bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative measurement of pH dynamics in living cells using the mCherry‐TYG red fluorescent protein as a lifetime sensor
Authors: Haynes Emily Patten , Tantama Mathew
Published: June 2021

pH regulation plays a crucial role in mammalian and bacterial cell metabolism, protein function, apoptotic signaling, and a wide range of other physiological processes. Monitoring intracellular pH after environmental perturbations would further knowledge about cellular activities in both healthy and disease states in mammalian cells, as well as mechanism of infection and antibiotic resistance in bacterial cells. Non‐optical techniques such as x‐ray crystallography and NMR cannot resolve live‐cell dynamics, and many fluorescent dye or protein‐based sensors are susceptible to concentration and intensity dependence of the fluorophore, sample thickness, and photo‐bleaching, making lab‐to‐lab translation difficult without extensive calibration. Therefore, there remains a need for options to quantitatively measure pH dynamics in live specimens. As an intrinsic property of fluorophores, fluorescence lifetime does not rely on protein concentration, method of measurement or fluorescence intensity. The utilization of FRET pairs in fluorescence lifetime imaging microscopy (FLIM), has shown applicability of lifetime measurement for dynamic quantitation, however dynamic range can be limited. Here we present in vitro and live‐cell characterization of a red monomeric pH sensitive mCherry mutant mCherry‐TYG for robust measurement of live bacterial and mammalian pH in real‐time. We were successfully able to translate lifetime measurements with ease, showing pH calibration of both isolated protein and bacterial suspensions overlaying with a 2 ns dynamic range between pH 5.5 and 9.0. We further tested applicability in monitoring environmental perturbations by quantifying changes in pH as a function of energy source in e. coli. Additionally, we are currently exploring application for monitoring receptor trafficking, endocytosis and degradation in mammalian cells. Our results demonstrate the possibility of red shifted protein‐based lifetime sensors as a robust tool for pH calibration in live cells.

Support or Funding Information

NIH/NINDS R21 NS092010, R21 NS106319

NIH/NEI R21 EY026425

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deciphering the mechanism of action of cholesterol analogs as inhibitors of Mycobacterium tuberculosis
Authors: Carrillo‐Alvarado Miguel Angel , Pardo Andrew , Alsaihati Laila , Noveron Juan C , Ouellet Hugues
Published: June 2021

Drug‐resistance continues to be a public health problem, with nearly half million people infected by multidrug‐resistant strain of Mycobacterium tuberculosis (Mtb) worldwide, urging the need to develop new antitubercular drugs. Cholesterol plays a critical role in phagocytosis by host macrophages. Mtb also utilizes cholesterol as a source of carbon and energy during infection and degrades it through three catabolic pathways acting on AB‐ and CD‐rings, and on the aliphatic side‐chain. These pathways produce acetyl‐ and propionyl‐CoA intermediates, which are then incorporated into cellular lipids and virulence factors. Cholesterol analogs with degradation‐resistant alkyl side‐chains, including 3β,16β,26‐trihydroxy‐cholesterol (16OHC), have been recently shown to effectively inhibit Mtb growth, but the mechanism of action remains unclear. In this study, we combined the use of synthetic cholesterol analogs with genetic and mass spectrometry approaches to demonstrate whether cholesterol side‐chain catabolic activities could be used as potential drug targets. Metabolomic profiling of Mtb wild‐type and mutant strains with decreased cholesterol side‐chain degrading activities fed with 16OHC revealed that this cholesterol analog is metabolized by all strains, except by Dcyp125 cells, to produce a metabolite that we identified as 3‐oxo‐chola‐1,4‐dien‐16β‐ol‐24‐oate methyl ester. Unexpectedly, our data showed that 16OHC is not an inhibitor of the cholesterol side‐chain degradation pathway, but rather an inhibitor of the AB‐ring degradation pathway. The inhibitory effect of 16OHC on selected recombinant Mtb AB‐ring degradation enzymes is being tested and data will be presented.

Support or Funding Information

Financial support from the Ralph & Kathleen Ponce de Leon Endowment at UTEP, NIH BBRC 5G12MD007592 and SC1 AI116567‐01A1 (NIH/NIAID/NIGMS) are gratefully acknowledged.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SOD2 Overexpression Affects Mouse Hepatic OXPHOS Complex Integrity
Authors: Harris Tyler J , Steyls Samantha , Christian Brooke
Published: June 2021

Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, are produced as byproducts of cellular respiration and can damage DNA, proteins, and lipids within a cell. To combat the production of superoxide, cells express superoxide dismutase 2 (SOD2), an antioxidant enzyme which converts superoxide into hydrogen peroxide. Mice that overexpress SOD2 were used to examine tissue‐specific ROS‐responsive genes. Microarray data indicates a disruption in genes encoding proteins that function in oxidative phosphorylation in SOD2 mice. BN‐PAGE analysis confirms the defect, but kinetic analysis of complexes 2 and 5 show no significant difference. We propose that the overexpression of SOD2 may destabilize assembled OXPHOS complexes, rendering them more sensitive to detergent. Current research is being conducted to test the stability of SOD2 oxidative phosphorylation complexes against oxidative stress, as well during aging.

Support or Funding Information

ASU Office of Student Research

ASU Department of Chemistry

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
4D cell biology: Big data image analytics and lattice light‐sheet imaging reveal dynamics of clathrin‐mediated endocytosis in stem cell‐derived intestinal organoids
Authors: Schöneberg Johannes , Dambournet Daphné , Liu Tsung‐Li , Forster Ryan , Hockemeyer Dirk , Betzig Eric , Drubin David
Published: June 2021

New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue‐scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high‐content imaging data. We expressed fluorescent protein fusions of clathrin and dyna‐ min2 at endogenous levels in genome‐edited human embryonic stem cells, which were differentiated into hESC‐derived intestinal epithelial organoids. Lattice light‐sheet imaging with adaptive optics (AO‐LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 μm xyz) at 5.7 s/frame. We developed an open‐source data analysis package termed pyLattice to process the resulting large (□60 GB) movie data sets and to track clathrin‐mediated endocytosis (CME) events. CME tracks could be recorded from □35 cells at a time, resulting in □4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO‐LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues.

Support or Funding Information

J.S. acknowledges a Moore/Sloan Data Science Fellowship from the Berkeley Institute for Data Science and a Siebel Fellowship from the Siebel Stem Cell Institute–UC Berkeley. This work was supported by National Institutes of Health (NIH) Grant R35GM118149 to D.G.D. D.H. is a Pew–Stewart Scholar for Cancer Research supported by the Pew Charitable Trusts and the Alexander and Margaret Stewart Trust. D.H. is supported by the Siebel Stem Cell Institute and NIH R01‐CA196884.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Short Variant of Mitochondrial Dynamin OPA1 Renders Improved Cell Survival under Stress Conditions
Authors: Lee Hakjoo , Yoon Yisang
Published: June 2021

The protein optic atrophy 1 (OPA1) is a dynamin‐related membrane remodeling protein associated with the inner mitochondrial membrane. OPA1 is a critical factor for mitochondrial structure and function by mediating mitochondrial inner membrane fusion and maintaining cristae structure. Under cellular stress associated with mitochondrial dysfunction, the inner membrane‐anchored long OPA1 (L‐OPA1) undergoes proteolytic cleavage to form short soluble OPA1 (S‐OPA1) lacking the transmembrane domain. The prevalent thought has been that S‐OPA1 is a functionally insignificant proteolytic product of L‐OPA1. Hence, the loss of L‐OPA1 and accumulation of S‐OPA1 by OPA1 cleavage under stress conditions is thought to be detrimental to cell survival. However, we recently demonstrated that S‐OPA1 is fully competent for maintaining energetics and cristae structure. Therefore, in this study, we used cells exclusively expressing L‐, S‐OPA1, or both, and tested the capacity of L‐ and S‐OPA1 in supporting cell survival under stress conditions. Under hydrogen peroxide‐mediated oxidant stress, we found that S‐OPA1‐expressing cells have better survival than L‐OPA1 cells. Interestingly, this difference in cell survival under oxidant stress was only observed in glucose‐free OXPHOS media but not in the presence of glucose, suggesting that S‐OPA1 may confer higher mitochondrial energetic activity compared with L‐OPA1. We also observed better survival of S‐OPA1‐expressing cells under nutrient starvation. Under apoptotic stress by treating cells with the transcription inhibitor actinomycin D, S‐OPA1 cells showed greatly decreased cytochrome c release compared to L‐OPA1 cells. These observations suggest that S‐OPA1 is more protective during apoptosis than L‐OPA1. Mechanistically, we found that S‐OPA1 cells have higher mitochondrial electron transport activity in nutrient depletion. Furthermore, by testing mitochondrial calcium retention capacity, we found that S‐OPA1 cells show increased resistance to mitochondrial permeability transition induced by mitochondrial calcium overload, compared with L‐OPA1 only cells. These data indicate that S‐OPA1 confers more efficient energy metabolism and more resistance to mitochondrial stress, allowing prolonged cell survival under stress.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Redox Regulation of TRPML1 Channel Activity and Lysosome Trafficking in Podocytes
Authors: Li Guangbi , Li Pin‐Lan
Published: June 2021

Recent studies have indicated that redox signaling is importantly involved in the control of exosome secretion from a variety of cells. However, the mechanism by which reactive oxygen species (ROS) regulates exosome release remains unknown. In the present study, we hypothesized that ROS attenuate TRPML1 channel‐mediated Ca2+ release in podocytes through inhibition of acid ceramidase (AC) and thereby reduce lysosome trafficking, leading to prolongation of MVB fate to promote exosome secretion. Using GCaMP3 Ca2+ imaging technique, it was found that ML‐SA1‐induced Ca2+ release from lysosomes in podocytes was almost completely blocked by H2O2. However, sphingosine as an AC product of ceramide hydrolysis increased TRPML1 channel‐mediated Ca2+ release from lysosome of podocytes, which was not prevented by pre‐treatment with H2O2. Given the fact that Hcy induces NADPH oxidase‐derived ROS production in podocytes, we tested whether Hcy‐induced endogenous ROS production inhibits TRPML1 channel activity in these cells. It was found that pre‐treatment of podocytes with Hcy for 24 hours completely blocked TRPML1 channel‐mediated Ca2+ release. In the presence of catalase, Hcy‐induced blockade of lysosomal TRPML1 channel activation was prevented. Moreover, inhibition of NADPH oxidase by gp91 ds‐tat peptide or diphenyleneiodonium also abolished the inhibitory effects of Hcy on ML‐SA1‐induced TRPML1 channel‐mediated Ca2+ release. By confocal microscopy, we observed that ML‐SA1 enhanced the lysosome‐MVB interaction in podocytes, which was diminished by pre‐treatment with H2O2. However, when podocytes were pretreated with sphingosine, ML‐SA1 remained to enhance lysosome‐MVB interaction in the presence or absence of H2O2. Functionally, ML‐SA1 was found to enhance lysosome trafficking in podocytes towards the perinuclear region, which was prevented by pre‐treatment of these cells with H2O2. From these results, we conclude that both exogenous and endogenous ROS inhibit TRPML1 channel activity in lysosome of podocytes through inhibition of AC, which may cause derangement of lysosome trafficking and lysosome‐MVB interaction, leading to increased exosome secretion.

Support or Funding Information

This study was supported by NIH grants DK54927, DK120491, and HL057244.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Overexpression of a Novel E3 Ligase leads to a Skeletal Muscle Myopathy through Alterations in Cytoskeleton Proteins and Enhanced Protein Degradation
Authors: Hughes David C. , Baehr Leslie M. , Driscoll Julia , Waddell David S. , Bodine Sue C
Published: June 2021

Multiple E3 ubiquitin ligases have been identified to be transcriptionally increased in skeletal muscle under atrophy‐inducing conditions. We have recently observed a novel E3 ligase, F‐box and Leucine‐rich protein 22 (FbxL22), to be elevated during denervation‐induced muscle atrophy. Little is known about the role of FbxL22 in skeletal muscle, with recent research highlighting its impact on cardiac muscle function and sarcomeric protein turnover (Spaich et al., 2012).

Subsequently, male C57BL/6 mice (12‐week old) were subjected to in vivo electroporation with a mouse specific FbxL22 plasmid (20ug) in the tibialis anterior (TA) muscle with the contralateral leg serving as an empty vector control (pcDNA3.1). After 14 days, TA muscles were sectioned for H&E stain and GFP visualisation to confirm plasmid incorporation. Muscle mass and cross‐sectional area (CSA) were determined. Western blotting was performed to determine intermediate filament, autophagy and poly‐ubiquitin protein levels in the transfected TA muscles.

Interestingly, we observed no difference in muscle mass after 14 days, but we did observe increased numbers of large and small fibers in the FbxL22 transfected muscle. At the protein level, there was a 75% reduction in alpha‐actinin‐2, which was accompanied by 4‐fold increase in desmin and vimentin. We additionally observed a 2‐fold increase in polyubquitination, and 1.5‐fold increase in autophagy markers (p62 and LCB II).

Our data highlights the powerful impact of FbxL22 on skeletal muscle structure. Future studies will seek to investigate the chronic effect of FbxL22 on skeletal muscle and characterize its role during the process of muscle atrophy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mTOR is a Mechanistic Target of Muscle and Cancer Cross‐Talk with Exercise
Authors: Goodenough Chelsea G , Davis Amanda R , Deaver J William , Riechman Steven E , Fluckey James D
Published: June 2021

INTRODUCTION

The mTOR signaling pathway regulates growth and cellular proliferation, which is vital for normal cell function. However, when this pathway is dysregulated, such as in cancer, it can lead to hyperactivation of signals which promote cell proliferation and protect against cell death. DEPTOR, a potent endogenous mTOR inhibitor, generally has low expression in many cancers. We have unique preliminary findings that myokines released during electrically‐stimulated muscle contraction of hemicorpus‐hindlimb perfused rats affect mTOR signaling and DEPTOR protein content in breast cancer cells. When MCF‐7 cancer cells were treated with perfusate collected during muscle contraction, a significant inhibition of proliferation was noted alongside diminished mTOR activity and elevated DEPTOR expression.

PURPOSE

To investigate changes in anabolic signaling and resultant protein synthesis rates in MCF7 human breast cancer cells exposed to pharmacologically administered mTOR inhibitors.

METHODS

MCF7 cells were cultured in 10cm plates until 60% confluence in standard Growth Media (GM). Cells were treated with either Rapamycin or Torin1 inhibitors (100nM or 250nM, respectively) in 10 ml of GM. Cells for protein synthesis analysis were additionally treated with heavy water (2H2O) to yield a 4% final volume. All groups were incubated in respective media for 24hrs and prepped for western blot or rates of protein synthesis analysis.

RESULTS

DEPTOR protein expression increased in both rapamycin and Torin1 treated cells (2‐fold & 7‐fold, respectively). Both Rapamycin and Torin 1 treated cells show decreased daily rates of protein synthesis compared to cell control (14.41, 9.09 vs 20.46 %/d, respectively).

CONCLUSION

Our results suggest that diminished DEPTOR protein content and concomitant mTOR hyperactivity are important contributors to MCF7 cellular proliferation, in culture. Pharmacological administration of mTOR inhibitors suppresses protein synthesis and consequent anabolic signaling, with a rescue of DEPTOR protein content, which, in part, is consistent with our previous findings using exercise perfusate.

Support or Funding Information

This work was funded in part by the College of Health and Education and the Huffines Institute for Sports Medicine at Texas A&M University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biostimulating Effect of 685 nm Low‐Intensity Laser Radiation on Cell Viability in Multicellular Spheroids Culture
Authors: Gastaldi Gabriela Gomes Cardoso , de Oliveira Juliana Paula , de Andréa Dernowsek Janaína , Rezende Rodrigo Alvarenga , da Silva Jorge Vicente Lopes , Parizotto Nivaldo Antônio , Amaral Creusa Sayuri Tahara , Amaral André Capaldo
Published: June 2021

Recent research evidences substantial morphological and physiological differences between cells maintained in vitro in two‐dimensional (2D) and three‐dimensional (3D) culture conditions. The current consensus is that 3D culture is better because it precisely mimics the cellular microenvironment in vivo. The biomodulatory effect of low‐intensity laser radiation (LILR) on cells in 2D cultures is already well established. On the other hand, the effect of this radiation on cells in 3D cultures has not yet been widely investigated. The aim of this work was to analyze the biomodulatory effect of LILR, at the wavelength (ƛ) of 685 nm, on the viability of osteogenic precursor cells (OPC) cultured as multicellular spheroids (MS). Agarose molds containning wells (300 μm diameter) were used for MS formation using the non‐adherent surface culture concept. A cell count (2×105) of the OPC lineage (MC3T3) was seeded in each mold and maintained under ideal growth conditions (Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum/37°C/95% humidified atmospheric air/5% CO2). The molds were irradiated for 5 consecutive days at doses of 0.5, 1.0 or 1.5 J/cm2 (triplicates), and the first irradiation was performed immediately after seeding. After this period, the cultures were submitted to cell viability assay and the results were expressed as a percentage of non‐irradiated control cultures (ANOVA ONE‐WAY, p≤0.05). The results showed that LILR, at ƛ 685 nm, provided a 79% increase in cell viability at the dose of 1J/cm2. The remaining doses exhibited similar viability rates that the control. These results confirm the dose‐dependent biostimulatory effect of LILR at ƛ 685 nm on MS culture and contribute to the understanding of the real influence of this radiation on cells under ideal conditions for in vitro simulation studies.

Support or Funding Information

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ CAPES ‐ Brazil

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transcriptional Upregulation of CaMKIIδ through the JAK/STAT3 Pathway is Necessary for the IL‐6‐Dependent Increase in Endothelial Cell Migration
Authors: O'Brien Brendan Joseph , Singer Harold , Adam Alejandro , Ginnan Roman
Published: June 2021

Multifunctional Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is a well‐characterized family of proteins comprised of four separate isoforms: α, β, δ, and γ. These isoforms are differentially expressed in a tissue‐specific manner and contribute to diverse physiological and pathophysiological conditions. However, little is known regarding CaMKII isoform expression and function in the endothelium. Previous data from our lab has shown that CaMKIIδ6 is the primary isoform expressed in human umbilical vein endothelial cells (HUVECs) and promote thrombin‐induced loss of barrier function. Interleukin 6 (IL‐6) is a proinflammatory cytokine that induces loss of barrier function in HUVECs through a JAK/STAT3 signaling cascade. The purpose of this study was to test if CaMKIIδ mediates IL‐6‐induced increase in endothelial barrier permeability. Using qPCR, we determined that CaMKIIδ is the most abundant isoform of the enzyme present in HUVECs. Upon treatment with IL‐6, significant increases in both CaMKIIδ transcription and protein expression were observed. Treatment of HUVECs with JAK inhibitor ruxolitinib and siRNA for STAT3 decreased CaMKIIδ expression. However, adenoviral overexpression of various mutant CaMKII isoforms or siRNA knock down of CaMKIIδ did not affect IL‐6‐dependent loss of barrier function as measured by electric cell‐substrate impedence sensing (ECIS). It has been reported that IL‐6 signaling not only induces a loss of endothelial barrier function, but also increases endothelial cell migration. Indeed, siRNA knock down of CaMKIIδ attenuated IL‐6‐induced endothelial cell migration in a scratch‐wound assay. For the first time, our findings thus far have identified transcriptional induction of CaMKIIδ by IL‐6 that is necessary for the subsequent increase in endothelial cell migration.

Support or Funding Information

RO1HL49426‐21 to Harold A. Singer

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Polarized Expression of Kir7.1 Channels in a 3D Organoid Culture Model
Authors: Beverley Katie , Shahi Pawan , Pattnaik Bikash
Published: June 2021

Background

Leber Congenital Amaurosis (LCA) is an autosomal recessive genetic condition of the retina that leads to blindness in infants and young children. Previous data indicate that mutations in Kir7.1, an inwardly rectifying potassium channel, located in the retinal pigmented epithelium apical membrane contributes to a LCA blindness subtype. The specific localization of the Kir7.1 in the polarized RPE cell is critical as it contributes to retina function. Using standard cell culture models it is impossible to reproduce Kir7.1 distribution, molecular interaction and hence function. Culturing cells in 3D‐organoids will likely provide an appropriate in vitro system to study Kir7.1 disease mechanism and develop therapeutics.

Hypothesis

We hypothesize that Kir7.1 protein will have a polarized distribution in differentiated Mardin Darby Canine Kidney (MDCK) 3D Cell Culture Model that allows us to study channel biology.

Methods

We isolated mRNA from cultured MDCK cells that were either transfected with Kir7.1 or non‐transfected. We then utilized PCR and ran the PCR products on an agarose gel. We have optimized a 3D cell culture model of MDCK cells. About 3×104 cells/ml were grown in a collagen cocktail (24 mM glutamine, 2.8 mM NaHCO3, 1X MEM alpha, 20 mM HEPES, 2 mg/ml Collagen I) for 5 days in a compartmentalized chamber coverslip. On Day 5 or when organoids were noticed, we transduced with GFP Kir7.1 through lentiviral particles (viral titer 3.54×109) 500 MOI. Approximately one week after transduction, we stained the organoids with Hoescht and WGA‐Alexa 594 for nucleus and membrane staining respectively. Using Nikon C2‐confocal microscope, z‐stack images were acquired and subjected to off‐line analysis.

Results

Our PCR results indicate that MDCK cells do not natively express Kir7.1 as it was only expressed in the transfected cells. After 5 days in culture, we began to observe well developed spherical organization of mature MDCK cells. A single layer of Hoescht positive cells could be visualized. Kir7.1 expression was mostly detected by GFP positive signal in the membrane lining the inner surface of the sphere. WGA‐Alexa 594, however, lined the outer membrane of the organoids.

Conclusion

The development of the MDCK 3D cell culture model allows for the study of polarized epithelium. Kir7.1 localization to the inner membrane of the organoid confirms that the basolateral membrane localization in MDCK cells corresponds with apical distribution in the RPE cells. Our data also indicates that MDCK cells are a good model for studying Kir7.1 because they do not natively express the protein. The experimental set up is thus robust and time efficient to study Kir7.1 channel biology in its most native setting.

Support or Funding Information

NIH T32HD041921 NIH EY024995

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Co‐localization of Hepatocellular Uptake Transporters in Detergent Resistant Membranes (Lipid Rafts)
Authors: Steiert Brianna , Idowu Jessica Y. , Dougherty Larissa , Hagenbuch Bruno
Published: June 2021

Background

We have previously shown that several transporters located in the basolateral membrane of human hepatocytes (OCT1, NTCP, OATP1B1, and OATP1B3) interact with each other. These interactions affect both surface expression and function. Currently, transporter studies are performed using cell lines that express a single transporter at a time without considering how transporter interactions will affect function. Determining how transporters co‐localize, e.g. in detergent resistant lipid rafts or via direct protein‐protein interactions, has far‐reaching implications for drug metabolism and pharmacological studies.

Aim

The purpose of this study is to test the hypothesis that these transporters co‐localize with well‐known lipid raft marker proteins.

Methods

Following homogenization in 1% Triton X‐100 on ice, lipid rafts were isolated from HEK293 cells expressing NTCP, OCT1, OATP1B1, and OATP1B3, or from frozen human liver samples. Homogenates in 40% sucrose were overlaid with a discontinuous 5% to 35% sucrose gradient. After centrifugation for 18 hours at 197,000 × gav, gradients were fractionated, and aliquots were analyzed using western blotting. The blots were probed with antibodies against positive (caveolin and Na+/K+ ATPase) and negative (clathrin) lipid raft markers, as well as with transporter‐specific antibodies.

Results

In human liver, caveolin, Na+/K+ ATPase, OATP1B1, OATP1B3, and OCT1 were detected in similar fractions. OCT1 also was found in clathrin‐positive fractions towards the bottom of the gradient. In HEK293 cells, NTCP and OCT1 co‐localized with caveolin in the middle of the gradient and again, OCT1 was also found towards the bottom of the gradient.

Conclusion

The detection of NTCP, OCT1, OATP1B1, and OATP1B3 along with the positive lipid raft markers caveolin and Na+/K+ ATPase indicates that co‐localization of these transporters occurs, at least in part, because of their residence in detergent resistant membrane domains. Future experiments are aimed at identifying the functional consequences of this co‐localization.

Support or Funding Information

P20 GM103549, P30 GM118247, and GM077336

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Breast Cancer in African American versus Caucasian Women: Difference in Gene Expression
Authors: Sumaida Dena , Hammamieh Rasha , Miller Stacy‐Ann , Day Agnes , Jett Marti
Published: June 2021

Breast cancer in African‐American (AA) women is more advanced and difficult to treat than in Caucasian women in the United States, but the reasons for theses difference are not yet known. A disparity in breast carcinoma survival between African‐American and Caucasian women has been noted over the past several decades. AA women have more advanced breast cancer at the time of diagnosis than Caucasian women. In addition AA women tend to have breast cancer tumor types that are more aggressive and have poorer prognosis. African American women, regardless of age were more likely to have tumor grade III or IV with fewer grade I and II tumors stages combined and for each individual stage group. The disparities observed in incidence trends and age at diagnosis, particularly according to race, highlight the need for further investigation of the differences between minority and the Caucasian populations. Cell lines were selected from both populations for both pre and post menopausal conditions and their gene profiles were compared using cDNA microarrays. A combined data filtering with statistical analysis revealed a set of genes that were able to distinguish between AA and Caucasian breast cancer cells. These genes are involved in a various pathways involved in cell proliferation and apoptosis as well as transcription and transportation.
Expression of Caveolin‐1 in Normal and Oviduct Pregnant Tissue
Authors: Wang Lin , Wang Qianhua
Published: June 2021

Objective To detect the expression of caveolin‐1 in normal and tubal pregnant tissue of human and explore its clinic significance.

Methods 20 fresh clinical operative tubal tissue specimens were collected from the women who were diagnosed oviduct pregnancy. 20 fresh normal oviduct tissues were collected from the women who were diagnosed uterine benign tumors and they were divided into two groups: the proliferative phase and secretory phase according to the morphological characters. The expression of caveolin‐1 was detected by immunohistochemical method and analyzed by image analysis technique.

Results The positive expression of caveolin‐1 in the normal human oviduct tissue of secretory phase was stronger than that of proliferative phase. And the positive expression of caveolin‐1 in tubal pregnant tissue was lower than that in the normal human oviduct tissue of secretory phase. The differences were significant (P<0.01). The positive expression of caveolin‐1 can be detected in cytotrophoblast cell but not in syncytotrophoblast cell of villus.

Conclusion The lower expression of caveolin‐1 in the oviduct pregnant tissue may play an important role in the oviduct pregnancy.
Dynamic Conformational Changes of Calmodulin when Bound to Nitric Oxide Synthase using FRET
Authors: Spratt Donald E , Taiakina Valentina , Guillemette J Guy
Published: June 2021

Calmodulin (CaM) is a ubiquitous Ca2+‐sensor protein that binds and activates the Nitric Oxide Synthase (NOS) enzymes (E.C. ). Although the structure of CaM has been determined when bound to a peptide derived from the CaM‐binding domain of endothelial NOS, the conformation of CaM when bound to the three mammalian NOS holoenzymes has yet to be determined. The objective of this study is to determine the dynamic conformational changes of CaM when bound to all three NOS isoforms through the use of Förster Resonance Energy Transfer (FRET). We have produced a double cysteine CaM mutant (T34C/T110C) using site‐directed mutagenesis for the performance of FRET measurements. Far‐red shifted fluorescent dyes were employed so that they could be selectively excited in the presence of the NOS cofactors that absorb light strongly between 250 and 500 nm. FRET studies were performed in the presence of a range of physiologically relevant free Ca2+ concentrations for CaM binding to the constitutive NOS enzymes. These studies will provide a better understanding of the differences between the Ca2+‐dependent and Ca2+‐independent binding of CaM to the NOS isozymes. This work is supported by NSERC (J.G.G.).
A novel role of the conserved glutamate in aldehyde dehydrogenase catalysis
Authors: Tsybovsky Yaroslav , Krupenko Sergey A.
Published: June 2021

Aldehyde dehydrogenase (ALDH) catalysis requires the NAD(P)+ coenzyme and involves two steps, acylation and deacylation. The active site of every ALDH has two strictly conserved residues, Cys302 and Glu268, which are essential for catalysis. While the role of the cysteine as a catalytic nucleophile is well established, the function of the glutamate is ambiguous. It has been proposed that the glutamate either deprotonates Cys302 during the acylation step or activates a water molecule in the deacylation step. Our crystallographic studies, aimed at clarifying the role of the glutamate, revealed that the substitution of an alanine for this residue in the ALDH domain of rat 10‐formyltetrahydrofolate dehydrogenase traps NADP+ in the active site, rendering the enzyme inactive. In this mutant, the coenzyme is bound so tightly that it cannot be removed by conventional techniques such as dialysis or chromatography, as well as by back‐soaking of protein crystals. At the same time, the conformation of the coenzyme remains the same as in the wild‐type enzyme. Thus, the glutamate is apparently required to prevent irreversible binding of NADP+ and to provide coenzyme mobility that is necessary for ALDH catalysis.
Molecular and Functional Characterization of a Unique Secretory Nuclease [LdNucs] from the Human Protozoan Pathogen, Leishmania donovani
Authors: Dwyer Dennis M. , Owings Joshua P. , Joshi Manju B.
Published: June 2021

Leishmania are parasites of humans that reside/multiply as promastigotes within the gut of their sandfly vectors and as obligate intracellular amastigotes in the phago‐lysosomal system of macrophages. These organisms are strict purine auxotrophs and must salvage such essential nutrients from their hosts. Here we identified, characterized and episomally‐expressed a gene (LdNucs) encoding a unique 35‐kDa, DTT‐sensitive, Class‐I “secretory nuclease” from L. donovani. Our results showed that LdNucs mRNA and its encoded secretory nuclease activity were constitutively expressed by both parasite developmental forms. Western blot and immunoprecipitation (IP) assays showed that an anti‐ LdNucs‐peptide antibody recognized both the native and LdNucs‐expressed enzymes. In addition, coupled IP‐enzyme activity assays demonstrated that this enzyme could hydrolyze various synthetic polynucleotides as well as, both ss‐ and ds‐DNAs and RNA. Further, IP analyses showed that sera of visceral leishmaniasis patients from multiple endemic foci all recognized and immunoprecipitated the LdNucs enzyme. The latter indicated that LdNucs was in fact expressed by amastigotes during the course of human infections. Cumulatively, our observations suggest that this “secretory” nuclease functions to hydrolyze host‐derived nucleic acids to satisfy the essential purine requirements of these parasites. Thus, the LdNucs must play critical role(s) in the survival, growth and development of these important human pathogens. This study was supported by the Intramural Research Program of the DIR, NIAID, NIH.
Exploiting small molecule and siRNA libraries to identify novel mechanisms for potential cancer therapeutic agents.
Authors: Lazo John Stephen , McDonald Peter R. , Wipf Peter
Published: June 2021

Historically phenotypic assays have provided some the most interesting and potent pharmacological compounds. The advent of small interference RNA (siRNA) libraries directed against known and potential drug targets affords the opportunity to exploit automated, phenotypic, high‐content cell–based assays for the unbiased detection of agents with novel mechanisms of actions. We used a 16,560 member siRNA library directed against druggable gene products to identify the biochemical networks and nodes that sensitize tumor cells to compounds affecting microtubule dynamics. A T98G human glioma cell line was interrogated with a synthetic lethal screen. We identified 219 and 115 siRNAs that enhanced the cytotoxicity of the natural products vinblastine and disorazole C1, respectively, with 30 siRNAs being common to both agents. These results suggest the two tubulin polymerization inhibitors could have different mechanisms of action or binding sites. Suppression of elements of the PI3K‐AKT‐mTOR pathway by siRNA resulted in enhanced cytotoxicity of the inhibitors of tubulin polymerization. Pharmacological inhibitors of this pathway also acted synergistically with vinblastine or disorazole C1 to increase cytotoxicity. These results illustrate the power of combining siRNA libraries with automated phenotypic assays to uncloak previously unknown drug pathways and to identify potential novel drug combinations.
Partnering for Drug Discovery and Development
Authors: Zivin Robert A
Published: June 2021

The products of academic research are well represented in today's medicines, representing many Billions in sales and the cutting edge of therapeutics. While some notable examples emerged essentially “completed” from the labs of their inventors, the general case is the identification of a target, a pathway or a chemical biology lead. In these latter cases, many of these advances “wandered in the desert for years” before being “discovered” and incorporated into drugs. The NIH and other organizations have recognized this problem, and have directed significant funding into translational development. With the challenges facing the Pharmaceutical Industry today, it is critical we also develop strategies to rapidly recognize and incorporate these advances and develop the partnerships which will allow us to most effectively address today's unmet medical needs.
The non‐coding RNA gadd7 is a regulator of lipotoxic‐induced ROS and ER stress
Authors: Thomas Rita , Listenberger Laura L , Harp Jeff D , Ory Daniel S , Schaffer Jean E
Published: June 2021

To elucidate molecular events in the lipotoxicity pathway, we used retroviral promoter trap mutagenesis to generate a mutant Chinese hamster ovary (CHO) cell line resistant to palmitate‐induced cell death. In this mutant, insertion of the provirus has disrupted one allele of the non‐coding RNA (ncRNA) growth arrest DNA‐damage inducible gene 7 (gadd7), creating a model of haploinsufficiency. Knockdown of gadd7 in wild type (wt) cells also causes palmitate‐resistance. Because reactive oxygen species (ROS) induces gadd7 and palmitate generates ROS, we hypothesized that palmitate induces gadd7 expression in a ROS‐dependent manner. Palmitate treatment of wt cells induces gadd7 expression, but this is diminished by co‐treatment with an antioxidant. Although gadd7 haploinsufficient cells generate ROS in response to palmitate, induction is decreased compared to wt cells. These data suggest a feed forward loop between gadd7 and ROS generation. Since ROS generation contributes to palmitate‐induced ER stress, we assessed gadd7's role in palmitate‐induced ER stress. Induction of ER stress related genes is significantly reduced in palmitate treated gadd7 haploinsufficient and knockdown cells, indicating that gadd7 expression is necessary for palmitate‐induced ER stress. Together these data implicate a ncRNA in propagation of oxidative stress and in regulation of ER stress responses.

NIH F31 (RT) and DK064989 (JES)
Pulmonary surfactant phosphatidyl glycerol inhibits Mycoplasma pneumoniae stimulated eicosanoid production from human alveolar macrophages and mouse RAW264.7 macrophages
Authors: Kandasamy Pitchaimani , Zarini Simona , Ed Chan , Leslie Christina C , Murphy Robert C , Voelker Dennis R
Published: June 2021

Mycoplasma pneumoniae (Mpn) is a common etiologic agent of respiratory infection in children and adults that has been associated with serious exacerbations of asthma. Mpn induces cyclooxygenase‐2 (COX‐2) and the generation of eicosanoids from human alveolar macrophages (ThromboxaneB2, PGD2 and PGF2α) and RAW264.7 cells (PGD2 and PGE2). Inhibition of the phosphorylation of p42/44 MAP kinase, or the activity of cytosolic phospholipase A2α, completely abolished Mpn induced AA release from macrophages. The minor pulmonary surfactant phospholipid palmitoyl‐oleyl ‐phosphatidylglycerol (POPG) antagonized the proinflammatory actions of Mpn by blocking the release of eicosanoids from the cells. The action of POPG reduced the phosphorylation of p42/44 and the expression of COX‐2 induced by Mpn. The effects of POPG were structure dependent and not reproduced by dipalmitoyl‐PG. These data suggest that POPG can play an important role in suppressing pathogen induced inflammation in the lung.
Reductive Metabolism of Phospholipid Aldehydes in Macrophages Enhance their Pro‐Inflammatory Potential
Authors: Ozhegov Evgeny A. , Ahmed Yonis , Li Xiao‐Ping , Srivastava Sanjay , Bhatnagar Aruni , Barski Oleg A.
Published: June 2021

1‐palmitoyl 2‐oxovaleroyl phosphatidyl choline (POVPC) is one of the major products of phospholipid (PL) oxidation generated in atherosclerotic lesions and other sites of chronic inflammation. The aim of the present study was to examine how metabolism of POVPC by macrophages affects its pro‐inflammatory potential. Using ESI/MS analysis we found that POVPC was rapidly metabolized in the THP‐1 derived macrophages (THP‐1DM) via 2 major pathways: hydrolysis to 1‐palmitoyl‐2‐lysophosphatidylcholine (lyso‐PC) and reduction to 1‐palmitoyl‐2‐hydroxyvaleroyl phosphatidylcholine (PHVPC). When the THP‐1DM were incubated with PHVPC only the hydrolysis product lyso‐PC was detected. Inhibition of phospholipase A2 (PLA2) by Pefabloc led to a significant increase in the reduction of POVPC to PHVPC and diminished the rate of PHVPC metabolism. As determined by qRT‐PCR exposure to POVPC resulted in time‐ and concentration‐dependent increase in TNFα, IL‐8, IL‐1β, IL‐6 and MCP‐1 mRNA. PHVPC was 3–8 fold more potent than POVPC, whereas lyso‐PC was as active as POVPC. Pefabloc increased cytokine induction by POVPC and PHVPC consistent with increased accumulation of PHVPC upon inhibition of PLA2‐mediated hydrolysis. Treatment with POVPC decreased the expression of CD‐36 and SRA. Decrease in the scavenger receptor expression was accompanied by a decrease in lipid accumulation as measured by oil red O staining. PHVPC was more potent in suppressing scavenger receptor expression and preventing lipid uptake than POVPC. We suggest that reductive metabolism could enhance the inflammatory effects of PL aldehydes and that PLA2, by preventing reduction of oxidized PL, might have a protective role against inflammation and atherosclerotic lesion formation. Funded by NIH grants HL65618, ES11860 and HL55477.
NF‐kappa B, a key intermediate in inflammatory gene transcription, is activated by estrogen in the immature rat uterus
Authors: Trivedi Shreya , Caruso Joseph , Gardner Russell , Russo Louise
Published: June 2021

The immature rat uterus has been used extensively as a model system to study estrogen‐induced regulation of endometrial remodeling and matrix metalloproteinase (MMP) activation. During estrogen‐induced uterine growth, an inflammatory‐like response has been documented involving fluid and leukocyte infiltration into the endometrium. Previous studies have shown that inhibition of inflammatory responses involving prostaglandins via pre‐treatment of animals with dexamethasone, the anti‐inflammatory glucocorticoid, or indomethacin, a COX enzyme inhibitor, prevents estrogen‐induced matrix turnover in the endometrial stroma as assessed via transmission electron microscopy. However the mechanism whereby hormone receptor activation induces a change in prostaglandin levels and the link to MMP activation are unknown. This study investigated the role of the inflammatory transcription factor, Nuclear Factor Kappa B (NF‐κB) in estrogen induced matrix turnover. NF‐κB is a cornerstone of inflammatory responses and is known to upregulate expression and secretion of inflammatory mediators including prostaglandins. The data show that uterine NF‐κB is activated within ten minutes of E2 treatment. Furthermore, inhibition of NF‐κB signaling was shown to fully block degradation of the endometrial collagen matrix. Inhibition of prostaglandin synthesis via pre‐treatment with a COX‐2 specific enzyme inhibitor blocked NF‐κB activation and E2‐induced matrix turnover clearly indicating a critical role for NF‐κB signaling in hormone induced tissue remodeling.
Crystallization of PKA regulatory subunit from Saccharomyces cerevisiae
Authors: Rinaldi Jimena Julieta , Yang Jie , Rossi Silvia , Ganapathy Sarma , Moreno Silvia , Taylor Susan
Published: June 2021

In mammals, the PKA holoenzyme exists as a complex of two catalytic subunits and a regulatory (R) subunit dimer. R subunits have a dimerization and docking domain at the N terminus; at the C terminus, two tandem cAMP‐binding domains and in between a flexible hinge region, including a substrate‐like inhibitor sequence that docks to the active site cleft of the C subunit. Much effort has been put in studying structure‐function relationships in mammalian PKAs. The aim of this work is to solve the structure of the R subunit from S. cerevisiae in order to learn which of the features of mammalian R are general and which are specific to this system. We over‐expressed and purified different deletion mutants of the protein: Δ85, and Δ167. The over‐expression and stability of the proteins was assessed both in yeast and in bacterial expression systems. Δ167 was crystallized at 21ºC in sitting or hanging drop in 15–18% w/v PEG 3350, 0.1M Tris‐HCl pH 8.5. These crystals were transferred to a cryoprtectant solution Images of each crystal trial were manually taken at 0, 2, 7, 14, 21 and 28 days after setup. X‐ray diffraction data of single crystals were collected at Stanford Synchrotron Radiaton Laboratory (SSRL).
Opposite Regulation of Human D1‐like Dopaminergic Responsiveness by Protein Kinase C is Shaped by Two Critical Serine Residues of the Third Intracellular Loop of Human D5 Dopaminergic Receptor
Authors: Plouffe Bianca , Tiberi Mario
Published: June 2021

Phorbol‐12‐myristate‐13‐acetate (PMA), an activator of serine/threonine protein kinase C, promotes in a strikingly opposite fashion sensitization and desensitization of the highly homologous human dopamine D1 and D5 receptors (D1R, D5R), respectively. We identified the third intracellular loop (IL3) as the main structural determinant controlling the opposite PMA‐mediated regulation of D1R and D5R responsiveness using a chimeric approach. Single‐point mutations were made to delineate the residues of D5R‐IL3 region implicated in PMA‐induced D5R desensitization. Dopamine responsiveness of cells transfected with wild‐type or mutant forms of D5R following PMA treatment was assessed using whole cell cAMP assays. Our results show that serine residues 261 and 271 of D5R‐IL3 (corresponding to alanine 230 and asparagine 240 in D1R‐IL3) impart PMA‐induced desensitization to D5R. In fact, D5R mutants harboring single serine‐to‐alanine substitution of residue 261 or 271 display a sensitized responsiveness following PKC activation that was reminiscent of D1R sensitization observed in PMA‐treated cells. Overall, our work suggests that the presence or absence of serine residues located at positions 261 and 271 of D5R‐IL3 or positions 230 and 240 of D1R‐IL3 control the fate of PMA‐mediated regulation of D1‐like responsiveness. Supported by CIHR grant (MOP‐81341 to MT) and FRSQ Graduate Scholarship (to BP).