEMPOL, selectively inhibited PDGF‐BB‐induced proliferation and ROS production in HA cells. PDGF‐BB induced activation of ERK1/2 and JNK1/2, but not p38 MAP kinase in both cell types. However, both NAC and TEMPOL attenuated PDGF‐BB‐stimulated heightened phosphorylation levels of ERK1/2 and JNK1/2 in HA cells only. Our results suggest that dramatic adventitial remodeling during the progression of HPH depends on PDGF‐BB‐induced AdFib proliferation mediated through ROS production and ERK/JNK MAP kinase activation.Funded by HL64917; P20RR016474
Improved Renal Tubular Function Detected by Magnetic Resonance Imaging after Revascularization of Experimental Renal Artery Stenosis
Authors: Warner Lizette , Woollard John R , Glockner James D , Krier James D , Zhu Xiang Y , Textor Steve , Lerman Lilach O 
Published: June 2021

Renal artery stenosis (RAS) may lead to hypertension and renal injury. Reversibility of renal tubular dysfunction in RAS upon revascularization is unclear. We tested the hypothesis that percutaneous transluminal renal angioplasty (PTRA) would restore tubular function assessed as medullary oxygenation response to furosemide, a diuretic that inhibits medullary tubular transport. This was studied in 16 pigs after 6 wks of RAS, using blood oxygen level dependent magnetic resonance imaging (BOLD MRI) before and after furosemide. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined with multidetector computed tomography. Pigs were then randomized for PTRA or sham (n=8 each) and studied again 4 wks later; 8 normal pigs served as controls.ResultsStenotic kidney RBF, GFR, and BOLD MRI responses were reduced in RAS shams (Figure). PTRA decreased the degree of stenosis (11±7 vs. 71±5% in untreated RAS) and blood pressure (108±3 vs. 130±9 mmHg, p<0.05). Renal and tubular function were improved in RAS+PTRA, although not to normal (Figure). Non‐stenotic kidneys showed no change in tubular function. Therefore, tubular solute transport‐related oxygen consumption can be partially restored and detected by BOLD MRI 4 wks after PTRA in post stenotic swine kidneys. This noninvasive method may be useful to assess the functional status of kidneys in RAS patients. Supported by NIH, HL085307.