bluuebunny/crossref_metadata_2025_split huggingface dataset:
TRPV5‐promotor driven expression of EGFP produces fluorescence in late DCT, CNT and early CCD
Authors: Praetorius Jeppe , Andrew Fenton Robert , Füechtbauer Ernst‐Martin , Vind Hofmeister Marlene 
Published: June 2021

TRPV5 (ECaC) is an apical Ca2+ channel of renal late distal convoluted tubules and connecting tubules (DCT2 and CNT, respectively), and is critically involved in regulated Ca2+ reabsorption. The protein was previously localized to CNT by immunohistochemistry and RT‐PCR analysis. Genetic disruption of TRPV5 leads to hypercalciuria.Transgenic mice were developed that express EGFP driven by the TRPV5 gene promoter 1) to verify and expand the expression‐profile of TRPV5 and 2) to generate a cell line of the renal tubules expressing this protein as a tool for further studies of TRPV5 biology. TRPV5 promoter‐driven EGFP fluorescence was detected in late DCT2 and CNT in isolated living tubules by confocal microscopy. In addition, EGFP was expressed in some cortical collecting ducts (CCD). Robust EGFP signal was confirmed in sections of paraffin embedded kidney, and the expression of EGFP in both late DCT2/CNT and CCD was verified by double‐fluorescence immunolabeling with various tubule markers. In preliminary analysis, TRPV5 protein expression in DCT2/CNT as well as in CCD using anti‐TRPV5 antibodies coincided with the expression of TRPV5 promoter‐driven EGFP.Thus, TRPV5 is expressed in DCT2/CNT and CCD, and an EGFP‐expressing transgenic mouse has been generated enabling easy isolation of TRPV5‐expressing Ca2+‐transporting renal tubules. Supported by the Danish National Research Foundation.
Sodium appetite and cocaine sensitization share similar mechanisms
Authors: Acerbo Martin Javier , Johnson Alan Kim 
Published: June 2021

Previous studies indicate that the augmented salt appetite induced by repeated sodium depletions may be related to psychostimulant‐induced sensitization. We further investigated this hypothesis by treating the same rats with DOCA to induce salt appetite and with cocaine to induce psychomotor sensitization. In Exp. 1, two groups of rats had either access to sodium (DS) or no access (DnS) and received daily injections of DOCA for 8 days. A third group with sodium access received vehicle (V) injections. All groups had access to distilled water. After a week off, rats were injected with cocaine and placed into cages to monitor activity. The group DnS displayed increased locomotion and showed an increased sensitivity to cocaine compared to V and DS groups. In Exp. 2, rats received daily injections of either cocaine (Coc) or saline (Sal) for 7 days. The Coc and Sal groups were then subdivided into 3 subgroups (DS, DnS, and V) and given DOCA treatment for 7 days. Finally, all rats were given access to sodium. DS rats with Coc history showed increased sodium appetite compared to rats with Sal history, whereas DnS rats with Coc or Sal history did not differ. In summary, DOCA‐induced salt appetite and cocaine‐induced psychomotor responses cross‐sensitized with one another suggesting that sodium appetite induced by DOCA and cocaine‐induced psychomotor sensitization share similar mechanisms.AHA 0625661Z; NIH DK66086 & HL14388
Proteomic validation: Searching for a heart of gold
Authors: Parsons Hannah L , Kulpa Jerzy , Brownsey Roger W. , Wambolt Richard W , Allard Michael F. 
Published: June 2021

Recent developments in proteomics techniques allow sensitive identification and relative quantitation of proteins in tissues and with increased sensitivity come the ability to detect small changes in protein expression. However, the means by which changes are validated remains incompletely defined. The proteome of mitochondria of hypertrophied hearts from rats with an abdominal aortic constriction (H) was quantitated using amine‐reactive isobaric tagging reagents (iTRAQ®) and tandem mass spectrometry. A small number (15 out of 250) were significantly increased and none decreased significantly. Increases ranged from 10 to 20%, as exemplified by voltage‐dependent anion channel‐1 (VDAC1), with only monoamine oxidase‐A (MAO‐A) showing a substantially greater increase (see table). Traditional immunoblot analysis revealed the significant increase in MAO‐A but not that of VDAC1. The discrepancy in results highlights the relative insensitivity of traditional immunoblot analysis and indicates that more sensitive approaches are essential.Supported by a grant from the Canadian Institutes for Health Research.
Long term exercise training exacerbates sarcopenia and only modestly attenuates apopotosis
Authors: Rowan Sharon , Hepple Russell T 
Published: June 2021

Apoptosis has been implicated in the age‐related loss of muscle known as sarcopenia. In addition, exercise training attenuates some markers of apoptosis. The purpose of this study was to evaluate the impact of a treadmill exercise training program initiated in late middle aged (LMA) rats and continued for 7 mo until senescence (SEN) (a period that spans a dramatic acceleration of muscle mass and functional decline) on gastrocnemius muscle (Gas) mass and markers of apoptosis. 62 male rats were randomly assigned to a training group (T) or sedentary group (S). Contrary to our expectation, following the 7 mo period the Gas mass was lower in T (890 ±60 mg) than C (1143 ± 85 mg; P<0.05). In situ immunolabeling experiments revealed that despite the fact that the loss of fast twitch fibers was markedly lower in T with aging, there was no difference between T and C in the fraction of nuclei that were TUNEL positive (3.0±0.5% vs 2.4±0.4%), or in the number of 8‐hydroxy guanosine positive spots per 100 fibers (1.3±0.3 vs 2.3±0.7). On the other hand, the fraction of fibers positive for activated caspase 3 was less in T (1.0±0.6%) vs C (2.3±0.5%; P<0.05). These results show that exercise training, although protecting fast twitch fibers with aging, had very modest impact on markers of apoptosis activation in Gas muscle of SEN rats.Supported by CIHR & AHFMR.
Hypoxia causes perinuclear mitochondrial clustering and nuclear oxidant stress in pulmonary artery endothelial cells (PAECS) via a dynein‐dependent molecular motor
Authors: Swiger Brad , Patel Mita , Pastukh Viktor , Gillespie Mark N. , Al‐Mehdi Abu‐Bakr 
Published: June 2021

Accumulating evidence suggests that mitochondria are motile, and may be recruited to subcellular domains where they participate in signal transduction. Mitochondria respond to hypoxia in PAECs by increasing oxygen radical generation, but whether hypoxia alters the disposition of mitochondria in PAECs is unknown, and if so, the molecular mechanism remains to be clarified. Accordingly, we mapped the disposition of Mitotracker‐stained mitochondria in cultured PAECs, and found that hypoxia caused a time‐dependent decrease in mitochondrial density near the plasma membrane and an increase in the perinuclear region. Interruption of the dynein‐dependent molecular motor using a specific siRNA prevented both the perinuclear mitochondrial clustering and, importantly, a hypoxia‐induced nuclear oxidant stress as detected by nuclear DCF fluorescence and by the level of oxidative base modifications in the VEGF promoter. Mitochondria were the source of the DNA‐modifying oxidants as evidenced by the finding that the respiratory chain inhibitor, myxothiazol, prevented the hypoxia‐induced nuclear DCF fluorescence without altering mitochondrial perinuclear clustering. These data suggest that hypoxia causes dynein‐dependent mitochondrial translocation to the perinuclear domain in PAECs that serves to create a nuclear oxidant stress important for hypoxic signaling.Supported by NIH
Maximal cutaneous blood flow is not affected by endurance training: implications for physiological interpretation of cutaneous blood flow
Authors: Ray Chester A , Kuipers Nathan T , Kearney Matthew L 
Published: June 2021

Cutaneous blood flow can be altered by non‐sympathetic influences such as hydration level, acclimatization, and training level. It is currently unknown what effect these factors have on maximal cutaneous blood flow. The purpose of this study was to investigate the effect of endurance training on maximal cutaneous blood flow. Fifteen healthy sedentary subjects (age, 25±1 years) had maximal cutaneous blood flow measured before and after eight weeks of endurance training or eight weeks of sedentary control. Arterial blood pressure (Finometer), brachial blood flow (Doppler ultrasound), and forearm cutaneous vascular conductance (laser Doppler flowmetry; flux/mean arterial pressure) were measured for 5 min at room temperature (22±1ºC). The arm was then heated for 30 min followed by an additional 30 min of measurements. A water perfused tube‐lined sleeve was used to heat the forearm skin to 41±1ºC. Exercise training increased VO2peak by 19.3%. Brachial blood flow and cutaneous vascular conductance increased with heating. Training did not alter brachial blood flow at rest (47±11 ml/min before training and 41±6 ml/min after training) or during maximal heating (217±28 ml/min before training and 217±28 ml/min after training). Training did not alter cutaneous vascular conductance at rest (0.010±0.003 ml/min/mmHg before training and 0.010±0.002 ml/min/mmHg after training) or during maximal heating (0.047±0.007 ml/min/mmHg before training and 0.051±0.009 ml/min/mmHg after training). These results suggest that endurance training does not alter maximal forearm cutaneous blood flow in humans.Research supported by NIH DC006459, HL077670, & M01RR10732
Role of Chylomicron Formation and Secretion in the Release of Diabetic Hormones GLP‐1 and GIP
Authors: Lu Wendell J , Yang Qing , D'Alessio David , Tso Patrick 
Published: June 2021

Glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP) are incretins produced in the intestine that play a central role in glucose metabolism and insulin secretion. Plasma GLP‐1 and GIP concentrations are low and difficult to assay in rodents. We have established a novel method of assaying GLP‐1 and GIP in vivo using the intestinal lymph fistula model. These studies further utilize this model to investigate the mechanism of lipid‐stimulated secretion of these incretins.Using this model, we have determined that peak GLP‐1 and GIP levels are 5–6 times higher in lymph than portal vein plasma. We further explored the role of chylomicron formation and secretion during lipid‐stimulated secretion of these incretins. Pluronic L‐81 (L‐81), a surfactant inhibiting chylomicrons, was given concurrently with Intralipid. The presence of L‐81 caused a significant decrease in lymph triglyceride level (P < 0.001). L‐81 plus Intralipid was shown to reduce lymph GLP‐1 secretion by 30 min (P < 0.001) and 60 min (P < 0.05), respectively, and the total GLP‐1 output over 5 hours was also significantly blunted (P < 0.046) compared to Intralipid stimulation alone. The effect of L‐81 was even greater with lipid‐stimulated GIP secretion which was completely abolished, and the decrease in total GIP output was more apparent (P < 0.008). We also demonstrated there was no direct correlation between free fatty acids levels and incretins secretion. We conclude chylomicron formation and secretion may be critical in the release of incretins in vivo during lipid ingestion, and GLP‐1 and GIP exhibit independent mechanisms of lipid‐stimulated secretion.
Increased Hsp32 Expression and Endothelial Dysfunction in Human
Authors: Rafiee Parvaneh , Nelson Victoria , Otterson Mary , Binion David 
Published: June 2021

INTRODUCTIONEndothelial dysfunction in IBD is associated with loss of NO generation and sustained levels of reactive oxygen species (ROS), which is linked to a loss of iNOS gene transcription. Hsp32 is associated with loss of NO generation via inhibition of iNOS gene expression in various disease, but little is known about its expression in IBD. We characterize Hsp32 expression in control and IBD endothelial cells, microvessels and submucosal gut tissues, to define the molecular mechanisms underlying the loss of NO generation,METHODS: Hsp32 and iNOS expression were assessed in freshly resected human gut tissues (normal and IBD) and human intestinal microvascular endothelial cells (HIMEC) by immunohistochemistry, RT‐PCR and Western blotting.RESULTS: Hsp32 expression was significantly increased in Ulcerative colitis (UC) and Crohn's disease (CD) compared to control tissue (n=9 for each group). Whereas the level of iNOS was decreased in the same samples. Strong staining for Hsp32 and a very weak iNOS staining in UC and CD microvessels compared to control tissue was observed. TNF/LPS resulted in increased iNOS expression in HIMEC.CONCLUSION: Increased Hsp32 expression in IBD microvessels may limit NO production leading to endothelial dysfunction. Hsp32 induction during chronic stress is an epigenetic mechanism for altered endothelial gene transcription and homeostasis in human IBD.
Modulation of the human paraoxonase gene family members by various effectors in Caco‐2 cells
Authors: Précourt Louis‐Philippe , Delvin Edgard , Amre Devendra , Sinnett Daniel , Seidman Ernest , Levy Emile 
Published: June 2021

The human paraoxonase (PON) gene family is composed of three members (PON1, PON2, PON3) aligned next to each other on chromosome 7. They are highly conserved in mammals, display similarities in their structural properties (~65% identity atthe amino acid level) and seem to play an important part in oxidative stress. Recently, PONs have been detected in the gastrointestinal tract, but their physiological roles and regulation remain obscure. Our objective was to examine the modulation of PON1, PON2 and PON3 in the Caco‐2 cell line. Fe2+‐ascorbate‐mediated lipid peroxidation promoted MDA formation without affecting sucrase activity, transepithelial resistance, DNA and protein content, as well as cell viability. The addition of the oxygen radical‐generating system reduced the gene expression of PON1, PON2 and PON3. Pre‐incubation with the antioxidant BHT or Trolox restrained the production of MDA and the inhibitory effect on PON mRNA levels. Similarly, the administration of CAPE, an inhibitor of NF‐kB activity, reversed the negative influence of inflammatory agents, including LPS, TNF‐α and INF‐γ. Rosiglitazone and DHA enhanced the gene expression of PON1 and PON3 without any influence on PON2 mRNA levels. Our data indicate that various effectors may impact on the gene expression of PON1, PON2 and PON3, sometimes with a distinct modulation.Supported by Crohn's and Colitis Foundation of Canada.
Hepcidin‐regulated and hepcidin–independent iron release pathways in hepatocytes and macrophages
Authors: Valore Erika V , Ganz Tomas 
Published: June 2021

Ferroportin (Fpn), a multi‐transmembrane protein, is the only known cellular exporter of Fe. In Fpn‐transfected cells or in macrophages, the iron‐regulatory hormone hepcidin (Hepc) binds to Fpn, induces its internalization and degradation, and thereby inhibits the release of Fe from cells. It is not clear whether the same mechanisms also regulate Fe efflux from hepatocytes. We examined Fe release from primary hepatocytes, hepatocyte cell lines (HepG2 and Hep3B), and macrophage cell lines (J774 and RAW264) with or without 7.5 μg/ml Hepc. After loading with 55Fe‐nitriloacetic acid, radioactive Fe release was monitored over 72h. In primary hepatocytes, Hep3B cells and macrophage cell lines, Hepc inhibited the total Fe release by up to 50% compared to control. The media were analyzed by gel‐filtration. For all cell types, 55Fe eluted in two fractions. The major fraction, ≈70 kD, eluted with transferrin, and its 55Fe content was decreased by Hepc‐treating the cells. A second fraction was > 220 kD and its 55Fe content was unaffected by Hepc‐treating the cells. On SDS‐PAGE, the 55Fe‐containing >220 kD fraction was unaffected by reducing conditions but was degraded by boiling. In summary, hepatocytes and hepatocyte‐derived cell lines exhibit Hepc‐regulated and presumably Fpn‐mediated Fe efflux. One or more alternative pathways for Fe export, not regulated by Hepc, also exist in hepatocytes and macrophage cell lines.Support provided by the Will Rogers Fund and NIH RO1 DK 065029
Survey of the GLUT2 translocation pore for substrate selectivity determinants
Authors: Witkowska Kate , Cheeseman Chris I. 
Published: June 2021

GLUT2 plays a major role in the absorption of nutrient hexoses, being expressed constitutively in the BLM and transiently in the apical membrane. This study's objective was to characterize motifs within TM 7 of GLUT2 which determine substrate specificity. When expressed in Xenopus oocytes, hGLUT1 transports fructose (FRU) at a rate of only 0.1% relative to glucose (GLU), while in GLUT2 they are equivalent. Mutating the NAV motif to NAI in GLUT1 increased FRU transport to approximately 0.9% (2.5 pmoles/oocyte/30 min with 100 μM substrate) relative to that of GLU. Mutating the molecular filter QLS in hGLUT1 to HVA also increased the relative rate of FRU transport (up to 0.5%) but to a lesser extent than the NAI mutation. Further, the combined hGLUT1 HVA‐NAI mutant had a relative FRU transport rate of 1.1 % (6 pmoles/oocyte/30 min at 100 μM substrate). Kinetic analysis for the hGLUT1 HVA‐NAI revealed that the Km for GLU was comparable to that of hGLUT1wt (1.5mM), while the Km for FRU was comparable to that for hGLUT2 (~70 mM).Thus, we have successfully introduced hGLUT2‐like FRU kinetics into hGLUT1, without affecting the GLU transport by this isoform. These results point to the presence of, at least two, selectivity filters within class I hGLUTs. NAV/NAI is the exofacial access filter, while QLS/HVA seems to allow FRU translocation beyond the substrate binding site without influencing GLU binding and/or translocation.
Cerebral Hypoperfusion Increases Neuronal Nitric Oxide Synthase Abundance in the Ovine Fetal Brainstem
Authors: Shah Maitreyee , Wood Charles E 
Published: June 2021

Nitric oxide (NO) is potent vasorelaxant in vascular smooth muscle and serves as a neurotransmitter in the brainstem, influencing the neural pathways that control cardiovascular function. Hypotension, and the resultant cerebral hypoperfusion, stimulates a series of cellular responses that modify the reflex responses to subsequent periods of hypotension. This study was performed to test the influence of brachiocephalic occlusion (BCO) on the abundance of nNOS in the fetal brainstem. Fifteen chronically catheterized, late gestation fetal sheep were subjected to a 10 minute period of cerebral hypoperfusion followed by reperfusion. Fetuses were euthanized 0, 0.5, 3, 8, and 18 hours after the onset of BCO (n=3 at each timepoint). Brain regions were rapidly dissected, snap‐frozen, and later processed for isolation of protein for Western Blot analysis. We tested for an upregulation in fetal brainstem nNOS abundance in response to brachiocephalic occlusion using anti‐nNOS antiserum (Transduction Labs) and anti‐β‐actin (Sigma) for normalization of protein loading. After normalization with β‐actin, nNOS protein abundance appeared to increase 3 hours after the onset of a 10‐min period of BCO.When analyzed by one‐way ANOVA, the apparent increase was not statistically significant (p=0.079). Analysis of a smaller data set, in which the 18 hour values were excluded yielded a statistically significant change (p=0.022). Analysis of the individual means by Bonferroni test revealed that the nNOS abundance was greater at 3 hours than at earlier or later times. We conclude that nNOS abundance is increased in fetal brainstem after a period of cerebral hypoperfusion, and we speculate that increased nNOS activity increases brainstem NO responses to subsequent periods of hypotension.
Characterization of stratification and tight junction formation in cultured human urothelial cells
Authors: Rickard Alice , Marentette John , McHowat Jane 
Published: June 2021

The urothelium is composed of stratified transitional epithelium of 3 cell types: basal, intermediate, and superficial. It has been proposed that urothelial cells acquire their mature characteristics through differentiation. The most differentiated, superficial cells develop tight junctions (TJs) that restrict urine movement and abnormalities in barrier function may be involved in syndromes such as interstitial cystitis. To further our understanding of urothelial differentiation, we used cells from normal human ureter and examined the zonula occludens (ZOs), “undercoat” scaffolding proteins in the cytosol that interact with claudins and actin filaments. Cell monolayers were stratified and differentiated using Ca and fetal bovine serum (FBS) and the process was studied using impedance, videomicroscopy, and confocal immunohistochemistry. With addition of Ca and FBS, impedance quickly increased by 2.5 fold. Withdrawal of the supplements returned impedance to baseline, suggesting the formation of tight junctions, rather than increased cell proliferation and immunohistochemistry demonstrated the appearance of ZO‐1 at cell junctions minutes after supplement addition. Observation by videomicroscopy suggested that cell division continued to occur after the cells had differentiated. Taken together, these studies increase our understanding of urothelial development.Supported by DK66119 (JM)
A novel constitutively active isoform of cGMP‐dependent protein kinase I (PKGI) is localized in the nucleus of vascular smooth muscle cells (VSMC)
Authors: Roberts Jesse David , Sugiura Takahiro , Nakanishi Hidehiko 
Published: June 2021

Objective: Pediatric pulmonary vascular disease (PVD) is associated with excessive VSMC proliferation. Because recent studies suggest that nitric oxide attenuates PVD and VSMC proliferation by stimulating PKGI activity, the mechanisms affecting PKGI's nuclear localization were investigated.Methods: PKGI was mapped to intracellular compartments using immunologic and biochemical methods and its catalytic activity was determined using a PKGI‐specific peptide substrate. The domain structure of nuclear PKGI was analyzed using antibodies and detailed using Edman‐based protein sequencing.Results: PKGI immunoreactivity was detected in the nuclei of VSMC, where its activity was cGMP‐independent. Immunoblotting revealed that the nuclear PKGI isoform (PKGIγ) is 18 kDa smaller than the cytosolic one and lacks a NH2‐terminal dimerization domain, which tethers it to cytosolic proteins. The PKGI dimerization domain was observed in the perinuclear region where it colocalized primarily with the endoplasmic reticulum. Sequencing of immunopurified endogenous nuclear PKGIγ confirmed the absence of a dimerization domain and revealed that its putative scissile amino acids are not encoded by DNA located near typical RNA splice donor‐acceptor guidance nucleotides.Conclusions: A novel constitutively active isoform of PKGI that appears to result from PKGI proteolytic processing is localized in the nuclei of VSMC.