bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of mucus layer on the transcellular absorption of lipophilic drugs in rat small intestine
Authors: KISHIMOTO HISANAO , MIYAZAKI KAORI , SHIRASAKA YOSHIYUKI , INOUE KATSUHISA 
Published: June 2021

PurposeThe mucus layer, which is adjacent to the intestinal wall, is considered to be a physiological barrier to intestinal drug absorption, since a drug must diffuse through this lipophilic layer prior to permeation across the intestinal epithelial cells. Thus, mucus layer permeation is suggested to be a rate‐limiting step in the intestinal absorption of lipophilic drugs. The major glycoprotein components of the mucus layer are mucins, which presumably play a pharmaceutical role in intestinal drug absorption, however, there have been no reports that describe the contribution of mucins to intestinal drug absorption at the molecular level. In this study, we examined the involvement of the mucus layer in the intestinal permeability of various lipophilic drugs in rat small intestine. Furthermore, we evaluated the regional distributions of mucins (MUCs) in rat gastrointestinal tract and explored the relationship between the effect of the mucus layer on passive transcellular transport and the expression pattern of mucins.MethodsDrug permeation experiments were conducted using the in vitro sac method. The amount of mucus glycoproteins liberated from the intestinal wall were determined by the alcian blue method. The mRNA and protein expression levels of mucins were evaluated by quantitative real‐time RT‐PCR (qPCR) and Western blotting, respectively.Results and DiscussionThe membrane permeability of the lipophilic drugs in the rat gastrointestinal tract was increased by pretreatment with dithiothreitol (DTT), which is commonly used as a mucus remover, and the alternation was remarkable in the upper part of the small intestine. Furthermore, the enhancing effects of DTT tended to be pronounced on the permeability of drugs with higher log D values in the case of neutral drugs with non‐dissociable groups. These results indicated that the effect of the mucus layer on intestinal permeability depends on the log D values and the physiological charge of the compound. On the other hand, the mRNA expression analysis of MUC genes revealed that the intestinal expression of MUC5AC is considerably higher in the duodenum, and those of MUC1, MUC2, and MUC3A are gradually increased toward the lower intestine in rats. Furthermore, Western blotting showed abundant MUC5AC protein in the mucus solution liberated from jejunum, but not from ileum and colon by the treatment with DTT. Therefore, these results suggest that rMUC5AC is involved in the absorption of lipophilic drugs in the upper part of rat small intestine.ConclusionsMUC5AC could be involved in the barrier function to limit the transcellular permeation of hydrophobic drugs in the upper part of the gastrointestinal tract.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of spontaneous DNA damage and single‐stranded DNA in generation of enlarged G2 phase cells in rad52 mutants of Saccharomyces cerevisiae
Authors: England Corbin J. , Weis Monica F. , Lewis L. Kevin 
Published: June 2021

The DNA of living creatures is continuously being damaged by endogenous and exogenous sources and different DNA repair pathways exist to mend the different types of damage. Double‐stranded breaks (DSBs) in the DNA of eukaryotes can be repaired by either homologous recombination (HR) or nonhomologous end‐joining (NHEJ). The initial steps of HR in the budding yeast S. cerevisiae lead to formation of long ssDNA tails at the broken ends. These tails arise through short resection by the Mrx and Sae2 proteins, followed by longer resection by Exo1 and Sgs1‐Dna2 and possibly other enzymes. The tails are subsequently coated with the single‐stranded DNA binding protein complex Rpa, which then recruits other proteins to the site such as DNA damage checkpoint sensor proteins and RAD52 group strand exchange proteins.After exposure to DNA damaging agents, yeast cell cultures exhibit an increase in G2 phase cells due to activation of the dominant DNA damage cell cycle checkpoint. rad52 haploid and diploid mutants grown in normal liquid cell culture display an increase in large‐budded cells. DAPI staining indicated that over 90% of these cells were in G2 phase. Creation of double mutant strains with RAD52 and any of seven different DNA damage checkpoint genes co‐inactivated resulted in loss of the high G2 cell phenotype, suggesting constitutive activation of DNA damage checkpoints in the mutants. rad52 cells were also found to have other unusual characteristics, including (a) larger average sizes than wildtype cells, especially among large‐budded cells, (b) faster cell sedimentation rates, and (c) strongly enhanced light scattering in aqueous suspensions. The role played by formation of long 3′ ssDNA tails at DSBs during HR has been investigated using rad52 double mutants incapable of forming long tails. DNA damage checkpoint protein and single‐stranded binding protein fusions with GFP are also being employed to analyze differences in foci formation within WT and rad52 cells.Support or Funding InformationNational Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Hydroxyproline to Measure Collagen in Teeth With and Without Whitening Strips
Authors: Meyer Seann , Fiorica Antonino , Keenan Kelly 
Published: June 2021

Billions of dollars are spent in the United States every year on tooth whitening. Whether it is done in a dentist's office or with over the counter products, the active ingredient is often hydrogen peroxide which has long been known to damage proteins. The outermost part of the tooth is the enamel and contains several proteins while dentin, the layer underneath, contains 90 to 95% collagen. This protein contains the modified amino acid hydroxyproline and the level of this amino acid has been used to measure the amount of collagen. Hydroxyproline can be detected using a colorimetric test: the original test required use of perchloric acid but the method was modified with use of hydrochloric acid replacing the perchloric acid. Teeth were purchased, placed in artificial saliva and treated with whitening strips following the manufacturer's instructions while other teeth did not receive the strips. Afterwards, the teeth were dialyzed against 10% EDTA, pH 9.0 for 2 weeks to remove calcium and then dialyzed against water to wash the teeth. They were then placed in liquid nitrogen and hammered into shards which were turned into a powder using a blender. The powder was suspended in an acetic acid solution to make the collagen fraction(COL). This fraction was treated with pepsin for four hours, neutralized with saturated Tris solution and centrifuged for 1 hour. The supernatant was the pepsin soluble fraction (PSC). Both COL and PSC were then hydrolyzed by adding 6 M HCl overnight at 95 °C. The resulting hydrolyzed samples were neutralized with 10 M sodium hydroxide and tested for hydroxproline. Results indicated a significant drop in hydroxproline content after one treatment and an even greater loss after three treatments. The drop in the level of hydroxyproline following treatment with whitening strips does suggest a loss of collagen from the dentin due to the whitening strips.Support or Funding InformationAuthors express gratitude to Research and Professional Development Grant from Stockton University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting Aspartate Transcarbamoylase in Staphylococcus aureus: A Novel Therapeutic Approach for Sepsis
Authors: Patel Chandni , Vaishnav Asmita , Brunzelle Joseph S. , Guy‐Evans Hedeel I. , Edwards Brian FP. , Evans David R. 
Published: June 2021

Sepsis is one of the leading causes of death in the United States. More than 1.5 million people get sepsis and 250,000 people die from sepsis each year. Staphylococcus aureus happens to be most common cause of healthcare‐associated infections. Research and development of new antibiotics against S. aureus is a high priority due to its multi‐drug resistance. The levels of pyrimidines in blood are too low to sustain the growth of bacteria, so they must rely on pyrimidine biosynthesis. Previous studies have shown that a defect in several pyrimidine biosynthetic enzymes resulted in 1000‐fold decrease in the titer of bacteria growing in blood. Aspartate transcarbamoylase (ATC) catalyzes the first committed step of the de novo pyrimidine pathway, making it a good drug target. S. aureus ATC containing his‐tag was overexpressed in Escherichia coli and purified using nickel affinity column chromatography. S. aureus ATC has a Km of 2.4 mM for carbamoyl phosphate and 4.5 mM for aspartate, with a maximal velocity of 296 μmol/min/mg. Cross‐linking data suggests that S. aureus ATC is a trimer of 33 kDa and it does not associate with DHO, the subsequent enzyme in the pathway, as seen in Pseudomonas aeruginosa, Aquifex aeolicus, and several other ATC. We have successfully solved the preliminary structure of S. aureus ATC at 2.27Å resolution. Once our preliminary structure has been further refined, it will serve as the basis for the design of novel non‐competitive inhibitors that block domain closure of ATC, an essential step in catalysis.Support or Funding InformationAmerican Heart Association Predoctoral Fellowship, Interdisciplinary Biomedical Sciences Fellowship, Wayne State University School of Medicine, Wayne State University Graduate School & Office of VP for ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Overexpression of Insulin‐like Growth Factor Binding Protein 1 (IGFBP1) Generates Tamoxifen Resistance in Breast Cancer Cells
Authors: Kieltyka Jacqueline , Zheng Yan , Hobbs Alvaro , Houston Kevin D. 
Published: June 2021

Tamoxifen, a selective estrogen receptor modulator and antagonist for estrogen receptor alpha (ERα) in breast tissue, is a commonly prescribed adjuvant treatment for patients presenting with ERα‐positive breast cancer. Acquired resistance to tamoxifen remains a clinically relevant challenge for many patients receiving treatment and some patients with ERα‐positive breast cancer are innately resistant to tamoxifen treatment. The molecular mechanisms underlying both acquired and innate tamoxifen resistance are not well understood. Previous studies have shown that, in addition to ERα inhibition, tamoxifen activates the G‐protein‐coupled estrogen receptor 1 (GPER1) and activation of this receptor is associated with the development of tamoxifen resistance. GPER1 mediates the extracellular accumulation of the insulin‐like growth factor binding protein 1 (IGFBP‐1) after tamoxifen treatment resulting in the inhibition of the IGF‐1R signaling in breast cancer cells. We hypothesize sustained exposure of breast cancer cells to IGFBP‐1 will result in the development of tamoxifen resistance. Stable transfection of an IGFBP‐1 expression vector was used for sustained exposure of MCF‐7 breast cancer cells. IGF‐1R expression was decreased in MCF‐7 cells stably transfected with the IGFBP‐1 expression vector similar to IGF‐1R expression in tamoxifen resistant breast cancer cells. Similar to tamoxifen‐resistant MCF‐7 cells, the IGFBP‐1 overexpressing MCF‐7 cells also have elevated levels of activated ERK1/2. Finally, cell viability assays revealed that sustained IGFBP‐1 overexpression decreased breast cancer sensitivity to tamoxifen treatment. Taken together, these results suggest that elevated levels of IGFBP‐1 contribute to the development of tamoxifen resistance in breast cancer cells.Support or Funding InformationResearch reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103451.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Hydrogen Sulfide Levels by Cannabinoids in the Bovine Isolated Retina
Authors: Bush Leah , Robinson Jenaye , Kutse Seun , Ebuzoeme Christabel , Onyenaka Collins , Akpan Idongesit , Opere Catherine , Ohia Sunny , Njie‐Mbye Ya Fatou 
Published: June 2021

Both endocannabinoids and hydrogen sulfide (H2S) have been reported to exert neuroprotective actions in neural tissues (Biermann et al., Mol Vis. 2011;17:1275–86; Nucci et al., Invest Ophthalmol Vis Sci. 2007;48(7):2997–3004). It remains to be determined if these modulators can interact with each other and/or share a common pathway in their neuroprotective actions.PurposeThe aim of the present study was, therefore, to investigate the pharmacological actions of endocannabinoids on the biosynthesis of H2S in isolated bovine neural retina.MethodsIsolated bovine retinae were homogenized and exposed to different concentrations of methanandamide (1 nM – 10 uM) and 2‐arachidonyl glycerol (2‐AG) (1 – 3 uM) for 90 minutes at 37ºC. Retinal homogenates were then incubated with ethylenediaminetetraacetic acid (EDTA) (1%), Zinc acetate (1%), and Borate buffer (pH 11) for 30 mins followed by 10‐minute incubation with N, N dimethyl p‐phenylenediamine (20 mM) and FeCl3 (300 mM). Samples were then centrifuged at 5000 g for three minutes and supernatant was assessed for H2S content using the well‐established Methylene Blue method.ResultsBoth methanandamide and 2‐AG elicited a concentration‐dependent decrease in basal H2S production in the isolated retina. For instance, methanandamide (10 μM) significantly (p <0.05) reduced basal production of H2S by 30% when compared to controls. Likewise, 2‐AG (10 μM) also elicited a 30% decrease in basal retinal H2S concentration when compared to controls.ConclusionsWe conclude that exogenously applied endocannabinoids can alter the basal production of H2S in the isolated neural retina. The observed effect of endocannabinoids on the pathway leading to H2S biosynthesis suggests that a cross‐talk exists between both neuromodulators in the retina.Support or Funding InformationSupported by NIH/NEI Research Grant 1R15EY022215‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The 4 Position of 5‐HT2A Structural Agonists Imparts the Anti‐Inflammatory Propensity Responsible for Reducing Airway Hyperrresponsiveness in Allergic Asthma
Authors: Flanagan Thomas Wayne , Landry Alexus Nichol , Hibicke Meghan , Nichols Charles David 
Published: June 2021

The 5‐HT2A receptor is the most widely expressed serotonin receptor in mammals. While distribution occurs largely in the central nervous system, the 5‐HT2A receptor has been detected in platelets, endothelial and epithelial cells, and lymphatic tissue. Most commonly viewed as the classical target of psychedelic compounds, our lab has made significant progress in understanding how activity at the 5‐HT2A receptor profoundly activates anti‐inflammatory pathways. Here we have generated a chronic asthma model to examine the therapeutic potential of 5‐HT2A receptor activation in persistent allergic airways disease. Our studies show that nasal administration of the 5‐HT2A agonist (R)‐DOI attenuates the elevated airway responsiveness, goblet cell hyperplasia, collagen deposition, and airway structural remodeling that accompanies persistent allergen challenge. Additionally, we have used an acute model in rodents to interrogate structural components of known psychedelics most critical for their anti‐inflammatory effects. Our studies indicate that the 4 position of 5‐HT2A structural agonists contains the chief mediator for reducing bronchial hyperresponsiveness in asthma. This ultimately suggests that a blood‐brain inpenetrant molecule can be developed that lacks the psychoactivity of classical psychedelics but maintains therapeutic efficacy.Support or Funding InformationThis work was supported, in part, by research funds from the American Asthma Foundation Scholar Grant Award to C.N., The Betsey Gordon Foundation, and the Eleusis Benefit Corporation, PBC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activity of Single and Double‐modified Salinomycin Analogs against Primary Acute Lymphoblastic Cells In Vitro
Authors: Urbaniak Alicja Joanna , Delgado Magdalena Rose , Antoszczak Michał , Borgström Björn , Strand Daniel , Huczyñski Adam , Chambers Timothy C. 
Published: June 2021

Salinomycin (SAL) and monensin (MON) are polyether ionophore antibiotics known from their anti‐cancer activity against various types of cancer cells, including those that display multi‐drug resistance as well as cancer stem cells. In order to increase the biological activity profile and reduce toxicity against normal cells, while retaining the activities in the micromolar range, a library of novel SAL analogs has been synthesized. This included esters and amides single‐modified in C1 position as well as double‐modified derivatives additionally acylated in the C20 position (Fig. 1). The aim of the study was to evaluate the activity of newly synthesized SAL derivatives (12 single‐modified and 12 double‐modified) towards primary acute lymphoblastic leukemia cells. MON (IC50 = 1.2 ± 0.8 μM) and nine SAL derivatives (IC50 in the range of 1.2 ± 0.02 to 1.9 ± 0.7 μM) were more potent than SAL (IC50 = 2.4 ± 0.7 μM) in cell viability assays. Moreover, the most potent SAL analogs induced characteristics of apoptotic cell death and increased expression of p53. In further studies, SAL acted synergistically with the Bcl‐2 inhibitor ABT‐263, whereas 2,2,2‐trifluoroethyl ester, the most active analog of SAL, antagonized ABT‐263. This is intriguing and suggests that the parent compound may differ mechanistically from its derivatives. The present findings indicate that SAL derivatives constitute promising lead compounds for targeting acute lymphoblastic leukemia. Further study and development of these novel cancer therapeutics with potential clinical application is warranted.Support or Funding InformationTCC acknowledges support from Arkansas Breast Cancer Research Program and Chancellors Circle Award from the UAMS College of Medicine. MA thanks the Polish Science Centre (NCN) for financial support by grant SONATA (2016/23/D/ST5/00242) and fellowship ETIUDA (2014/12/T/ST5/00710). We thank the Swedish Research Council, and the Cancer Foundation for funding.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Amino Acids in Muscle and Blood from Patients with Pancreatic Cancer‐ Induced Cachexia
Authors: Talbert Erin E. , Chakedis Jeffery , Guttridge Denis C. 
Published: June 2021

Cachexia, or weight loss, is a profound side effect of cancer. Patients that suffer from cachexia have poor treatment tolerance, poor post‐surgical outcomes, poor survival, and poor quality of life. Despite decades of research, we lack a pharmacological intervention to treat cachexia, as mechanisms driving cachexia remain incompletely understood. Further, while the significance of cancer cachexia is clear, this syndrome remains poorly recognized, and a biomarker of cancer cachexia would be a valuable tool to improve the recognition of this syndrome. Because cachexia is ultimately a metabolic syndrome, we hypothesized that a metabolomics approach could identify new potential mechanisms of cachexia while also identifying potential biomarkers. Using a targeted mass spectrometry approach, we sought to distinguish cachectic pancreatic cancer patients from those without cancer or weight stable pancreatic cancer patients. Surprisingly, amino acid content of muscle from both cachectic and weight stable cancer patients was pronouncedly decreased compared to control patients. Similarly, plasma amino acid content was also generally decreased in both weight stable and cachectic cancer patients. Only differential levels of gamma‐amino‐N‐butryic acid (GABA) distinguished cachectic cancer patients from weight stable cancer patients and controls. In summary, the majority of changes we detected were specific to pancreatic cancer rather than cachexia, emphasizing the need to include cancer patients with and without cachexia in the study design.Support or Funding InformationSupport was provided through a Pilot and Feasibility Grant from the Mayo Clinic Metabolomics Resource Core through grant number U24DK100469 from the National Institute of Diabetes and Digestive and Kidney Diseases. Additional funding was provided by NIH R01CA180057 (DCG) and American Cancer Society Postdoctoral Fellowship PF‐15‐156‐01‐CSM (EET).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of the mechanosensitive ion channel Piezo1 in Autosomal Dominant Polycystic Kidney Disease (ADPKD)
Authors: Chebib Fouad , Beyder Arthur , Wang Xiaofang , Alcaino Constanza , Ehrlich Barbara , Torres Vicente 
Published: June 2021

Disruption of intracellular calcium likely underlies the upregulation of cyclic AMP (cAMP) signaling and the proliferative and secretory phenotype of the cystic epithelium in ADPKD. However, the mechanisms by which polycystins reduce cytoplasmic calcium remain elusive. Impaired flow‐induced calcium entry through ciliary polycystin complex has been previously proposed as a pathogenic mechanism, but newer evidence suggests the involvement of polycystins in modulation of other mechanosensitive ion channels such as Piezo1, the newly‐identified stretch‐activated non‐selective cation channel. We hypothesized that polycystins, through their physical and functional interaction with Piezo1, modulate various cellular responses to mechanical stimulation.Piezo1 was expressed in both wild type mouse inner medullary collecting duct cells (mIMCD3) cells and PKD2 null cells (~3 fold higher than wild type). Wild type mIMCD3 cells were stably transfected with genetically encoded biosensors, jRGeco1a and Flamindo2, to study spatiotemporal dynamics of calcium and cAMP during live cell imaging, respectively. Piezo1 activation by Yoda1 increased cytoplasmic calcium and simultaneously lowered forskolin‐induced cAMP (Figure 1A). Additionally, exerting physiological fluid shear stress (FSS) (ibidi pump system) led to reduction in cAMP (Figure 1B). Both Yoda1 and FSS lead to an increase in ATP release in mIMCD3 cells (Figure 1C). Furthermore, Yoda1 significantly reduced forskolin‐induced in vitro cystogenesis by 68% in WT and by 48% in PKD2 null mIMCD3 cells (Figure 1D).These results suggest that Piezo1 is involved in renal cystogenesis by controlling FSS‐induced calcium entry. Further studies are required to determine in vivo effect of Piezo1 on renal cystogenesis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.