bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of active site residues essential for the metabolism of phenacetin to acetaminophen in human 2A cytochromes P450
Authors: DeVore Natasha M , Smith Brian D. , Urban Michael J. , Nikaeen Naseem , Blake Linda C. , Scott Emily E. 
Published: June 2021

Human cytochromes P450 from the 2A subfamily have differing abilities to metabolize carcinogens and procarcinogens, including the nicotine‐derived 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK), aflatoxin B1, and 4‐aminobiphenyl. Recently, Fukami et al. established that phenacetin, a hepatocarcinogen and known substrate for the CYP1A family, is even more efficiently metabolized by human lung cytochrome P450 2A13 (CYP2A13), while the human liver cytochrome P450 2A6 (CYP2A6) has almost no activity despite being 94% identical in amino acid sequence. To determine the basis for substrate specificity between these two closely related human 2A enzymes, site directed mutagenesis was used to construct eleven CYP2A13 single point mutants with mutations at positions in or near the active site where CYP2A13 and CYP2A6 wild type enzymes differ. Phenacetin metabolism kinetics were determined for the wild type enzymes CYP2A13 and CYP2A6, and eleven CYP2A13 mutants. Two CYP2A13 mutants, A117V and L366I, each increase phenacetin metabolism activity greater than four‐fold. Four amino acid substitutions, S208I, F300I, A301G, and G369S, virtually abolished phenacetin metabolism in CYP2A13. The CYP2A6 enzyme is being modified by substitution of one or more 2A13 active site residues to determine those required to incorporate phenacetin metabolism into CYP2A6.NIH GM76343
Upregulation of Adenosine A1 Receptor in Coronary Atherosclerosis in the Metabolic Syndrome and in the in Vitro Organ Culture Model of Coronary Atherosclerosis
Authors: Long Xin , Edwards Jason M , Neeb Zachary , Alloosh Mouhamad , Sturek Michael 
Published: June 2021

Ossabaw miniature swine fed excess atherogenic diet develop metabolic syndrome (MetS) and coronary atherosclerosis similar to humans. Organ culture is a useful in vitro model of arterial injury/atherosclerosis. Our lab previously made the novel discovery that adenosine A1 receptor (A1R) signaling via ERK phosphorylation mediates smooth muscle cell proliferation in intact coronary rings from Ossabaw pigs. Thus, we hypothesized that A1R will be upregulated in atherosclerotic coronary artery of MetS Ossabaw pigs and also in the organ culture model. Coronary intimal thickness in MetS pigs (n=7) increased 2.4‐fold compared to control pigs (n=6) (p<0.05). A1R mRNA and protein increased 4‐fold and 3‐fold, respectively, in MetS compared to control pigs (p<0.05). A1R mRNA increased 5‐fold in the 2‐day organ cultured coronary compared to cold stored ones (p<0.05), and A1R protein increased 2‐fold in the 4‐day organ cultured coronary compared to cold stored. In an in vitro organ culture model of arterial injury, A1R stimulated ERK phosphorylation increased 8‐fold compared to non‐injured artery segments.CONCLUSION: Upregulation of A1 receptor expression and ERK signaling may contribute to coronary atherosclerosis in MetS and in the in vitro organ culture model of arterial injury.
EXPERIMENTAL STUDY ON DIFFERENTIATION OF BONE MARROW STROMA CELLS TO CARDIOMYOCYTES‐LIKE
Authors: Bai Xiaojuan , Zhou Ying , Wang Bo 
Published: June 2021

Objective To investigate the ultrastructure and cell factor changes of BONE MARROW STROMA CELLS(BMSCs) after induction and differentiation into cardiomyocytes‐like, in order to provide theory foundation for the therapy of dilated cardiomyopathy(DCM) with BMSCs transplantation.Method BMSCs were isolated and proliferated in vitro, after induction and differentiation by 5‐azacytidine(5‐aza), they were observed by electromicroscopy to see the ultrastructure changes; RT‐PCR were also used to check cardiac‐special factors including ANP, BNP, α‐MHC, β‐MHC, comparing with the cardiomyocytes in primary culture in vitro.Result BMSCs were verified by immunohistochemistry that they differentiated into cardiomyocytes‐like; under electromicroscopy, glycogensome and myofibril can be seen in cell plasm, which was similar to cardiomyocyte in primary culture. RT‐PCR showed that BMSCs expressed cardiac‐specific factors, including ANP, BNP, α‐MHC, β‐MHC after induction and differentiation.Conclusion After induction by 5‐aza, BMSCs were similar to cardiomyocytes in primary culture in ultrastructure and cardiac‐specific factors expression, which suggested that they can be used for the therapy of DCM with BMSCs transplantation.
ATP released from pulmonary neuroepithelial bodies activates Clara‐like cells in the NEB microenvironment via P2Y2 receptors
Authors: De Proost Ian , Pintelon Isabel , Wilkinson William J , Goethals Sofie , Brouns Inge , Van Nassauw Luc , Riccardi Daniela , Timmermans Jean‐Pierre , Kemp Paul J , Adriaensen Dirk 
Published: June 2021

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine cells, shielded from the airway lumen by Clara‐like cells (CLCs). In ex vivo mouse lung slices, NEB cells respond with a [Ca2+]i rise to stimulation with high K+, followed by a delayed [Ca2+]i increase in CLCs, indicative of an indirect activation. Aim was to unravel the mechanism of this potential interaction between NEBs and CLCs. Because accumulation of ATP is known for NEB cells, we focused on a possible purinergic signaling pathway. Using ‘sniffer patching’ to detect ATP release, stimulation with high K+ was shown to elicit quantal ATP release from NEBs. Use of enhanced ATP hydrolysis and the P2 receptor blocker suramin confirmed that ATP was responsible for activating CLCs following application of high K+. A combined pharmacological and Ca2+ imaging approach revealed the involvement of functional P2Y2 receptors, and immunohistochemistry verified the expression of P2Y2 receptors on CLCs. It is concluded that stimulation of NEBs in ex vivo lung slices evokes exocytosis of ATP and subsequent selective purinergic activation of surrounding CLCs in the NEB microenvironment, which recently has been assigned a potential pulmonary stem cell niche.Support: FWO grants G.0085.04 and G.0081.08 (DA); UA grants NOI BOF 2003, GOA BOF 2007 (DA) and KP BOF 2006 (IB); BBSRC grant BB/D01591X/1 (PJK, DR)
SNARE Proteins And SNARE Regulators Mediate Aquaporin2 Exocytosis In Renal Cells
Authors: Procino Giuseppe , Barbieri Claudia , Tamma Grazia , De Benedictis Leonarda , Pessin Jeffrey , Svelto Maria , Valent Giovanna 
Published: June 2021

Aim: The expression and the functional involvement of SNARE proteins and SNARE‐regulating proteins (Munc18b and Munc18c) in catalyzing aquaporin 2 vesicles fusion at the apical membrane is investigated in a mouse cortical collecting duct cell line.Methods: Localization of SNARE and SNARE‐regulating proteins was performed using a combination of confocal microscopy and cell fractionation. SNARE complex formation was demonstrated by co‐immunoprecipitation. Functional involvement of each protein was demonstrated coupling RNA interference to apical surface biotinylation in order to quantify the effect of protein knockdown on AQP2 exocytosis in response to FK stimulation.Results: Both VAMP2 and VAMP3 were found associated with AQP2 immunoisolated vesicle. Syntaxin 3 was found associated with the apical plasma membrane along with its functional partner Munc18b. Upon FK stimulation, Synataxin 3 is able to dissociate from Munc18b and to form a stable complex with VAMP2, VAMP3 and SNAP23. siRNA experiments indicate that Syntaxin 3, VAMP2, VAMP3 and SNAP23 are required for AQP2 vesicle fusion at the apical membrane. Moreover, Munc18 knockdown induced accumulation of AQP2 at the apical plasma membrane.Conclusion: These findings, taken together, propose a complementary set of SNARE proteins and SNARE‐regulators responsible for AQP2 storage vesicle fusion into the apical membrane in the renal collecting duct.
Identification of a regulatory phosphorylation site in a cell cycle and swelling regulated ClC Cl‐ channel
Authors: Falin Rebecca , Morrison Rebecca , Ham Amy‐Joan L , Strange Kevin 
Published: June 2021

clh‐3 encodes two splice variants, CLH‐3a and CLH‐3b, of a C. elegans voltage‐gated ClC Cl‐ channel. CLH‐3b is expressed in the worm oocyte and is activated during meiotic cell cycle progression or in response to cell swelling. Channel activation is brought about by serine/threonine dephosphorylation mediated by the type 1 phosphatases GLC‐7α and GLC‐7β. GCK‐3 is a Ste20 kinase and homolog of mammalian PASK/SPAK, which regulates cation‐coupled Cl‐ cotransporters. GCK‐3 binds to a 101 amino acid C‐terminal splice insert unique to CLH‐3b. When co‐expressed with CLH‐3b, GCK‐3 dramatically inhibits channel activity and alters gating kinetics and voltage sensitivity. Knockdown of GCK‐3 in worm oocytes constitutively activates CLH‐3b. Using mass spectrometry, we identified a serine residue (S747) located within the 101 amino acid splice insert that is phosphorylated in the presence of GCK‐3. S747 and surrounding amino acids conform to the Ste20 kinase phosphorylation motif. Mutation of S747 to alanine prevents GCK‐3 mediated channel inhibition and alterations of channel voltage‐dependent properties. We conclude that phosphorylation of S747 is critical for regulation of CLH‐3b activity. S747 may be phosphorylated directly by GCK‐3 or other downstream kinases.Supported by NIH R01 grant DK51610
Caveolar endocytosis is essential for tumor necrosis factor (TNF) ‐induced occludin internalization in vivo
Authors: Marchiando Amanda M , Shen Le , Schwarz Brad T. , Guan Yanfang , Watson Alastair JM , Montrose Marshall H. , Turner Jerrold R. 
Published: June 2021

TNF causes epithelial barrier dysfunction in vitro and in vivo and is accompanied by redistribution of the tight junction protein occludin. Mechanistic characterization of this vesicular trafficking process and associated tight junction regulation has been limited by the inability to image occludin internalization in real time. Our aim was to characterize the route and mechanisms of TNF‐induced occludin endocytosis in vivo.We developed mice expressing EGFP‐occludin and mRFP1‐ZO‐1 in intestinal epithelium, and both fusion proteins colocalized with their endogenous counterparts. Jejunal mucosa was imaged by confocal microscopy of live anaesthetized mice.TNF (5μg i.p.) triggered focal intra‐junctional occludin aggregation followed by occludin endocytosis. The small endocytic vesicles contained caveolin‐1 but not clathrin heavy chain. Occludin endocytosis was prevented by dynasore, a dynamin II inhibitor; L‐t‐LacCer, a synthetic lipid inhibitor of caveolar endocytosis; and cholesterol chelation with MβCD; while neither macropinocytosis nor clathrin‐mediated endocytosis inhibitors blocked internalization. Occludin endocytosis was also defective in caveolin‐1 knockout mice, though TNF activated myosin normally.These data are the first to image occludin protein trafficking in vivo and demonstrate the pivotal role of caveolin‐1‐dependent caveolar endocytosis in this process.Supported by NIH.
Evaporative Heat Loss and Changes in Core Temperature Set‐Point during Different Absolute Workloads
Authors: Jay Ollie , Kenny Glen 
Published: June 2021

Previous work suggests changes in core temperature set‐point during exercise are determined by relative workload (i.e. % of VO2max). Concurrent rates of metabolic heat production (M‐W), whole‐body evaporative (EHL) and dry heat loss (DHL) were measured by whole‐body direct calorimetry in 5 males throughout 90‐min of cycling at 3 absolute oxygen uptakes of 1.82±0.05 (hi), 1.26±0.05 (med) and 0.65±0.03 (low) L‐min’ at 30°C and 30% RH. Due to the variation in VO2max between participants (3.24 to 4.44 L·min−1) these workloads corresponded to a range of relative workloads within each condition. Rectal temperature (Tre) was also measured throughout. Sustained levels of M‐W obtained throughout exercise were 500±17 (hi), 348±10 (med) and 203±10 W (low). After 90‐min, Tre was steady at 0.96±0.13, 0.70±0.10 and 0.23±0.03°C above rest for hi, med and low respectively. EHL was 423±18, 290±10 and 150±16 W respectively and DHL was 41±10, 45±15 and 58±14 W. Within condition, EHL varied little between participants despite relative workloads differing by 15–20%. End‐exercise EHL appears to be determined by absolute metabolic workload rather than relative workload. Variations in core temperature set‐point within condition are attributed to individual differences in sweating sensitivity during the early stages of exercise; and effects of body mass and composition upon the ‘passive’ system.Supported by NSERC Grant.
Nutritional energy in the regulation of human muscle mTOR signaling following resistance exercise
Authors: Glynn Erin L , Fry Christopher S , Drummond Micah J , Dreyer Hans C , Dhanani Shaheen , Volpi Elena , Rasmussen Blake B 
Published: June 2021

Resistance exercise (Ex) stimulates the mammalian target of rapamycin (mTOR) signaling pathway. The ingestion of leucine‐enriched essential amino acids and carbohydrate (EAA+CHO) 1 hr following Ex further stimulates mTOR signaling. The purpose of this study was to determine whether the addition of nutritional energy to a EAA+CHO stimulus following Ex enhances mTOR signaling. Sixteen male subjects were randomized to two groups containing an equivalent amount of EAA (∼20g) but different amounts of CHO. Both groups ingested the nutrient solution 1 hr after a bout of Ex. mTOR signaling was assessed by immunoblotting from muscle biopsy samples at baseline, immediately following, and 1 and 2 hr post‐Ex. There were no differences between groups for any proteins measured prior to nutrient ingestion. At 2 hr post‐Ex (following nutrient ingestion) the phosphorylation status of mTOR and its downstream effectors S6K1 and 4E‐BP1 increased with no differences between groups (P>0.05). However, Akt/PKB phosphorylation was higher in the extra energy group, likely due to increased blood insulin concentration (P<0.05). We conclude that mTOR signaling is not further enhanced, despite increased Akt activation, by the availability of additional energy (carbohydrates) following Ex as compared to a stimulus containing the same amount of EAA and approximately half the caloric energy.Funded by NIH Grant # 5R01AR049877
Relationships between oxidative stress, HIF‐1α transcription, erythropoietin and vascular endothelial growth factor during sustained hypoxia in humans
Authors: Pialoux Vincent , Mounier Rémi , Brown Allison D , Steinback Craig D , Poulin Marc J 
Published: June 2021

The aim of this study was to investigate the relationships between reactive oxygen species (ROS), hypoxia inducible factor (HIF‐1α) transcription, and HIF‐1α target genes erythropoietin (EPO) and vascular endothelium growth factor (VEGF) in humans. Five healthy men (32±7 yrs, mean±SD) were exposed to 12 h of sustained hypoxia (PetO2=60 Torr). DNA oxidation (8‐Hydroxy‐2′‐deoxyguanosine, 8‐OHdG) oxidation protein products (AOPP), EPO and VEGF were measured in plasma and HIF‐1α mRNA was assessed in leucocytes before, and after 1, 2, 4, 6, 8, 10, 12 h of exposure to hypoxia.HIF‐1α mRNA amount increased during the first 2h of hypoxic exposure (+ 68%; p=0.03), then returned to baseline levels. VEGF increased at 4h (+121%; p=0.02) whereas EPO increased progressively from 4h to 12h (+114%; p<0.01). AOPP increased continuously from 4 h (+ 69%, p=0.04%) to 12h (+ 216 %, p=0.03) of hypoxic exposure while 8‐OHdG increased after 6 h (+ 78%, p<0.01) and remained elevated until 12h. During the “acute” increase phase of HIF‐1α (i.e., between 0 and 2h), 8‐OHdG was positively correlated with HIF‐1α (r=0.55, p=0.02).These findings demonstrate that hypoxia induces oxidative stress via an overproduction of ROS. Finally, this in vivo study in humans corroborates the previous in vitro findings demonstrating that ROS is involved in the stabilization of HIF‐1α transcription.Supported by Alberta Heritage Foundation for Medical Research, Heart & Stroke Foundation of Canada, Canadian Institutes of Health Research.
Exercise training decreases the size and alters the composition of the neointima in a porcine model of coronary restenosis
Authors: Fleenor Bradley Stephen , Ivey Jan , Casati Jennifer , Bowles Douglas K. 
Published: June 2021

Exercise training reduces adverse events following coronary angioplasty in humans. Therefore, our aim was to determine if exercise reduces the size or alters the composition of the neointima (NI) in a porcine balloon coronary injury (BCI) model. Miniature Yucatan swine were randomly assigned to cage confinement (SED, n=7) or exercise (EX, n=8) training on a treadmill. After 4 months the animals underwent BCI of the left anterior descending (LAD) and left circumflex (LCX) arteries. The animals recovered for 2 days and resumed the previous protocol of SED or EX. Additionally on day 12 following the BCI, each animal received an intravenous injection of BrdU to label proliferating cells. At 28 days post BCI the animals were euthanized and the LAD and LCX were excised. Injured and uninjured sections from each artery underwent standard histological procedures and were stained with VVG, pico Sirius red and antibodies for BrdU and fibronectin. Our results demonstrate that exercise decreased lesion size and NI proliferation (−48%) in the LAD (p<.05) but not the LCX. Exercise significantly increased NI collagen (5.9%) and decreased NI fibronectin (−7.9%) in both arteries (p<.05). However, in the uninjured media collagen decreased in the EX (−5.2%, p<.05) with no effect on fibronectin. In conclusion, exercise may decrease post angioplasty events by reducing lesion size and altering NI composition.Supported by: NIH HL 52490
Evidence for NK‐1 Receptors in the Thermal Hyperemic Response in Human Skin
Authors: Wong Brett J , Minson Christopher T 
Published: June 2021

We tested the hypothesis that functional NK1 receptors are required for normal expression of the thermal hyperemic response in human skin. Nine subjects were equipped with four microdialysis fibers on the forearm. Sites received: 1) Ringer's solution as a control; 2) L‐NAME to inhibit NO synthase; 3) substance P (SP); or 4) SP plus L‐NAME. Skin blood flow was measured via laser‐Doppler flowmetry (LDF) and local heaters were used to control skin temperature. Sites 2 and 4 were perfused with L‐NAME for 30 minutes. Sites 3 and 4 were then perfused with SP and SP plus L‐NAME, respectively, for 15 minutes and the SP‐induced vasodilation was allowed to return to baseline. Once a stable baseline was achieved, local heaters were increased from 33°C to 42°C (0.5°C/5s). Cutaneous vascular conductance (CVC) was calculated as LDF/MAP and scaled to maximal vasodilation via nitroprusside. The initial peak in L‐NAME (43 ± 3 %CVCmax) and L‐NAME + SP sites (53 ± 3 %CVCmax) were significantly reduced compared to control (79 ± 3 %CVCmax) and SP sites (79 ± 2 %CVCmax; p<0.001 for both). The secondary plateau in L‐NAME (36 ± 4 %CVCmax) and SP + L‐NAME sites (56 ± 6 %CVCmax) were significantly reduced compared to control (86 ± 3 %CVCmax) and SP sites (91 ± 1 %CVCmax; p<0.001 for both). L‐NAME sites were significantly reduced compared to SP + L‐NAME sites (p<0.01). These data demonstrate NK1 receptor desensitization, alone or with NO synthase inhibition, significantly alters the cutaneous thermal hyperemic response.Supported by NIH HL‐70928
Decreased sustained potassium current contributes to different firing patterns of celiac neurons in DOCA‐salt hypertensive and normotensive Rats
Authors: Wang Xiaohong , Kreulen David L 
Published: June 2021

It is not known if changes in the electrophysiological properties of sympathetic ganglia neurons contribute to increased sympathetic activity in hypertension. Using whole‐cell patch clamp dissociated celiac neurons from hypertensive and normotensive rats were divided into three groups based on their action potential (AP) firing patterns to a constant suprathreshold depolarizing current (100pA; 400 ms). Phasic neurons fired several APs and adapted quickly; tonic‐phasic neurons fired several APs and adapted slowly; tonic neurons fired continuously. The distribution of these three groups was significantly different in neurons from hypertensive rats. There were more phasic neurons, less tonic‐phasic neurons and similar amount of tonic neurons in hypertensive rats. The firing frequency in tonic neurons was higher in hypertensive rats compared to normotensive rats. Potassium channel currents were tested to explore the reason of these neuronal firing changes in hypertension. Sustained potassium current was significantly lower in celiac neurons from hypertensive rats compared to those from normotensive rats. The M‐type and A‐type potassium currents were not different between hypertensive and normotensive rats. In normotensive rats, 23.44mM tetraethylammonium (TEA) reduced the amplitude of sustained potassium currents by 50%. TEA also decreased the firing frequency in tonic neurons and converted tonic‐phasic neurons to phasic neurons. The different celiac neuronal firing patterns contributed by decreased sustained potassium current may play a compensatory role in elevated sympathetic activity in hypertension.Support HL70687
Long‐term cytokine blockade attenuates hypertensive response and protects against renal injury in spontaneously hypertensive rats
Authors: Elks Carrie M. , Haque Masudul , Mariappan Nithya , Guggilam Anuradha , Francis Joseph 
Published: June 2021

Introduction: Recent findings support a role for cytokines in renal dysfunction. In this study, we examined whether cytokine blockade protects against renal injury in spontaneously hypertensive rats (SHR).Methods: After measurement of baseline blood pressure, male SHR and WKY controls were treated with etanercept (ETN, 1 mg/kg/day), a recombinant protein which binds circulating TNF‐α; pyrrolidine dithiocarbamate (PDTC, 100 mg/kg/day), a NF‐κB blocker; or vehicle for 15 weeks. Blood pressure was measured weekly using tail plethysmography. Renal function was measured, rats were sacrificed, and renal tissue was collected for structural and functional mitochondrial assays. Oxidative stress in kidney medulla was measured using electron paramagnetic resonance (EPR). mRNA levels of cytokine and oxidative stress genes in renal tissue were measured using real time RT‐PCR.Results: Untreated SHR had higher mean arterial pressure and demonstrated glomerular injury and decreased renal function. These animals also had increased expression of cytokine and oxidative stress genes, accompanied by mitochondrial dysfunction. Treatment with ETN or PDTC in SHR attenuated hypertension, renal damage, and mitochondrial damage.Conclusion: Long‐term cytokine blockade protects against renal injury in SHR.
Tissue Doppler indices of cardiac contractile function during whole‐body heat stress
Authors: Brothers R Matthew , Bhella Paul S. , Shibata Shigeki , Wingo Jonathan E. , Levine Benjamin D. , Crandall Craig G. 
Published: June 2021

Previously we demonstrated that heat stress increases ejection fraction, suggestive of improved contractile function. However ejection fraction is highly load dependent and may be an imprecise measure of contractile function. Therefore, we sought to use tissue Doppler mitral annular systolic velocity as a myocardial specific and less load dependent index to test the hypothesis that heat stress improves contractile function when compared to normothermic conditions. Peak systolic septal and lateral mitral annular systolic velocities were measured during normothermic and heat stress (i.e., Δ internal temperature 1.0 ± 0.05°C) conditions in six healthy male volunteers. Normothermic measurements were performed following a 45‐minute supine rest period and always preceded the heat stress measurements. Heat stress significantly increased septal mitral annular systolic velocity (normothermia: 8.8 ± 0.4 cm/s; heat stress: 11.1 ± 0.8 cm/s; P=0.01) and lateral mitral annular systolic velocity (normothermia: 10.4 ± 0.9 cm/s; heat stress: 13.9 ± 1.5 cm/s; P=0.03) relative to normothermic conditions. These data support our previous findings suggesting that the contractile state of the left ventricle is improved during heat stress conditions.Supported by NIH Grant NIH‐HL61388 & HL84072