bluuebunny/crossref_metadata_2025 huggingface dataset:
Uric Acid Activates Aldose Reductase and the Polyol Pathway for Endogenous Fructose Production and Fat Accumulation in the Development of Fatty Liver
Authors: Lanaspa Miguel A. , Andres‐Hernando Ana , Johnson Richard , Sanchez‐Lozada Laura G
Published: June 2021

Dietary sugars containing fructose have been strongly associated with the development of fatty liver, but recent studies also suggest fructose can be produced in the liver via the polyol pathway where it may also induce hepatic fat accumulation. Here we investigate whether uric acid regulates aldose reductase, a key enzyme in the polyol pathway. We evaluated whether soluble uric acid regulates aldose reductase expression both in cultured hepatocytes (HepG2 cells) and in the liver of hyperuricemic rats, and whether this stimulation was associated with endogenous fructose production and fat accumulation. Uric acid dose stimulated the expression of aldose reductase in HepG2 cells in association with endogenous fructose production and triglyceride accumulation. The mechanism was mediated by uric acid‐induced oxidative stress with the stimulation of the transcription factor NFAT5. Uric acid could amplify the effects of elevated glucose levels to stimulate hepatocyte triglyceride accumulation. Hyperuricemic rats also showed elevated hepatic aldose reductase expression, endogenous fructose accumulation and fat accumulation that was significantly reduced by co‐administration of the xanthine oxidase inhibitor, allopurinol. These studies suggest that uric acid, which is generated during fructose metabolism, may act as a positive feedback to stimulate endogenous fructose production by stimulating aldose reductase in the polyol pathway. These studies provide an amplifying mechanism by which soft drinks rich in glucose and fructose can induce non‐alcoholic fatty liver disease.

Support or Funding Information

Supported by NIH Grant R01 DK108859

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered Small Intestinal Morphology and Whole‐Body Energy Homeostasis in the Intestinal Fatty Acid Binding Protein (IFABP) Knockout Mouse
Authors: Lackey Atreju I , Chen Tina , Zhou Yin X , Bottasso Arias Natalia M , Gajda Angela M , Joseph Laurie B , Storch Judith
Published: June 2021

Intestinal‐fatty acid binding protein (IFABP; FABP2) is a 15 kDa intracellular protein present in high abundance in the cytosol of the small intestinal (SI) enterocyte. High fat (HF) feeding of IFABP−/− mice resulted in lower weight gain and reduced fat mass relative to WT mice, revealing that ablation of this intestine‐specific protein leads to dramatic whole‐body phenotypic alterations (Gajda et al., JBC 2013 288:30330–30344). To understand the mechanisms leading to these differences in systemic energy homeostasis, we examined intestinal properties that may underlie the observed lean phenotype. Fecal lipid analysis demonstrated that IFABP−/− mice did not differ in their fecal lipid content, suggesting they are not malabsorbing dietary fat. However, the total excreted fecal mass for the IFABP−/− mice was significantly increased relative to WT mice. Moreover we found reduced intestinal transit time in the IFABP−/− mice, which underlies the increased fecal excretion. During tissue collection it was noted that the small intestine of IFABP−/− mice appeared to be more fragile, thus we questioned whether they may have alterations in intestinal structure. Histological analysis demonstrated that IFABP−/− mice have a shortened average villus length, reduced goblet cell density, and a reduced Paneth cell abundance relative to WT mice. BrdU proliferation assays demonstrated that the amount of proliferating cells in the crypts of IFABP−/− mice does not differ from that of WT mice, suggesting that the blunt villi phenotype is likely not due to alterations in proliferation. IHC analyses revealed increased staining for markers of inflammation, which is often observed in animals that have increased intestinal permeability. Taken together, while the effects of IFABP ablation within the enterocyte may be relatively modest in terms of lipid, there are clear changes in gut motility and morphology which likely contribute to the dramatically different phenotypes occurring at the whole‐body level. Thus, these results suggest that IFABP is likely involved in dietary lipid sensing and signaling, influencing intestinal motility, nutrient absorption, and intestinal structure, thereby impacting systemic energy metabolism.

Support or Funding Information

This work was supported by National Institutes of Health Grant DK‐38389 and NJAES (to J. S.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alterations in Nuclesome Core Particle Dynamics in the Context of Paraganglioma
Authors: Amato Matthew , Maher Louis Jim , Becker Nicole , Botuyan Maria Victoria , Zhao Debiao Jim
Published: June 2021

Paraganglioma (PGL) is a rare form of neuroendocrine cancer for which the underlying molecular basis is poorly understood. A mysterious hallmark of many PGL tumors is the inactivation of succinate dehydrogenase (SDH), a mitochondrial enzyme of the TCA cycle. Why does SDH loss induce cancer in certain cells? As a result of SDH loss, PGL tumor cells accumulate succinate, which may have epigenetic consequences. For example, certain lysine residues on histone proteins in nucleosomes are subject to succinylation by succinyl‐CoA as a post translational modification (PTM) that converts the lysine cation to an anion. We hypothesize that the structure and stability of the nucleosome core particle (NCP) is affected by succinylation PTMs through weakening of electrostatic histone‐DNA and histone‐histone interactions. Proteomic analysis of an SDH‐loss cell line model of PGL previously demonstrated that the greatest increase in nucleosomal lysine succinylation maps to H4K79 when compared to a control cell line. The mammalian H4K79 residue lies within the nucleosome core near wrapped DNA. We specifically hypothesize that the charge inversion conferred by H4K79 succinylation, destabilizes the NCP. Point mutations at H4K79 have been generated as lysine succinylation mimics at this site. Biophysical and in vitro transcription assays are being utilized to validate the assembly of NCPs incorporating lysine succinylation mimics at this site and to interrogate their resulting stabilities. Analyses of alterations in NCP dynamics in variant NCP will help to elucidate the role of lysine succinylation of histones in the altered chromatin landscape observed during PGL oncogenesis.

Support or Funding Information

GM75148‐09‐10 PREP

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Energetics and Interfacial Interactions of Spliceosomal Protein Dib1 Predicted with MD Simulations
Authors: Goldstein Rachel , Cheng Sara , Orr Gabby , Schreib Christian , Cheng Kwan , Maeder Corina
Published: June 2021

The spliceosome is the highly dynamic macromolecular complex responsible for catalyzing the excision of introns from pre‐messenger RNA. The spliceosome consists of five small nuclear ribonucleoproteins as well as several additional proteins. As part of a complex assembly pathway, the essential protein Dib1 must leave the spliceosome. Experimental analysis has found a temperature sensitive phenotype of Dib1 (TS‐Dib1, F85A) that hinders splicing activity. This phenotype has also been hypothesized to perturb spliceosome assembly. Our current work uses atomistic molecular dynamic (MD) simulations to study the molecular interactions in the region of the spliceosome around Dib1 in order to determine what energetic and structural changes result from this point mutation.

Starting with recently published cryo‐EM structures, the spliceosome was reduced to only include components in close proximity to Dib1 (either native or F85A). The reduced Dib1 structure consists of three snRNAs (U4, U5, and U6) and four proteins (Prp6, Prp8, Brr2, Prp31). Both systems, containing either native Dib1 or mutant Dib1, were simulated using atomistic MD simulations to identify energetic differences between native Dib1 and mutant TS‐Dib1. The simulation conditions mimicked the biological conditions of previous bulk splicing assays, with an aqueous solvent, 0.1 M salt, and temperature differences that have experimentally shown TS‐Dib1 inactivation. The MD simulations revealed a lower global binding energy in the TS‐Dib1 system compared to the native Dib1 system. Additionally, interfacial hydrogen bonding patterns displayed increased hydrogen bonding among Prp6, Prp8, and Dib1 in the native Dib1 system that were not present in the TS‐Dib1 system, which could contribute to the lower global binding energy. The results from the MD simulations support the experimentally observed temperature sensitivity in the TS‐Dib1 system and could help explain the role of Dib1 in spliceosome assembly.

Support or Funding Information

NIH R15GM120720, Robert A Welch Foundation Grant W‐1905

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antioxidants prevent cytotoxic and mutagenic effects of hexavalent chromium on human and bacterial cell cultures
Authors: Mayotte Timothy , Himes Ryan
Published: June 2021

Cr(VI) (hexavalent chromium) has recently been found in the drinking water of over 250 million Americans. It is a powerful oxidizing agent, and is known to cause cancer, although the specific mechanism has yet to be elucidated. There is no known preventative treatment for Cr(VI) exposure, and the US EPA is currently determining what concentration of Cr(VI) in drinking water can be safely tolerated. This study sought to test the hypothesis that Cr(VI) cytotoxicity can be prevented by various antioxidants. We tested this hypothesis by exposing human embryonic kidney and human intestinal epithelial cells to Cr(VI), with and without the presence of the antioxidants vitamin C, or epigallocatechin gallate (EGCG). Cr(VI) was found to be significantly toxic to human cell cultures at concentrations of 200 parts per billion (ppb) and higher. This effect was blocked by 10 parts per million (ppm) vitamin C and by 15 ppm EGCG. No cytotoxic effects were observed on cells treated with vitamin C or EGCG alone. We also used an Ames test to determine if these antioxidants can prevent Cr(VI)‐induced mutagenesis on bacterial cells. The Ames test revealed that Cr(VI) causes DNA mutations in bacterial cells at concentrations of at least 20 ppb. This effect was blocked by 20 ppm vitamin C. No mutagenic effect was observed with vitamin C alone. Taken together, the results suggest that Cr(VI) is toxic to human cells through an oxidative mechanism at a concentration of at least 200 ppb. Additionally, Cr(VI) may also cause DNA mutations at concentrations of 20 ppb or more. These effects can be abrogated with antioxidant co‐treatment. While further study is merited, these results should help to inform government agencies tasked with monitoring drinking water quality.

Support or Funding Information

This research was made possible by funding from the Craighton T. and Linda G. Hippenhammer Faculty Scholarship Grant, the Elbert Pence and Fanny Boyce Undergraduate Summer Research Fund, and the Olivet Nazarene University Honors Program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RECK suppresses interleukin‐17/TRAF3IP2‐mediated MMP‐13 activation and human aortic smooth muscle cell migration and proliferation
Authors: Mummidi Srinivas , Das Nitin A , Carpenter Andrea J , Yoshida Tadashi , Mostany Ricardo , Izadpanah Reza , Higashi Yusuke , Sukhanov Sergiy , Delafontaine Patrice , Siebenlist Ulrich , Randy Scott Rector , Bysani Chandrasekar
Published: June 2021

Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular proliferative/occlusive disorders. The proinflammatory cytokine interleukin (IL)‐17 signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various transcription factors critical for inflammatory responses, including AP‐1 and NF‐κB. Here we investigated whether IL‐17A/TRAF3IP2 signaling promotes MMP‐13‐dependent human aortic smooth muscle cell (SMC) migration and proliferation, and whether RECK overexpression blunts its mitogenic and migratory potential. Indeed, IL‐17A treatment induced (i) AP‐1, NF‐κB, CREB and p38 MAPK activation, (ii) miR‐21 induction, (iii) miR‐27b and miR‐320 inhibition, (iv) MMP‐13 expression and activation, (v) RECK suppression, and (vi) SMC migration and proliferation, all in a TRAF3IP2‐dependent manner. RECK (REversion‐inducing‐Cysteine‐rich protein with Kazal motifs) is a membrane‐anchored MMP inhibitor, and forced expression of TRAF3IP2 suppressed its expression, but induced MMP‐13 expression and activation. Importantly, forced expression of RECK promoted IL‐17‐mediated MMP‐13/RECK physical association, reduced MMP13 activity and inhibited MMP‐13‐dependent SMC migration and proliferation. Further supporting its causative role in vascular proliferative diseases, addition of recombinant MMP‐13 stimulated SMC migration and proliferation, in part via ERK activation. These results indicate that increased MMP‐13 and decreased RECK contribute to IL‐17A/TRAF3IP2‐mediated SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases.

Support or Funding Information

VA‐I01‐BX002255IK6BX004016‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selenium status influences selenoprotein H interactions with other selenoproteins in the response to oxidative stress
Authors: Cao Lei , Lee Sanggil , Cheng Wen‐Hsing
Published: June 2021

Selenium (Se) is an essential trace element exerting its biological functions mainly through selenoproteins. Evidence suggests that selenoprotein H (SELONOH), a nuclear protein suppressing oxidative stress and cellular senescence, is the most prominent selenoprotein responding in parallel to dietary Se deficiency and age in telomere‐humanized mice. On the other hand, SELENOH is one of the few low‐hierarchy selenoproteins whose expression is very sensitive to fluctuations in Se status. The objective of this study was to explore impacts of SELENOH on the expression of other selenoproteins and redox regulation upon changes in Se status. A plasmids expressing wild‐type SELENOH was constructed and transfected into human HEK293T cells. Adding Se (Na2SeO3, 0–200 nM) to cell culture medium dose‐dependently increased protein levels of overexpressed SELENOH and other selenoproteins including glutathione peroxide 1 and thioredoxin reductase 1; however, their mRNA transcripts were not affected by Se status and SELENOH suppressed protein expression of the other two selenoproteins. Supplementing Se (100 nM) enabled overexpressed SELENOH to protect against paraquat‐induced senescence and declines in cellular viability. Analyses of SELENOH‐associated proteins through LC‐MS‐MS co‐identified nucleolar and glycosylation proteins. Altogether, these results suggest that SELENOH could compete with other selenoproteins for available Se but protects against oxidative stress together with them.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Establishing the Role of Rieske Protein Reduction Potential in the Formation of Reactive Oxygen Species in Complex III
Authors: Henderson Victoria , Hunsicker‐Wang Laura
Published: June 2021

The factors that modulate the reduction potential of complex III's catalytic Rieske metalloprotein subunit (RP) have been the subject of much study. During the bifurcated Q‐cycle, RP transports one electron from quinol to cytochrome c1. Quinol's other electron is passed from heme bL to heme bH, which reduces a quinone bound at the QN site. If the Q‐cycle is disrupted, electrons may interact with molecular oxygen to produce reactive oxygen species (ROS). For example, if RP's reduction potential is too low, it may never accept quinol's electron or do so inefficiently. The latter scenario may shift Q‐cycle equilibria against RP movement towards cytochrome c1, instead favoring RP electron release and interaction with molecular oxygen to produce ROS. Thus, we propose that a mismatch in reduction potential between RP and quinol may serve as a possible source of ROS generated from complex III. Understanding the sources of ROS in the electron transport chain may lead to better treatments for diseases that arise from high levels of ROS.

We are investigating this hypothesis by (1) measuring the reduction potential of isolated Saccharomyces cerevisiae (yeast) RP expressed in Escherichia coli (E. coli) cells (ScRieske) and (2) measuring the ROS produced by intact yeast complex III expressed in yeast cells (ScComplexIII). We created a wild‐type ScComplexIII yeast strain (JPJ1‐RIP1) by restoring respiratory function to an anaerobic RP‐deletion strain (JPJ1) via complementation with a RP expression vector. Now, we are measuring JPJ1‐RIP1 and JPJ1 ROS production by fluorometry using dichlorofluorescein (DCF) as a ROS indicator. We have also expressed and purified wild‐type ScRieske in E. coli and have characterized its structure using circular dichroism (CD) and UV‐visible spectroscopy. Furthermore, we have probed the electronics of ScRieske's [2Fe‐2S] cluster via chemical modification experiments with diethyl pyrocarbonate (DEPC). Lastly, we have obtained E. coli and JPJ1‐RIP1 expression vectors for ScRieske mutants Y185F, S183A, and S183T. Ultimately, we will use our ROS assay to quantify ROS production of ScRieske mutants with modulated reduction potentials to study the relationship between RP reduction potential and ROS production in complex III.

Support or Funding Information

Trinity Chemistry and Neuroscience Departments, Welch Foundation, The San Antonio Area Foundation, 2017 Murchison Summer Research Fellowship, 2017 ASBMB Undergraduate Research Award

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Implication of Cholesterol in Regulating the Pore‐formation Mechanism of Vibrio Cholerae Cytolysin, a β‐barrel Pore‐forming toxin
Authors: Kathuria Reema
Published: June 2021

Vibrio cholerae cytolysin (VCC) is a β‐barrel pore‐forming toxin with potent cell‐killing cytolytic/cytotoxic activity. To exert it's cytolytic effect, VCC binds to the target cell membranes and forms transmembrane oligomeric β‐barrel pores. Earlier studies have indicated that cholesterol might play a critical role in regulating the mode of action of VCC. However, exact mechanistic basis of the process still remains unknown. In the present study, we have explored and examined the roles of cholesterol in the different steps of VCC mode of action. Employing the membrane lipid bilayer of lipid vesicles or liposomes containing varying amount of cholesterol, we have shown that cholesterol governs the efficient interaction of the toxin with the membrane lipid bilayer that in turn facilitates subsequent oligomeric pore‐formation process. We have also studied the physiological relevance of membrane cholesterol in the context of the biomembranes of the eukaryotic cells. Our results have shown that the depletion of membrane cholesterol from erythrocytes compromises the pore‐forming activity of VCC. In the biomembrane of erythrocytes, where accessory interactions are available, binding of VCC is not affected to any significant extent. However, upon depletion of membrane cholesterol in the erythrocytes membranes, membrane‐bound fraction of VCC remains trapped in the form of abortive oligomeric assembly, and does not form the functional pores. Our study has also revealed that VCC shows a marked tendency to associate with the cholesterol‐rich membrane micro‐domains on erythrocytes that, in turn, appears to facilitate cholesterol‐dependent pore‐formation mechanism of the toxin. In sum, our results suggest that the presence of cholesterol in the target membranes play critical role for the pore‐formation mechanism of VCC.

Support or Funding Information

We acknowledge the support through funding from the Department of Biotechnology (DBT), Govt. of India [DBT grant no. BT/HRD/NBA/37/01/2014 (x)] and also the support through funding under the Centre of Excellence (COE) in Frontier Areas of Science and Technology (FAST) program of the Ministry of Human Resource Development, Govt. of India, in the area of protein science, design, and engineering (to K.C.). We also thank the Indian Institute of Science Education and Research Mohali for the support.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Handover mechanism of the growing pilus by the bacterial outer‐membrane usher FimD
Authors: Du Minge
Published: June 2021

Pathogenic bacteria such as Escherichia coli assemble surface structures termed pili, or fimbriae, to mediate binding to host‐cell receptors. Type 1 pili are assembled via the conserved chaperone usher pathway. The outer‐membrane usher FimD recruits pilus subunits bound by the chaperone FimC via the periplasmic N‐terminal domain of the usher. Subunit translocation through the β‐barrel channel of the usher occurs at the two C‐terminal domains (which we label CTD1 and CTD2) of this protein. How the chaperone–subunit complex bound to the N‐terminal domain is handed over to the C‐terminal domains, as well as the timing of subunit polymerization into the growing pilus, have previously been unclear. Here we use cryo‐electron microscopy to capture a pilus assembly intermediate (FimD–FimC–FimF–FimG–FimH) in a conformation in which FimD is in the process of handing over the chaperone‐bound end of the growing pilus to the C‐terminal domains. In this structure, FimF has already polymerized with FimG, and the N‐terminal domain of FimD swings over to bind CTD2; the N‐terminal domain maintains contact with FimC–FimF, while at the same time permitting access to the C‐terminal domains. FimD has an intrinsically disordered N‐terminal tail that precedes the N‐terminal domain. This N‐terminal tail folds into a helical motif upon recruiting the FimC‐subunit complex, but reorganizes into a loop to bind CTD2 during handover. Because both the N‐terminal and C‐terminal domains of FimD are bound to the end of the growing pilus, the structure further suggests a mechanism for stabilizing the assembly intermediate to prevent the pilus fibre diffusing away during the incorporation of thousands of subunits.

Support or Funding Information

R01GM062987

Van Andel Institute Graduate School

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Pharmacology of a Conformational‐Specific Extracellular Nanobody Against CXCR4
Authors: Soave Mark , Heukers Raimond , Smit Martine J , Briddon Stephen J , Hill Stephen J
Published: June 2021

Camelid single‐domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15kDa immunoglobulin domain[1]. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes[2]. Here, we have used the nanobody VUN400[3], which recognises the second extracellular loop of the human CXCR4 chemokine receptor, as a probe to monitor specific CXCR4 conformations. VUN400 was fused on its C‐terminus to the 11‐amino acid HiBiT tag (VUN400‐HiBiT) which, in the presence of the LgBiT protein, complements to form a full length functional NanoLuc luciferase. Here, we use complemented luminescence to detect binding of the VUN400‐HiBiT to specifically recognise N‐terminally tagged LgBiT‐CXCR4 expressed in living HEK293 cells (KD 59.1±11.2 nM; n=4). The binding of VUN400‐HiBiT to CXCR4 could be prevented by the orthosteric ligand CXCL12 and allosteric antagonists AMD3100 and IT1t (Table 1), allowing VUN400‐HiBiT to be used as a probe to detect specific conformations of CXCR4. These data demonstrate that the high specificity offered by extracellular‐targeted nanobodies can be utilised to probe receptor pharmacology.

Support or Funding Information

The authors would like to thank the MRC (grant code MR/N020081/1) for financial support.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CP‐25 Inhibits Methotrexate‐Induced Apoptosis in Renal Tubular Epithelial Cells
Authors: Wang Chun , WU Yi‐jin , TANG Hao , WANG Bin , WANG Yong , SUN Wei , XIAO Feng , WEI Wei
Published: June 2021

Objective

Methotrexate (MTX), known as nephrotoxic drug, frequently causes renal injury via inducing apoptotic death in human renal tubular epithelial cells(RTEC). Paeoniflorin‐6′‐O‐benzene sulfonate (CP‐25) is active compound obtained by modifying the structure of Paeoniflorin. We have previously showed CP‐25 inhibited chronic inflammation and effectively improved symptoms of arthritis and Sjogren syndrome in experimental animal models. This study was to investigate effects of CP‐25 on MTX‐induced apoptosis in RTEC lines in vitro and the related mechanisms.

Methods

NRK‐52E and NK‐2 were treated with various concentration of MTX in the presence or absence of CP‐25. After treatment, the proliferation of cells were measured by MTT assay, and apoptotic death of the cells were detected by Annexin V/PI dual staining. The expression of apoptotic related protein were measured by western blotting.

Results

Treatment of MTX significantly inhibited proliferation of NK‐2, NRK‐52E in dose‐and time‐dependant manners. Moreover, MTX could significantly induce apoptotic death in NK‐2 and NRK‐52E cells. Further study revealed that MTX exposure to the cells could activate intrinsic and extrinsic apoptotic signal pathways, which was evidenced by increased expression of Bax, cyt‐c, clea‐caspase‐3, clea‐caspase‐8, and clea‐caspase‐9, and decreased expression of Bcl‐2 and XIAP. Interestingly, we found that co‐treatment of MTX with CP‐25 could significantly reduce MTX‐induced apoptotic death in the cells. As expected, CP‐25 inhibited MTX‐induced activation of intrinsic and extrinsic apoptotic pathways via regulating abnormal expression of apoptosis‐related proteins. Finally, we found MTX exposure to the cells significantly increased nuclear translocation of NF‐κB coupled with increase in phosphorylated Iκ‐B, which was significantly inhibited by CP‐25.

Conclusion

CP‐25 could inhibit MTX‐induced pro‐apoptotic effects on RTEC. The underlying mechanism is associated with inhibitory effects of CP‐25 on the activation of intrinsic/extrinsic apoptotic pathways and NF‐κB nuclear translocation. Our data indicate CP‐25 maybe a promising therapeutic drug for the treatment of MTX‐induced renal damage.

Support or Funding Information

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 Rutaecarpine on the Benzo(a)pyrene‐Induced DNA Damage In Vitro
Authors: Li You , Park Miki S. , Chan William K. , Livesey John , Zhou Zhu
Published: June 2021

Benzo[a]pyrene (BaP) is one of the most potent mutagens and carcinogens known. The metabolic pathway of BaP involves several steps initiated by cytochromeP450 (CYP) enzymes such as CYP1A1 to give the ultimate carcinogen benzo(a)pyrene‐7,8‐diol‐9,10‐epoxide (BPDE). BPDE is responsible for genotoxic effects by alkylating the DNA at the N2 position of guanine bases. Rutaecarpine, a pentacyclic indolopyridoquinazolinone alkaloid isolated from the unripe fruit of Evodia rutaecarpa, has been used in traditional medicine to treat various diseases. It has been reported as a CYP1 family inducer. Our objective is to determine if co‐administered BaP and rutaecarpine would increase the formation of BPDE, which will ultimately increase the DNA damage. We utilized ethoxyresorufin‐O‐deethylase (EROD) assay to measure CYP1A1 enzyme activity and Muse™Multi‐Color DNA Damage Kit to examine DNA damage levels. The results showed that BaP (5 μM) and rutaecarpine (1.25–10 μM) significantly induced CYP1A1 enzyme activity (p<0.005) when administered separately in mucoepidermoid pulmonary carcinoma (H292) cells, with induction by rutaecarpine occurring in a concentration‐dependent manner. Interestingly, co‐administration of rutaecarpine, especially at a high concentration (10 μM), with BaP caused induction of CYP1A1 to a lesser extent as compared with either compound alone. Similar results were observed in hepatocellular carcinoma (Hep3B) cells. It was subsequently confirmed by the analysis of DNA damage in both H292 and Hep3B cells, in which the percent of DNA damage decreased by more than 2‐fold after combined concurrent administration of rutaecarpine and BaP, compared with BaP alone. In summary, our data supports the possibility of a protective effect of rutaecarpine against BaP‐induced DNA damage in vitro.

Support or Funding Information

This research was supported by a Scholarly and Artistic Activity Grant from University of the Pacific.

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 miR‐503‐5p exacerbates hypoxia/reoxygenation injury in H9C2 cardiomyocytes
Authors: He Yanjing , Cai Yin , Liu Hao , Irwin Michael G , Xia Zhengyuan
Published: June 2021

Acute myocardial infarction (MI) is a leading cause of death worldwide, and the most effective therapy for reducing acute myocardial ischemic injury is myocardial reperfusion, while reperfusion itself can lead to further cardiomyocyte death, called ischemia/reperfusion injury (IRI). A number of microRNAs have been shown to be involved in IRI, however, the role of miR‐503‐5p, which is implicated in cancers, diabetes mellitus or pulmonary arterial hypertension, remains unknown in myocardial IRI. This study was aimed to investigate the effects of miR‐503‐5p in myocardial IRI. Cardiac miR‐503‐5p expression was measured in mice subjected to myocardial IRI achieved by occlusion of left anterior descending artery for 30 minutes followed by reperfusion for 2 hours. H9C2 cardiomyocytes hypoxia/reoxygenation (H/R) was achieved by exposing the cells to glucose deprivation and hypoxia for 10 hours followed by reoxygenation 6 hours. We found that cardiac miR‐503 expression level was down‐regulated after induction of IRI compared with control, and it was also down‐regulated in H9C2 cells after exposure to H/R. In vitro, cell viability was decreased after H/R treatment, and lactate dehydrogenase (LDH) activity was increased. Administration of an agomir designed to overexpress miR503‐5p in cells before inducing H/R further reduced cell viability and elevated LDH activity. Moreover, proapoptotic Bax protein showed no significant change after overexpression of miR‐503‐5p in both normal and H/R states, while anti‐apoptotic Bcl protein decreased significantly. We analyzed putative miR‐503 targets using the TargetScan software, and found that the phosphoinositide‐3‐kinase regulatory subunit 1(PI3K p85) was one of the target genes. After overexpression of miR‐503‐5p, we found that PI3K p85 was decreased in both normal and H/R states. In addition, Akt1 and Akt2 as well as Phospho‐Akt were decreased in H/R state, and were further decreased after miR‐503‐5p overexpressed. Furthermore, we also found that prosurvival protein phosphorylation of signal transducer and activator of transcription (STAT3) was decreased after exposure to H/R, and declined further after overexpression of miR‐503‐5p. It is concluded that miR‐503‐5p regulates H9C2 cardiomyocyte injury during H/R, and exacerbates cell injury by inhibiting the activation of prosurvival protein Akt and STAT3.

Support or Funding Information

Supported by Hong Kong RGC(17158616 M) and HMRF (05161826)grants.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calibration and characterization of intracellular Asante Potassium Green probes, APG‐2 and APG‐4
Authors: Rana Priyanka S. , Gibbons Brandon A. , Alexy Vereninov A. , Yurinskaya Valentina E. , Clements Robert , Model Timothy M. , Model Michael A.
Published: June 2021

The response of fluorescent ion probes to ions is affected by intracellular environment. To properly calibrate them, intracellular concentration of the measured ion must be made equal to its extracellular concentration. In the first, computational, part of this work, we show, using the example of potassium, that the two requirements for ion equilibration are complete dissipation of membrane potential and high membrane permeability for both potassium and sodium. In the second part, we tested the ability of various ionophores to achieve potassium equilibration in Jurkat and U937 cells and found a combination of valinomycin, nigericin, gramicidin and ouabain to be the most effective. In the third part, we applied this protocol to two Asante Potassium Green probes, APG‐4 and APG‐2. APG‐4 shows good sensitivity to potassium but its fluorescence is sensitive to protein density; therefore, calibration buffer had to be supplemented with 50 mM sucrose to keep cell volume constant. With these precautions taken, the average potassium concentrations in U937 and Jurkat cells were measured as 132 mM and 118 mM, respectively. The other tested probe, APG‐2, is non‐selective for cations; this is, however, a potentially useful property because the sum [K+] + [Na+] determines the amount of intracellular water.

Support or Funding Information

The research was partly supported by the Kent State University Research Council (MM) and by the Russian Fedration grant 0124‐2018‐0003 (VY and AV)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heparin‐avidin interaction interferes with the XPerT permeability assay
Authors: Furman William , Garrett Joshua
Published: June 2021

The endothelium is a monolayer of cells that line the vasculature and act as a semi‐permeable barrier controlling what passes from the lumen of blood vessels into the interstitium. Acute changes in endothelial permeability are commonly seen during inflammation. There are several in vitro assays which allow the researcher to look at changes in endothelial permeability under various inflammatory conditions, one of which is known as the XPerT assay. In this assay, endothelial permeability is assessed using fluorescence microscopy and is based on high affinity interactions between ligand (Alexa fluor‐488‐conjugated avidin) added in the culture medium, which permeates cell monolayer to reach substrate‐bound acceptor (biotinylated gelatin). Interestingly, heparin is known to strongly bind avidin and is also commonly found in different formulations of endothelial media in wildly differing concentrations. From this, we hypothesized that media with high concentration of heparin would interfere with the XPerT assay. To test this, we added increasing amounts of heparin to the XPerT assay and found that high doses impeded the ability of the avidin to interact with the biotinylated gelatin. Furthermore, we found that when high doses of heparin were present, the avidin was more likely to bind directly to the endothelial cells and not the biotinylated gelatin. Taken together, our data suggests that using a low heparin containing endothelial medium is optimal for using the XPerT assay to test endothelial permeability.

Support or Funding Information

This work was supported in part through the Hartwick College Faculty Research Grants Program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stromal Interaction Molecule and Gelsolin Mediate Pathological Cardiac Remodeling
Authors: Mancarella Salvatore
Published: June 2021

Pathological cardiac remodeling is characterized by a series of poorly understood changes in myocardial microstructure involving changes in the cardiomyocytes cytoskeleton. Stromal interaction molecule 1 (STIM1) resides mostly in the sarco/endoplasmic reticulum where is senses intraluminal Ca2+ levels and activates the Orai channels on the plasma membrane to initiate Ca2+ influx. We have previously shown that STIM1 is involved in the dynamic remodeling of the actin cytoskeleton. However, the downstream effectors of STIM1 leading to cytoskeletal remodeling are not known. Proximity‐labeling technique (BioID) can capture weak and transient protein‐protein interactions. Hence, in the present study we investigated the STIM1 interactome used the BioID technique. A promiscuous biotin ligase BirA* was fused to the cytoplasmic C‐terminous of STIM1 and generated MEF stable cell line expressing STIM1‐BirA*. Biotin was added to the culture medium of MEF expressing STIM1‐BirA as well as in control cells not expressing STIM1‐BirA. Cells were harvested after twenty‐four hours and total lysates were incubated with streptavidin magnetic beads to pull down the biotinylated proteins which were subsequently identified by mass spectrometry. STIM1 interactors were validated in neonatal rat cardiac myocytes (NRCM) coupled with biochemical, immuno‐staining techniques and confocal microscopy. Screening of biotinylated proteins identified several high confidence known and previously unknown interactors of STIM1. One of the novel interactors was Gelsolin (GNS), a Ca2+‐dependent actin‐severing protein that is a key regulator of actin filament assembly and disassembly. STIM1 and GSN relationship was further investigated using NRCM. We show that STIM1 upregulation in NRCM was enough to induce upregulation of GNS protein levels. Immunostaining confirms colocalization between STIM1 and GSM. Furthermore, STIM1 overexpression in NVCM show altered actin dynamic and tubulin overexpression probably in response to cytoskeletal instability. We report the first use of BioID to characterize the STIM1 interactome. Our work unravels an unprecedented role of GSN as a part of the STIM1 interactome and its role in the modulation of the actin cytoskeleton.

Support or Funding Information

We gratefully acknowledge funding from the the National Heart, Lung and Blood Institute (award HL114869, to Dr. S.M.), and from UTHSC seed funding.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Decreased adiposity and enhanced glucose tolerance in Zyflamend treated mice
Authors: Alquraishi Mohammed , Donohoe Dallas , Voy Brynn H. , Whelan Jay , Bettaieb Ahmed
Published: June 2021

Obesity is a growing epidemic that presents a major health problem worldwide. Obese individuals are at a higher risk of chronic diseases including cardiovascular diseases, nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. Zyflamend is a poly‐herbal supplement derived from the extracts of ten different herbs that effectively activates AMPK in‐vitro in multiple cancer cell lines. When activated, AMPK is instrumental in inhibiting lipogenesis and enhancing catabolic pathways that generate ATP, such as fatty acid oxidation. The objective of this study is to investigate the effects of Zyflamend on adiposity and glucose homeostasis in vitro and in‐vivo. To that end, we fed mice chow, high fat diet, or high fat diet supplemented with Zyflamend over a period of 24 weeks and we examined alterations in body weight and glucose homeostasis. In addition, we treated human and mouse pre‐adipocytes with Zyflamend and examined changes in adipogenesis and key signaling pathways were determined in vitro. Mice fed High fat diet supplemented with Zyflamend displayed decreased weight gain and resistance to HFD‐induced glucose intolerance. Further, Zyflamend treatment decreased HFD‐induced hepatic dyslipidemia. These findings correlated with decreased adipogenesis of mouse and human adipocytes in‐vitro. Additionally, Zyflamend enhanced adipose insulin signaling as evidenced by increased tyrosyl phosphorylation of the insulin receptor and enhanced downstream signaling.

Conclusion

These studies identify Zyflamend as a potential regulator of systemic glucose tolerance, energy balance and adiposity.

Support or Funding Information

The work was supported by NIH R00DK100736 (AB)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepatic ZIP8‐mediated selenium transport contributes to normal liver function
Authors: Liu Liu , Liu Zijuan
Published: June 2021

ZIP8 is a multi‐functional electroneutral membrane transporter with substrates including both essential and toxic divalent metals (e.g. zinc, manganese, iron and cadmium) and micronutrient selenite. Despite that ZIP8 is found to be associated with metabolic disorders by GWAS, the physiological and pathological roles of ZIP8 remain to be characterized and its connection to any metabolic pathways has not been identified. Our preliminary results demonstrate that moderate or acute decreases in ZIP8 activity resulted in hepatic pathology. Chronic moderate ZIP8 deficiency in transgenic mice led to spontaneous liver neoplastic nodules with increased oxidative stress, inflammation and fibrosis. And acute ZIP8 decrease in mice via adenovirus induced significant hepatomegaly, injury, inflammation and proliferation in liver. In addition, we found that elevation of ZIP8 induced significant glycogen accumulation while downregulation of ZIP8 decreases the glycogen level in primary hepatocytes and liver. Along with these liver pathology, the selenium level and selenoproteins (including GPX1 and GPX2) significantly decreased after ZIP8 deficiency. Thus ZIP8 plays an important role in maintaining normal hepatic function, likely through regulating selenium homeostasis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diverse Regenerative Strategies of the Intestinal Epithelium
Authors: Klein Ophir
Published: June 2021

Epithelial surfaces form critical barriers to the outside world and are continuously renewed by adult stem cells. Whereas epithelial stem cell dynamics in homeostasis are increasingly well studied, how stem cells are redirected from a tissue‐maintenance program to initiate tissue repair after injury remains incompletely understood. Our initial work focused on the interrelationship between stem cell populations in the gut, and in this talk I will present more recent studies examining the response of the intestinal epithelium to damage. Using a parasitic helminth that disrupts tissue integrity during its life cycle as a model, we found that tissue damage leads to re‐initiation of a developmental program, thus representing a fundamental and novel mechanism by which the intestinal crypt can remodel itself to sustain function after injury. I will also discuss our experiments using live imaging of adult intestinal organoids, which show that nascent daughter dispersal occurs when neighboring cells insert between them during cytokinesis. Our data suggest that the mechanics of progenitor cell division provide a driving force for cell mixing in elongated mammalian epithelia.

Funding provided by NIH and CIRM.

Support or Funding Information

Funding provided by NIH and CIRM.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Conformational Resilience of Protein Disulfide Isomerase
Authors: Guyette Jessica , Evangelista Baggio , Tatulian Suren A. , Teter Ken
Published: June 2021

Protein disulfide isomerase (PDI) is a 57 kDa protein with both oxidoreductase and chaperone activities that is mainly localized to the endoplasmic reticulum. It is a U‐shaped protein with an abb'xa’ structural organization where the a and a′ domains have conserved CGHC active sites, the b and b′ domains are involved with substrate binding, and x is an unstructured linker. PDI exhibits substantial structural flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin, suggesting PDI can regain a folded, functional conformation after exposure to stress conditions. To determine whether the unfolding‐refolding cycle of PDI is a substrate‐induced process or an intrinsic physical propensity of PDI, we used circular dichroism to examine the structural properties of PDI subjected to thermal stress. PDI exhibited remarkable conformational resilience that is linked to its redox status. In the reduced state, PDI exhibited a 54°C unfolding transition temperature (Tm) and regained 84% of its native structure after 70% thermal denaturation. Oxidized PDI had a lower Tm of 48°C and only regained 44% of its native conformation after 70% denaturation. Additional studies documented increased thermal stability of a PDI deletion construct lacking the a′ domain and decreased thermal stability of a construct lacking the a domain. Both constructs were able to regain a portion of their native conformation after exposure to thermal stress and, like full‐length PDI, were more stable in the reduced form than the oxidized form. A bb'x PDI construct lacking both a and a′ domains was highly stable, with a 60°C Tm in the reduced state and a 56°C Tm in the oxidized state. Both reduced and oxidized forms of the bb'x construct could return to the native state after heating to 90°C. These findings provide new insight into the physical foundation of the biological function of PDI.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hsp90 Recognizes a Conserved Motif in the A Chains of ADP‐Ribosylating Toxins that Move from the Endoplasmic Reticulum to the Cytosol
Authors: Kellner Alisha , Teter Kenneth
Published: June 2021

Cholera toxin (CT) enters the cell via receptor‐mediated endocytosis and travels in a retrograde fashion to the endoplasmic reticulum (ER). The catalytic A1 subunit is then displaced from the rest of the holotoxin, unfolds, and is exported to the cytosol where it regains an active conformation for the ADP‐ribosylation of its G‐protein target. We have shown that the cytosolic chaperone Hsp90 is required for CTA1 translocation to the cytosol. We have also shown that Hsp90 is able to independently bind and refold CTA1. Using libraries of CTA1‐derived peptides, we have identified two distinct Hsp90 binding sites within the 192 amino acid protein, an N‐terminal RPPDEI (CTA111–16) motif and a C‐terminal LDIAPA (CTA1153–158) motif. Peptides corresponding to either site were able to block Hsp90 from binding to full‐length CTA1, and both binding sites were required for translocation of CTA1 to the cytosol. The LDIAPA motif is unique to CTA1, but an RPPDEI‐like motif is present in four other ER‐translocating ADP‐ribosylating toxins: pertussis toxin, Pseudomonas aeruginosa exotoxin A, Escherichia coli heat‐labile toxin, and Salmonella Typhimurium ADP‐ribosylating toxin. Using site‐directed mutagenesis to further investigate the RPPDEI motif, we found that a modification of either proline residue blocks CTA1 translocation to the cytosol. Our work has identified, for the first time, specific amino acid sequences that are recognized by Hsp90 and are essential for toxin translocation from the ER to the cytosol.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.