bluuebunny/crossref_metadata_2025_split huggingface dataset:
Compartmentalized cAMP Response to Prostaglandin EP2 Receptor Activation in Human Airway Smooth Muscle Cells
Authors: Harvey Robert D , Miyashiro Kathryn , Agarwal Shailesh R , Latt Htun , Ostrom Rennolds S 
Published: November 2023

It has been previously demonstrated that prostaglandin EP2 receptors selectively couple to adenylyl cyclase type 2 in non‐lipid raft domains of human airway smooth muscle (HASM) cells, where they regulate specific cellular responses. In the present study, we used FRET‐based biosensors targeted to different subcellular locations to determine exactly where EP2 receptor production of cAMP occurs in these cells. The Epac2‐camps biosensor, which is expressed throughout the cell, was used to measure bulk cytoplasmic responses, while MyrPalm‐Epac2 and CAAX‐Epac2 were targeted to lipid raft and non‐lipid raft regions of the plasma membrane, and NLS‐Epac2 was targeted to the nucleus. All probes were able to detect cAMP responses produced by direct activation of adenylyl cyclase with forskolin or exposure to the beta‐adrenergic receptor agonist isoproterenol. On the other hand, butaprost, an EP2 receptor agonist, elicited a response that could be detected by CAAX‐Epac2, but not MyrPalm‐Epac2. The butaprost response could also be detected by Epac2‐camps, as well as NLS‐Epac2. Exposure to rolipram, a selective inhibitor of phosphodiesterase type 4 (PDE4), produced no effect on its own that could be detected by any of the probes. However, in the presence of rolipram, exposure to butaprost produced a response that could then be detected by the MyrPalm‐Epac2 probe. Rolipram also enhanced the butaprost responses detected by Epac2‐camps and CAAX‐Epac2. These results support the conclusion that EP2 receptor activation leads to cAMP production in subcellular compartments associated specifically with non‐lipid raft regions of the plasma membrane. Furthermore, the activity of PDE4 appears to play a role in maintaining the integrity of this compartmentalized response.Support or Funding InformationSupported by NIH (GM107094)
Heat Shock Protein 90 Regulates μ‐Opioid Receptor Signaling In Vitro and in Different Regions of Mouse Brain
Authors: Lei Wei , Mullen Nate , McCarthy Sarah , Brann Courtney , Richard Philomena , Cormier James , Lindros Kayla , Edwards Katie , Bilsky Edward , Streicher John Michael 
Published: November 2023

Heat shock protein 90 (Hsp90) is a ubiquitous and highly expressed regulator for various signaling pathways and proteins. Only 2 previous studies suggested that Hsp90 might be involved in the regulation of opioid receptor signaling, which is one of the primary targets for drug discovery for chronic pain. This study was designed to determine the regulatory activities of Hsp90 on μ‐opioid receptor (MOR) signaling in various types of MOR expressing cells and in mouse brain. MOR expressing CHO, HEK, U2OS, and SH‐SY5Y neuroblastoma cells were treated with an Hsp90 specific inhibitor (17‐AAG) for 24 hrs followed by treatment with the MOR full agonist DAMGO. The expression and activation of signaling proteins was investigated by Western blot. We found that the expression of MOR, βarrestin2, and Akt was reduced in some cell types by inhibiting Hsp90. In contrast, the expression of Hsp70 was increased by the treatment of 17‐AAG in all cell types, as expected. The activation of ERK MAPK was also altered by Hsp90 inhibition in a cell context dependent manner – whether increasing DAMGO stimulated activation, increasing the baseline, or blunting activation. The strongly context dependent nature of Hsp90 regulation indicated that we would need to determine the role of Hsp90 in regulating the MOR in vivo. We thus injected CD‐1 mice intracerebroventricularly with 17‐AAG for 24 hrs, followed by injection of vehicle or DAMGO into the brain. Interestingly, we found an increased ERK signaling baseline and abolished DAMGO induction in the periaqueductal grey, along with increased Hsp70 expression. Preliminary results in the brain stem suggest that ERK MAPK activation in response to DAMGO is abolished by 17‐AAG, without increasing the baseline. Furthermore, 17‐AAG treatment showed a modest decrease on the acute anti‐nociceptive tail‐flick response to DAMGO, and increased urine and feces output after naloxone precipitated withdrawal in models of acute and chronic dependence. These findings demonstrate that Hsp90 has a strong regulatory role in MOR signaling both in vitro and in vivo, and suggest that Hsp90 activators or other modulators could be potentially used as a co‐therapy for improving the therapeutic index of opioid drugs.Support or Funding InformationWork supported by institutional startup funds from the University of Arizona, along with an internal UNE Mini‐Grant and a Pilot Grant from a UNE Centers for Biomedical Research and Excellent (COBRE) grant (Ian Meng PI, #P20GM103643).
Internalization of Protease‐Activated Receptor 4 is AP‐2 Dependent and Utilizes a Non‐canonical Sorting Motif
Authors: Smith Thomas Horace , Coronel Luisa , Nieman Marvin , Trejo JoAnn 
Published: November 2023

Protease‐activated receptor‐4 (PAR4) is a GPCR that is activated by proteolytic cleavage of its N‐terminus by thrombin and other proteases. Due to this irreversible proteolytic mechanism of activation, the mechanisms that control signaling by PAR4 are important for regulating the magnitude and duration of the signaling response. While the processes that control signaling of PAR1, a related GPCR activated by thrombin, have been extensively characterized, the mechanisms that regulate PAR4 signaling remain poorly understood. Unlike PAR1, activated PAR4 is not phosphorylated and exhibits prolonged signaling. These findings raised the question of whether PAR4 signaling is regulated by intracellular trafficking, a process which remains poorly defined for the receptor. The focus of this study is to delineate the mechanisms that control PAR4 intracellular trafficking. To examine the mechanisms that control PAR4 endocytosis, we expressed FLAG‐PAR4 in HeLa cells, a cell model system used extensively to characterize clathrin‐ and dynamin‐mediated endocytosis. We showed that upon activation with the agonist peptide AYPGKF, ~25% of PAR4 internalized from the cell surface over one hour. We next employed immunofluorescence confocal microscopy to visualize the endocytic sorting of activated PAR4 and found that the majority of activated and internalized PAR4 localized to early endosomes by 30 minutes and then sorted to lysosomes between 60 and 90 minutes. We utilized siRNA knockdown of clathrin and immunofluorescence microscopy to show that PAR4 undergoes clathrin‐mediated endocytosis following activation. Consistent with clathrin‐mediated endocytosis, expressing a dominant‐negative dynamin K44A mutant also blocked activated PAR4 internalization. Interestingly, however, unlike most GPCRs, PAR4 internalization occurred independently of both β‐arrestin 1 and 2 and the C‐tail domain. Rather, we identified a highly conserved potential AP‐2 binding motif in the receptor's third intracellular loop, and thus hypothesized that PAR4 internalization was mediated by the clathrin adapter protein AP‐2. Using siRNA knockdown of AP‐2, we confirmed that the adapter protein was necessary for PAR4 endocytosis. We also observed that a PAR4 mutant lacking the putative AP‐2 binding motif exhibited a defect in agonist‐induced internalization. To confirm our findings we examined endogenous PAR4 internalization in the Dami megakaryoblastic cell line. In ongoing studies, we are investigating the effect of dysregulated PAR4 trafficking on signal regulation.Support or Funding InformationFunding for this project was provided by NIH/NHLBI F31 HL116187 (T. Smith).
Increased Tubular Dysfunction and Injury Response Biomarkers in the Absence of Overt Clinical Nephrotoxicity in Cancer Patients Receiving Cisplatin Therapy
Authors: George Blessy , Wen Xia , Johnston Nickie , Gomez Madeleine , Ellison Lucas , Joy Melanie , Aleksunes Lauren 
Published: November 2023

Novel urinary proteins have emerged as more sensitive indicators of acute kidney injury compared to serum creatinine and may provide clues regarding the mechanism and anatomic location of nephrotoxicity. We sought to characterize urinary concentrations of biomarkers that reflect an acute injury response (KIM‐1, clusterin, NGAL, TFF3), glomerular and tubular dysfunction (cystatin C, calbindin, GST‐π, albumin, β2MG) and inflammation (IL‐18, MCP‐1, osteopontin) after exposure to cisplatin. Patients (n=57) with solid tumors and scheduled for chemotherapy with cisplatin ≥ 25 mg/m2 were recruited. Urine was collected at baseline, 3, and 10 days post infusion and analyzed using a multiplex protein assay. Markers of tubular dysfunction (β2MG, cystatin C, albumin) were elevated at day 3 by 330%, 185%, and 147%, respectively. By day 10, levels of calbindin and GST‐π were increased by 827% and 160%, respectively. Markers of injury and inflammation (KIM‐1, clusterin, and MCP‐1) were also elevated at day 10. Although reported to be down‐regulated in tubular injury in rats, levels of TFF3 in cisplatin‐treated patients were increased at days 3 (170%) and 10 (203%). Interestingly, serum creatinine and urinary concentrations of pro‐inflammatory proteins IL‐18 and osteopontin were not altered. ROC curves calculated from pre‐ and post‐cisplatin infusion concentrations revealed that β2MG and TFF3 had the highest AUC for day 3 (0.79 and 0.68, p<0.002), while calbindin, TFF3, KIM‐1, and MCP‐1 AUCs (0.76, 0.69, 0.68 and 0.68, p<0.003) were highest at day 10. These data suggest that novel urinary protein biomarkers may reveal mechanisms and time‐dependent subclinical kidney injury in the absence of clinically detectable damage in patients receiving cisplatin.Support or Funding InformationSupported by an AFPE Fellowship, DK080744, DK093903, and CA072720.
N‐acetylcysteine decreases the susceptibility to Diclofenac‐induced nephrotoxicity in diabetic patients
Authors: Yeh Ching Hua 
Published: November 2023

Diabetes patients are at high risk for chronic kidney diseaseor end stage renal disease which leading to dialysis because of long‐term taking medicine. Diclofenac (DLF) is a commonly prescribed NSAID. DLF‐induced nephrotoxicity may involve production of reactive oxygen species leadingto oxidative stress and cell death in the kidney. DLF could induce nephrotoxicity by inhibiting the activity of Nrf‐2 has been discovered in our previous research. The present study clarified whether the dysfunction of Nrf‐2 mediated oxidative stress under high‐glucose oxidative stress is related to the high susceptibility to DLF induced‐nephrotoxicity in diabetes patients. In vivo, normal or STZ‐diabetic mice were intraperitoneally injected with DLF 200 mg/kg for continuous three days or 5 mg/kg for four weeks to induce acute or chronic renal damage respectively. N‐acetylcysteine (NAC; 600 mg/kg) was administrated by oral gavage to DLF‐injected mice. Tissue injury, oxidative stress and fibrotic changes were observed in the kidney of these mice. The expression changes of antioxidative genes, Nrf‐2, SOD, GPx, and BMP‐7 in normal and STZ‐diabetic mice were compared. In vitro, the oxidative stress and the gene expression changes in NRK52E cell exposed to high glucose combined with DLF stimulation were analyzed. Both acute and chronic DLF‐injection increased the BUN and creatinine levels of mice. The DLF‐induced nephrotoxicity was severe in STZ‐diabetic mice. The antioxidant‐NAC treatment could attenuate the DLF‐induced acute or chronic kidney injury as well as decrease the TGF‐β and fibronectin expression in the kidney of STZ‐induced diabetic mice. The renal expressions of BMP‐7 and Nrf‐2 as well as SOD and GPx in the DLF‐injected STZ mice were increased by NAC treatment. DLF cause high cytotoxicity when NRK52E cell was exposed to high glucose medium. The NAC treatment could decrease the significant cell death by increasing the activity of Nrf‐2 and inhibiting oxidative stress induced by high glucose combined with DLF stimulation. The present study demonstrated that NAC treatment could decrease the susceptibility of DLF‐induced kidney injury in diabetic mice by increasing the Nrf‐2‐triggered antioxidative effects in the kidney. We suggest that the low Nrf‐2 activity and extremely high oxidative stress is the main reasons of susceptibility in diabetic patients why they are more prone to the development of kidney injury than others.Support or Funding InformationThe present study was supported by grants MOST103‐2320‐B‐212‐001 from the Ministry of Science and Technology, ROC.
Pharmacokinetic Analysis of Aerosol Propellant HFA‐134a Elimination in the Human Breath
Authors: Shin Hye‐Won , Landaw Elliot M. , Barletta Barbara , Meinardi Simone , Aizik Shlomit , Leu Szu‐Yun , Blake Donald , Cooper Dan M. 
Published: November 2023

The goal of the study is to model the elimination kinetics of Hydrofluoroalkane (HFA‐134a) in the exhaled breath following a typical single asthma inhaler administration. HFA‐134a is the most commonly used aerosol propellant in metered dose inhalers. We previously demonstrated that our technical capabilities allow us to measure breath HFA‐134a levels at 24 to 48 hours post asthma inhaler administration, and thus breath HFA‐134a is a promising biomarker for identifying asthma inhaler compliance (Clinical and Translational Science 2015).Exhaled breath gases were collected in evacuated electro‐polished stainless steel canisters from healthy participants at baseline and at 5 min, 7 min, 10 min, 15 min, 30 min, 1hr, 2hr, 3hr, 4hr, 6hr, 8hr, 12hr. 24hr, 36hr, and 48hr ‐post inhaled corticosteroids administration (Flovent HFA, Glaxo Smith Kline). The corresponding ambient HFA‐134a levels were also measured. HFA‐134a levels in the breath and ambient air samples were quantified using gas chromatography with a quadrupole mass spectrometer detector. The Institutional Review Board at the University of California, Irvine approved the study, and informed consent was obtained from the participants.HFA elimination in the breath followed a three‐exponential pharmacokinetic model. The estimated time constants (mean (SD)) for the three components were 5.5 (0.5) minutes, 62.4 (11.1) minutes, and 664 (140) minutes, with an overall mean retention time (MRT) of 71.8 (40.4) minutes. The shortest and longest half‐life components comprised 66 (17) % and 9 (7) % of the total area under the curve, respectively. Breath carbon dioxide (CO2) levels were also monitored as a reference breath gas to ensure that the gas sampled was alveolar gas. The mean (SD) CO2 level was 4.7 (0.6) % of all breath samples.This study demonstrates that the breath HFA‐134a elimination in healthy control humans can be described by a 3rd order exponential equation. In earlier studies, we modeled bicarbonate distribution kinetics and found similar time constants (2.2 min, 11.9 min, and 83.3 min; MRT 65 min.; Barstow et al. Am. J. Physiol. 259: R163, 1990). We speculate that the first two time constants reflect compartmental kinetics with the lung and circulation while the slowest primarily reflects distribution to body tissues. The kinetics for HFA‐134a appear to be slower than for bicarbonate. The calculated HFA‐134a elimination parameters (i.e., rate constants or corresponding half‐lives) may be useful in conjunction with breath HFA‐134a levels for accessing asthma inhaler medication compliance and further to understand physiological complexity of the lungs that affect HFA‐134a elimination.Support or Funding InformationThis work was supported by NIH grants: Translational Pilot Research Award (subaward of UL1 TR000153) and Program Project Grant P01HD048721
A novel CXCR4‐ARF1‐MAPK signaling pathway controls prostate tumorigenesis
Authors: Davis Jason E , Wu William H , Surrao Kristen , Teng Yong , Wu Guangyu 
Published: November 2023

Prostate cancer is one of the most common cancers in men and is the second leading cause of cancer‐related mortality in men. It is well known that enhanced activation of the MAPK pathway Raf‐MEK‐ERK1/2 directly correlates with the progression, androgen independence and poor prognosis of prostate cancer. Recent studies have also shown that the chemokine receptor CXCR4 is highly expressed in prostate cancer and contributes to prostate tumor growth and metastasis. However, the molecular mechanisms underlying the hyper‐activation of MAPK and the function of CXCR4 remain poorly elucidated. Here, we demonstrated that CXCL12‐induced CXCR4 activation markedly enhanced the activation of ADP‐ribosylation factor 1 (ARF1), a Ras‐related small G protein, in a time‐ and concentration‐dependent fashion, and siRNA‐mediated depletion of ARF1 dramatically inhibited MAPK activation by CXCR4 in prostate cancer cells. More interestingly, we found that ARF1 can mimic Ras to directly and activation‐dependently interact with Raf1, specifically its Ras‐binding domain. Mutations that disrupted ARF1 activation considerably attenuated ARF1‐Raf1 interaction and MAPK activation. These data suggest that, through its direct interaction with Raf1, ARF1 regulates the MAPK activation by CXCR4 in prostate cancer cells.We then investigated the role of ARF1 in prostate tumorigenesis. We found that ARF1 expression and activation were markedly elevated in human prostate cancer cells and tissues. Depletion of ARF1 significantly inhibited prostate cancer cell proliferation in vitro and xenograft tumor growth in vivo, whereas over‐expression of ARF1 produced opposing effects. These data have revealed an unappreciated function of ARF1 in prostate cancer. Altogether, our studies have identified a novel CXCR4‐ARF1‐MAPK signaling pathway which plays an important role in prostate tumorigenesis and may represent a key molecular target for therapeutic innovation (R01GM076167).Support or Funding InformationR01GM076167
MRTF‐A and YAP exert dual control in GPCR and RhoA‐mediated transcriptional regulation and cell proliferation
Authors: Yu Olivia M. , Miyamoto Shigeki , Brown Joan Heller 
Published: November 2023

The ability of a subset of G‐protein coupled receptors (GPCRs) to activate RhoA endows them with unique growth regulatory properties. Two transcriptional pathways are activated through GPCRs and RhoA, one utilizing the transcriptional co‐activator MRTF‐A and SRF, the other the transcriptional co‐activator YAP and TEAD. These pathways have not been compared for their relative importance and potential interactions in RhoA target gene expression. GPCRs for thrombin and S1P on human glioblastoma cells robustly couple to RhoA and induce the matricelluar protein CCN1. Knockdown of either MRTF‐A or YAP abrogates S1P stimulated CCN1 expression, demonstrating that both co‐activators are required. MRTF‐A and YAP are also both required for transcriptional control of other S1P regulated genes in various cell types and for S1P stimulated glioblastoma cell proliferation. Interactions between MRTF‐A and YAP are suggested by their synergistic effects on SRE.L and TEAD‐luciferase expression. Moreover, MRTF‐A and YAP associate in co‐IPs from S1P stimulated cells. ChIP analysis of the CCN1 promoter demonstrates that S1P increases co‐activator binding at their canonical transcription factor sequences. Unexpectedly, SIP also enhances MRTF‐A binding at TEA sites. Our findings reveal that GPCR and RhoA‐regulated gene expression requires dual input and integration of two distinct transcriptional pathways.Support or Funding InformationThis work was supported by NIH Grant GM036927, HL028143, and PPG/HL085577 to JHB, and by HL097037 and AHA Grant 15GRNT22970009 to SM. OY was supported by T32 GM007752 (Graduate Training in Cellular and Molecular Pharmacology) and T32 DK007541 (Endocrinology and Metabolism Training Grant).
MULTI DYNAMICS MATHEMATICAL FUNCTION AND MATRIXES IN STIMULATION IMMUNE SYSTEM
Authors: Izunobi Felicia Ukamaka , Einstein George P 
Published: November 2023

One of the most frequent complications of people with diabetes is foot disorders, specially foot ulcers or wounds. These wounds can easily become infected, and are known as a diabetic foot infections (DFIs). If they are not treated, the infection can progress rapidly, involving deeper tissues and threatening survival of the limb. Sometimes these infections conclude with the affected limb needing to be amputated. It is unclear whether any particular antibiotic is better than any another for curing infection or avoiding amputation.Materials and MethodsThis report focuses on Multi Dynamics Mathematical Function and Matrixes for Infections of diabetic's patients. First Dynamic Mathematical function and Matrixes presented successful G. P. Einstein Sigma Quantum Biophotonics (GPEB) Therapy Model. Second Multi Dynamic Function and Matrixes presented Sigma Quantum Natural Medicine I.F. Ukamaka Model (IFUN). In Multi Dynamics models the independent variables are time and Quantum Absorbed Energy for stimulation the immune System by Biophotonics Spectrum GPEB or Natural Medicine (IFUN).ResultsThe research of investigating the marks of the cytokines profile of mononuclear layers of cells that remove infections for patients with diabetic type two. Expression Analysis mRNA (PCR): Complementary DNA test (cDNA) was carried out on synthetic matrix/dye RNA (1μg) by application of Transcriptor First Strand cDNA on Synthetic Kit (Roche). IL‐1, IL‐6, Il‐10 IL‐12 TNF‐α IFN γ.ConclusionMulti Dynamics Math Function and Matrixes creates hope to extend the treatments for diabetics type 2 patients with infections resistant to antibioticsSupport or Funding InformationEinstein Medical Institute USA
Sex‐based Differences in the Aortic Function of UCD‐T2DM Rats: A Novel Rat Model of Type 2 Diabetes Mellitus
Authors: Akther Farjana , Shaligram Sonali , Karimi Mujibullah D. , Graham James L. , Stanhope Kimber L. , Havel Peter J. , Rahimian Roshanak 
Published: November 2023

UC Davis type 2 diabetes mellitus (UCD‐T2DM) Rat is a validated model of type 2 diabetes mellitus. UCD‐T2DM rats are characterized by polygenic, adult‐onset obesity and spontaneous β‐cell failure and, as a result, more closely models the pathophysiology of type 2 diabetes in humans than other rodent models of the disease. The objective of this study was to investigate alterations in aortic endothelial function in the UCD‐T2DM Rat model and to determine whether there is a sexual dimorphism in diabetes‐induced aortic dysfunction. Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10−8 to 10−5M) was measured in intact aortic rings pre‐contracted with phenylephrine (PE, 2μM). Endothelium‐independent vasodilation to sodium nitroprusside (SNP, 10−9 to 10−5 M) was assessed in endothelium‐denuded rings pre‐contracted with PE (2μM). Furthermore, constrictor response curves to PE (10−8 to 10−5 M) were generated.Diabetes significantly impaired relaxation responses to ACh and SNP in aortic rings from female UCD‐T2DM Rats, however, potentiated the relaxation in males. Moreover, diabetes significantly shifted PE contractile responses to the left in aortic rings of both sexes. Both maximal contraction and sensitivity to PE in aortic rings from diabetic females were significantly higher than those in control rats. In males, diabetes did not affect the maximal contraction to PE.These data, for the first time, show that the vascular function in aortic rings of UCD‐T2DM is altered in both sexes. The decreased sensitivity of vascular smooth muscle to NO along with the enhanced contractile responsiveness to PE may in part contribute to the attenuated relaxation response to ACh in diabetic female rats. On the other hand, an elevated sensitivity of vascular smooth muscle to NO may partially explain the potentiation of ACh responses in diabetic males. The overall results indicate the presence of sex‐based differences in the aortic function of UCD‐T2DM Rats.