bluuebunny/crossref_metadata_2025 huggingface dataset:
ER Stress Induced Apoptosis of Smooth Muscle in Pulmonary Hypertension is Regulated by the Sodium‐Hydrogen Exchanger
Authors: Huetsch John , Yun Xin , Jiang Haiyang , Shimoda Larissa
Published: June 2021

Objective

Altering the apoptosis‐resistant phenotype of pulmonary arterial smooth muscle cells (PASMCs) may be key to reversing pathologic pulmonary vascular remodeling in pulmonary hypertension (PH). Endoplasmic reticulum (ER) stress and sodium‐hydrogen exchanger (NHE) activity have each been implicated in vascular remodeling in PH. We studied the effect of NHE activity upon ER stress‐induced apoptosis in PASMCs isolated from the SU5416‐hypoxia (SuHx) rat model of PH.

Methods

PH was induced in male Wistar rats via exposure to the vascular endothelial growth factor receptor inhibitor SU5416 plus 3 wk of 10% oxygen followed by 2 wk of normoxia (SuHx model), while control rats were exposed to vehicle and 5 wk of normoxia. PASMCs were isolated from SuHx and control rats and expanded in culture. Apoptosis was measured using Hoechst stain. Thioflavin fluorescence was quantified as a measure of ER stress. Protein levels of the ER stress‐induced transcription factor, CCAAT/enhancer‐binding protein homologous protein (CHOP), were measured via immunoblot. To measure NHE activity, PASMCs were incubated with pH‐sensitive fluorescent dye and the Na+‐dependent recovery of intracellular pH following acidification by ammonium pulse challenge was measured via fluorescence microscopy. Cell stress was induced with 250 μM H2O2. NHE was pharmacologically inhibited with 10 μM ethyl‐isopropyl amiloride.

Results

ER stress, a known stimulus of apoptosis, was increased by H2O2 in control PASMCs and was elevated at baseline in SuHx PASMCs. H2O2 exposure increased apoptosis in control PASMCs. Despite increased ER stress, SuHx PASMCs exhibited low levels of apoptosis both at baseline and in response to H2O2. CHOP protein expression, which is induced by ER stress and triggers the apoptotic cascade, was increased by H2O2 in control cells, but remained low in H2O2‐treated SuHx PASMCs. NHE activity was increased in PASMCs from SuHx rats, and pharmacologic NHE inhibition in SuHx PASMCs increased both basal and H2O2‐induced CHOP expression, and also increased apoptosis.

Conclusions

PH PASMCs are resistant to ER‐stress induced apoptosis due to decreased CHOP expression. NHE inhibition increases CHOP expression in PH PASMCs and restores ER‐stress induced apoptosis, suggesting a novel mechanism to control vascular remodeling in PH.

Support or Funding Information

NIH K08HL133475 & R01HL073859

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Zinc binding to Muscle LIM Protein regulates its structure and interaction with binding partners
Authors: Boateng Samuel , Atkins Jennifer , Hawanatu Sesay , Sorensen Thomas , McGuffin Liam
Published: June 2021

Muscle LIM protein (MLP) is a member of the cysteine‐rich protein family, localising predominantly to the Z‐disk of the myofilaments in cardiac myocytes. MLP is a mechanosensor and functions through its translocation to the nucleus from the myofilaments and through protein‐protein interactions in the cytoplasm. Mechanosensing enables the heart to adapt appropriately to alterations in hemodynamic load, whilst mutations in the MLP gene lead to cardiomyopathy and heart failure. MLP contains two LIM domains each consisting of a double zinc‐finger motif. Rather than interacting with DNA, LIM domains mediate protein‐protein interactions associated with cellular signalling.

The aim of this investigation is to determine how zinc binding regulates the structure of MLP and determine the impact on its interaction with binding partners. The analysis was performed using a combination of circular dichroism (CD), bioinformatics tools and online servers. CD is the difference in absorption between left‐handed and right‐handed circularly polarised light, occurring when a molecule contains light‐absorbing groups. It can be used to study the secondary, tertiary and quaternary structure of proteins, as well as studying its conformational stability under different conditions.

In this study, we used CD to determine structural changes to MLP in the presence and absence of zinc. MLP is predominantly helical (~36%) with a small percentage of beta‐strands of ~8%, 34% of unordered and 21% of turns. In the presence of excess zinc, the helical content was reduced to ~19% with a gain of beta‐strand component to 30%. The presence of EDTA caused MLP to lose some helical conformation to 22% with an increased in the beta‐strand content to 16%. These data suggest that zinc increases the structural folding and organization of MLP.

The full length wildtype MLP sequence was submitted to a variety of servers along with the C58G mutation known to reduce zinc binding in the first LIM domain. This mutation in humans leads to heart failure. Heart failure is defined as a progressive condition in which the heart is unable to pump enough blood to meet the body's needs for oxygen and nutrients. Bioinformatic protein‐protein analysis indicated that the C58G mutation inhibits interaction with NRAP, TCAP, alpha‐actinin and cofilin 2 which would interfere with MLP binding to the z‐disk and the myofilaments within cardiac myocytes. This would have a significant negative impact on mechanosensing in the heart and explains why the C58G mutation in MLP leads to heart failure.

Support or Funding Information

University of Reading and Diamond Light Source

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proliferation and osteogenic differentiation of human mesenchymal stem cells on cross‐linked polypeptide multilayer films
Authors: Yuan Chia‐Sui , Hung Yu‐Han , Yoneda Takashi , Cheng Yung‐Hsin , Lo Chun‐Min
Published: June 2021

Polyelectrolyte multilayer film is an emerging method for substrate coating to regulate cellular behaviors. This material allows for modulation of thickness, stiffness, and adhesiveness. In this study, human mesenchymal stem cells (hMSCs) were cultured on polypeptide multilayer films built up by the alternate deposition of cationic poly‐L‐lysine (PLL) and anionic poly‐L‐glutamic acid (PLGA), cross‐linked with EDC/sulfo‐NHS, and coated with type I collagen. The in‐vitro degradation of the cross‐linked PLL/PLGA films in the presence or absence of hMSCs were followed by fluorimetry after labeling of the outermost PLL layer with FITC. Cell proliferation was measured by CyQuant proliferation assay and osteogenic differentiation was evaluated by q‐PCR of osteogenic specific genes such as ALP, OCN, and OPN. The results showed that these cross‐linked films, with or without hMSCs on top of them, were quite stable in the culture medium and not subjected to degradation at least for a few weeks. While hMSCs maintained similar attachment and spreading behavior regardless of layer number (1–6 layers) of films, they demonstrated better proliferation rate on even number layer of films, where the top layers are PLGA films. In addition, compared with cells cultured on polystyrene culture wells and treated with osteogenic induction medium, hMSCs cultured on 6‐layer cross‐linked PLL/PLGA films showed a significant increase in the osteogenic differentiation rate. Our observations indicate a feasible way to manipulate stem cell behavior on polypeptide multilayer films by changing the number of layers, the outermost polypeptide layer, and the concentration of cross‐linkers.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulating the Effect of Electrostatic Steering for Protein‐Protein Interactions via Circular Permutation
Authors: Chen Xingyu , Gao Meng , Huang Yongqi
Published: June 2021

Assembly of protein complexes proceeds via nonspecific encounter complexes whose formation is facilitated by long range electrostatic interactions. Mutation of charged residues and change of salt concentration are two strategies widely applied to modulate the effect of electrostatic steering. Furthermore, electrostatic interactions between two proteins may be also influenced by their conformations. Consequently, the electrostatic steering effect during binding of an unfolded protein to its target may be different from that during binding of a folded protein to its target. In this study, we employed sequence circular permutation and molecular dynamics simulations to investigate the influence of protein conformation on the encounter process. We used the well‐studied barnase/barstar complex as a model system and the coarse‐grained structure‐based Gō‐model and Debye‐Hückel function to describe interactions. Binding simulations were performed at two different temperatures such that the WT and permutated barnase were folded at the lower temperature and they were unfolded at the higher temperature. The ensembles of encounter complex and the binding rate constants were analyzed for WT barnase as well as seven permutants. Our results showed that the ensembles of encounter complex of the unfolded barnase were much diverse than those of the folded barnase. We also found that circular permutation modified the structure ensemble of unfolded barnase, resulting in difference among their ensembles of encounter complex and binding rates. Our results suggest that the protein binding process can be engineered by modulating the protein conformation ensemble.

Support or Funding Information

This work was supported by National Natural Science Foundation of China (Grant number: 21603121) and Hubei University of Technology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of eNOS ser‐600 Phosphorylation: A Link to the Cell Cycle
Authors: Patel Kevin , Lecher Julia , Chrestensen Carol
Published: June 2021

Nitric oxide (NO) is a gaseous, free‐radical signaling molecule produced by nitric oxide synthases (NOS). NO has a variety of cellular and physiological functions ranging from maintaining cardiovascular homeostasis to neurotransmission. In low concentrations NO is deemed physiologically crucial and in high concentrations NO is understood to be pathophysiological. The role of NO in the cell cycle is less understood, it is likely cell type specific and concentration dependent, in many cases low levels of NO promote proliferation and prevent apoptosis; at high levels NO has been shown to arrest cells and cause apoptosis. Endothelial nitric oxide synthase (eNOS) is one of three isoforms in the NOS family, it is the most regulated of the isoforms. Regulations include protein‐protein interactions and posttranslational modifications. The most commonly studied and well understood posttranslational modification of eNOS is phosphorylation. This work builds on our laboratories discovery of the ser‐600 phosphorylation site in the auto‐inhibitory loop of eNOS. Our work confirms this site is modulated in cells and links it to the cell cycle using immunofluorescence, flow cytometry, western blot analysis, in vitro kinase assays, and in vitro eNOS activity assays.

Support or Funding Information

This work was supported by NIH grant # R15 GM110634‐01A1 to Dr. Carol Chrestensen.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect Of Loading Method On Metabolism Of Foreign Molecules In Intact Cells Of Dictyostelium Discoideum
Authors: Chadha Rahuljeet (RJ) , Kalminskii Grigorii , Tierney Allison J. , Kovarik Michelle L.
Published: June 2021

Loading foreign molecules inside living cells allows one to monitor cellular metabolism, track various metabolic pathways and alter activities of the cell. However, the effects of loading methods on the fate of these foreign molecules are poorly understood. In this experiment, we examined three promising loading methods including pinocytosis, electroporation and myristoylation to insert a peptide substrate reporter in cells of Dictyostelium discoideum. These methods were optimized to maximize cell viability, and factors such as loading efficiency, loading duration, localization and uniformity were characterized. Capillary electrophoresis was used to determine how each loading method affected the enzyme activity on the peptide substrate reporter. It was observed that these loading methods had strong effects on the stability and degradation of the peptide. Myristoylation was found to be the most effective method at transporting the peptide into cells while electroporation produced the most uniform loading within cells suggesting cytoplasmic loading. The peptide was degraded most rapidly after loading by myristoylation (half‐life = 12 ± 1 min), while pinocytic loading produced slightly slower degradation (half‐life = 19 ± 2 min). Phosphorylation of the peptide was only observed in cells loaded with electroporation. This experiment suggests that the choice of loading method influences the stability and metabolism of foreign molecules in cells. Our future work includes investigating the specificity of different peptide substrate reporters in Dictyostelium lysates.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Primary cilia on LECs play a crucial role in lymphatic vasculature development and remodeling
Authors: Paulson Delayna Marie , Leddy Rebecca , Ward Cody , Latterell Mackenzie , Peterson Chase , Ford Khalil , Ekle Courtney , Pazour Gregory , Fink Darci
Published: June 2021

Primary cilia are non‐motile organelles which project from the cell surface and function to receive and process molecular signals. We have recently discovered that lymphatic endothelial cells (LECs) display primary cilia in vitro and in vivo in both developmental and inflammation/wound healing contexts. We have induced primary cilia formation in both immortalized mouse and primary adult human LECs by serum starvation protocols and detected them by immunofluorescence microscopy for ciliary markers ADP ribosylation factor like GTPase 13B (ARL13B) and acetylated alpha tubulin. We have used the mouse corneal suture model to create in vivo conditions of inflammation and wound recovery and found that primary cilia incidence both in LECs and globally in cornea is dependent on inflammatory status. Primary cilia assembly and transport of ciliary cargo is governed by a set of protein complexes made up of intraflagellar transport (IFT) proteins. To ascertain the role of intraflagellar transport in lymphatic development, we developed a conditional knockout (KO) mouse model of IFT20. At the gross level, E16.5 IFT20 KO mice embryos show hydrops, polydactyly, and craniofacial abnormalities. These embryos also possess a malformed, red blood cell‐filled lymphatic vessel (LV) phenotype. Using two photon microscopy of whole mount embryonic skin, we have found the IFT20 KO embryos to have higher lymphatic vessel density and larger LV diameter in comparison to negative littermate controls. We have found that the difference in LV diameter is not due to increased LEC proliferation. Differences in dorsal skin lymphatic vessel patterning across the midline have also been identified in the IFT20 KO. We have also confirmed decreased incidence of primary cilia in IFT20 KO embryonic skin using spinning disk confocal microscopy. Ongoing studies in these embryos are designed to characterize differences in smooth muscle actin coverage of collecting LVs, intercellular LEC junction protein expression and organization, and lymphatic valve formation and possible insufficiency. We have also generated a lymphatic‐specific conditional deletion mouse model of IFT20 loss and are working to understand whether the LV phenotypes present in the global IFT20 KO mouse are due to LEC‐intrinsic defects or to secondary effects on lymphatic vasculature due to defects in other cell types following loss of IFT20. The discovery of primary cilia on LECs may fundamentally change what is known about how extracellular signals are processed by lymphatic endothelium to direct development and remodeling of the lymphatic vasculature.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Interaction between Regulator of G‐protein Signaling (RGS10) and STIM2 Identifies New Mechanism for the Regulation of COX‐2 in Microglia
Authors: Wendimu Menbere Y. , Alqinyah Mohammed , Freeman Paul , Vella Stephen , Moreno Silvia NJ , Hooks Shelley
Published: June 2021

Regulator of G‐protein Signaling 10 (RGS10) mediates strong anti‐inflammatory and neuroprotective effects. RGS10 is highly enriched in microglia where it suppresses the expression of many proinflammatory genes, including the key inflammatory enzyme cyclooxygenase 2 (COX‐2). The neuroprotective and anti‐inflammatory effects of RGS10 position it as a potential therapeutic target in neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. Our previous work demonstrated that microglial RGS10 strongly suppresses lipopolysaccharide (LPS)‐induced COX‐2 and tumor necrosis factor alpha (TNFα) expression, and this suppression is independent of its canonical G protein‐targeted mechanism. In this study, we further define the G protein‐independent anti‐inflammatory mechanism of RGS10 in microglia, focusing on the regulation of COX‐2. We hypothesized that novel binding partners link RGS10 to LPS‐stimulated inflammatory signaling pathways. Using an unbiased RGS10 co‐immunoprecipitation approach coupled with LC‐MS/MS, we identified the endoplasmic reticulum (ER)‐localized calcium sensor STIM2 as a major RGS10 interacting protein. STIM2 is an essential component of the store‐operated calcium entry (SOCE) machinery, which functions to detect Ca2+ depletion from the ER, and subsequently activate extracellular Ca2+ entry through plasma membrane Orai channels. Orai‐mediated Ca2+ entry is implicated in regulating inflammatory signaling in microglia through activation of the Ca2+‐dependent phosphatase, calcineurin, and its downstream pro‐inflammatory transcription factor target, NFAT. We have validated the RGS10‐STIM2 interaction in microglia and defined the specificity of interaction and its regulation by calcium and G protein signaling. To delineate the role of STIM2 and downstream calcineurin signaling in mediating the anti‐inflammatory effect of RGS10, we determined the effect of STIM2 knockdown and calcineurin inhibition on LPS‐stimulated COX‐2 expression in wildtype and RGS10 knockout microglial cells. Our findings demonstrate that LPS‐stimulated COX‐2 amplification in RGS10 knockout microglial cells is blocked by inhibition of either STIM2 expression or calcineurin activity. Furthermore, the increased inflammatory signaling observed in RGS10 knockout microglia correlates with enhanced SOCE, as measured by Ca2+ imaging using a fluorescent calcium indicator. Collectively, these results suggest that RGS10 strongly suppresses LPS‐stimulated inflammatory signaling in microglia by inhibiting STIM2‐induced calcium entry and the subsequent activation of calcineurin‐dependent inflammatory gene expression.

Support or Funding Information

National Institute of Neurological Disorders and Stroke (NINDS)[Grant NS101161]

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using CYP3A4 NMR derived structural information to mitigate time‐dependent inhibition due to diltiazem metabolism
Authors: Wilderman Ross , Halpert James
Published: June 2021

Human CYP3A4 metabolizes a significant fraction of clinically relevant drugs. CYP3A4‐mediated metabolism of the vasodilator diltiazem leads to time‐dependent inhibition (TDI) and clinically relevant drug‐drug interactions. We hypothesized that introduction of steric bulk into the CYP3A4 active site by site‐directed mutagenesis will disrupt active site contacts, alter diltiazem orientation in the CYP3A4 active site, and decrease TDI, as previously seen with midazolam. A model of diltiazem in the CYP3A4 active site was produced using distance restraints generated from longitudinal T1 NMR relaxation experiments. CYP3A4‐ligand interactions were also monitored using spectral binding titrations. Evaluation of diltiazem‐mediated CYP3A4 TDI was performed using the reporter substrate 7‐ethoxy‐4‐(trifluoromethyl)coumarin. The diltiazem model revealed that the dimethylamino group that undergoes CYP3A4‐mediated N‐demethylation is oriented toward the heme iron with the aromatic ring of the benzothiazepine core near active site residues I369 and L373, the ester moiety near L216, and the p‐hydroxyphenyl group near S119. Diltiazem caused a Type II spin shift upon binding to CYP3A4, indicating interaction of the amine with the heme iron. Substitution of I369 with a larger residue decreased CYP3A4 TDI, and an NMR derived model of diltiazem in complex with CYP3A4 I369W revealed that the p‐hydroxyphenyl group was toward the heme iron instead of the dimethylamino group. In summary, a hybrid NMR‐computational approach was used to rapidly generate a model of diltiazem in complex with CYP3A4. Introduction of steric bulk into the CYP3A4 active site by mutagenesis altered the orientation of diltiazem and mitigated TDI. The next step is to add steric bulk to the cognate part of the compound. These results indicate that NMR‐derived models of protein‐ligand interactions provide a structure‐based approach for decreasing metabolic liabilities of CYP enzyme substrates.

Support or Funding Information

This work was supported by internal funding from the University of Connecticut to James Halpert.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sphingosine‐1‐phosphate Enhances Mitochondrial Integrity and Endothelial Barrier Function Following Hemorrhagic Shock
Authors: Alves Natascha Guimaraes , Yuan Sarah , Breslin Jerome
Published: June 2021

Excessive microvascular permeability is a serious complication involved in traumatic injury and inflammatory diseases. Sphingosine‐1‐phosphate (S1P) has known endothelial barrier‐protective properties, and has been shown to ameliorate microvascular leakage in a model of combined alcohol intoxication and hemorrhagic shock and resuscitation (HSR). However, the mechanisms of S1P‐induced barrier protection during HSR are unknown. In the current study, we tested the hypothesis that S1P could enhance endothelial barrier function during hemorrhagic shock by preserving the integrity of junction proteins and the endothelial glycocalyx, and protecting mitochondrial function. We used an established in vivo rat model of conscious HSR and assessed microvascular leakage, endothelial glycocalyx integrity, and mitochondrial function by intravital microscopy. Assessment of adherens junction protein localization was done by immunofluorescence confocal microscopy after HSR. Cultured rat intestinal microvascular endothelial cell (RIMEC) monolayers were used to test the ability of S1P to protect against glycocalyx shedding and endothelial barrier dysfunction caused by direct disruption of mitochondrial integrity due to inhibition of mitochondrial complex III. Our in vivo results show that S1P protects against HSR‐induced hyperpermeability, preserves the expression of adherens junctional proteins, and protects against glycocalyx degradation. S1P treatment during HSR also protects against mitochondrial membrane depolarization. Besides that, S1P protects RIMECs against mitochondrial dysfunction‐induced endothelial barrier dysfunction and glycocalyx degradation by blocking complex III inhibition. Our results show that S1P protects against HSR‐induced mitochondrial dysfunction in endothelial cells, which in turn improves the structure of the endothelial glycocalyx after HSR and allows for better junctional integrity, preventing excess microvascular permeability.

Support or Funding Information

Supported by NIH grants R01HL098215, R01GM120774, R01HL070752 and R01GM097270.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TGFβ1 Regulates KIM‐1 and Caveolin‐1 Expression through the Smad3 and ERK1/2 Signaling Pathways in Renal Cancer Cells
Authors: Zhang Yuanyuan , Chen Xiaoming , Zhao Xueying
Published: June 2021

Kidney injury molecule‐1 (KIM‐1) is a type I transmembrane glycoprotein that is rapidly upregulated at the gene and protein level following various types of injury to the kidney. KIM‐1 expression is increased not only in acute and chronic kidney injury diseases but also in renal cell carcinoma. It may play an important role in carcinoma process. Caveolin‐1, the principal component of caveolae, is regarded as an important inhibitor of multiple signaling molecules. The cytokine transforming growth factor‐β1 (TGF‐β1) has been extensively studied in tumor biology and is believed to serve a variety of functions in tumor progression. In this study, we investigated the regulatory mechanism of TGF‐β1 on KIM‐1 and caveolin‐1 expression in 769‐P cells, a human renal cell adenocarcinoma cell line. Western blot analysis indicated that TGF‐β1 treatment resulted in a downregulation of KIM‐1 and an upregulation of caveolin‐1 in 769‐P cells. Specific knockdown of Smad3 with siRNAs significantly increased basal KIM‐1 expression and attenuated TGF‐β1‐induced KIM‐1 down regulation, which was associated with increased activation of extracellular signal‐regulated kinase (ERK) 1/2 and signal transducer and activator of transcription 3 (STAT3) signaling pathways. Interestingly, pre‐treatment of 769‐P cells with ERK inhibitor U0126 abolished TGF‐β1‐induced caveolin‐1 upregulation and partially restored KIM‐1 protein level. We also observed a significant decrease in KIM‐1 in 769‐P cells administered with caveolin‐1 scaffolding domain peptides. Taken together, our data demonstrate that TGF‐β1 may differentially regulate KIM‐1 and caveolin‐1 expression in renal cancer cells via both Smad3 and ERK1/2 signaling pathways.

Support or Funding Information

NIH SC1DK112151; NIH/NCRR/RCMI 8G12MD007602 & 8U54MD007588

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pretreatment With Water Kefir Promotes A Decrease In Ulcer Development In An Ethanol‐Acidified Ulcer Model
Authors: Brasil Girlandia Alexandre , Andrade Moraes Flavia S. , Prucoli Falsoni Raiana M , Resende Mahira Sabino , Andrade Tadeu Uggere , Lima Ewelyne Miranda
Published: June 2021

Gastric ulcer are one of the most prevalent diseases in the world, the treatment are expensive and produce many side effects, so the research of new therapies are valuable. To evaluate the gastroprotective and antioxidant effects of pretreatment with water kefir (WK) on ulcer induction with HCL/ethanol. All pretreatments were maintained for 14 days. Male mice (C57BL/J6) were separated into five groups: the control (C) group received vehicle without ulcer induction; the ulcerated (U) group received vehicle; the lansoprazole (L) group received 30 mg/kg/day lansoprazole; the water kefir (WK15 and WK30) groups received WK at a dose of 0.15 or 0.30 ml/kg/day, respectively. Gastroprotection was measured by ulcer area, ulcer index and ulcer reduction percentage. The antioxidant effects were quantified by measuring advanced oxidized protein products (AOPPs), superoxide dismutase (SOD), and catalase activity in the stomach.

Pretreatment with WK at both doses exerted gastroprotection against HCL/ethanol ulcer induced similar to pretreatment with lansoprazole (ulcer area: C: 14477±6204; U: 127578±33260; L: 39958±13921; K15: 79357±23814; K30: 63252±22435 μm2. p<0.05). In addition, WK promoted decreased protein oxidation (C: 0.018±0.004; U: 0.036±0.004; L: 0.021±0.004; WK15: 0.014±0.006; WK30: 0.023±0.003 μM of chloramine T/mg of protein) and increased SOD activity (C: 21.75±8.16, U: 2.91±1.63, L: 27.27±2.97, WK15: 28.79±4.84, and WK30: 28.25±8.69 USOD/mg protein) and catalase activity (C: 5.73±1.63, U: 0.78±0.23, L: 4.29±1.21, WK15: 4.28±0.74, and WK30: 3.56±1.23 ΔEmin/mg protein−1).

We conclude that pretreatment with water kefir for 14 days promotes increased antioxidant enzyme activity, preventing gastric lesions against HCl/ethanol ulcer model by maintaining antioxidant performance in gastric tissue.

Support or Funding Information

This work was partially financed by FAPES (#72662319) and UVV.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effects of taurine depletion on bile acid composition and its amino acid‐conjugation in the bile of cats
Authors: Miyazaki Teruo , Sasaki Sei‐ich , Toyoda Atsushi , Shirai Mutsumi , Ikegami Tadashi , Matsuzaki Yasushi , Honda Akira
Published: June 2021

Aim

The most established effect of functional amino acid‐like compound taurine is the conjugation with bile acids. In addition, taurine ameliorates hypercholesterolemia through promotion of hepatic bile acid synthesis from cholesterol by enhancing of CYP7A1. On the other hand, the conjugations of bile acids with taurine as well as glycine and the biosynthesis of taurine are also promoted by bile acids through the nucleus farnesoid X receptor. These findings imply that there is an interactive relationship on the metabolism of taurine and bile acids. Contrary to rodents, the ability of taurine biosynthesis is very poor in humans, and therefore, the body pool of taurine in humans depends on the oral ingestion. Furthermore, the ratio of conjugation of taurine, but not glycine, with bile acids also depends on taurine ingestion. This study evaluated the influence of depletion by taurine non‐ingestion on the metabolism of bile acid in bile in cats with poor taurine‐synthesizing ability as in humans.

Method

Control (N=4) and Depletion(N=6) groups of adult cats were fed the soy protein‐based diet containing 0.15% and 0% taurine, respectively, for 30 weeks. Thereafter, taurine in the serum and tissues and bile acids in the bile were measured by LC‐MS/MS system.

Result

Serum taurine concentration in the Depletion group significantly decreased to one tenth compared to that in the Control group after the 2nd week, and was undetectable since the 4th week. After 30 weeks, taurine in the various tissues was depleted in the Depletion group. Over 99.5% of bile acids in the bile were conjugated with taurine in the Control group. In the Depletion group, taurine‐conjugated bile acids significantly decreased to 35.2%, and instead, unconjugated bile acids significantly increased to 62.9%. Bile acid concentration in the bile significantly decreased to approximately 40% compared to that in the Control group. Bile acid types in cats were same to those in humans. In the composition of bile acids, cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acids (CDCA) were 83%, 12%, and 5%, respectively, in the Control group. On the other hands, CA significantly increased to 98%, and DCA and CDCA significantly decreased to 0.6% and 1.2%, respectively, in the Depletion group.

Conclusion

Taurine depletion caused the decrease of taurine conjugated bile acids in the bile, and instead, the increase of unconjugated bile acids. Consequently, the level of bile acids significantly decreased and the composition has changed. These results suggested that the synthesis of bile acids in humans might be regulated by taurine ingestion through the direct effects of taurine or the state of taurine conjugation with bile acids.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Novel Calreticulin Reporter for Determination of Immunogenic Cell Death
Authors: Bocian Rebecca , Franco April , Gurzell Eric , Soendergaard Mette
Published: June 2021

Immunogenic cell death (ICD) initiates a series of damage associated molecular patterns (DAMPs) that are able to activate an immune response. Of these, translocation of calreticulin (CRT) from the endoplasmic reticulum (ER) to the outer leaflet of the cell membrane is a hallmark event that leads to activation of antigen presenting cells (APC), thus priming an immune response. Induction of ICD in cancer cells may provide a manner of activating an immunological anti‐tumor response, which may be utilized in immunotherapy. Currently, a few chemotherapeutic drugs, such as doxorubicin, are known to induce ICD and have been proposed to be repurposed for this use. Thus, it is likely that other known drugs may exhibit ICD induction and may be utilized in immunotherapy. However, to evaluate such drugs, there is a need to develop a high throughput method of detecting ICD.

Here, a novel ICD reporter based on translocation of CRT and the HiBit‐tag expression system is described. This system utilizes a recombinant HiBit‐tag that associates with cell impermeable LgBit, thereby forming a bioluminescent protein complex. HiBit‐tagged CRT will complex with LgBit when translocated to the outer leaflet of the cell membrane, which may be detected bioluminescently by addition of D‐luciferin. Thus, the intensity of bioluminescence may be correlated to translocation of CRT, a hallmark of ICD.

To create the novel ICD reporter, the human CRT gene was amplified using polymerase chain reaction (PCR) from plasmid HsCD00322958 (Harvard Medical School, MA), and inserted onto the 3′ end of the HiBit tag (pBiT3.1‐N, Promega, WI) using restriction enzyme digestion (PspXI and SbfI) followed by ligation. The recombinant HiBiT‐CRT plasmid was transformed into competent DH5α E. coli cells using the heat‐shock method. The HiBit‐CRT cassette was validated by DNA Sanger sequencing (Genscript, Piscataway, NJ). In order to transfect human ovarian adenocarcinoma cells (SKOV‐3) with the ICD reporter, the optical concentration of geneticin was determined by performing a kill curve. In brief, SKOV‐3 cells were maintained in McCoy's 5A cell medium supplemented with 10 % fetal bovine serum (FBS) at 37°C and 5% CO2, and then treated with varying concentrations of geneticin (0.05 mg/mL, 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL, 1.25 mg/mL, 1.5 mg/mL, 1.75 mg/mL, 2 mg/mL). The cell viability was evaluated daily for 7 days using bright field microscopy. The optimal concentration was determined to be 1.25 mg/mL. Next, SKOV‐3 cells will be transfected with the ICD reporter, and used to screen the Approved Oncology Drug Set VI (kind gift from the National Cancer Institute) for compounds that induce ICD.

Support or Funding Information

Western Illinois University Research Council Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Biochemical Program of Extreme Terminal Differentiation
Authors: Finley Daniel
Published: June 2021

As cells undergo extreme forms of terminal differentiation, they are able to accumulate specific proteins to exceptionally high levels–hundreds of milligrams per ml in the case of globins and crystallins. During the time in which these proteins are being generated in high amounts, almost all other components of the cell, numbering many thousands, are eliminated. The mature red blood cell has one of the simplest cellular proteomes known, with hemoglobin concentrated to a remarkable ~98% of soluble protein by some estimates. How can cells carry out such vast programs of biosynthesis and degradation simultaneously? We proposed in 1995 that the ubiquitin‐proteasome system (UPS) may play a central role in this process. We have addressed this problem through quantitative global proteomics in collaboration with the laboratories of Drs. Mark Fleming, Mitch Weiss, and Peng Ji. We found that ubiquitinating factor UBE2O, which is strongly induced in late erythroid differentiation, functions as an E2–E3 hybrid enzyme to mediate the elimination of ribosomes and myriad other proteins in maturing red cells. UBE2O recognizes substrates directly, and its unexpectedly broad specificity appears to reflect the presence of multiple substrate recognition domains within the enzyme. Additional ubiquitinating factors–TRIM10, TRIM58, and UBE2H–are, like UBE2O, specifically induced in late erythroid differentiation (during this period, most UPS components disappear). Applying quantitative global proteomics to mouse mutants in these enzymes, we find that the ubiquitin ligase TRIM10 eliminates dynactin, many myosins, actin crosslinking proteins, and CopI vesicles, while in contrast TRIM58 eliminates dynein and centrosome components, and UBE2H eliminates ER components. In general we find that erythroid lineage inducible ubiquitinating factors have quite distinct specificities, as if each were tasked to eliminate its own part of the cell. Together the eliminate many of the basic functionalities of cell biology. The UPS has been conceived of as having two major types of function: the selective degradation of regulatory proteins, and protein quality control. Our work indicates a new capacity of the UPS, to effect global and developmentally controlled proteomic remodeling. We propose that ubiquitin‐dependent proteome remodeling is likely to be an important new aspect of the biology on eukaryotic cells, critical for the generation of highly differentiated cell types.

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R01 HL125710

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screening Tools for Efficient Delivery of siRNA and Plasmid into Mammalian Cells
Authors: Collins Michelle , Jordan Elizabeth , Terefe Joseph , Pineda Maxinne , Ugozzoli Luis , Rubio Teresa
Published: June 2021

Modulating gene expression is essential to achieve a better understanding of gene function. The transfer of exogenous nucleic acids such as plasmids or small interfering (si)RNAs into mammalian cells is an important technique for the study and analyses of gene function, expression, regulation, and mutation, and it has advanced basic cellular research, enabled drug target identification, and validation. Electroporation is a well‐established gene transfer method, and an effective means of transferring nucleic acids into cells. The shift of today's research to more scientifically relevant cell lines has created a void for efficient delivery tools. In addition, the wide adoption of RNAi technology, has led to a demand of screening tools to identify the most effective siRNA. In order to obtain consistent downstream results, it is critical that delivery conditions be highly efficient.

Here, we show data that demonstrates the benefits of using a high throughput electroporation system to develop optimal conditions for delivery of plasmids and (si)RNA into mammalian cells. We demonstrate that the use of this instrument allows for rapid screening as well as the ability to identify the most efficient delivery conditions into a difficult‐to‐transfect cell line. Finally, we present quantitative PCR data that validates the benefits of this technique.
The Ontogeny of poll, fbxw4, sufu mRNA expression during Development of the Chick Limb
Authors: Chryst‐Ladd Megan N , Everman David , Maze Jennifer
Published: June 2021

Split‐hand/split‐foot malformation (SHFM), also known as ectrodactyly, is a congenital limb malformation that is characterized by absence or reduction of the central digital rays of the hands and feet. To date there are five well established loci for SHFM. At one of these loci, SHFM3 at chromosome 10q24, tandem duplications have been associated with the SHFM phenotype in humans. The smallest reported duplication includes the genes POLL, BTRC, DPCD, and a portion of FBXW4. Another gene near the duplicated region, SUFU, has been shown to be overexpressed in SHFM3 patients. RNA collected from chicken embryo limb buds (E2‐15) was analyzed for expression of poll, fbxw4, and sufu by reverse‐transcription polymerase chain reaction (RT‐PCR). Preliminary studies demonstrate that fbxw4 and sufu are first detected at E5 and are no longer detected after E13, which is consistent with the normal timing of digit patterning in the chicken. No poll expression was observed in limb bud RNA, which is not surprising, given that there is no previous correlation with limb development. These results suggest that both fbxw4 and sufu are expressed in the limb during the period of digit patterning, and while poll is in the commonly duplicated region, it is not expressed in a similar fashion. Future studies will explore the effects that overexpression of fbxw4 and sufu have on limb development in chicken and investigate the expression of other genes from the duplicated region.
Alteration of the substrate specificity of ketosteroid isomerase from Pseudomonas putida Biotype B
Authors: Cha Hyung Jin , Jang Do Soo , Shin Han Seop , Choi Kwan Yong
Published: June 2021

Most enzymes are quite specific for their natural substrates. Therefore, it is often necessary to modify or broaden their substrate selectivity for industrial or biotechnology applications. However, there are many difficulties to change the substrate specificity, since the relationship between substrate specificity and structure is too complex to understand. Especially, there are few examples that report changing the substrate specificity in steroid‐binding proteins by mutagenesis. Utilizing a ketosteroid isomerase (KSI) from bacterial source, we studied about the substrate specificity. We focused on role of the end of steroid–binding cleft, which interact with D‐ring derivative steroids. Therefore, we made following mutants such as M90A, M90G, and G60L, to alter the substrate specificity by broadening or narrowing this region. KSI catalyzes the isomerization of various Δ5‐3‐Ketosteroid to Δ4‐3‐Ketosteroid. Steroids such as 5‐Androsten‐3,17‐dione, 5(10)‐estren‐3,17‐dione, and 5‐pregnene‐3,20‐dione was used as the substrate. We found that the catalytic activity of M90A mutant against 5‐Pregnene‐3,20‐dione was increased about 2.5‐fold relative to that of WT.
Computer modeling and synthesis of small molecule gp41 inhibitors
Authors: Smith Kellie Nicole , Smith Marilyn , Smith Richard
Published: June 2021

The process of membrane fusion of the Human Immunodeficiency Virus Type 1 (HIV‐1) to the host cell has recently been a target for small molecule inhibitors. With inhibitors such as peptidic T20, viral entities are unable to infect host cells and HIV infection is suppressed. Based on the success of T20, non‐peptide small molecule inhibitors of gp41 (HIV‐1 envelope glycoprotein) will be investigated to determine if the same HIV‐suppression results are obtained. Previous attempts by others include two N‐carboxyphenylpyrrole ring derivatives. Their analysis has led to the development of a novel class of inhibitors, the phenylalkyl phenylacetic acids (PAPAs). In this work, derivatives of the PAPA class of compounds were modeled. Each derivative underwent conjugate gradient energy minimization followed by Monte Carlo simulation. Results gave energetic and structural information about the potential compounds. Synthetic procedures were carried out for one analog, PHO2Cm. The synthetic procedure was accomplished and will be used in the future for compounds that show computational inhibitory activity.
A proteomic analysis of CBA‐Pk‐1slc/Pk‐1slc mice with Red Blood Cell Type Pyruvate Kinase deficiency using 2DE together with MALDI–TOF‐MS analysis
Authors: Kedar Prabhakar , Shimizu Kazuyuki , Ghosh Kanjaksha , Fujji Hisaichi , Colah Roshan , Kanno Hitoshi
Published: June 2021

SLC3 is a Friend erythroleukemic cells line from the Pk‐1slc mouse erythrocyte pyruvate kinase(PK)deficiency. SLC3 undergo spontaneous apoptosis leads to ineffective erythropoiesis. It was showed that glycolytic inhibition by PKLR gene mutation augmented oxidative stress, leading to the activation of HIF‐1 as well as downstream proapoptotic gene expression. We studied proteomic response of SLC3 was quantitatively analyzed two‐dimensional gel electrophoresis (2DE) followed by identification with matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF‐MS). We detected significant differences between SLC3 and a control Friend cell. 72% of the total proteins studied were significantly down regulated in SLC3 and only 28% of proteins were up regulated. Several membrane proteins are significantly down‐regulated in SLC3 may cause reduced stability and integrity of PK deficient RBC. Oxidative stress proteins in SLC3 mice were up‐regulated and antioxidant proteins were down regulated. These proteins related to generation of ROS. For the refolding of mutant protein, molecular chaperon is expectedly unregulated in SLC3. Upregulation of two chaperons are presumably an adapted reaction in SLC3. Heat‐shock protein (HSP70) and mortalin, A novel mediator of erythropoietin signaling. We speculated that down‐regulation of mortalin might be attributable to disturbance of EPO signaling, resulting in premature death of cells. The proteomic approach makes an important contribution to characterizing the proteome of the SLC3.
An actin‐based wave generator organizes cell motility
Authors: Weiner Orion David
Published: June 2021

Although many of the regulators of actin assembly are known, we do not understand how these components act together to organize cell shape and movement. To address this question, we analyzed the spatial dynamics of a key actin regulator–the Scar/WAVE complex–which plays an important role in regulating cell shape in both metazoans and plants. We have recently discovered that the Hem‐1/Nap1 component of the Scar/WAVE complex localizes to propagating waves that appear to organize the leading edge of a motile immune cell, the human neutrophil. Actin is both an output and input to the Scar/WAVE complex: the complex stimulates actin assembly, and actin polymer is also required to remove the complex from the membrane. These reciprocal interactions appear to generate propagated waves of actin nucleation that exhibit many of the properties of morphogenesis in motile cells, such as the ability of cells to flow around barriers and the intricate spatial organization of protrusion at the leading edge. We propose that cell motility results from the collective behavior of multiple self‐organizing waves.
Rap1 promotes Prostate Cancer metastasis
Authors: Bailey Candice L , Casey Patrick J
Published: June 2021

Elucidating the mechanisms of prostate cancer (CaP) survival and metastasis are critical to the discovery of novel therapeutic targets. Increasing evidence demonstrates significant roles for integrins and matrix metalloproteinases (MMP) in CaP metastasis, making them attractive targets for therapy. The monomeric G protein, Rap1 activates integrins and has been implicated in cancer tumorgenesis. Rap1 signals to pathways involved in cell adhesion, migration, and survival, suggesting Rap1 may promote several processes associated with CaP metastasis. Thus, we sought to explore the functional significance of Rap1 activation in CaP by examining its impact on invasion and migration of CaP cells during metastasis.

Results from in vitro assays demonstrate activation of Rap1 increases CaP cell migration toward bone‐secreted factors. Similarly, results show activation of Rap1 enhances CaP cell invasion, and may involve modulation of MMPs. Inhibition studies indicate that integrin activation is a mechanism of Rap1‐mediated CaP migration and invasion. Additional studies will explore whether Rap1 influences MMPs directly or through its subsequent activation of integrins. The in vivo impact of Rap1 in CaP is being evaluated in the mouse and preliminary results demonstrate activated Rap1 drives the rate and incidence of CaP metastasis.

Support for CLB: Department of Defense PCRP #DAMD17‐0510560