bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimization of Breast Cancer Resistance Protein (BCRP) Expression in the Yeast Pichia pastoris
Authors: Oliveira Maisa C. , Lavigne Collette A. , Tran Brandon M. , Fowler Michael E. , Okwuone Dakota D. D. , Farokhnia Roxana , Wise John G. , Vogel Pia D. 
Published: June 2021

Multidrug resistance (MDR) has been linked to the failure of chemotherapy, and poor prognoses and reduced survival rates in patients in several cancer types. MDR cancers often become resistant to pharmacologically and structurally diverse drugs by overexpressing members of the family of ATP Binding Cassette (ABC) transporters. The breast cancer resistance protein (BCRP) is expressed in many tissues, including those with barrier or detoxifying functions such as blood‐brain barrier, placenta, and is responsible for the active export of a wide variety of toxins across the plasma membrane. Given its role in MDR, inhibitors of BCRP have been studied for years. To search for BCRP inhibitors as potential leads for the development of co‐therapeutics to combat MDR, we used high‐throughput in silico ligand docking similar to studies performed previously in the search for inhibitors of the P‐glycoprotein (Brewer et al., Mol. Pharmacol. 86, 716–726, 2014). A number of drug like molecules were identified that reversed multidrug resistance of a BCRP‐overexpressing breast cancer cell line generated in our lab (Nanayakkara, unpublished). To understand the mechanism of inhibition of BCRP by these compounds, biochemical assays assessing ATP hydrolysis activity as well as other biochemical properties of BCRP are required. To obtain protein amounts that are large enough for biochemical and biophysical evaluation of inhibitor action, a gene for human BCRP was designed with optimized codon usage for translation and expression in P. pastoris. Maximum expression of BCRP in the PichiaPink™ system was observed after 9 hours of growth on a methanol based medium. Protein expression was confirmed by Western blot analysis using both BCRP and anti‐His antibodies. ATP hydrolysis assays indicated that the protein expressed was catalytically active and that the ATP hydrolysis activity could be stimulated by the BCRP transport substrate, Hoechst 33342. Here we also report our attempts to purify BCRP from PichiaPink™ membranes and characterize the protein both as solubilized in mixed micelles as well as upon reconstitution into membrane nanodiscsSupport or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, Ms. Lynn McBee and New England Biolabs, the Communities Foundation of Texas, a private gift from Ms. Suzy Ruff of Dallas, Texas, as well as the Hamilton Undergraduate Research Scholarship the SMU Undergraduate Research Assistantship Programs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of ABC Transporters in Multidrug Resistant Cancer Cell Lines
Authors: Holcomb‐Webb Kasi L. , Nanayakkara Amila K. , Aljowni Maha , Lippert Alexander R. , Vogel Pia D. , Wise John G. 
Published: June 2021

ATP‐binding cassette (ABC) transporters are transmembrane proteins that act as drug efflux pumps and are associated with multi‐drug resistance (MDR) in cancers. Two specific proteins that belong to this protein superfamily are P‐glycoprotein (P‐gp) and breast cancer resistant protein (BCRP). These two proteins are known to pump chemotherapeutics out of cells, rendering treatments of some cancers less efficient. Certain cancer cell lines overexpress these proteins, leading to MDR. If a compound can be identified that inhibits these efflux pumps, then sensitivity to chemotherapeutics can be restored. The levels of expression of P‐gp and BCRP were quantitatively measured in four cancer cell lines that are used by us to screen for inhibitors of P‐gp or BCRP. The expression of P‐gp, BCRP, and MRP1 (multidrug resistance‐associated protein 1) was measured using quantitative One‐Step qPCR in a prostate cancer cell line, DU145, and compared to an MDR derivative line that overexpresses P‐gp, DU145 TXR. We found that DU145 TXR expresses P‐gp, BCRP, and MRP1 by about 14,000‐fold, 4‐fold, and 0.75‐fold, respectively, when compared to the parental cell line, DU145. A breast cancer cell line, MCF‐7, and its mitoxantrone‐resistant derivative, MCF‐7 M100, were also analyzed. The MCF‐7 M100 cell line was found to express BCRP, P‐gp, and MRP1 by about 600‐fold, 0.3‐fold, and 1.6‐fold, respectively, compared to the parental cell line, MCF‐7. The results were qualitatively confirmed at the protein level using Western Blot analyses. In other experiments, we demonstrated that P‐gp and BCRP inhibitors identified by us that reversed multidrug resistances in the MDR prostate and breast cancer cell lines, DU145 TXR and MCF‐7 M100, did not down‐regulate expression levels of P‐gp or BCRP, suggesting that reversal of MDR was accomplished by inhibiting transport processes and not transporter expression levels.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM09477102] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas, as well as the Hamilton Undergraduate Research Scholars Program and the SMU Summer Research Assistantship and Undergraduate Research Assistantship Programs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genetic and Bioinformatic Analysis of Streptomyces Bacteriophage Cluster BF
Authors: McClendon‐Mosss Tianeaka , Hughes Lee 
Published: June 2021

Streptomyces bacteria are at the forefront of the world's battle to control infectious bacteria. More than 70% of the antibiotics currently in use were derived from the secondary metabolites of various Streptomyces bacterial species. In the search to find new ways of combating antibiotic‐resistant infectious bacteria, bacteriophage‐derived genetic tools are quickly emerging with promising possibilities. Research to identify and characterize new Streptomyces bacteriophages is currently underway with the hopes of creating new genetic engineering tools that can be used to introduce variety into the genetic material of the host, thereby causing the production of new secondary metabolites. In this study, 8 Streptomyces bacteriophages in Cluster BF were analyzed genetically using bioinformatic tools such as DNA Master, Glimmer, GeneMark, and Starterator software. The bacteriophages in this study were isolated from soil samples gathered by student researchers at the University of North Texas using Streptomyces griseus 10137 as the host. Isolation techniques used were developed by the Science Education Alliance‐Phage Hunters Advancing Genomics and Evolutionary Science (SEA‐PHAGES) program. The average genome length of the bacteriophages in this cluster was approximately 46000 base pairs and each contains between 62 and 64 putative genes. The average G+C content for the phages in this cluster is 59.7%. Low G+C content, while common among the Cluster BF phages, is highly unusual given the fact that most phages tend to reflect the genomes of their hosts, and Streptomyces bacteria are high G+C organisms at approximately 70% or higher G+C content. Cluster BF members also contain an abnormally high number of tRNA genes, ranging in number from 20 to 22. Host range experiments on eight other Streptomyces species were also conducted. The results of this work show that the Cluster BF phages have interesting genomes that merit additional study. Future experiments will be designed to explore mechanisms that allow these low G+C phages to infect a high G+C host like Streptomyces, with particular emphasis on the role that the tRNA genes play.Support or Funding InformationThis research was funded by the University of North Texas Department of Microbiology. A portion of this project was funded by the Science Education Alliance‐Phage Hunters Advancing Genomics and Evolutionary Science (Sea‐Phages) Program and a grant from the Howard Hughes Medical Institute HHMI‐Transition Students ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing a Loss of Function Mutation in the NOD2 gene in Puerto Rico
Authors: Acevedo‐Sanchez Yamilex , Martínez‐Cruzado Juan Carlos 
Published: June 2021

Loss‐of‐function (LoF) mutations are genetic variants predicted to truncate the structure of protein coding genes, nullifying their ability to fulfill their biological role. The distribution of these variants was outlined in Phase 1 of the 1000 Genomes Project, which included 55 individuals from the Puerto Rican population. Here, a frameshift mutation in the NOD2 gene, which has been previously associated with Crohn's Disease, was identified, having an allelic frequency of 0.029 in the Puerto Rico sample set of the 1000 Genomes Project, Phase 3. NOD2 encodes a protein that has a role in immune response via activation of the nuclear factor‐kappa‐B. The purpose of our study was to identify the distribution and frequency of this mutation in the Puerto Rican population. Genomic DNA obtained from saliva samples was genotyped using the polymerase chain reaction‐restriction fragment‐length polymorphism (PCR‐RFLP) technique. The presence of the mutant allele introduces an ApaI recognition site when a mismatched primer is used for PCR‐amplification, resulting in two fragments of 92bp and 18bp instead of one fragment 110 base pairs long. Fragment length is analyzed by agarose gel electrophoresis. Preliminary results show that from 42 samples analyzed, there is one heterozygous individual for the mutant allele in the northeast region of Puerto Rico. Further analysis is needed to provide an accurate estimation of the frequency and distribution of this polymorphism.Support or Funding InformationResearch reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number T34GM008419This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
C‐terminal domain of Mad1 interacts with Mad2 to catalyze O‐C Mad2 conversion in coordination with mitotic checkpoints
Authors: Nguyen Nhan Do Minh , Ahmad Ejaz , Liu Song‐Tao 
Published: June 2021

Aneuploidy occurs when chromosomes are unequally separated. It is important that all chromosomes' kinetochores attach to spindle microtubules before the cell starts to divide; if not, the chromosomes will be distributed unevenly between the two daughter cells. In order to prevent chromosome missegregation, the mitotic checkpoint complex (MCC) prevents chromosomes that are unattached to spindles from division. Human MCC is composed of multiple proteins: Bub3, BubR1, Cdc20, and Mad2. Kinetochores that are unattached to spindles give out the signal that triggers the MCC to assemble. Once formed, the MCC is able to inhibit anaphase‐promoting complex/cyclosome (APC/C). Without the activation of APC/C, the anaphase progression is temporarily stopped. In our study, we investigated Mad2, which is a critical part of the MCC. There are two functional conformations of Mad2, closed and opened, also known as C‐Mad2 and O‐Mad2, respectively. In the MCC, Mad2 exists in closed form (C‐Mad2). Therefore, to assemble the MCC, O‐Mad2 needs to be converted to C‐Mad2. Our research focuses on the pathway where Mad1 recruits O‐Mad2 and converts it to C‐Mad2. Previous research was concentrated on the middle part of Mad1 (Mad1‐MIM), and it was posited that Mad1 could not independently recruit O‐Mad2 without forming a complex with C‐Mad2. However, our research on the C‐terminal domain (CTD) of Mad1 indicates that Mad1‐CTD binds to both O‐ and C‐Mad2. This result suggests that CTD‐Mad1 could potentially recruit O‐Mad2, and convert it to C‐Mad2 which then assembles into the MCC.Support or Funding InformationThe work was carried out during the summer of 2017 in the Department of Biological Sciences at the University of Toledo in Dr. Song‐Tao Liu's lab with support from the Department of Chemistry at Wabash College.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transient Receptor Potential Vanilloid 4 Channel Regulates TGFβ1‐Mediated Fibroblast Differentiation And Airway Remodeling Through RAC/NADPH Oxidase 4 Axis
Authors: Ghebreigziabher Samrawit , Al‐Azzam Nosayba , Snyderman Matthew , Thodeti Charles , Paruchuri Sailaja 
Published: June 2021

Transient Receptor Potential Vanilloid 4 (TRPV4) is a stretch‐activated, mechano‐sensitive calcium channel. We have shown recently that TRPV4 regulates TGFβ1‐mediated fibroblast differentiation in vitro and D. farinae‐induced airway remodeling in vivo. Here, we present a unique finding that this regulation requires NADPH oxidase 4 (NOX4), a member of the NADPH oxidases. Our results reveal that TGFβ1 transcriptionally up‐regulated NOX4 gene expression and this expression was sensitive to TRPV4 inhibition. Further, TGFβ1‐induced fibrotic gene expression was inhibited by NOX4 antagonist (GKT137831), antioxidants and NOX4 siRNA. Interestingly, inhibition of Rac, a member of small family GTPases attenuated TGFβ1‐induced NOX‐4 upregulation and fibroblast differentiation, highlighting a novel TRPV4‐RAC‐NOX4 interaction. Importantly, TRPV4 KO mice which were protected from D. farinae‐induced asthma also exhibited lower levels of NOX4 gene expression, suggesting that TRPV4 promotes airway remodeling through NOX4. Our data unravels a unique role for TRPV4‐RAC‐NOX4 interaction in TGFβ1‐mediated fibroblast differentiation and lung remodeling.Support or Funding InformationThis work is supported by James Foght Assistant Professor support.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Disruption of a Short Open Reading Frame (sORF) In The mRNA 5′ Leader Sequence (5′LS) of the Type 1 Angiotensin Receptor (AT1R) Increases Angiotensin II (Ang II)‐ Induced AT1R Internalization and Signaling through the Extracellular Signal‐Regulated Kinases (ERK1/2) Pathway
Authors: Kadam Parnika , Ji Hong , Pai Amrita V. , Speth Robert , Mueller Susette , Sandberg Kathryn 
Published: June 2021

Antagonists of the AT1R are widely prescribed clinically to treat hypertension and associated cardiovascular and renal diseases. We investigated the effects of a sORF in exon (E) 2 in the AT1aR mRNA 5′ leader sequence on Ang II‐induced AT1aR internalization and signaling through the ERK1/2 signal transduction pathway. The start codon of this sORF was disrupted [E1,2(‐108T),3‐AT1aR] and along with the unmutated sORF [E1,2,3‐AT1aR] were cloned into an expression plasmid containing enhanced green fluorescent protein [EGFP‐N2]. Human embryonic kidney‐293 (HEK) cells were transfected with intact or disrupted sORF (AT1aR‐EGFP‐N2) plasmids. Radioligand binding studies with 125I‐Ang II (300 pM) in transfected HEK cells demonstrated disruption of the sORF increased AT1aR binding by 1.4‐fold after 30 min of Ang II stimulation [AT1aR (cpm): E1,2,3‐AT1aR, 5865±1040 vs. E1,2(‐108T),3‐AT1aR, 8201±878; p<0.05; n=2;] and 1.3‐fold more AT1aR was internalized after 15 min of Ang II treatment [AT1aR (cpm): E1,2,3‐AT1aR, 9576±399 vs. E1,2(‐108T),3‐AT1aR, 12,440±292; p<0.05, n=2]; however, the rate of Ang II‐induced AT1aR internalization did not differ between the two constructs. Ang II (10 nM) stimulation rapidly increased ERK1/2 activation with peak activity occurring between 5–10 min in HEK cells expressing both the disrupted and intact sORF. ERK1/2 activity rapidly declined between 10–40 min in cells expressing the intact sORF. In contrast, disruption of the sORF led to sustained ERK1/2 activation up to at least 40 min. In addition, a dose response curve indicates that disruption of this sORF increases ERK1/2 signaling induced by Sar1, Ile4, Ile8‐Ang II even when normalized to AT1aR Bmax determined by radioligand binding. This AT1aR biased agonist selectively activates the β‐arrestin‐ERK1/2 signaling cascade. Thus, this study supports previous findings that a seven amino acid peptide (PEP7) encoded by this sORF is a selective inhibitor of AT1R‐mediated ERK1/2 activation. PEP7, therefore, may serve as a valuable tool in dissecting the physiological and pathophysiological role of β‐arrestin‐dependent and ‐independent AT1R signaling cascades.Support or Funding InformationFunding agency & grant #: NIH/NHLBI 1R01‐HL121456 to KSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Beta‐Adrenergic Receptor Signaling in Distal Convoluted Tubules
Authors: Cheng Lei , Wu Qi , Fenton Robert A. 
Published: June 2021

The sympathetic nervous system (SNS) plays a central role in blood pressure regulation. Recent studies have shown that cells of the distal convoluted tubule (DCT) can be stimulated directly via the beta‐adrenergic receptor resulting in activation of the NaCl cotransporter NCC. Whether these effects are mediated by the beta1‐ or beta2‐adrenergic receptor is unclear, and the signaling cascades rapidly activated by beta‐adrenergic receptor stimulation in the DCT are unknown. RT‐PCR identified alpha1B‐, alpha1D‐ and beta2‐adrenergic receptors, but no beta1 receptors, in mpkDCT cells (a model of the DCT). Treatment of mpkDCT cells with the short‐acting beta2‐adrenergic receptor agonist salbutamol (1μM) for 15 min resulted in significantly increased cAMP levels, and phosphorylation of VASP at a PKA‐dependent site. Global changes in signaling networks following 15 min of salbutamol treatment of mpkDCT cells were assessed using stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics. A total of 43773 unique phosphopeptides containing 13599 confidently assigned phosphorylation sites were identified, 9209 of which were suitable for additional statistical analysis. Salbutamol increased phosphorylation at 81 sites; with kinases including PAKB, CLK, p70S6K and PKA predicted to be responsible. 71 sites, including several targets for CDK and GSK3 kinase groups, were decreased in phosphorylation. The predicted kinases fit well with motif analysis, where sites increased in phosphorylation were basophilic (RXXS), whereas the downregulated motif (SXXXS) is predicted to be a target for GSK3. Pathway analysis predicted that CREB and AKT are also important pathway nodes altered by salbutamol. Phosphorylation of the NCC‐modulating kinase OSR1 was increased at S339, a site previously shown to be regulated by PI3K‐mediated signaling. In summary, beta2‐adrenergic receptor signaling in mpkDCT cells involves PI3K and cAMP‐mediated pathways. Incorporation of our data into known signaling networks may provide novel clues linking the sympathetic nervous system, DCT regulation and NCC activity.Support or Funding InformationFunding is provided by the Novo Nordisk Foundation, the Lundbeck Foundation, Marie Skłodowska‐Curie Individual Fellowship and the Danish Medical Research Council.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐fat diet augments muscle atrophy by changes of muscle atrophy‐related genes in mice
Authors: Goto Ayumi , Tsuzuki Takamasa , Yoshihara Toshinori , Furuichi Shinya , Tsukioka Kei , Kakigi Ryo , Naito Hisashi 
Published: June 2021

IntroductionRecently, it has been reported that obesity accelerates muscle atrophy and protein degradation induced by 14‐days denervation in mice. Muscle atrophy primarily results from accelerated protein degradation and is associated with increased expression of two muscle‐specific ubiquitin ligases: MuRF1 and atrogin‐1. PGC1α is the master transcription regulator that stimulates mitochondrial biogenesis, and expression level of PGC1α was reduced in obesity and muscle atrophy. However, the expression level of these genes in response to muscle atrophy on obesity is still unclear. The purpose of this study was to investigate the expression level of MuRF1, atrogin‐1 and PGC1α during muscle atrophy in mice fed a high‐fat diet.MethodsThis study was approved by the Juntendo University Animal Care Committee (H28‐14). Fifty‐two male C57BL/6J mice (6 weeks old) were randomly divided into two groups: 1) normal diet (ND, n=26), 2) high‐fat diet (HFD, n=26). HFD were fed a high‐fat diet (60% calories from fat) for 16 weeks, while ND received a chow diet (10% calories from fat). Following 16 weeks of each experimental diet intake, all mice were lightly anaesthetized with the inhalant isoflurane and one hindlimb was immobilized in the plantar flexion position with casting tape. After immobilization for 1, 3, and 7 days, the soleus and plantaris muscles were removed. Statistical significance was analyzed by using two‐way (diet and time) analysis of variance (ANOVA). Statistical significant level was set at P < 0.05.ResultsBody weight and epididymal fat pad mass were significantly higher in the HFD group compared with the ND group. In the 7 days, the HFD significantly augmented loss of mass rate as compared with ND. The expression level of MuRF1 mRNA 3d in soleus and plantaris muscles was significantly higher in the HFD group compared with the ND group. The expression level of atrogin‐1 mRNA was significantly increased in response to immobilization, but no changes in ND and HFD. The expression level of PGC1α mRNA was significantly reduced in response to immobilization and was lower in HFD compared with ND in soleus and plantaris muscle.ConclusionHFD augments muscle atrophy by immobilization, at least in part by increasing the expression level of MuRF1 mRNA.Support or Funding InformationThis study was supported in part by Japan Society for the Promotion of Science KAKENHI Grant (16H07182), President's Grant for Interfaculty Collaboration, Juntendo University (Goto A: K1613, K1708) and MEXT‐Supported Program for the Strategic Research Foundation at Private Universities.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.