bluuebunny/crossref_metadata_2025 huggingface dataset:
Floating cultured melanoma cells are a distinct subpopulation enriched for cancer stem cell biomarkers
Authors: Huang John , Lee Chung‐Wei , Chuang Ya‐Ting , Murphy George F
Published: June 2021

Among the heterogeneous cancer cell subpopulations, slow‐cycling and drug‐resistant cancer stem cells are characterized by self‐renewal, tumorigenic growth in vivo, and spherogenic growth in vitro. Melanoma cells in culture grow either as adherent, or as non‐adherent, ‘floating’ cells. We have determined that the subpopulation of viable floating cells express a slow‐cycling phenotype, and thus reasoned that cancer stem cells may be enriched in this fraction. Melanoma floating cells were found to be more capable than adherent cells of spherogenic growth in vitro (p<0.05). Moreover, they were selectively resistant to the cytotoxic effects of dacarbazine treatment (p<0.01). Additionally, floating cells were capable of generating both cells with adherent and floating phenotypes, in keeping with stem cell capacity both for self‐renewal and for giving rise to non‐stem cell progeny. To characterize further the differences between floating and adherent cells, mRNA expression of cancer stem cell markers was analyzed. Floating cells exhibited distinctive profiles for ABCB5, SOX10, PAX3, FOXD3, and MITF. However, SOX2, CD20, and CD133 were not differentially expressed. Accordingly, floating melanoma cells express a distinctive phenotype in which certain properties shared by cancer stem cells preferentially reside, and thus may represent a novel subpopulation for study of melanoma stem cell biology. Funded by NIH NCI R01CA138231.
cGMP‐dependent protein kinases (PKG‐Iβ and PKG‐II) modulate β‐catenin/TCF pathway in cancer cells
Authors: Sellak Hassan , Wu Songwei , Lincoln Thomas M.
Published: June 2021

The β‐catenin signaling pathway plays an essential role in the development and in diseases such as cancer. Cyclic GMP‐dependent protein kinase (PKG) has been reported to be implicated in β‐catenin signaling. However, the precisemechanism by which PKG modulates β‐catenin pathway isnot completely understood since most of cancer cells loose endogenous PKG expression. The present study was undertaken to determine the effects of ectopic overexpression of PKG on colon cancercell lines. PKG levelswere manipulated in human colon cancer cells by stable transfectionof full‐length PKGcDNA (PKG‐Iβ, PKG‐II) and β‐catenin levels as well as TCF‐luciferase activities were analyzed. In an electromobility shift assay (EMSA), PKG‐I and PKG‐II expression in SW480 colon cancer cells resulted in an increase of TCF, CREB, Ap1, and NF‐kβ binding but not Ap2 or Oct1. Both PKG isoforms stimulation with 8pCPTcGMP induced phosphorylation of VASP (serine239), GSK3β (serine9), and CREB (serine133), while Sox9 phosphorylation (serine181) was only achieved by activated PKG‐II isoform. The results also show that while the overexpression of PKG did not alter total β‐catenin levels, nuclear β‐catenin was slightly increased. The nuclear β‐catenin was concomitant to an increase of TCF‐luciferase activity and binding in PKG overexpressing cells compared to control. These results show that reintroducing PKG (PKG‐Iβ, PKG‐II) in cancer cells affects β‐catenin/TCF pathway and other signals. We are currently exploring the possible contribution of these changes in cell migration, proliferation, and new vessels formation.
p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment
Authors: Marengo Barbara , Ricciarelli Roberta , De Ciucis Chiara , Furfaro AnnaLisa , Colla Renata , Canepa Elisa , Traverso Nicola , Marinari Umberto Maria , Pronzato Maria Adelaide , Domenicotti Cinzia
Published: June 2021

Neuroblastoma (NB), the second most common pediatric tumor, is characterized by biological and clinical heterogeneity. Considering the limited success of chemotherapy, the aim of our study is to identify and target key molecular pathways associated with NB chemoresistance. In this context, we analyzed the main survival and death pathways triggered by etoposide, a commonly used chemotherapeutic agent, in HTLA‐230, a MYCN‐amplified NB cell line isolated from a high‐risk patient. Etoposide induced a concentration‐dependent reduction of cell viability and, at very high doses, totally counteracted cell tumorigenicity and neurosphere formation. In addition, etoposide activated p38 Mitogen‐activated protein Kinase (MAPK), AKT and c‐Jun N‐terminal kinase. Therefore, the treatment with etoposide combined with SB203580, an inhibitor of p38MAPK activity, decreased cell viability and tumorigenicity, counteracted neurosphere generation and slowed down the cell migration and invasion. In this context, the expression of COX‐2, ICAM‐1 and CXCR4 was down regulated, the formation of capillary‐like structures was prevented, by generating a phenotype inadequate for tumor development.. Collectively, our results suggest that p38MAPK inhibitors, in combination with standard chemotherapy, could be a novel strategy to counteract NB resistance and relapse.

Grants: PRIN 2009M8FKBB_002; Genoa University.
Thrombospondin‐1 (TSP1) Impairs Vasorelaxation via Signal Regulatory Protein (SIRP)‐α‐Mediated Activation of NADPH Oxidase 1 (NOX1)
Authors: Yao Mingyi , Csanyi Gabor , Rodriguez Andres , Roberts David , Pagano Patrick , Isenberg Jeffrey
Published: June 2021

The secreted matricellular protein TSP1 acutely controls vascular tone by inhibiting relaxation of vascular smooth muscle cells (VSMC). However, the exact mechanism involved remains unknown. TSP1 is the soluble ligand for the cell surface receptor CD47, whereas CD47 has been reported to be a ligand for signal inhibitor regulatory protein SIRP‐α. Both CD47 and SIRP‐α are expressed in VSMC and SIRP‐α was associated with reactive oxygen species (ROS) production in macrophages. This suggested that TSP1, via SIRP‐α, inhibits vasodilation by promoting ROS production in VSMC. We herein show that TSP1 binds to SIRP‐α and activates its intracellular signal transducer Src homology 2 domain‐containing tyrosine phosphatase‐1 (SHP‐1). Knocking down CD47 or antibody blockade of CD47 in VSMC does not alter TSP1‐mediated activation of SIRP‐α. TSP1 phosphorylates the p47phox subunit of Nox1 and increases O2•− production in VSMC, which is decreased following siRNA silencing of SIRP‐α. In functional bioassays TSP1, in a SIRP‐α‐dependent manner, inhibits vasorelaxation through Nox1‐stimulated O2•− production. These data identify the TSP1‐SIRP‐α signaling axis as a novel activator of Nox1‐derived O2•− in VSMC for inhibition of arterial vasodilation, and further suggest that targeting SIRP‐α may offer therapeutic potential to mitigate pathologic ROS and enhance tissue blood flow in cardiovascular disease.
Pharmacological Interventions to Overcome Reactive Oxygen Species in Hemorrhagic Shock
Authors: Doursout Marie‐Francoise , Liang Yangyan , Vandelaar Laura
Published: June 2021

In previous studies it has been shown that hemorrhagic shock (HS) leads to the production of reactive oxygen species (ROS). ROS cause lung injury and vascular endothelium injury. It is also known that Nitric oxide (NO) reacts with superoxide anion to form peroxynitrite, a powerful oxidant that damages organ tissues. This study was to identify the effectiveness of different pharmacological interventions in overcoming ROS induced hypotension and lung injury in rats subjected to HS. The drugs tested were NG‐methyl‐L‐Arginine (L‐NMA) a NOS inhibitor, N‐acetylcysteine (NAC) a conventional ROS inhibitor, ODQ a sGC specific guanylyl cyclase inhibitor. The study consisted of 5 groups: control (HS with no pharmacological intervention), HS+L‐NMA, HS+L‐NMA+NAC, HS+ODQ, HS+ODQ+NAC. The rats were anesthetized, catheterized, and then blood was drawn until the blood pressure reached 30 mmHg in order to subject them to HS. Rats were monitored for 6 hours, with blood samples taken simultaneously to test NO and cytokine production. At 1 hour post HS a drug or drug combination was given. At completion, lung samples were taken for wet‐to‐dry, immunochemistry and pathology. While all drug combinations tested seemed to create an improvement in heart rate and BP, the L‐NMA+NAC and the ODQ+NAC groups showed the greatest recovery in restoring BP by decreasing ROS.
Cystathionine Gamma‐Lyase Deficiency Impairs H2S Biosynthesis And Vessel Reactivity In Type‐2 Diabetes
Authors: Velmurugan Gopal V , White Carl
Published: June 2021

Together with the precursor L‐cysteine, cystathionine gamma‐lyase (CSE) is a key enzyme involved in the biosynthesis of hydrogen sulphide (H2S), a potent gaseous vasodilator. Since impaired vasodilation is a hallmark of diabetes the purpose of the current study was to assess the role of H2S in diabetes‐induced vascular dysfunction. Mesenteric arterioles from type‐2 diabetic (db/db) mice were found to express ~30% less CSE protein and 5 fold less CSE mRNA compared to nondiabetic (db/m) controls. In pressure myography experiments, addition of L‐cysteine (1 – 50 μM), to arterioles pre‐contracted with phenylephrine, dose‐dependently induced vasorelaxation in both db/m and db/db vessels, however, the efficacy of L‐cysteine was significantly lower in db/db. The CSE inhibitors D,L‐propargylglycine (PAG) and amino‐oxyacetic acid (AOAA) completely ablated L‐cysteine‐induced relaxation confirming that L‐cysteine operated through the H2S pathway. Incubating db/db vessels overnight with sulforaphane (500 nM), a phytochemical found in cruciferous vegetables, doubled its mRNA level. Taken together, these data suggest that reduced CSE expression and impaired H2S production may contribute to altered vascular reactivity in type‐2 diabetes and hint at the potential for sulforaphane to therapeutically correct H2S deficiency.
Comparative analysis of the vascular sympatho‐inhibition to moxonidine in control or streptozotocin‐induced diabetic rats
Authors: Centurion David , Moreno‐Vázquez Eber , Villamil‐Hernández Ma. Trinidad , Alcántara‐Vázquez Oscar , Acosta‐Cota Selene J. , Sánchez‐López Araceli
Published: June 2021

Moxonidine peripherally inhibits sympathetically‐induced vasopressor responses in pithed rats by α2‐adrenoceptors and I1 receptors. During diabetes, autonomic failure can occur and peripheral sympatho‐inhibition by moxonidine could be modified. This study has investigated in streptozotocin‐induced (STZ) diabetic rats the alterations of: (1) the vasopressor responses to sympathetic stimulation or exogenous noradrenaline; and (2) the inhibition to moxonidine on the vascular sympathetic outflow. Male Wistar rats were pre‐treated with STZ (60 mg/kg, i.p.) or vehicle. 14 days later, the rats were pithed and prepared for measurement of diastolic blood pressure and heart rate. Then, the vasopressor responses induced by either selective electrical stimulation (2 ms, 60 V, 0.03–3 Hz) of the vascular sympathetic outflow (T7‐T9) or i.v. bolus injections of exogenous noradrenaline (0.03–3 μg/kg) were determined before and during i.v. infusion of moxonidine (3 and 10 μg/kg min) or saline. In rats pretreated with STZ or vehicle, sympathetic stimulation or exogenous noradrenaline elicited frequency‐dependent or dose‐dependent vasopressor responses, respectively. The vasopressor responses were less marked in rats pretreated with STZ than in rats treated with vehicle. In rats pretreated with STZ or vehicle, i.v. continuous infusion of moxonidine (but not saline) significantly inhibited the vasopressor responses to sympathetic stimulation but not those induced by exogenous noradrenaline. The sympatho‐inhibition was less marked in rats pretreated with STZ. These results suggest that STZ‐induced diabetes produced a reduction of the vasopressor responses and the sympatho‐inhibition to moxonidine probably due to a decrease of density of receptors or decoupling of transductional mechanisms.
Reduced Nitric Oxide–cGMP Signaling Contributes to Vascular Dysfunction Induced by the Adipokine Chemerin.
Authors: Neves Karla , LOBATO NUBIA , LOPES RHEURE , ZANOTTO CAMILA , FILGUEIRA FERNANDO , OLIVEIRA ANA MARIA , TOSTES RITA
Published: June 2021

The new adipokine Chemerin has been implicated in the cardiovascular complications associated with obesity and metabolic syndrome. The present study was designed to test the hypothesis that the adipokine chemerin induces vascular dysfunction by decreasing nitric oxide (NO)–cGMP signaling. Isometric contraction was recorded in rat thoracic aortic rings incubated with chemerin (0.5ng/mL or 5ng/mL; 1 hour) or vehicle. Chemerin markedly reduced both sodium nitroprusside [pD2: 8,6±0,1 vs. chem 0,5ng/mL=7,4±0,1; chem 5ng/mL=6,9±0,1] and acetylcholine [Emax: vehicle=93,1±11,1; chem 0,5ng/mL=66,1±3,1; chem 5ng/mL= 63,8±12,6]‐induced relaxation in rat aorta. Chemerin increased eNOS phosphorylation [a.u.: 1 vs. 1.9±0.1], but also induced NOS uncoupling, as demonstrated by a decreased eNOS dimer/monomer ratio [a.u.: 1 vs. 0,5±0,1]. Chemerin decreased vascular expression of soluble guanylate cyclase [a.u.: 1 vs. 0.6±0.1] as well as cGMP levels [pmol/mg: 1±0,2]. Chemerin increased superoxide anion generation [rlu: 100 vs. 120,5±8,1], determined by lucigenin chemiluminescence assay in cultured VSMCs from rat aorta, an effect that was abrogated by incubation with L‐NAME [120,5 vs. 71,6±9,4] or tempol [120,5 vs. 73,3±7,5]. In conclusion, chemerin decreases NO‐cGMP signaling via ROS generation, NOS uncoupling and decreased GC activity. Our studies may contribute to a better understanding of the role of factors released by the visceral adipose tissue on vascular function and, consequently, on the vascular dysfunction in obesity and obesity‐associated diseases.
Modulation of vascular response by high salt intake depends on the presence or absence of adenosine A2A receptor using A2A AR‐null mice
Authors: Pradhan Isha , Mustafa Jamal S , Zeldin Darryl C , Ledent Catherine , Falck John R , Nayeem Mohammed A
Published: June 2021

High salt (HS) has been shown to modulate adenosine‐induced vascular response through A2AAR. We hypothesized that HS enhances adenosine‐induced relaxation through cyp‐epoxygenases in the presence of A2AAR, but exaggerates contraction in the absence of A2AAR. Organ bath and western blot studies were conducted with 4% (HS) and 0.18% NaCl (NS) diet fed A2AAR−/− and A2AAR+/+ mice. Non‐selective adenosine analog (NECA) produced significantly higher relaxation in HS‐A2A AR+/+vs. NS‐A2A AR+/+, NS & HS‐A2AAR−/−. Also, CGS 21680, selective A2A AR agonist, enhanced relaxation in HS‐A2A AR+/+vs. NS‐A2AAR+/+ which was blocked by an EET antagonist (14,15‐EEZE) but not by L‐NAME (NOS inhibitor) and indomethacin (COX inhibitor). NECA induced contraction in NS & HS‐A2AAR−/−was reversed by A1AR antagonist, DPCPX. Also, CCPA, a selective A1AR agonist yielded higher contraction in NS & HS‐A2AAR−/−vs. NS & HS‐A2AAR+/+ mice. Compared with NS‐A2AAR+/+, HS‐A2AAR+/+ demonstrated upregulation of A2AAR (60%), cyp2c29 (64%) and downregulation of A1AR (17%) & cyp4a (46%). A1AR (20 %) & cyp4a (50%) were upregulated and cyp2c29 (54%) was downregulated in HS‐A2AAR−/−vs. HS A2AAR+/+. Our data suggest HS elicited enhanced A2AAR‐induced relaxation through cyp‐epoxygenases in A2AAR+/+is abolished in the A2AAR−/−, further exaggerating contraction via A1AR. Supported (BGF‐WVU, STF‐WVU, HL027339, HL094447, GM31278 & z01 ES025034).
The Effect of a Hydrogen Sulfide (H2S)‐Releasing Non‐ Steroidal Anti‐Inflammatory Drug (NSAID) on Gastric and Renal Damage in Streptozotocin (STZ)‐Induced Diabetic Rats
Authors: Chan Melissa , Blackler Rory , McKnight Webb , Wallace John
Published: June 2021

Non‐steroidal anti‐inflammatory drugs (NSAIDs) induce gastrointestinal (GI) and renal damage, which is exacerbated in diabetes. Hydrogen sulfide (H2S) counteracts adverse GI effects, so we investigated the GI and renal effects of naproxen and an H2S‐releasing NSAID, ATB‐346, in a model of diabetes. Diabetes was induced in male Wistar rats (n = 4–5) by streptozotocin (STZ) treatment. After two weeks of hyperglycemia, rats received vehicle, naproxen sodium (10 mg/kg) or ATB‐346 (14.5 mg/kg) for 9 doses over 4.5 days. Stomach and small intestine (SI) were removed to score hemorrhagic damage. Blood urea nitrogen (BUN) and creatinine analysis were performed in serum and urine, respectively. mRNA levels in renal tissue were assessed by quantitative RT‐PCR. No gastric damage was observed in any group, though extensive SI damage was observed in the naproxen‐treated group (p<0.05). No difference in markers of renal dysfunction after dosing with any drug. A 3‐ to 4‐fold increase in eNOS mRNA expression was observed in all groups compared to vehicle and we found a >;7‐fold increase in COX‐2 mRNA expression in kidneys of both naproxen and ATB‐346–treated rats. Together, these data indicate that ATB‐346 treatment confers a protective effect on the SI in STZ‐induced diabetic rats, whilst not inducing any more renal changes than naproxen. Supported by the Canadian Institutes of Health Research.
Both supraspinal and spinal mechanisms may be involved in the maintenance of gastric mucosal integrity in the rat.
Authors: Gyires Klara , Tóth Viktoria , Király Kornél , Barna István , Zádori Zoltán Sándor
Published: June 2021

Several neuropeptides given intracerebroventricularly (i.c.v.) or into motor nucleus of vagus induce gastric mucosal defense in the rat.

Aim

To study 1. if central modulation of the level of endogenous gastroprotective compounds may affect gastric mucosal defense, 2. if gastroprotection can be elicited also by microinjection of neuropeptides into lateral hypothalamus (LH) or intrathecallally (i.t.).

Methods

Gastric mucosal damage was induced by acidified ethanol. β‐endorphin level was determined by RIA.

Results

1. Gastroprotection was induced via inhibition of the degradation of anandamide and 2‐arachidonoyl‐glycerol by URB 597 (1.5–29.5 nmol i.c.v) and JZL 184; (0.3–1.3 nmol i.c.v.) resp., as well as by the inhibition of the degradation of endomorphins by diprotin A (1 μmol i.c.v.). 2. Neonatal monosodium glutamate treatment reduced significantly the immunoreactive β‐endorphin content in the nucleus of the solitary tract and the mucosal protective effect of opioid peptides. 3. Gastric mucosal protection was elicited both by i.t. administration of endomorphins and β‐endorphin (10 pmol) and by microinjection of different neuropeptides into LH.

Conclusion

1. Elevation of the level of endocannabinoids and endomorphins resulted in gastric mucosal defense, while reduction of the level of β‐endorphin counteracted the gastroprotective action of opioid peptides. 2. Gastroprotection could be elicited also by spinal mechanism.
Macrophage (M ϕ) infiltration and oxidative stress in rat ileum cause loss of nitrergic inhibitory neurons in DOCA‐salt hypertensive rats
Authors: Thang Loc , Fried David , Galligan James
Published: June 2021

Alterations in gut motility are caused by inflammation. Hypertension is associated with vascular inflammation especially in the mesenteric circulation. We studied intestinal inflammation and gut motility in vitro in the deoxycorticosterone acetate (DOCA)‐salt model of hypertension in rats. Using immunohistochemistry, we found that there is an increased number of Mϕ and oxidative stress (O2−) in the myenteric plexus of hypertensive compared to normotensive rats. Electrical field stimulation (EFS) of whole caused larger neurogenic longitudinal muscle contractions in ileal segments from hypertensive compared to normotensive rats (P<0.05). Nitro‐L‐arginine (NLA, 100 μM), a nitric oxide synthase inhibitor, increased neurogenic longitudinal muscle contractions by 70% and 20% in ileal segments from sham and DOCA‐salt rats respectively. Acetylcholine and bethanechol also caused larger longitudinal muscle contractions in ileal segments from DOCA‐salt rats. Our data support the hypothesis that Mϕ‐associated oxidative stress in the Ileal myenteric plexus of DOCA‐salt hypertensive rats compromises nitrergic inhibitory neuromuscular transmission leading to increased smooth muscle contractility. (Supported by P01HL070687)
Redox regulation of the KATP channel complex in colonic inflammation
Authors: Kang Minho , Gade Aravind R , Akbarali Hamid I
Published: June 2021

Hydrogen sulfide (H2S), like nitric oxide (NO) is an endogenous gaseous mediator. Sulfhydration of cysteine residues and tyrosine nitration are post‐translational modifications that alter cellular function. The objective of this study was to define the interaction of tyrosine nitration and cysteine sulfhydration within the KATP channel in experimental colitis. A modified biotin switch assay was performed to determine sulfhydration of the KATP channel subunits in CHO transfected cells and in native mouse colonic tissues. NaHS (a donor of H2S) significantly enhanced sulfhydration of SUR2B but not Kir6.1 subunit. Treatment with dithiothreitol reversed both the basal and the NaHS‐induced sulfhydration. The basal sulfhydration of SUR2B was significantly enhanced in the mouse colon following inflammation. SIN‐1 (a donor of peroxinitrite) induced nitration of Kir6.1 subunit but not SUR2B. NaHS reduced the nitration of Kir6.1 by SIN‐1. Two specific mutations within SUR2B, C24S and C1455S, prevented sulfhydration by NaHS and failed to decrease tyrosine nitration. The studies described herein suggest that sulfhydration of SUR2B prevents the nitration of Kir6.1. These data suggest that the increase of H2S production observed in colonic inflammation might be a protective mechanism triggered by the protection of nitration of Kir6.1 by sulfhydration of SUR2B.
Identification of a novel activator of GOA‐1, a trimeric G protein critical for early stages of C. elegans development
Authors: Coleman Brantley David , Nguyen Lien Thi , Garcia‐Marcos Mikel
Published: June 2021

Classical activation of trimeric G proteins occurs via guanine nucleotide exchange factor (GEF) activity of GPCRs. However, it has become recently evident that a set of non‐receptor proteins can also activate G proteins. Among these non‐receptor GEFs, GIV is the first one that activates G proteins via a defined motif, i.e., the Gα‐Binding and Activating (GBA) motif. We investigated if a similar GBA motif capable of activating G proteins is present in other unrelated proteins. Our bioinformatics searches revealed a putative GBA sequence in an uncharacterized protein highly expressed in early stages of C. elegans development. Here we demonstrate that this protein, that we call ceGBA, binds directly to GOA‐1, a G protein critical for C. elegans development. By using a combination of site directed‐mutagenesis, homology modeling and biochemistry we demonstrate that ceGBA utilizes its GBA motif to bind to the switch II/α3 helix hydrophobic pocket of inactive GOA‐1 and subsequently activates the G protein by accelerating the rate of nucleotide exchange. These results not only suggest a novel receptor‐independent mechanism of GOA‐1 activation during organismal development but also demonstrate that the GBA motif is a functional module for G protein activation conserved among divergent proteins across evolution.
Neuroadaptations in D2‐like autoreceptor function following AMPH self‐administration
Authors: Calipari Erin S , Sun Haiguo , Jones Sara R , Chen Rong
Published: June 2021

Exposure to amphetamine (AMPH) self‐administration (0.187 mg/kg/inj, 5 days) results in neuroadaptations in the mesolimbic dopamine system. Dopamine D2‐like autoreceptors are critical modulators of dopaminergic signaling but an understudied topic in drug addiction research. We report a marked subsensitivity of D2 autoreceptors following chronic AMPH. After escalation of AMPH self‐administration in rats, we found that the ability of D2 autoreceptors to inhibit dopamine release was reduced in AMPH rats (IC50 = 200 nM) compared to controls (30 nM), as assessed by voltammetry in the nucleus accumbens core. The reduced function was not due to reduced expression, because AMPH increased D2 short form (presynaptic D2 form) mRNA (46%). To determine the mechanism of D2 subsensitivity, we examined gene expression of the inhibitory subunit of G protein (Gαo) and regulator of G protein signaling 4 (RGS4). Gαo couples to the D2 autoreceptor for signaling, and RGS4 negatively regulates Gαo‐mediated D2 receptor function. We found decreased Gαo mRNA (23%) and increased RGS4 mRNA (27%) in the ventral tegmental area, which likely contribute to the decreased function of D2 autoreceptors following repeated AMPH administration. It is possible that changes in Gαo and RGS4 signaling may be involved in early stages of addiction, increasing dopamine release and contributing to compulsive AMPH‐taking behavior.
RGS2 regulates urotensin II‐induced intracellular Ca2+ elevation and contraction in glomerular mesangial cells
Authors: Adebiyi Adebowale
Published: June 2021

Urotensin II (UII), a kidney‐localized vasoactive peptide regulates renal functions, including renal blood flow, glomerular filtration rate, urine output, and electrolyte excretion. In the present study, the physiological functions of UII and regulation of UII receptor (UTR) in glomerular mesangial cells (GMCs) were investigated. RT‐PCR, Western immunoblotting, and immunofluorescence indicated the expression of UTR in murine GMCs. Mouse UII (mUII) elevated intracellular Ca2+ concentration ([Ca2+]i) and decreased murine GMC surface area by ~ 55 and 20%, respectively. mUII‐induced [Ca2+]i elevation and contraction in the cells were attenuated by SB 657510, a UTR antagonist, araguspongin B, an inositol 1,4,5‐trisphosphate receptor inhibitor, thapsigargin, a sarco/endoplasmic reticulum Ca2+‐ATPase inhibitor, and La3+, a store‐operated Ca2+ channel blocker. mUII‐induced [Ca2+]i elevation in murine GMCs was reduced by ~ 82% in Ca2+‐free medium. However, nimodipine, an L‐type Ca2+channel blocker did not alter mUII‐induced [Ca2+]i elevation and contraction in the cells. Co‐immunoprecipitation, co‐localization, and in situ ligation proximity assay demonstrated that UTR interacts with regulator of G‐protein signaling (RGS) 2 in murine GMCs. Biotinylation indicated that RGS2 is predominantly localized intracellularly in murine GMCs. However, mUII treatment elevated RGS2 surface expression in the cells by ~ 55%. siRNA‐mediated knockdown of RGS2 in murine GMCs increased mUII‐induced [Ca2+]i elevation and contraction by ~ 35 and 31%, respectively. These findings indicate that mUII induces store‐operated Ca2+ entry and contraction in murine GMCs. These data also suggest that mUII stimulates recruitment of RGS2 protein to GMC plasma membrane as a negative feedback mechanism to regulate UTR activation.

Supported by the National Institute of Health
Structural Studies of RGS Inhibitors
Authors: Higgins Colin Anthony , Fuentes Ernesto , Roman David
Published: June 2021

Regulators of G‐protein signaling (RGS) proteins regulate G‐protein coupled receptor (GPCR) signal transduction cascades by acting as GTPase accelerating proteins upon binding activated Gα subunits. RGS proteins express in cell‐type specific manners, regulate specific GPCRs, and are overexpressed in pathologies from cancer to Parkinson's disease. As such, they are attractive drug targets. Small‐molecule RGS4 inhibitor screens have yielded a number of covalent thiol modifiers that act allosterically at Cys148, far from the RGS/Gα interface. Thus far, the structural basis of RGS4 inhibition is unknown, so in the current study we investigate the structural consequence of RGS4's covalent modification that leads to inhibition of Gα binding. We purified recombinant RGS4 from Escherichia coli and obtained nuclear magnetic resonance spectra for free and modified RGS4. Overlaid 15N‐1H HSQC spectra show significant changes in RGS4 structure upon small molecule modification. Data from circular dichroism, dynamic light scattering, and other techniques provide further evidence that the pocket may be amenable for rational drug design. Project funded by 5T32GM067795–08 and pre‐doctoral fellowship from AFPE.
PKCε‐mediated P‐Rex1 downregulation suppresses breast cancer cell proliferation
Authors: Wong Chuu‐Yun A. , Xie Yan , Wolff Dennis W. , Abel Peter W. , Tu Yaping
Published: June 2021

P‐Rex1, a Rac1 selective activator, is upregulated and promotes tumorgenesis and metastasis of various cancers. We recently found that phorbol‐12‐myristate‐13‐acetate (PMA) downregulates P‐Rex1 in breast cancer cells. The present study was to investigate the importance and mechanism underlying PMA downregulation of P‐Rex1. P‐Rex1 mRNA and protein expression were determined by real‐time RT‐PCR and Western blot analysis, respectively. Cell proliferation was analyzed by counting cell numbers. We found that P‐Rex1 expression is elevated in breast cancer MCF‐7, T47D and BT‐474 cell lines compared to normal breast epithelial cells. PMA treatment decreased P‐Rex1 expression and proliferation of breast cancer cells by over 70%. Restoring P‐Rex1 expression attenuated the PMA inhibition of cell proliferation. Go6983, a protein kinase C (PKC) inhibitor, blocked the PMA inhibition of P‐Rex1 expression and breast cancer cell proliferation. Breast cancer cells express PMA‐sensitive PKCδ, PKCε and PKCη. Overexpression of constitutively active PKCε, but not PKCδ or PKCη, significantly suppressed P‐Rex1 expression and breast cancer cell proliferation. In conclusion, PMA downregulates P‐Rex1 to suppress breast cancer cell proliferation via a PKCε signaling pathway, which could be a new therapeutic target for development of a novel breast cancer treatment. Supported by NIH CA125661 and Nebraska State LB595.
Effect of D3 Dopamine Receptor Signaling Properties on Rodent Locomotion
Authors: Cote Samantha Rae , Li Ying , Kuzhikandathil Eldo
Published: June 2021

The dopaminergic system is known to be involved in locomotion, and has been implicated in Parkinson's disease. The D3 dopamine receptor (D3R) exhibits a tolerance property wherein repeated agonist stimulation elicits a progressively decreasing response. In vitro studies have shown that among others, the tolerance property is observed in the D3R‐Mitogen Activated Protein Kinase (MAPK) signal transduction pathway. Previous in vitro and computer modeling studies have shown that D3R tolerance is agonist dependent. However, it is not known if these tolerance and non‐tolerance inducing D3R agonists elicit different effects in vivo. In this study our objective was to test whether the D3R tolerance property affects rodent locomotion, since it is known that D3R stimulation leads to a decrease in locomotor activity (LMA). Drd3‐ EGFP mice that report expression of endogenous D3R were administered one or multiple doses of tolerance causing (PD128907) or non‐tolerance causing (ES609) agonists and LMA measured. D3R‐induced activation of MAPK signaling was also determined. Mice administered ES609 exhibited decreased LMA after each administration. In contrast, while mice administered a single dose of PD128907 showed decreased LMA, a second administration did not significantly reduce LMA. At the signaling level, D3R‐MAPK tolerance occurred in mice administered multiple doses of PD128907 but not ES609. Immunohistochemical analyses of Drd3‐EGFP brain slices from mice administered ES609 or PD128907 showed that the tolerance‐induced changes in phosphorylated MAPK occur in D3R positive cells and their post‐synaptic targets. These results suggest that agonists that modulate D3R tolerance property have distinct signaling and behavioral effects in vivo.
Inhibition of both FAAH and MAGL, but not either separately, produces delta‐9‐THC like discriminative stimulus effects
Authors: Hruba Lenka , Niphakis Micah J. , Cravatt Benjamin F. , Lichtman Aron H. , McMahon Lance R.
Published: June 2021

The CB1 receptor‐mediated in vivo effects of delta‐9‐THC (THC) were compared to those of anandamide and 2‐AG and their respective degradative enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) in male C57BL6/J mice discriminating THC (5.6 mg/kg i.p.). THC dose‐dependently increased THC appropriate responses with an ED50 value of 3.3 mg/kg. Mice did not respond as though they received THC when instead they received anandamide, 2‐AG, or inhibitors with selectivity for FAAH (PF‐3845) or MAGL (JZL‐184). Mice also did not respond as though they received THC when anandamide (1–10 mg/kg) was combined with PF‐3845 (10 mg/kg) or when 2‐ AG (0.32–10 mg/kg) was combined with JZL‐184 (40 mg/kg). In contrast, the same amount of THC appropriate responding as that produced by THC itself was produced by a combination of PF‐ 3845 and JZL‐184 and also by drugs that inhibit both FAAH and MAGL, including SA‐57 (ED50 value = 2.4 mg/kg) and JZL‐195 (ED50 value = 27 mg/kg). The CB1 receptor‐selective antagonist rimonabant attenuated the effects of both SA‐57 and JZL‐195. According to these results, degradation of both anandamide and 2‐ AG is needed to produce CB1 receptor agonism of the type produced by THC, perhaps reflecting more widespread CB1 receptor stimulation in brain as compared with inhibition of the degradation of anandamide or 2‐AG alone. Supported by: USPHS grants DA17259, DA19222, and DA32933.
Green Tea and Cancer: A Qualified Health Claim Case Study
Authors: Berhaupt‐Glickstein Amanda , Hallman William K. , Nucci Mary , Hooker Neal
Published: June 2021

In 2005, the Food and Drug Administration (FDA) released an enforcement letter for qualified health claims (QHC) about the ingestion of green tea (food and supplement forms) and the reduced risk of breast cancer or prostate cancer. The QHCs for green tea were enforced shortly after the FDA release of the Consumer Health Information for Better Nutrition Initiative (CHIBN), which expanded the enforcement of QHCs from dietary supplements to conventional foods. Green tea and breast/prostate cancer was identified as a case study to assess the progression of QHCs since the CHIBN expansion. An in‐depth review of court cases, FDA enforcement letters, and correspondence between FDA and green tea manufacturers was conducted. The investigation demonstrated that QHC statements about green tea and breast/prostate cancer have undergone multiple iterations and as a result, are a potential source of confusion for food and supplement designers and manufacturers. The progression of QHC statements for green tea and breast/prostate cancer reflects other enforced QHC diet‐disease categories. The evidence suggests that current FDA enforcement of QHCs is ineffective and in need of repair.

Grant Funding Source: Robert Wood Johnson Foundation Investigator Awards in Health Policy Research
Neuropeptide Y (NPY) infusion attenuates development of PTSD‐like symptoms to traumatic stress in rats
Authors: Serova Lidia , Tillinger Andrej , Alaluf Lishay G , Laukova Marcela , Sabban Esther L
Published: June 2021

NPY has anxiolytic properties associated with resilience to stress‐related disorders, such as PTSD. However, due to its hypertensive effects, peripheral administration is undesirable, thus we used intranasal (IN) infusion. Rats were pretreated with single IN NPY or vehicle before exposure to single prolonged stress (SPS) animal model of PTSD and compared to untreated controls. Thirty min after SPS the elevation of plasma ACTH and corticosterone was not as pronounced in NPY‐infused rats and the induction of tyrosine hydroxylase (TH) in locus coeruleus (LC) was attenuated. Seven days after SPS, they displayed reduction in development of anxiety, depressive‐like behavior and hyperarousal. Plasma ACTH, corticosterone, and hippocampal glucocorticoid receptor levels were significantly above controls only in the vehicle, but not IN NPY‐treated. Baseline TH mRNA levels in LC did not differ among groups, but increased with forced swim in the vehicle‐, but not NPY‐pretreated animals. Administration of IN NPY immediately post‐SPS led to a similar reduction in PTSD associated behaviors. The results show that IN NPY can alter stress‐triggered dysregulation of the HPA axis and activation of central noradrenergic activity and provide proof of concept for potential of IN NPY as a non‐invasive prophylactic treatment or early intervention in response to traumatic stress. Supported by US army grant DM102881.
Age‐related changes in anxiety‐like behavior modified by expression of α4 subunit containing nicotinic acetylcholine receptors
Authors: Anderson Shawn , Brunzell Darlene
Published: June 2021

Anxiety disorders are reduced in the geriatric population. Prior studies show that nicotinic acetylcholine receptors containing the α4 subunit (α4*nAChR; *other subunits co‐assemble to form a receptor) decline with age and are expressed in brain regions that regulate anxiety‐like behavior. This study evaluated 6, 12 and 24 month old (mo) wild type (WT) and heterozygous (α4HET; 1/2 WT α4*nAChR expression) male mice in a light‐dark box assay. Anxiety‐like behavior was reduced in 24 mo WT mice; they had reduced latencies to enter the light chamber and spent more time there compared to 6 mo WTs. Injection of a selective α4*nAChR antagonist (DHβE) and comparison to α4HET mice resulted in similar trends. Conversely, 24 mo α4HETs showed an increase in these anxiety‐like behaviors compared to 6 mo α4HET mice and injection of DHβE resulted in elevated latencies. Genotype‐and drug‐associated changes in exploratory behavior were not due to altered locomotor activity. Together with previous studies, these data suggest that reduced expression of α4*nAChRs may contribute to age‐related decreases in anxiety, but further suggest that inhibition of α4*nAChRs when basal levels are already decreased could elevate anxiety phenotype. These studies were supported by a Jeffress Memorial Trust research grant J‐951 and NIH R01 grant DA031289 to D.H. Brunzell. Training support for S.M. Anderson was provided by T32 DA00702736 to W.L. Dewey.
Chronic administration of the antipsychotic olanzapine (olz) dose‐dependently enhances sleep phenotypes in a rat model of metabolic syndrome
Authors: Radzikowski Sylvia , Shortal Brenna P , Watson Christopher J , Koch Lauren G , Britton Steve L , Baghdoyan Helen A
Published: June 2021

Schizophrenia is characterized by disrupted sleep (Am J Psychiatry 164:483, 2007). Olz, an effective treatment for schizophrenia, enhances sleep but causes daytime drowsiness and excessive weight gain that increases risk of metabolic syndrome (Mol Psychiatry 17:242, 2012). Using a rat model of metabolic syndrome (Trends Cardiovasc Med 22:29, 2012), this study is testing the hypothesis that olz alters sleep, EEG power, and locomotor activity. Olz was delivered by subcutaneous osmotic pumps at doses of 5 (n=4 rats) or 7.5 (n=3) mg/kg/day for 7 days. Dependent measures were quantified before (control) and during olz treatment. During the 12‐h dark phase of the 24‐h recording period, olz (7.5 mg) decreased time spent awake (12%) and increased time in non‐REM (44%) and REM (40%). Olz (5 mg) had no effect on sleep. In the dark phase, olz increased EEG theta power (5–6 Hz) during waking by 39% and 34% (5 and 7.5 mg, respectively), and decreased locomotor activity by 49% (both doses). In the light phase, olz increased waking EEG theta power by 42% and 57% (5 and 7.5 mg) and decreased locomotor activity by 35% and 60%. Blood levels of olz were in the therapeutic range (JPET 305:625, 2003), at 111 and 144 ng/mL (5 and 7.5 mg). These results are consistent with human data showing that olz increases sleep (Psychopharmacology 190:507, 2007) and EEG slowing (ibid 150:216, 2000). Support: MH45361 (HAB), RR17718 (LGK, SLB), Dept Anesthesiology.
Simultaneous, in vivo monitoring of 10 neurotransmitters in rat prelimbic cortex (PrL) reveals that systemic and local administration of the atypical antipsychotic olanzapine (olz) differentially altered only serotonin (5HT) levels
Authors: Norton Christine C. , Mabrouk Omar S. , Shatsman Anna , Radzikowski Sylvia , Kennedy Robert T. , Baghdoyan Helen A.
Published: June 2021

Prefrontal cortex function is disrupted in patients with schizophrenia (J Neurosci 32:3022, 2012). 5HT receptors are known to be of key importance in the mechanism of action of atypical antipsychotics (Curr Opin Pharmacol 11:59, 2011). This study is testing the hypothesis that systemic and direct PrL administration of olz alters neurotransmitter levels in the PrL. Adult, male Sprague‐Dawley rats were anesthetized with isoflurane and received olz by IV injection (n=4, 1 mg/kg) or directly into the PrL by reverse dialysis (n=4, 100 μM). Histological analysis confirmed that transmitter measures were obtained from the PrL. IV olz significantly (P<0.05) altered only levels of dopamine (+108%), 5HT (‐39%), and adenosine (‐25%). Mean plasma level of olz during dialysis sampling of the PrL was in the therapeutic range (194 ng/mL; JPET 305:625, 2003). Direct delivery of olz to the PrL significantly altered levels of dopamine (+146%), 5HT (+31%), adenosine (‐39%), glycine (‐38%), and aspartate (‐50%). Whereas both routes of olz delivery increased dopamine, IV olz decreased 5HT but direct PrL delivery of olz increased 5HT. This differential response of 5HT suggests that IV olz decreases 5HT in the PrL by activating 5HT1A autoreceptors on brainstem 5HT neurons. Support: NIH grants MH45361 (HAB), D007268 (OSM), EB003320 (RTK), Dept Anesthesiology
Lost in translation ‐ challenges for academic scientists in writing clinical protocols
Authors: Seymour E Mitchell , Wright Jeanne , Moore Mona , Reisdorph Bill , Weatherwax Kevin
Published: June 2021

Writing clinical protocols is often challenging for early‐career scientists because it requires knowledge and perspectives uncommonly taught during academic training. This presentation from an academic scientist, IRB reviewer, and regulatory professional will focus on challenges in translating a NIH grant proposal into a Phase I/II clinical drug protocol. Numerous required elements in a clinical protocol are not emphasized in grant proposals; these elements will be compared and contrasted. Elements commonly deficient in clinical protocols include: detailed knowledge of FDA‐approved drug labels including risks and indications; inclusion/exclusion criteria that enable timely recruitment while limiting confounders; distinction of primary versus secondary outcomes and statistical power for the primary endpoint; inclusion of primary and secondary safety endpoints; and inclusion of a detailed trial monitoring plan and risk mitigation approaches. In addition, several logistical realities of clinical studies are novel to basic scientists, and these elements affect clinical trial design and execution. In translational research settings, formal training in writing clinical protocols is desirable for junior faculty to facilitate regulatory review and study execution. The author is supported by National Institutes of Health grant number UL1TR000433.
17β‐Estradiol (E2) induces sulfotransferase (SULT) 2A1 expression through estrogen receptor (ER) α
Authors: Li Wei , Ning Miaoran , Kim Heesue , Jeong Hyunyoung
Published: June 2021

Our recent microarray study showed that E2 increases mRNA levels of SULT2A1 in human hepatocytes. SULT2A1 sulfonates dehydroepiandrosterone and oxysterol, playing an important role in maintenance of steroid and lipid homeostasis. The objective of this study is to elucidate regulatory mechanisms underlying SULT2A1 induction by E2. In HepG2‐ER cells (HepG2 stably expressing ERα) and human hepatocytes, E2 induced SULT2A1 expression at both mRNA and protein levels (1.8–2.7‐fold). Co‐treatment of ERα‐degrading antiestrogen ICI182,780 abrogated the induction, indicating that ERα is required for SULT2A1 induction by E2. Results from luciferase reporter assays in HepG2 cells showed that E2 increased SULT2A1 promoter activity by 13‐fold. Transient transfection of ERα mutants harboring point mutations in the DNA‐binding domain significantly decreased the induction, suggesting involvement of the classical mechanism of ERα action in SULT2A1 regulation. Results from deletion assays revealed that the cis‐element responsible for the ER action is located in −256/+140 of SULT2A1. Together, the results demonstrated that E2 induces transcription of SULT2A1 via ERα, potentially through direct binding of ERα to the promoter region. The induction of SULT2A1, which may alter the intracellular concentration of oxysterol, potentially provides insights into the known lipid‐lowing effects of estrogen.

This work was supported by the National Institute of Child Health and Human Development (HD065532).
A novel method to estimate AF‐219 levels in bladder interstitium derived from drug excreted into the urine
Authors: Smith Steven A , Black Dan , Song Di , Ford Anthony P
Published: June 2021

The P2X3 antagonist AF‐219, being studied in bladder disorders, is eliminated by renal clearance. Local renal delivery of AF‐219 to the bladder may confer a therapeutic advantage, supplementing & prolonging effect of systemic (p.o.) administration. We therefore sought to estimate the bladder venous concentration of a drug, assuming that when a drug is instilled into the bladder, the sole source of any systemic exposure must be via diffusion through to the veins that drain it. Thus by comparing the plasma levels following continuous intravenous infusion or intra‐vesicular instillation, we can estimate the concentration of drug in the veins, approximating the interstitial levels in the bladder wall itself. This approach was tested with AF‐219, methylnaltrexone, tiotropium, antipyrine, & AF‐353, co‐administered in cassette format to rats. The local concentration of each compound in the blood draining the bladder was calculated from plasma concentrations, infusion rate, & bladder blood flow. With this method, the local concentrations in the blood draining the bladder were determined to be approximately 1.1%, 1.7%, 2.0%, 2.4%, and 10% of the urine concentration of AF‐219, methylnaltrexone, tiotropium, antipyrine, and AF‐353, respectively.

Conclusion

due to its high urinary concentration, AF‐219 can achieve a pharmacologically relevant exposure in bladder wall beyond that driven systemically.
Celecoxib induces apoptosis by the intrinsic pathway in HT‐29 colon carcinoma and A375 melanoma cells
Authors: Schneider Ryan A , Eckles Kalee G , Kelty Vincent C , Palmisano Lauren R , Strozewski Karina A , Teckmeyer Jordan S , Kinder David H
Published: June 2021

Chronic use of non‐steroidal anti‐inflammatory drugs (NSAIDs) reduces the incidence of cancers of the colon, skin and other organs. The risk of adverse effects associated with NSAIDs precludes the use of many of these agents in a chemoprevention strategy. We and others have demonstrated direct cytotoxic effects of the COX‐2 selective NSAID, celecoxib, in human cancer cell lines. We sought to further elucidate the cellular mechanisms involved in celecoxib's cytotoxicity in HT‐29 colon carcinoma and A375 melanoma cells. HT‐29 and A375 cells were treated for 48 hours with increasing concentrations of celecoxib followed by the XTT assay to establish an IC50 value for each cell line. The IC50 values for celecoxib were 55 μM and 60 μM in the HT‐29 and A375 cell lines respectively. Western blotting was performed to assess induction of apoptotic markers subsequent to celecoxib treatment. HT‐29 and A375 cells were treated with celecoxib at IC50 and 2× IC50 doses respectively for 24 hours. Celecoxib at the 2× IC50 doses increased expression of cleaved caspases‐3 and ‐7 and poly ADP ribose polymerase (PARP) in both HT‐29 and A375 cells. Induction of intrinsic apoptosis by celecoxib was demonstrated in both cell lines indicated by increased expression of cleaved caspase‐9. These results demonstrate that celecoxib elicits a direct cytotoxic effect via induction of the intrinsic apoptosis pathway in HT‐29 and A375 cells.