bluuebunny/crossref_metadata_2025_split huggingface dataset:
Critical Role of R7 Binding Protein in the Sensation of Acute and Pathologic Itch
Authors: Pandey Mritunjay , Zhang Jian‐hua , Adikaram Poorni , Mishra Santosh K , Harris Benjamin , Kahler John F , Loshakov Anna , Sholevar Roxanne , Genis Allison , Hoon Mark , Neubig Richard , Simonds William F 
Published: November 2023

Itch is a common irritating sensation, which triggers a desire to scratch. Itch sensation could be triggered due to potentially harmful agents such as insects, parasites and certain plant alkaloids or in a variety of diseases conditions such as eczema, dry skin itch or jaundice or peripheral neuropathies.In the present study we identified R7BP, a palmitoylated membrane anchoring protein that facilitates Gαi/o –mediated GTPase activating protein (GAP) activity, diminishes scratching in response to a number of pruritogens administerd either intradermally in the nape of the neck or in the central nervous system by lumbar puncture. However, when tested to a number of sensory nociceptive modalities which included mechanical, heat and chemically induced pain, the R7BP KO mice did not show any significant changes except in response to the heat induced pain. The pruriceptive defect in R7bp KO was reversed in a double knock out mice also lacking OPRK1, encoding the Gαi/o coupled kappa opioid receptor.A similar pruriceptive phenotype was observed in a knock‐in of GAP‐insensitive mutant mice with a Gαo (Gnao1 G184s/+) mutation. Further in a model of atopic dermatitis, R7BP KO mice showed diminished scratching behavior and enhanced sensitivity to kappa opioid agonists as compared to wild type littermates.Based on our observations, R7BP has an important role to play in somatosensory itch sensation and probably it functions by modulating the kappa opioid receptor. Our findings also suggest that Gαo has a significant role in itch perception and it could be involved regulation of kappa opioid receptor activity to modulate itch sensation.Taken together, our results indicate that R7BP is a key regulator of itch sensation and suggest that the potential targeting of R7BP – dependent GAP activity as a novel therapeutic strategy for pathologic itch.Support or Funding InformationThis research was supported by the Intramural Research Programs of the National Institute of Diabetes and Digestive and Kidney Diseases and the National Institute of Dental and Craniofacial Research
Identification of Novel Small Molecule Stabilizers of RGS4 Protein Levels
Authors: Karaj Behirda , Sjogren Benita , Neubig Richard R. 
Published: November 2023

G protein‐coupled receptors (GPCRs) are one of the largest membrane protein families and represent one of the most common class of drug targets, with 30% of the currently approved drugs on the market. Regulators of G protein Signaling (RGS) are crucial regulators of GPCRs that act as GTPase accelerating proteins (GAPs) on active, GTP‐bound, Gα subunits of heterotrimeric G proteins, thereby attenuating downstream signaling. RGS4 protein levels has been shown to be decreased in conditions such as breast cancer, non‐small cell lung cancer, and schizophrenia. Given the implications that low RGS4 protein levels can have in many diseased states, our study intends to identify small compounds that stabilize the levels of RGS4 protein.To identify small molecules that enhance the RGS4 protein levels, we screened library collections of over 100,000 compounds in a cell based high‐throughput assay. 29 hits identified from the screen were further confirmed in dose response with fresh powder. Nine compounds were selected for follow up and their effects on RGS4 mRNA, protein half‐life and endogenous protein levels were tested. Our current data shows that all nine compounds enhance RGS4 protein through a post‐transcriptional mechanism as they do not significantly increase the RGS4 mRNA levels, but do extend RGS4 protein half‐life. Representative compound 10 and 11 (numbered based on the original 29 hits) are the most potent enhancers of RGS4 protein levels with EC50 values of around 200 nM. These, as well as all other hits tested, also showed a significant and dose‐dependent increase in endogenous RGS4 protein levels in SH‐SY5Y cells.Of high interest are two structurally related groups that show effects on RGS4 protein levels when tested in both a recombinant system and endogenous system (SH‐SY5Y cells). Their effect on the proteasomal degradation as well as mechanism of action is under further investigation. Further development of these compounds could lead to development of novel drugs that can help stabilize RGS4 protein levels.Support or Funding InformationSupported by: The National Institutes of Health (R01 GM110195)
RGS2 modulates the selection of Gαi/o subtype involved in dopamine D2 receptor signaling
Authors: Luessen Deborah J , Hinshaw Tyler P , Sun Haiguo , Chen Rong 
Published: November 2023

IntroductionRegulator of G‐protein signaling (RGS) proteins modulate GPCR activity by terminating G protein signaling through acceleration of GTP hydrolysis. It is believed that RGS proteins are recruited by and bind to activated Gα subunits to exert GTPase‐activating (GAP) activity. However, recent literature has indicated that certain RGS proteins bind to GPCRs and this binding is critical for receptor‐mediated G protein signaling. Previously, we reported that RGS2 is a negative modulator of dopamine D2 receptor‐mediated Gαi/o signaling. The present study aimed to investigate the mechanism underlying the interaction of RGS2 with D2Rs and Gαi/o proteins.MethodsIn the present study, we utilized a neuroblastoma 2a cell line stably expressing the short form of dopamine D2 receptors (N2A‐D2S). RGS2 N‐terminus truncation (partial and full truncation) mutants, RGS2 siRNA and corresponding controls were transiently transfected into N2A‐D2S cells. Immunoprecipitation was performed to evaluate the coupling between D2Rs and RGS2. Immunocytochemistry and confocal microscopy were utilized to study agonist (quinpirole) stimulated recruitment of Gαi/o subtypes to surface D2Rs. [35S] GTPγS binding was performed to measure D2R‐stimulated Gαi/o activation.ResultsOur data indicate that RGS2 interacts with D2Rs via its N‐terminus and modulates D2R‐stimulated recruitment of Gαi/o subtypes. Specifically, we found that: 1) RGS2 couples to D2Rs via its N‐terminus. RGS2 N‐terminus truncation abolishes the co‐localization of RGS2 with surface D2Rs 2) RGS2 N‐terminus truncation attenuated D2R‐stimulated Gαi/o activation; and 3) RGS2 knockdown alters the selection of Gα subtype recruited to activated D2Rs. D2R stimulation by quinpirole (5 min, 1 μM) induced translocation of Gαi2, not Gαo, towards the plasma membrane in the control cells. However, RGS2 knockdown by siRNA resulted in the translocation of Gαo, not Gαi2, towards the plasma membrane after D2R stimulation.These results suggest that the interaction between RGS2 and D2Rs mediates the selection of the Gαi/o subtype engaged in D2R‐mediated signaling. The finding of this study highlights a novel mechanistic action of RGS2 in regulation of D2R‐mediated Gαi/o signaling.Support or Funding InformationThis work is supported by NIH NIAAA training grant T32 AA007565, DA006634 and a pilot grant from Center for Molecular Communication and Signaling at Wake Forest University.
Stable Gastric Pentadecapeptide BPC 157 Antagonizes Hydrogen Peroxide Induced Oxidative Stress in Drosophila melanogaster
Authors: Filosevic Ana , Waldowski Rozi Andretic , Vidovic Tinka , Sikiric Predrag , Drmic Domagoj 
Published: November 2023

In this work we hypothesize novel anti‐oxidative role for the stable gastric pentadecapeptide BPC 157 and we test this using a model organism, Drosophila melanogaster. BPC 157 was originally an anti‐ulcer peptide used in trials for ulcerative colitis and now is in trials for the treatment of multiple sclerosis where it largely interacts with NO‐system (Curr Pharm Des 2014;20(7):1126–35). BPC 157 is also thought to be a novel mediator of Robert's cytoprotection in rat studies, where its endothelium and mucosal protection is believed to be related to oxidative stress injury (Curr Pharm Des 2014;20(7):1126–35).To explore anti‐oxidative properties of BPC 157 we used the animal model of hydrogen peroxide (H2O2) induced oxidative stress in Drosophila melanogaster. Addition of increasing concentrations of H2O2 to regular Drosophila medium dose‐dependently shortened the lifespan of wild type (wt) Canton S Drosophila males (maximal survival on H2O2 is 8 days, compared to average lifespan of 80 days on regular fly food medium). Longevity of H2O2 fed males increased when they were grown on H2O2 medium supplemented with BPC 157. At 7 days, 54% of H2O2 wt males survived, compared to 84% of 0.6% H2O2 + 3.5 ng/ml BPC 157; at 12 days there was no survival on 0.6% H2O2, and 4% survival on 0.6% H2O2 + 3.5 ng/ml BPC 157.These results shows that; first, BPC 157 is biologically active in this invertebrate model system, and second, that that BPC 157 and H2O2 likely act on similar molecular pathways regulating longevity. Doses effective in promoting increased survival on H2O2 were extrapolated from biologically active doses in mammals. In conclusion, this suggests likely conservation of mechanisms by which BPC 157 antagonizes Reactive Oxidative Species (ROS) in invertebrates and vertebrates. Based on these intriguing observations we will investigate mechanisms by which BPC 157 exerts its anti‐oxidant actions. Because of the significant genetic and mechanistic conservation between Drosophila and vertebrates, discoveries made in this simple model organism will have significant translational potential and relevance for human health.Support or Funding InformationUniversity of Zagreb, Zagreb, Croatia (Grant number BM099)
Regulation of Hepatic CYP3A4 by 3‐Methylcholanthrene in Humanized PXR‐CAR‐CYP3A4/3A7 Mice
Authors: Crosby Michael , Riddick David S. 
Published: November 2023

Polycyclic aromatic hydrocarbons (PAHs) are a broad class of priority environmental pollutants. These chemicals exert a wide array of health effects through activation of the aryl hydrocarbon receptor (AHR). A well‐documented response to AHR activation is the pronounced induction of cytochrome P450 1A1 (CYP1A1), a drug‐metabolizing enzyme involved in phase I drug biotransformation. Interestingly, less is known about the regulation of human cytochrome P450 3A4 (CYP3A4) by PAHs. Alterations of CYP3A4 expression and activity are of tremendous importance to drug metabolism, since this enzyme's abundance and central role in drug metabolism rank it among the most important human cytochromes P450. As such, CYP3A4 is a common target for drug interactions. Our laboratory has previously shown that treatment of male C57BL/6 mice with 3‐methylcholanthrene (MC), a model PAH, suppressed the activity and expression of hepatic Cyp3a11, the mouse counterpart of human CYP3A4. Controversially, studies in primary human hepatocytes and liver‐derived continuous cell lines report both inductive and suppressive effects of PAHs on human CYP3A4 expression. The current study used MC to study the effects of PAHs on CYP3A4 activity and expression in a novel humanized PXR‐CAR‐CYP3A4/3A7 mouse model [Hasegawa et al. Mol Pharmacol 80: 518–28, 2011]. Adult male and female mice were treated by intraperitoneal injection with MC (80 mg/kg), or corn oil vehicle, and euthanized 24 or 72 hours after injection. Livers were subsequently harvested for analysis of CYP3A4 catalytic activity, protein expression and mRNA expression. Marked induction of mouse hepatic Cyp1a1 expression by MC at both 24 and 72 hours was confirmed as a positive control response. Basal hepatic CYP3A4 expression at the catalytic activity, protein, and mRNA levels was significantly higher in female vs. male mice, a sexual dimorphism not reported in the original published characterization of this humanized mouse strain. A similar sex difference in hepatic CYP3A4 expression was documented previously in a CYP3A4/3A7‐transgenic mouse line [Cheung et al. J Pharmacol Exp Ther 316: 1328–34, 2006]. Further, MC decreased CYP3A4 activity and expression in female mice 72 hours after injection. Despite much lower basal hepatic expression of CYP3A4 compared to females, similar trends for CYP3A4 suppression by MC were observed in male mice; these trends did not achieve statistical significance. Our results suggest that this humanized mouse model will be useful for further investigations of the underlying mechanisms and functional consequences of hepatic CYP3A4 suppression by PAHs in an in vivo system.Support or Funding InformationCanadian Institutes of Health Research
Tipping the Balance of Hepatic CYP3A4 Activity Through Integrin‐Mediated Outside‐In and Inside‐Out Signaling
Authors: Jonsson‐Schmunk Kristina , Croyle Maria 
Published: November 2023

Study objectiveWe have previously shown that engagement of integrin receptors by adenovirus infection or an uninfectious RGD peptide significantly suppresses hepatic cytochrome P450 3A4 (CYP3A4) and that removal of RGD epitopes from the adenovirus capsid reverses this effect. Integrin receptors are activated by two different signaling pathways. “Outside‐in” signaling is initiated when a ligand binds to the extracellular domain of the integrin receptor and “inside‐out” signaling is activated by intracellular proteins binding to the cytoplasmic domain of the β subunit of the integrin receptor. Because both pathways could be impacted by virus infection, the objective of this study is to determine how key moderators of each pathway impacts hepatic CYP3A4 expression and function.MethodsThe impact of outside‐in signaling was assessed in the human hepatocyte cell line HC‐04 through transfection of siRNA specific for the alpha and β subunits of the integrin receptor while impact of inside‐out signaling was assessed by transfection of siRNA for talin, an intracellular protein that connects the integrin β subunit to actin. CYP3A4 activity (P450‐Glo™), gene expression (qRT‐PCR) and protein levels (Western Blot) were determined after silencing was confirmed. Changes in protein levels of talin were determined by Western Blot after 24–72 hours of infection with a recombinant adenovirus (AdlacZ). Changes in gene expression pathways associated with integrins and CYP3A4 in response to silencing of integrins or talin was assessed by PCR.ResultsSilencing of the β3 subunit reversed the suppression induced by adenovirus infection. This was not observed when the alpha subunit was silenced. In contrast, silencing of talin 1 resulted in a 2.3 fold increase in CYP3A4 activity in the absence of virus infection. However, talin protein levels were reduced by 80% 48 hours after infection with AdlacZ. Changes in gene expression pathways were detected for pathways associated with integrins, talin and CYP3A4.ConclusionsOur results suggest that integrins play an important role in the regulation of hepatic CYP3A4 through the inside‐out singling pathway. Silencing of talin alone resulted in an increase in CYP3A4 activity, suggesting that the inside‐out pathway may play a role in the maintenance of baseline hepatic CYP3A4 activity and may contribute to variation in hepatic CYP3A4 activity in the human population. Understanding the interplay between integrins, talin and CYP3A4 could be used to modulate drug metabolism to improve therapeutic outcomes in healthy patients and those with disease states influenced by integrin expression such as osteoporosis, ulcerative colitis and cancer.Support or Funding InformationThis project was supported by Grant U01AI078045 (M.A.C.), a special research grant from the University of Texas Vice President for Research (M.A.C.) and a University of Texas Continuing Graduate Fellowship (K.J.‐S.).
In Vitro Assessment of Drug Interaction Potential of Licorice Extracts
Authors: Tonsing‐Carter Alyssa , Li Guannan , van Breemen Richard 
Published: November 2023

Although hormone therapy remains the standard of care for women experiencing vasomotor symptoms related to menopause, concerns over an increased risk of stroke and breast cancer have women considering botanical dietary supplements for management of these symptoms. Licorice (Glycyrrhiza sp.) is popular among women as an alternative for hormone therapy, and it has a long history of use in Traditional Chinese Medicine. To ensure the safe use of licorice dietary supplements, we investigated their potential for drug interactions. Standardized licorice extracts of Glycyrrhiza glabra, G. inflata, and G. uralensis, the three most common species used in dietary supplements, were studied for possible inhibition or induction of cytochrome P450 enzymes involved in drug metabolism. In studies using human liver microsomes, and a cocktail probe substrate assay, all three licorice species showed inhibition of CYP2B6, CYP2C8, CYP2C9 and CYP2C19. G. inflata also inhibited CYP1A2, CYP2D6 and CYP3A4. Induction qualified cryopreserved human hepatocytes were used to predict effects of the extracts on cytochrome P450 enzyme induction. CYP3A4 was unaffected by all Glycyrrhiza sp, and CYP1A2 and CYP2B6 were induced in hepatocytes by G. uralensis and G. inflata. Due to these data, dietary supplements containing G. uralensis and G. inflata are more likely than G. glabra to cause clinically relevant drug interactions.Support or Funding InformationNational Institutes of Health grant P50 AT000155 from the Office of Dietary Supplements and the National Center for Complementary and Integrative Health and training grant T32 AT007533 from the National Center for Complementary and Integrative Health.
RAGE upregulation confers a proliferative phenotype to PANC‐1 pancreatic cancer cells
Authors: Swami Priyanka , Vetter Stefan W. , Leclerc Estelle 
Published: November 2023

Pancreatic cancer is one of the deadliest cancer and is characterized by late diagnosis, aggressive clinical course and resistance to existing therapies. Recent efforts have been concentrated on identifying new targets and molecular pathways which contribute to the development and progression of this lethal disease, and which could be used to establish new therapeutic approaches.Several reports have shown that the Receptor for Advanced Glycation End products (RAGE) contributes to the progression of pancreatic cancer. RAGE is demonstrated to be involved in processes like apoptosis, autophagy, and tumor cell bioenergetics. It is reported that RAGE is required for optimal mitochondrial function in tumors for ATP production. However, the effect of RAGE expression on the proliferative and migratory properties of pancreatic cancer cells is currently not known. A better understanding of the mechanisms of RAGE signaling could lead to the development of new therapeutic strategies in pancreatic cancer. Our objective was to investigate the effect of RAGE up‐regulation on the proliferation and migration properties of the PANC‐1 pancreatic cancer cell‐line. We generated cells that overexpress RAGE four‐fold compared to non‐transfected cells. We then compared the proliferation, migration and invasion properties of the new cells with those of the wild‐type cells. We also compared the signaling pathways affected by RAGE overexpression in these cells. We showed that four‐fold up‐regulation of RAGE in PANC‐1 cells resulted in increased cell proliferation to the expenses of cell migration. Both Boyden chamber and wound healing supported the reduced migratory properties of the RAGE overexpressing cells. Further analysis showed significantly reduced levels of integrins in the RAGE transfected cells. We suggest that in the PANC‐1 cells, which exhibit a mixed epithelial – mesenchymal phenotype, RAGE expression switches the phenotypic balance toward a proliferative phenotype. We are currently exploring how RAGE up‐regulation affects the expression of its ligands in these cells.Support or Funding InformationThis work is partially supported by the National Institutes of Health grant 1P20GM109024 and the NDSU College of Health Professions.
Induction of Autophagy and Apoptosis in Triple‐Negative Breast Cancer Cells by LT‐IIc, a Type II Heat‐Labile Enterotoxin
Authors: Heakal Yasser , Berlingeri Sofia Girald , Hu John , Federowicz Matthew , Connell Terry D , Masso‐Welch Patricia 
Published: November 2023

Despite the recent advances in breast cancer treatment, triple negative breast cancer (TNBC) remains a serious health problem with poor prognosis and limited therapeutic options. Although hampered by development of resistance, chemotherapy‐based regimens remain the first‐line adjuvant choice for the treatment of TNBC. To combat development of resistance, it is essential to identify agents that induce cell death by independent induction of multiple cell death pathways. In this report, we screened cholera toxin (CT), a type I heat‐labile enterotoxin (HLT) of Vibrio cholerae, and LT‐IIa and LT‐IIb, type II HLT of Escherichia coli, for capacity to induce cell death specifically in triple‐negative MDA‐MB‐231 breast cancer cells. While CT, LT‐IIa and LT‐IIb had little effect on the cells, LT‐IIc reduced cell viability by ~50%, concomitant with development of extensive intracellular vacuolation. LT‐IIc‐induced cytotoxicity was not observed in MCF10A, a non‐tumorigenic breast epithelial cell line, or in MCF7, an estrogen receptor‐positive breast cancer cell line. LT‐IIc independently and simultaneously stimulated autophagy and apoptosis in MDA‐MB‐231 cells. Neither knockdown of ATG5, a key mediator of cellular autophagy, nor activation of caspase 3/7 disrupted LT‐IIc‐mediated cell death. Similarly, pharmacological disruption of autophagy using bafilomycin A1, did not interfere with LT‐IIc‐mediated cell death. In contrast, treatment of MDA‐MB‐231 cells with rapamycin, a known inducer of autophagy, enhanced LT‐IIc mediated cell death. Collectively, these findings indicate that LT‐IIc uniquely induces cell death through the divergent induction of autophagy and autophagy‐independent apoptotic pathways.Support or Funding InformationD'Youville College School of Pharmacy and NIH
Role of transient receptor potential canonical 5 (TRPC5) channels in vascular tone regulation
Authors: Liang Cai 
Published: November 2023

Intracellular Ca2+ concentration in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) plays an important role in vascular tone control and blood pressure regulation. Previously, most research focused on voltage‐dependent Ca2+ channels which mediate Ca2+ influx and vascular contraction. However, recent focus has been shifted to TRP channels. Reports show that several TRP channels, including TRPC1 and TRPC3, have important regulatory role in vascular tone control. However, there are relatively few studies about the role of TRPC5 channels in vascular tone regulation.In present study, we determined the expression of TRPC5 in mouse carotid arteries, aorta and mesenteric arteries. Immunohistochemical staining results demonstrated that TRPC5 is expressed in both ECs and VSMCs. Consistently, Western blot results also showed TRPC5 expression. Isometric tension of arteries was measured using wire myograph. In carotid arteries, selective TRPC5 inhibitor clemizole attenuated endothelium‐dependent contraction (EDC) in a dose‐dependent manner; another TRPC5 inhibitor ML204 also dramatically inhibited EDC. Importantly, EDC in carotid arteries from TRPC5 knockout (KO) mice was substantially lower compared with that of wild type (WT) mice. These results indicate that TRPC5 contributes to EDC response in mouse carotid arteries. Attempts were made to determine which cyclooxygenase (COX) mediates EDC in carotid arteries. In both TRPC5 WT and KO mice, selective COX‐2 inhibitor NS‐398 dramatically reduced EDC, while selective COX‐1 inhibitor VAS only slightly inhibited it. Non‐selective COX inhibitor indomethacin abolished EDC. Similar studies were performed using small mesenteric arteries. We found that ANG II‐induced contraction in KO mice was lower than in WT mice. This difference disappeared after removal of endothelium layer. Furthermore, clemizole significantly inhibited ANG II‐induced contraction. Taken together, our studies suggest that TRPC5 contributes to EDC response in mouse carotid arteries, and it also plays a role in ANG II‐induced contraction in mouse small mesenteric arteries.