bluuebunny/crossref_metadata_2025_split huggingface dataset:
How EGF and Insulin Activate Their Receptors
Authors: Leahy Dan 
Published: November 2023

Receptors for Epidermal Growth Factor (EGF) and Insulin are Receptor Tyrosine Kinases (RTKs), which consist of an extracellular ligand‐binding region, a single transmembrane (TM) region, and a cytoplasmic kinase. RTKs play essential roles in the development and maintenance of normal organisms, but abnormal RTK activity is frequently associated with cancer and many successful anti‐cancer agents target RTKs. Ligand‐dependent dimerization is an important feature of RTK activation, and structural and biophysical studies of EGFR and other RTKs provide elegant visualization of many ligand‐dependent RTK dimers. The Insulin Receptor and its homologs are constitutive disulfide‐linked dimers, however, that appear to require a ligand‐dependent conformational change to activate. Recent results show that the extracellular regions of both the EGF and Insulin receptors prevent subunit TM regions from interacting in the absence of ligand but that the TMs associate in the presence of ligand. These results update the ligand‐dependent dimerization paradigm for RTK activation by demonstrating (i) the importance of autoinhibition RTK activity in the absence of ligand and (ii) the role of TM separation in governing RTK activity.Support or Funding InformationNIH GM099321 and NIH GM099092
FFA3 activation stimulates duodenal bicarbonate secretion and prevents NSAID‐induced enteropathy via the GLP‐2 pathway in rats
Authors: Said Hyder , Akiba Yasutada , Kaji Izumi , Kaunitz Jonathan D 
Published: November 2023

The duodenal mucosa expresses the short‐chain fatty acid (SCFA) receptors FFA2 and FFA3, implicating SCFAs generated by foregut luminal flora in the pathological condition termed small intestinal bacterial overgrowth (SIBO) in the genesis of functional dyspepsia symptoms. FFA2 activation stimulates duodenal bicarbonate secretion (DBS) via 5‐HT4 receptor activation, but, when cyclooxygenase (COX) is inhibited, injures the mucosa via 5‐HT3 receptor activation. To further explore the effects of SCFAs on the foregut mucosa, we investigated the effects of FFA3 activation on DBS and nonsteroidal anti‐inflammatory drug (NSAID)‐induced enteropathy.We measured DBS in vivo in a duodenal loop perfused with the selective FFA3 agonists N‐[2‐methylphenyl]‐[4‐furan‐3‐yl]‐2‐methyl‐5‐oxo‐1,4,5,6,7,8‐hexahydro‐quinoline‐3‐carboxamide (MQC) or AR420626 (AR) while measuring released GLP‐2 into the portal vein (PV). Intestinal injury was induced by indomethacin (IND) with or without MQC (1 – 10 mg/kg) or AR (0.01 – 0.1 mg/kg) treatment.Luminal perfusion with MQC or AR (0.1 – 10 μM) dose‐dependently increased DBS accompanied by GLP‐2 release into PV. The effect of MQC was inhibited by co‐perfusion of an FFA3 antagonist N‐(2‐methylphenyl)‐4‐[5‐(2‐trifluoromethoxy‐phenyl)‐furan‐2‐yl]‐2‐methyl‐5‐oxo‐1,4,5,6,7,8‐hexahydro‐quinoline‐3‐carboxamide (CF3‐MQC, 30 μM). AR‐induced increased DBS was reduced by iv injection of the GLP‐2 receptor antagonist GLP‐2(3–33) (3 nmol/kg). IND‐induced small intestinal ulcer formation was dose‐dependently reduced by intragastric administration of MQC or AR. The GLP‐2 receptor antagonist GLP‐2(3–33) (1 mg/kg, ip) or CF3‐MQC (1 mg/kg, ig) reversed AR‐induced protection of intestinal mucosa from NSAID‐induced enteropathy.These results suggest that in contrast to FFA2 activation, which injures the mucosa via the 5‐HT pathway, FFA3 activation enhances mucosal defenses and prevents NSAID‐induced enteropathy via the GLP‐2 pathway. Dual effects of SCFA receptor activation may help explain the diversity of functional symptoms related to foregut bacterial overgrowth (SIBO).Support or Funding InformationVA Merit Review, NIH R01 DK54221
Butyrate Enhances MCT1 Association with CD147 via GPR109A Activation‐Dependent Mechanisms
Authors: Chatterjee Ishita , Kumar Anoop , Anbazhagan Arivarasu N , Alrefai Waddah A , Borthakur Alip , Dudeja Pradeep K 
Published: November 2023

Butyrate, a key short‐chain fatty acid (SCFA) produced by colonic luminal bacteria, serves as the primary fuel for the colonocytes and exerts multiple beneficial effects on gut epithelial health. Efficient intracellular absorption of butyrate is important for these effects. Monocarboxylate transporter 1 (MCT1) plays a major role in intestinal butyrate absorption. Localization of MCT1 on the luminal membrane of colonocytes is critical for its optimal absorptive function. We have earlier shown substrate (butyrate)‐induced enhancement of butyrate absorption in human intestinal epithelial C2BBE cells. This enhancement was secondary to increased abundance of MCT1 protein on the apical plasma membrane following butyrate‐induced activation of GPR109A, a Gαi‐coupled receptor (GPCR), involving nutrient sensing mechanisms. Since membrane targeting of MCT1 has been shown to be governed by its interaction with CD147, an ancillary glycoprotein, current studies examined whether butyrate increases MCT1/CD147 interactions via GPR109A‐dependent mechanisms.MethodsMCT1 function was measured as p‐chloromercuri‐benzene sulfonate [pCMBS (MCT1 inhibitor)]‐sensitive 14C‐butyrate uptake, MCT1/CD147 interactions by co‐immunoprecipitation, MCT1 phosphorylation by immunoblotting of immunoprecipitated MCT1, GPR109A silencing by siRNA.ResultsButyrate (10 mM, 1 hr) significantly increased MCT1‐CD147 interaction in human intestinal C2BBE cells. GPR109A silencing or inhibition by pertussis toxin (PTX) abrogated these effects. GPR109A activation is known to decrease intracellular cyclic AMP that could alter MCT1 phosphorylation, and in turn could modulate its interaction with CD147. Therefore, we examined MCT1 phosphorylation in response to butyrate and role of GPR109A. Butyrate (10 mM, 1 hr) increased serine‐phosphorylated MCT1 that was attenuated in response to PTX inhibition of GPR109A. Further, okadeic acid, a protein phosphatase inhibitor, augmented butyrate enhancement of MCT1 function, presumably via maintaining MCT1 phosphorylation status critical for its membrane targeting.ConclusionOur results highlight distinct role of GPR109A as a SCFA sensor in mediating substrate‐induced enrichment of MCT1 apical membrane abundance via modulating MCT1 phosphorylation and increasing MCT1 interaction with CD147.Support or Funding InformationSupported by NIH(NIDDK) and Department of Veteran Affairs
Modulation of CD4+ T Cell Differentiation by the Probiotic Lactobacillus reuteri
Authors: Collins Fraser , Britton Robert A , McCabe Laura R , Parameswaran Narayanan 
Published: November 2023

Probiotic bacteria are micro‐organisms that when taken in adequate amounts have beneficial effects on the host. Previous studies have demonstrated that probiotic bacteria and specifically Lactobacillus reuteri have important immunoregulatory effects; regulating differentiation of T cells through interactions with dendritic cells or in cultures of peripheral blood mononuclear cells (PBMC). However, it is not known whether or how L. reuteri or its secreted factors can directly influence T cell differentiation. To test this, we isolated Naïve CD4+CD62L+ T cells from the spleen of male C57BL/6 mice by magnetic isolation and measured their cytokine response to treatment with live, dead or whole and fractionated conditioned medium (components separated based on size) from L. reuteri (ATCC 6475). For live bacterial studies, cells were treated with multiplicity of infection (MOI) of 1, 10 or 100. For conditioned media studies cells were treated with whole, >100kD, 50–100kD, 30–50kD, 10–30kD, 3–10kD and <3kD fractions. After 4 days, cells were stained and analyzed by flow cytometry for expression of the cytokines IL‐10, IL‐17A, IFNγ and TGFβ (Latency Associated Peptide). Live L. reuteri significantly increased expression of IL‐10, IL‐17A and IFNγ but not TGFb in a dose dependent manner. Heat killed L. reuteri did not affect expression of these cytokines except for IL‐10 and IL‐17A at the highest dose. Compared to these studies, L. reuteri conditioned media significantly increased expression of IL‐10, IL‐17A, IFNγ and TGFb. Examination of fractionated conditioned media (by size, 3Kd to >100Kd) indicated that all fractions elicited increased cytokine expression compared to control with the greatest effect observed in the >100kD fraction. Taken together, our data demonstrate that live probiotic L. reuteri and its secreted factors are able to directly influence naïve T cell differentiation. Dead bacteria only affected cytokine expression at high doses suggesting an important role for the secretory products of live L. reuteri in the regulation of T‐cell differentiation and cytokine secretion.Support or Funding InformationThis project was supported by funding from NIH NCCIH 1RO1AT007695‐01
Protective Actions of 5‐HT4 Receptors in the Colonic Epithelium
Authors: Spohn Stephanie N. , Scott Rachel , O'Neill Conor , MacNaughton Cathy , Dicay Michael , Bianco Francesca , Bonora Elena , Lavoie Brigitte , Wilcox Rebecca L , De Giorgio Roberto , MacNaughton Wallace , Sharkey Keith A , Mawe Gary M 
Published: November 2023

5‐HT4 receptors are expressed in colonic epithelium, and activation with 5‐HT4 receptor agonists causes mucus secretion from goblet cells, chloride secretion from enterocytes, and 5‐HT release from enterochromaffin cells. We tested whether this receptor could serve a protective role in models of colitis and under basal conditions. CD‐1 mice were given DSS (4% w/v for 5 days in drinking water) or TNBS (7.5 mg/mL) on day 0. 5‐HT4 receptor agonist (Tegaserod, 1mg/kg), vehicle, or antagonist (GR 113808) plus agonist was delivered by enema daily day 1–7 (prevention) or day 6–15 (recovery) to determine the effects of epithelial 5‐HT4 receptor activation in colitis. To test the actions of 5‐HT4 receptors under basal conditions, GR 113808 was administered to normal mice by enema for 10 days. Colitis was evaluated using disease activity index (DAI) and histological damage scores (HDS). Possible protective actions such as cell proliferation in colonic tissue and migration and resistance to oxidative stress in CaCo‐2 cell culture were also explored. To investigate function, we evaluated propulsive motility in the guinea pig distal colon. Intraluminal tegaserod treatment in both DSS and TNBS‐inflamed animals significantly attenuated the development of, and accelerated the recovery from, colitis in an antagonist‐sensitive manner. However, administration of Tegaserod (1 mg/kg) by intraperitoneal injection had no effect. TNBS‐induced dysmotility in guinea pigs was reversed by 5‐HT4 receptor agonist treatment, and this action was blocked by the atagonist. Increased cell proliferation was detected in the form of increased Ki‐67 immunoreactivity in crypts of inflamed animals treated with Tegaserod. We also observed areas of accelerated healing of scratches and increased resistance to H2O2 induced oxidative stress in CaCo‐2 cells. Interestingly, antagonist treatment alone in normal mice resulted in significant increases in DAI, HDS, and bacterial translocation in mice, as well as disrupted motility patterns in guinea pig colon. In summary, activation of 5‐HT4 receptors on the colonic epithelium reduces the development of, and accelerates recovery from, colitis. It is likely acting through increased wound healing by increased cell migration and cell proliferation, as well as resistance to apoptosis. Additionally, 5‐HT4 receptor activation protects colonic motility. Taken together, these data suggest that epithelial 5‐HT4 receptors have a protective effect in the normal and inflamed colon.Support or Funding InformationFunded by DK62267
Elevated D‐2‐Hydroxyglutarate during colitis drives progression to colorectal cancer
Authors: Theiss Arianne , Han Jie , Genta Robert , Sweetman Lawrence 
Published: November 2023

Although the association between inflammatory bowel diseases (IBD) and an increased risk of developing colorectal cancer is well‐established, the pathogenesis of colitis‐associated cancer is poorly understood. Our previous study of quantitative metabolic profiling of urine serially collected from mice during azoxymethane (AOM)‐dextran sodium sulfate (DSS)‐induced colitis‐associated cancer identified that urine D‐2‐hydroxyglutarate (D2HG) levels during colitis directly correlated with severity of polyp formation. Levels of D2HG are normally low and accumulation of D2HG has been linked to oncogenesis in glioma and acute myeloid leukemia. Here, we determined whether elevated D2HG during colitis drives progression to colorectal cancer. Wild‐type C57BL/6 male mice were i.p. injected with 7.6 mg/kg AOM followed by 2 cycles of 3% DSS (1 week DSS and 3 weeks recovery). During DSS, mice were i.p. injected with 25 mg/kg D2HG or vehicle once daily. Body weight, severity of diarrhea, and presence of blood in the stool were monitored daily during DSS administration and twice per week during recovery phases. Excised colons were observed for polyp number/size and embedded for dysplasia scoring. Mice injected with D2HG exhibited similar body weight loss, severity of diarrhea, and presence of blood in the stool during DSS as vehicle‐injected mice. However, mice injected with D2HG during colitis exhibited delayed recovery of body weight and significantly more and larger (>6mm in diameter) colonic polyps with the majority of polyps being high‐grade dysplasia or intramucosal adenocarcinoma. These results suggest that elevated D2HG during colitis enhances progression to colon cancer.Support or Funding InformationCancer Prevention Research Institute of Texas (CPRIT) RP150638
Estrogen receptor beta (ERβ) activation plays a therapeutic role in murine models of inflammatory bowel disease (IBD) via inhibiting P2X3 and P2X7 receptors
Authors: Ma Bei , Jiang Qian , Li Wenxin , Li Zhaoshen 
Published: November 2023

IBD is an inflammatory condition of the bowel system, represented by cumulating and recurring abdominal pain, diarrhea and bloody stools. ERβ has been shown to play a therapeutic role in IBD, while P2X3 receptor (P2X3R) and P2X7 receptor (P2X7R) have been known to promote the initiation and exacerbation of IBD. However, the mechanism how ERβ exerts therapeutic effects and its relationship with P2X3R and P2X7R in IBD are still unknown. In our study, animal behavior tests, histological staining, western blotting, ELISA, and visceromotor reflex recording were used to determine whether the therapeutic effect of ERβ in IBD was through down‐regulation of the expression and function of P2X3R and P2X7R. Based on the successful model of IBD characterized by more weight loss, higher DAI scores and significant inflammatory responses, the expressions of P2X3, P2X7 and ERβ in different tissues including rectocolon, dorsal root ganglia (DRG) and dorsal horn of spinal cord (SCDH) were examined. We found that in colitis group, the expressions of ERβ in rectocolon, DRG and SCDH were all significantly reduced, while that of P2X3R were significantly increased in rectocolon and DRG, but not SCDH, and P2X7R was significantly increased only in rectocolon, but not DRG and SCDH, compared with control group. After administration of A317491 or BBG, antagonists of P2X3R and P2X7R, the weight loss, DAI scores and inflammatory responses of colitis rats in antagonists‐treated groups were all significantly reversed compared with those of control group. Likewise, when agonists of ERβ, DPN and ERB‐041 were given, there was also such a therapeutic role, and at the same time, so did ERβ transfection with recombinant lentivirus. As to the expressions of P2X3R and P2X7R, DPN could significantly decreased P2X3R in all the three fields, namely rectocolon, DRG and SCDH, while P2X7R was decreased only in rectocolon. However, ERB‐041 could decrease P2X3R and P2X7R both in rectocolon only, but not DRG. Furthermore, DPN inhibited visceromotor reflex by colonic extension and secretion of proinflammatory factors, IL‐1b, TNF‐a and IL‐6, in colitis rats. In brief, these results suggest that ERβ may play an important therapeutic role in IBD through down‐regulation of P2X3R and P2X7R. Therefore, it will provide certain evidences and new therapeutic strategies for clinical treatment towards IBD.Support or Funding InformationThis study is supported by National Natural Science Foundation of China (No, 31471103).
Transfection of Rat Intestinal Epithelial Cells with a Stable Mitogen‐Activated Protein Kinase Phosphatase (MKP)‐1 Mutant
Authors: Talavera Maria Margarita , Crowell Sara , Nelin Leif Daniel , Liu Yusen 
Published: November 2023

MAPK phosphatases (MKPs) are critical modulators of the innate immune response in both immune cells and intestinal epithelial cells (IECs). MKP's regulate the innate immune response via MAPK inactivation by dephosphorylation of specific Tyr‐Xaa‐Thr motif, where Xaa represents amino acid residues specific to a MAPK subfamilies. We have shown that peak MKP‐1 expression in LPS stimulated rIEC‐6 cells was 60 minutes, limiting the regulation of the inflammatory response to a short interval. The objective of this study was to create a unique transfected rIEC‐6 cell line that expresses a more stable MKP‐1 mutant protein. rIEC‐6 cells were transfected with one of three plasmids using either Fugene or Viofectin transfection reagent and a fluorescent tag (EGFP‐Cl). The three plasmids were designated either empty vector control or WT‐Flag‐tagged MKP‐1 plasmid or Flag‐tagged MKP‐1 S359/364D plasmid. The Flag MKP‐1S/D mutant carries a specific serine to aspartate mutation in the C‐terminus created using site‐directed mutagenesis. Successful clone selection was achieved with increasing concentration of G418 over a period of 14 days. Clones were grown in DMEM growth media for 24 hours. Cell lysate harvested for protein and vector expression quantified using Western blot analysis. Results demonstrated an 18× fold increase in Flag MKP‐1S/D expression compared to WT‐Flag MKP‐1 protein in rIEC‐6 cells. In conclusion, expression of a more stable MKP‐1 protein in intestinal epithelial cells will enable further study of the critical role of MKP‐1 in the intestinal inflammatory response.Support or Funding InformationLiu (PI) R21 AI113930 NIH/NIAID
Zinc Deficiency Contributes to Alcohol Consumption‐Induced Defect of Mitochondrial Electron Transport Chain and Overproduction of Reactive Oxygen Species in the Live of Rats
Authors: Sun Qian , Zhong Wei , Zhang Wenliang , Sun Xinguo , Zhou Zhanxiang 
Published: November 2023

Clinical studies demonstrated that patients with alcoholic liver disease (ALD) have decreased hepatic zinc levels; however the effect of zinc deficiency on the function of hepatic mitochondria has not been well studied. The present study was undertaken to determine if zinc deficiency causes mitochondrial electron transport chain (ETC) defect and if defected ETC function could increase hepatic oxidative stress. Adult male Wistar rats were pair‐fed with the Lieber‐DeCarli control or ethanol liquid diet for 5 months. Chronic alcohol exposure significantly increased hepatic levels of triacylglycerol and free fatty acid and induced apoptotic cell death. Zinc levels in both the whole liver and the purified hepatic mitochondria were significantly reduced by alcohol exposure in association with perturbed expression of zinc transporters. Chronic alcohol exposure also increased the levels of 4‐hydroxynonenal (4HNE) in the whole liver as well as the purified mitochondria. The expression of hepatic mitochondrial respiratory complex I, III, IV, and V were repressed by chronic alcohol exposure, which was in consistence with a significant decrease of the hepatic ATP production. Examination of the mitochondrial biogenesis markers demonstrated that chronic alcohol exposure significantly decreased AMPK, PGC1α, NRF1 and TFAM levels in association with downregulation of mitochondrial DNA expression. In order to define the link between zinc deficiency and the defect of mitochondrial respiratory complexes, HepG2 cells were treated with zinc chelator, TPEN, for 6h. Experimental zinc deficiency significantly decreased the protein levels of mitochondrial respiratory complex I, III, and IV. In addition, p‐AMPK, PGC1α, NRF1 and TFAM levels as well as mitochondrial DNA expression were significantly decreased by TPEN treatment. Consequently, mitochondrial respiratory complex I, III, or IV was silenced by corresponding shRNA, respectively. HepG2 cells with decreased expression of complex I, III, or IV by gene manipulation exhibited a decrease in mitochondrial membrane potential and an increase in free radical production compared to the cells transfected with scrambled shRNA. These results suggest that hepatic zinc deficiency critically mediates alcohol‐diminished mitochondrial biogenesis and mitochondrial DNA levels which, at least partially, accounts for alcohol‐induced defect of mitochondrial ETC and overproduction of reactive oxygen species.Support or Funding InformationR01AA020212 and 2R01AA018844
Liver‐Directed Knockout and Transgene Delivery of Arginase‐1 in Mice
Authors: Funk Colin D , Ballantyne Laurel , Schulze Andreas 
Published: November 2023

Arginase‐1 (Arg1) catalyzes the distal step in the liver‐based urea cycle that converts arginine to urea and ornithine. Genetic deficiency of this enzyme in humans, unlike proximal urea cycle disorders that lead to hyperammonemia and neonatal crisis, leads to neurological deficits (e.g. spastic diplegia) that manifest around 2–4 years of age and progress throughout childhood. We previously generated a tamoxifen‐inducible Arg1 deficient mouse model (Arg1‐Cre) that leads to a phenotype much more severe than in humans with lethality occurring ≈2 weeks after loss of enzyme function. The purpose of this study was to evaluate if liver‐selective Arg1 loss is sufficient to recapitulate the phenotype observed in global Arg1 knockout mice, as well as to gauge the effectiveness of a gene therapy protocol to rescue the phenotype. Liver‐selective Arg1 deletion was induced by using an adeno‐associated viral (AAV)‐thyroxine binding globulin (TBG) promoter‐Cre recombinase vector (1.5×1011 genome copies (g.c.), intraperitoneally (i.p.) injected) administered to mice (n=6) with loxP sites flanking exons 7 and 8 of the Arg1 gene (Arg1 floxed mice; Arg1fl/fl). An AAV vector expressing an Arg1‐enhanced green fluorescent protein (Arg1‐eGFP) transgene from a strong promoter (hybrid cytomegalovirus enhancer/chicken β‐actin) was used in a gene therapy approach to “rescue” tamoxifen‐treated Arg1‐Cre mice. The results indicate that liver‐selective loss of Arg1 (>95 % deficient) leads to a phenotype resembling the whole body knockout of Arg1 with lethality around 2 weeks after Cre‐induced gene disruption in all mice. Delivery of Arg1‐eGFP AAV (n=10 male; 1.5×1011g.c. i.p. administered 2 weeks prior to tamoxifen treatment) rescues ≈ half of Arg1 global knockout mice (survival >4 months) but a significant proportion still succumb to the enzyme deficiency even though liver expression and enzyme activity of the fusion protein reach levels approaching that found in wild‐type animals. This raises a conundrum relating to liver‐specific expression of Arg1. On the one hand, loss of expression in this organ appears to be both necessary and sufficient to explain the lethal phenotype of the genetic disorder in mice. On the other hand, the gene therapy rescue studies suggest that loss of extra‐hepatic Arg1 expression factors into disease etiology. Further studies will be necessary to illuminate the detailed mechanisms for pathogenesis of Arg1‐deficiency.Support or Funding InformationCanada Research Chairs Program