bluuebunny/crossref_metadata_2025_split huggingface dataset:
Physiological Study of the Neurotoxic Actions of Manganese on Dopamine Cell Signaling in Crassostrea virginica, Downstream of D2‐Like Receptor Activation
Authors: Robertson Cheyanne , Carroll Margaret A. , Catapane Edward J. 
Published: June 2021

Dopamine and serotonin are important neurotransmitters in invertebrates and have been well studied in bivalves with respect to gill and heart. Lateral gill cell cilia of Crassostrea virginica are controlled by a reciprocal dopaminergic‐serotonergic innervation from their ganglia. Dopamine slows down and stops beating of gill lateral cell cilia while serotonin accelerates cilia beating rates. Previously we found that dopamine postsynaptic receptors involved in controlling gill lateral cell cilia are D2‐like (D2DR). The D2DR signaling pathway involves steps that include inhibition of adenylyl cyclase and activation of phospholipase C (PLC). While the effects of dopamine on cAMP levels in oyster gill is well known, the activation of PLC by dopamine D2DR signaling has not been well studied. PLC activation increases cellular inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG are both second messengers. IP3 binds to IP3 receptors and increase cytoplasmic calcium levels. High cytoplasmic calcium levels have been shown to slow down gill lateral cell cilia beating. DAG activates protein kinase C. Manganese is a neurotoxin causing Manganism in people, a Parkinson's‐like syndrome. The dopamine induced cilio‐inhibition of gill lateral cell cilia activity in C. virginica is disrupted by manganese. Earlier work of our lab showed the site of action of manganese is the D2DR receptor. We hypothesize IP3 and DAG would be cilio‐inhibitory but not be effected by manganese treatments. We studied this by conducting acute experiments testing the effects of IP3, a DAG analog, a DAG kinase inhibitor, and manganese on gill lateral cell cilia beating. Ciliary activity of gill lateral cells was measured by stroboscopic microscopy and expressed as beats/min ± sem. The results showed that IP3, the DAG analog and the DAG kinase inhibitor each produced a dose dependent (10−6 – 10−4 M) decrease in gill lateral cell cilia beating. Acute treatments with 500 μM of manganese did not reduce the effectiveness of IP3 and the DAG analog to reduce cilia beating. This study provides new knowledge of the physiological actions of the PLC, IP3 and DAG components of the D2DR pathway on gill lateral cell cilia of C. virginica and demonstrates that manganese did not affect those aspects of the D2DR pathway. The study also provides a foundation to further study the physiological roles of PLC in bivalve gill as well as the neurotoxic actions of manganese on the physiology of the D2‐like receptors in the gill.Support or Funding InformationSupported in part by a Carnegie Foundation award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interrogating Purinergic Signaling: From Extracellular ATP to Intracellular Second Messenger Dynamics
Authors: Tantama Mathew , Conley Jason , Valentino Stephen , Cholger Daniel 
Published: June 2021

Purinergic signals, such as extracellular adenosine triphosphate (ATP), mediate cell‐to‐cell communication during fundamental processes such as neural transmission, inflammation, and chemotaxis. Furthermore, the aberrant release of extracellular ATP can contribute to injury and disease, such as in epilepsy, cancer, and infections. Despite its importance in both physiological and pathophysiological conditions, it has remained a challenge to quantitatively measure the spatial and temporal dynamics of extracellular ATP because of the broad ranges of concentrations, distances, and timescales over which it acts. In order to address this challenge, we are developing genetically‐encoded biosensors that enable the visualization of the release and clearance of extracellular ATP. When extracellular ATP is released, it can activate P2 receptors, including P2X ligand‐gated ion channels and P2Y G‐protein coupled receptors, that lead to downstream intracellular signaling via second messengers such as calcium and cyclic adenosine monophosphate (cAMP). Thus, in order to interrogate purinergic signal transduction across the plasma membrane, we are also developing multiplex imaging methods that simultaneously monitor spectrally compatible sensors that correlate the dynamics of extracellular ATP release, purinergic receptor activation, and intracellular second messenger signaling. Using this integrative live‐cell imaging approach, we study both activity‐dependent and injury‐dependent purinergic signaling in neural systems.Support or Funding InformationWe gratefully acknowledge support from the Ralph W. and Grace M. Showalter Trust, the Purdue Research Foundation, and from National Institutes of Health grants R21 NS092010 and R21 EY026425.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mTORC2 Regulates the Maturation of Protein Kinase C by Folding and Phosphorylation
Authors: Baffi Timothy R. , Gould Christine M. , Wang Zhiyong , Gutkind Silvio , Newton Alexandra C. 
Published: June 2021

Protein Kinase C (PKC) is primed by a series of ordered phosphorylations and conformational transitions to yield an autoinhibited enzyme that is positioned to respond to second messengers. For most PKC isozymes, this maturation to produce a signaling‐competent enzyme depends on the mammalian Target of Rapamycin Complex 2 (mTORC2); however, the molecular mechanisms by which mTORC2 regulates PKC are unknown. Here we use a variety of Förster resonance energy transfer (FRET)‐based live cell imaging and pulse‐chase biochemical assays to examine the role of mTORC2 in the the folding, phosphorylation, and activity of overexpressed PKCbII. We show that in cells lacking Sin1, a critical component of mTORC2, PKC exists in an unprimed, unphosphorylated, and open conformation, with fully‐exposed membrane‐targeting modules. In contrast, overexpression of PKCbII in cells with functional mTORC2 results in accumulation of a fully‐phosphorylated and autoinhibited enzyme. Introduction of Glu at the two phosphorylated residues on the C‐terminal tail is insufficient to bypass the requirement for mTORC2 in the folding or rate‐limiting processing step of PKC. We also show that PDK‐1 is involved in the mTORC2‐regulated rate‐limiting step of PKC processing, as overexpression of PDK‐1 in the absence of mTORC2 results in phosphorylation of the hydrophobic motif, but not the turn motif. Finally, analysis of chimeric proteins of an mTORC2‐dependent (PKCbII) and mTORC2‐independent (PKCd) enzyme, coupled with peptide array experiments, identify two critical regions in the kinase domain and C‐terminal tail, respectively, that each confer mTORC2‐dependence. Taken together, our data support a model in which mTORC2 serves two independent functions in the processing of PKC: 1) as the direct kinase of the turn motif, and 2) in the regulation of the rate‐limiting conformational change that allows the enzyme to autophosphorylate at the hydrophobic motif and adopt an autoinhibited conformation.Support or Funding InformationThis work was funded by NIH GM43154 to AN, NIH Pharmacological Sciences Training Grant NIH T32GM007752 to TB (AN), and PhRMA Foundation Pre Doctoral Fellowship in Pharmacology/Toxicology to TB (AN).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CDK1‐dependent Phosphorylation of the Tumor Suppressor Phosphatase, PHLPP1, Regulates the Mitotic PHLPP1 Interactome
Authors: GRZECHNIK AGNIESZKA Teresa , Wong Cassandra , King Charles C. , Gingras Anne‐Claude , Newton Alexandra C. 
Published: June 2021

PH domain Leucine‐Rich Repeat Protein Phosphatase 1 (PHLPP1) is a tumor suppressor originally discovered for its ability to directly dephosphorylate and inactivate the pro‐survival kinase Akt, a key transducer of growth factor signaling. In the decade since this discovery, a number of other PHLPP1 targets have been elucidated, but still little is known about the molecular mechanisms governing the function and regulation of PHLPP1 itself. Here we report that PHLPP1 is hyperphosphorylated during mitosis in a CDK1‐dependent manner, and that this hyperphosphorylation regulates its interaction with mitotic proteins. Specifically, we show that PHLPP1 undergoes an electrophoretic mobility shift in mitotic cells that is lost with lambda phosphatase treatment, and is prevented by CDK1 inhibition. This mobility shift can be recreated in vitro using recombinant CDK1‐Cyclin B. Mass spectrometry and biochemical analysis reveals that these phosphorylations modify the N‐terminus of PHLPP1, a functionally uncharacterized region. A proximity dependent biotin identification (BioID) interaction screen revealed that mitotic PHLPP1 interacts with components of the mitotic spindle apparatus and the kinetochore. Additionally, the data suggest that the N‐terminus is required for the dissociation of PHLPP1 from interphase scaffolds, such as Scribble, during mitosis. Our data are consistent with a model that mitotic phosphorylation of PHLPP1 may play a key role in regulating binding to its mitotic partners. Reversible protein phosphorylation, orchestrated by kinases and phosphatases, regulates proper progression through mitosis, which is essential as errors in division can result in aneuploidy, a hallmark of cancer. The finding that PHLPP1 binds mitotic proteins in a cell cycle and phosphorylation‐dependent manner may have relevance to its tumor suppressive function.Support or Funding InformationUCSD Graduate Training Program in Cellular and Molecular Pharmacology NIH T32 GM007752 (to ATG), Frontiers of Innovation Scholars Program at UCSD (to ATG), NIH R35 GM122523 (to ACN)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heavy Metals' Analysis of Leachate From Municipal Solid Waste Landfill in Nekede, Owerri, Nigeria
Authors: Ibeh Olusola , Okafor Ikechukwu , Udochi Solomon 
Published: June 2021

Although municipal solid waste landfills have become an indispensable part of modern‐day societies, the presence of heavy metal in leachate from such pose a serious threat of contamination to water bodies, especially when the landfill is unlined. The assessment of heavy metals in leachate from the unlined landfill in Nekede, Owerri in Imo state of Nigeria was carried out. Samples were collected from actual leachate streams in the landfill and analyzed using standard procedures. Results show that certain heavy metals in the leachate were above the drinking water and waste water discharge standards by NESREA, NEMA and WHO. Ca, Hg, Se, As, Mn, Fe, Zn, Cd and Pb were all above the permissible limits in concentrations of 127.6, 1.57, 0.63, 1.46, 1.57, 68.21, 11.18, 0.31 and 1.15 respectively, all in mg/l. In view of the potential of the leachate to pollute the surrounding surface and ground water sources in the vicinity of the landfill it is pertinent that appropriate regulatory bodies intervene to rehabilitate the landfill and also to continuously monitor the ground water sources around the area in order to contain any contamination that might occur.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Characterization of KRAS with a Novel Interactor, Argonaute 2
Authors: Waninger Jessica , Shankar Sunita , Siebenaler Ronald F. , Beyett Tyler S. , Tesmer John J. , Chinnaiyan Arul M. 
Published: June 2021

The RAS family of small GTPases is one of the most commonly mutated oncogenes in human cancer. Mutations usually allow the accumulation of the GTP‐bound active form of RAS, thus resulting in the aberrant activation of MAPK and PI3K pathways that control cell growth, survival, and differentiation. While much has been learned about the major downstream pathways and effectors of RAS, little progress has been made in successfully targeting mutant RAS in the treatment of cancer. Recently, we identified a functional interaction between KRAS and Argonaute 2 (AGO2), a core component of the RNA‐induced silencing complex (RISC). Further interrogation of this interaction revealed that the switch II domain of KRAS (both WT and mutant) binds to the regulatory N‐terminal (N‐t) domain of AGO2. Mutant KRAS‐AGO2 binding resulted in attenuation of the RNAi function of AGO2 by preventing microRNA unwinding, a necessary step in the formation of the mature RISC complex. In recent studies using genetically engineered mouse models, AGO2 was found to be essential for mutant KRAS driven pancreatic cancer development, foreshadowing a critical role for the KRAS‐AGO2 interaction in vivo. Further, it has previously been identified that the Tyr393 residue in AGO2 is phosphorylated by the EGFR kinase leading to decreased AGO2 function. We observed that AGO2 phosphorylated at Tyr393 disrupts its interaction with WT but not with mutant KRAS. Differences in binding of WT and mutant KRAS with or without AGO2 phosphorylation can provide valuable information on potential residues/regions in KRAS and AGO2 that can be exploited for targeting the KRAS‐AGO2 interaction, providing a basis for differential regulation of the KRAS‐AGO2 interaction through growth factor activation. In this study, we will biophysically characterize the binding interface between KRASG12D and the N‐t fragment of AGO2. In addition, we will examine the structural changes induced by EGFR phosphorylation of AGO2. Structural characterization of this interaction will provide valuable information on potential residues/regions in KRAS and AGO2 that can be exploited for targeting this interaction in therapeutic development.Support or Funding InformationHHMINIHMSTP Training GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of exogenous ouabain on endothelium‐dependent vascular function
Authors: Chunrong HE , Vanhoutte Paul Michael , Leung Wai Sum Susan 
Published: June 2021

Endogenous ouabain is an inhibitor of sodium‐potassium adenosine triphosphatase (Na+/K+‐ATPase, also known as sodium‐pump). It is released by high sodium chloride concentrations from the hypothalamus and the adrenal cortex. Upon its release in the circulation, endogenous ouabain increases arterial constriction and peripheral resistance hence arterial blood pressure. In the clinical setting, digoxin, targeting the sodium pump as well, is an orally active agent exerting inotropic effects to treat heart failure or atrial arrhythmias. But toxicity often occurs due to overdose. The present study aimed to investigate the local effects when the sodium pump is inhibited by exogenous ouabain in acute settings. Aortae isolated from male Sprague Dawley rats (12 weeks old) were cut into rings and incubated in the organ chambers with Krebs‐Ringer solution for isometric tension recording, in the absence or presence of ouabain. They were contracted with phenylephrine and relaxed with increasing concentrations of endothelium‐dependent and ‐independent vasodilators, acetylcholine (ACh) and DETA NONOate, or exogenous arachidonic acid (AA). Ouabain inhibited endothelium‐dependent relaxations to ACh and AA in a concentration‐dependent manner. It reduced the maximal relaxation to ACh in aortae with endothelium where the nitric oxide signaling pathway plays a dominant role in vasodilatation, and increased the half maximal effective concentration (EC50) of DETA NONOate. The inhibitory effect of ouabain on AA‐induced relaxations was not observed in the presence of indomethacin (cyclooxygenase inhibitor). Ouabain potentiated the relaxations to AA when endothelial potassium channels involved in endothelium‐dependent hyperpolarization (EDH) signalings [namely intermediate‐ and small‐conductance calcium‐activated potassium channels (IKCa and SKCa, respectively)] were blocked by TRAM‐34 and UCL1684, respectively; the relaxation was not affected by the inhibition, with barium, of inwardly‐rectifying potassium channels (KIR, which also play a role in EDH pathway). Taken in conjunction, ouabain likely impairs the nitric oxide and/or cyclooxygenase pathways to reduce endothelium‐dependent relaxations. When the major mechanisms for the generation of endothelium‐derived relaxing signals (namely, nitric oxide, prostanoids and EDH) are inhibited, ouabain unmasks the relaxation to AA that is independent of KIR activation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bypassing Occlusion: Abdominal Aorta Occlusion in Rats and the Therapy with the Stable Gastric Pentadecapeptide BPC 157
Authors: Kasnik Katarina , Kralj Tamara , Pavlov Katarina Horvat , Drmic Domagoj , Blagaic Alenka Boban , Seiwerth Sven , Sikiric Predrag 
Published: June 2021

AimWe focused abdominal aorta occlusion in rats and the therapy with the stable gastric pentadecapeptide BPC 157. Effective in perforated stomach lesion and perforated caecum lesion as well as ischemic colitis, ischemia and reperfusion, BPC 157 rapidly activates vessels ”running“ toward the perforated defect (stomach perforation (Gastroenterology 2017; 152, Issue 5, Supplement 1, S889–S890; Gastroenterology 2017; 152, Issue 5, Supplement 1, S890–S891) or to bypass vessel obstruction (ischemic colitis) (World Journal of Gastroenterology, in press, 2017) and largely attenuates lesions course. In this study, we investigated the effect of pentadecapeptide BPC 157 on rapid activation of collateral circulation after acute occlusion of the abdominal aorta in rats.MethodsFemale Wistar Albino rats, 300 ± 20 g b.w. were used. Animals were randomized to control and pentadecapeptide BPC 157 treated group. Deeply anesthetised (diazepam 6 mg/kg i.p., thiopental 40 mg/kg i.p.) rats underwent abdominal aorta ligation with surgical suture at the level between a right iliolumbar artery and the bifurcation of the aorta. Medication was 1 ml bath, either BPC 157 10 μg/kg or saline, given immediately after abdominal aorta ligation at the site of the ligation. The digital subtraction angiography (DSA) assessment was at the 1 h ligation time. A fluoroscopy unit (Shimadzu type C‐Vision Plus, Tokyo, Japan) and a nonionic contrast medium (Iohexol; Omnipaque 350, GE Healthcare, Chicago, USA) given as 2 ml, 0.3 ml/sec in abdominal aorta closely above ligation site. Collateralization (presentation of superior and inferior mesenteric arteries and their interconnection (arch of Riolan), right and left ovarian arteries, lumbar arteries) was estimated at end of arterial DSA phase just before venous phase initiation, analyzed using the ISSA (VAMSTEC‐ Software Company, Zagreb, Croatia) image program.ResultsControls. Regularly rats underwent abdominal aorta occlusion between a right iliolumbar artery and the bifurcation of the aorta exhibit abdominal aorta failure with occlusion presenting with the very poor presentation of the collaterals, exhibiting only limited presentation of the superior mesenteric artery and arch of Riolan. BPC 157. We noted an immediate presentation of superior and inferior mesenteric arteries and their interconnection (arch of Riolan), right and left ovarian arteries, lumbar arteries in all rats underwent abdominal aorta occlusion that received BPC 157 therapy (Fig. 1).ConclusionStable gastric pentadecapeptide BPC 157 may be the therapy of abdominal aorta occlusion in rats.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phenotypic alteration of vascular smooth muscle cells as a sequence of mild hyper‐caloric intake in the absence of hyperglycemia: Potential modulation by antidiabetic drugs
Authors: SLEIMAN FATIMA , Mroueh Ali , Eid Ali , El‐Yazbi Ahmed F. 
Published: June 2021

Diabetic patients remain at a high risk of atherosclerotic cardiovascular disease in addition to hypertension and microvascular complications. A significant proportion of diabetic patients present with vascular abnormalities upon initial diagnosis implicating an early vascular insult in the course of development of diabetes. A contractile‐to‐synthetic vascular smooth muscle cell (VSMC) phenotypic switch is associated with different diseases including atherosclerosis, diabetes, hypertension, and cancer. In the present study, we examined whether early metabolic derangement in the course of diabetes development was associated with VSMC phenotypic alteration before hyperglycemia develops. For this purpose, we used a high‐calorie diet (HC) fed rat model with delayed development of hyperglycemia. Up to 12 weeks of feeding, neither increased serum glucose nor elevated mean arterial pressure was detected. Control and HC rats were sacrificed at 4, 8, and 12 weeks, and their aortae were dissected for isolation of primary VSMCs. VSMC morphology changed whereby the typical hill‐and‐valley culture pattern was gradually substituted by densely packed areas of rhomboid cells in explants for aortic tissues from HC rats. This changed appeared to be dependent on the duration of feeding. VSMC proliferation and migration increased in cultures derived from HC‐fed rats compared to controls at the same age and passage number. Moreover, VSMC contractile phenotype markers smooth muscle alpha actin and calponin gradually decreased in cells cultured from HC‐fed rats in a manner proportional to the feeding duration. In vitro, exposure to pioglitazone, but not metformin was associated with a reduction in VSMC proliferation in cells derived from HC‐fed rats. On the contrary, similar exposure to increased oleiate and palmitate concentrations in the culture medium resulted in an increased proliferation of VSMCs from control rats. Our results indicate that VSMC phenotype and function is potentially affected by the early metabolic derangement associated with the development of diabetes regardless of the blood glucose concentration. Studies to investigate the effect of chronic exposure of HC‐fed rats to metformin and pioglitazone on VSMC phenotype are underway.Support or Funding InformationSupported by AUB MPP fund #320148This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.