bluuebunny/crossref_metadata_2025_split huggingface dataset:
C16‐Ceramide directly binds and activates p53 in response to cellular stress
Authors: Jeffries Kristen , Krupenko Natalia 
Published: June 2021

Ceramide is a key bioeffector in cellular adaptation to stress, and increased ceramide generation often functions as a pro‐apoptotic signal. Another key mediator of apoptosis, the p53 tumor suppressor protein, responds to diverse stress stimuli through its role as a transcription factor. We observed that folate withdrawal leads to CerS6 up‐regulation and C16‐ceramide accumulation in a p53‐dependent manner as a pro‐apoptotic cue, and hypothesized that p53 activation could result from the direct binding of C16‐ceramide to p53, which disrupts the protein interaction with its negative regulator, MDM2. Pulldown with biotinylated C16‐ceramide and membrane binding assays confirmed that p53 binds C16‐ceramide with high affinity and selectivity towards ceramide acyl chain length. In agreement, C16‐ceramide treatment of cultured cells disrupted the interaction between MDM2 and p53 in bimolecular fluorescence complementation experiments. The physiological relevance of this mechanism of p53 activation was demonstrated in serum starved cells where CerS6‐dependent C16‐ceramide generation mediated the stress response. Strikingly, the siRNA silencing of CerS6 prevented both p53 and p21 accumulation in response to serum starvation and partially rescued cells from G0/G1 arrest. These findings establish C16‐ceramide as the first physiologic metabolite activating p53 through the direct binding to the protein. Future experiments will determine the mechanism for the loading of C16‐ceramide onto p53 in cellular stress response.Support or Funding InformationNIH CA193782‐01This 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 Cks2 localization and interaction with Cdk1 in mitotic cells of Xenopus laevis embryos
Authors: Lu Zhiye , Kim Yeaseul , Sandquist Joshua C. 
Published: June 2021

Although mitotic regulation has been extensively studied, several of the mechanisms behind this process have yet to be fully understood. Our group is particularly interested in mechanisms regulating the timing of mitotic exit and how such mechanisms coordinate with other mitotic cues such as spindle orientation. Recent work in our lab has shown that spindle movements in epithelial cells of the developing frog embryo correlate with mitotic exit and perturbations in normal spindle movements lengthen metaphase. Further, these changes in mitotic progression are accompanied by increases in Cdk1 Tyr15 phosphorylation, specifically at cell junctions. We propose that the link between spindle movement and mitotic progression is an important part of the mechanisms influencing spindle orientation in the frog epithelium. Our current work examines the role of Cks2 in the aforementioned mechanism. Cks2 serves as a targeting subunit for the mitotic kinase Cdk1. Specifically, work in yeast suggests Cks2 may direct Cdk1 toward the anaphase‐promoting complex (APC) and interact with both complexes, facilitating the phosphorylation of Cdk1 and activation of the APC and thus help control mitotic exit. In this study, we characterize Cks2's mitotic function in Xenopus laevis. Our aim is to characterize Cks2 distribution throughout mitosis and determine whether perturbing Cks2 function alters mitotic progression in the gastrula‐stage epithelium. We hypothesize that if Cks2 influences Cdk1 function in mitosis, then the two proteins will exhibit similar localization patterns in mitotic cells. We visualized endogenous Cks2 and also expressed GFP‐tagged Cks2 in fixed epithelial tissues of Xenopus laevis embryos through confocal microscopy. Our early data show that Cks2 localization overlaps with that of a phosphorylated Cdk1(pY15) at cell junctions and overexpression of GFP‐tagged Cks2 results in increased levels of Cdk1‐pY15. In addition, the assay expressing a dominant negative version of Cks2 suggests that depletion of endogenous Cks2 activity may contribute to decreased Cdk1‐pY15 at cell junctions and increased cell area. Therefore, these data suggest a link between Cks2 and Cdk1 in mitotic regulation and are a first step toward a greater characterization of Cks2's role in regulating mitotic progression in vertebrate organisms.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Zyflamend Induces Apoptosis in Pancreatic Cancer Cells via Modulation of the JNK Pathway
Authors: Bettaieb Ahmed Bettaieb , Puckett Dexter , Chahed Samah , Whelan Jay 
Published: June 2021

Pancreatic cancer is the fourth leading cause of death in the United States with over 53,670 new cases and 80% mortality in 2017. The overall prognosis and survival rate of this diagnosis are grim, and in the vast majority of cases, terminal. Current pharmacological therapies and treatments targeting pancreatic cancer have proven ineffective far too often. Therefore, there is an urgent need for alternative therapeutic approaches. Zyflamend, an anti‐inflammatory herbal compound, has proven effective in various in‐vitro and in‐vivo cancer platforms, shows promise. However, its effects on pancreatic cancer in particular, remain largely unexplored. The objective of this study is to investigate the effects of Zyflamend on pancreatic cancer cells survival and decipher the underlying molecular mechanisms. Pancreatic cancer cells were treated with Zyflamend and key signaling pathways involved in the survival and proliferation were investigated. Our studies show that Zyflamend treatment caused a dose‐dependent decrease in cell proliferation, consistent with an increase in apoptotic cell death and activation of the JNK pathway. Additionally, Zyflamend in conjunction with chemotherapeutic compounds exhibited a synergistic effect against pancreatic cancer. Inhibition of JNK abrogated the pro‐apoptotic effects of Zyflamend. These studies identify Zyflamend as a potential novel approach to treat pancreatic cancer via modulation of the JNK pathway.Support or Funding InformationThis work was supported by a grant from NIH/NIDDK R00DK100736 to ABThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Multi‐Resistance Bacteria by Acetone Extract of Cola acuminata
Authors: Telles Caroline M , McKinsey Cayla , Gray Wesley 
Published: June 2021

The rapid emergence of multidrug resistant pathogens has created an urgent need for discovering novel antibiotics with natural plant products as potential antimicrobial agents. Cola acuminata (Bizzy Nut), possesses a variety of bioactive compounds which exhibit important bioactive activities against the growth of certain mammalian cell lines, bacteria and fungi. However, the specific chemicals responsible for the antimicrobial bioactivity have not been identified. Therefore, we investigated a bioactive extract from Cola acuminata for the presence of antimicrobial activity using two multidrug resistant pathogenic bacterial species, Staphylococcus aureus and Klebsiella pneumoniae. The specific bioactive compounds present in C. acuminata was partially purified using sequential extraction with solvents of increasing polarity, followed by SPE purification. The antimicrobial activity associated with each extraction was evaluated using the agar diffusion and MIC assay screened with Staphylococcus aureus as a model multidrug resistant pathogenic organism. An enriched antimicrobial activity (Biz‐3w) obtained from the acetone extract (Biz‐3) and SPE purification was effective against both Gram‐positive and Gram‐negative bacteria. We observed a dose and time dependent inhibition of Staphylococcus aureus, Bacillus subtilis and Enterococcus faecalis with an effective dose (ED50) of 4 ug/ml, 3 ug/ml, and 3.2ug/ml respectively. The order of potency of Biz‐3w was Bacillus subtilis ≥ Staphylococcus aureus > Enterococcus faecalis. The results obtained in this study provide preliminary evidence of the presence of secondary metabolites in C. acuminata capable of inhibiting multidrug resistant bacteria.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcium‐sensing Receptor Internalization is β‐arrestin‐dependent and Modulated by Allosteric Ligands
Authors: Bräuner‐Osborne Hans , Mos Iris , Jacobsen Stine Engesgaard , Foster Simon Richard 
Published: June 2021

G protein‐coupled receptor (GPCR) internalization is a complex process responsible for the transport of GPCRs from the cell‐surface into intracellular compartments. Receptor internalization is crucial for the termination of GPCR activity and in some cases linked to the initiation of G protein‐independent signalling pathways and endosomal receptor signalling. Internalization has been widely studied in class A GPCRs, where the most prevalent mechanism after receptor activation is initiated by kinase‐mediated phosphorylation of intracellular loops and the C‐terminus, followed by β‐arrestin recruitment and dynamin driven internalization via clathrin‐coated pits. However, the mechanism and clinical relevance in class B and C GPCRs remain largely unknown.The extracellular calcium‐sensing receptor (CaSR) is a prototypical class C GPCR that plays a pivotal role in calcium homeostasis due to continuous sensing of small changes in blood ionized calcium (Ca2+) levels. Impressively, CaSR is able to tightly control its activity despite being constantly exposed to its endogenous agonist. Previous studies investigating CaSR internalization were based on functional assays, and microscopy‐ or flow cytometry‐based analyses. However, there is no general agreement on the pathway(s) involved in CaSR internalization. Overall, the mechanism of CaSR remains to be poorly understood.In this study, we aimed to investigate CaSR internalization using the recently developed TR‐FRET‐based real‐time internalization assay for the first time. We demonstrate that Ca2+ induces CaSR internalization in a concentration‐dependent manner with an EC50 of 3.3 mM, which aligns well with the IP1 response with an EC50 of 3.2 mM. We show that internalization can be positively modulated by NPS R‐568 and negatively modulated by NPS 2143. Through the usage of CRISPR‐Cas9 edited HEK293 cells, we demonstrate that CaSR internalization is β‐arrestin dependent.Support or Funding InformationThis project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement NO 675228, the Lundbeck Foundation, the Carlsberg Foundation, the Augustinus Foundation and the Toyota Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Osteogenic and chondrogenic differentiation effect of neohesperidin dihydrochalcones in human adipose‐derived stem cells
Authors: Heo Hee Sun , Cha Ji Hyun , Lee Jong Hun 
Published: June 2021

Reactive oxygen species (ROS) are highly reactive which cause damage to various cellular functions such as proliferation, differentiation, and survival. Nuclear factor erythroid 2‐related factor‐2 (Nrf2) promotes expression of antioxidant enzyme such as heme oxygenase 1 (HO‐1) and NAD(P)H:Quinone Oxidoreductase 1 (NQO‐1). Neohesperidin is a compound originated from peel of citrus fruits and its hydrogenated form, neohesperidin dihydrochalcone (NHDC) has antioxidant activity. Human adipose‐derived stem cells (hADSC) are multipotent adult stem cells responsible for the regeneration of fat, bone and cartilage. Thus, we observed the antioxidant effects of NHDC derivatives on hADSC through Nrf2 activation. Upregulation of ROS inhibits osteogenic differentiation of hADSCs, in part through inhibition of signaling pathway, which is essential for bone development and maintenance. ROS levels regulate inhibition of proliferation and modulate initiation of the hypertrophic changes in chondrocytes. Cellular viability and compounds' proper concentrations were set by MTS assay. The effects of these compounds on suppressing oxidative stress and hADSCs differentiation into osteocytes and chondrocytes was investigated using Alizarin Red staining, Alcian Blue staining, reverse‐transcriptase polymerase chain reaction (RT‐PCR), and Western blotting. The results demonstrate that NHDC derivatives play an efficacious role in inducing osteogenic and chondrogenic differentiation of hADSCs through reducing cellular oxidative stress.Support or Funding InformationThis research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education 2018R1D1A1B07049077This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessment of Cellular Respiration and Oxidative Phosphorylation in Cocks Semen after Freezing
Authors: Nikitkina Elena , Musidray Artem , Timofeeva Svetlana , Krutikova Anna , Plemyashov Kirill , Pleshanov Nikolay 
Published: June 2021

The biochemical processes associated with the generation of energy (ATP) ‐ oxidative phosphorylation and glycolysis ‐ occupy a central place in the vital activity of cells. Numerous studies have established that the ratio of respiration and phosphorylation is a sensitive indicator of the functional state of the cell and the whole organism. Oxidative phosphorylation (OXPHOS) is the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing energy which is used to produce adenosine triphosphate (ATP). OXPHOS is the terminal process of cellular respiration (CR). Aim of the study was to evaluate the ratio of respiration and phosphorylation in cocks sperm after freezing. Semen from 10 cocks was frozen. CR was assessed by the polarographic method. OXPHOS can be evaluated by the reaction CR on adding 2,4‐dinitrophenol (2,4 ‐ DNP ) to semen sample. 2,4 ‐ DNP disrupt the proton gradient by carrying protons across a membrane and uncouples proton pumping from ATP synthesis because it carries protons across the inner mitochondrial membrane. As a result it increases respiration rate. If the respiration rate increases – there is good OXPHOS. Results are depicted as ranges, percentages of totals and averages ± SEs. The addition of 2,4 ‐ DNP to sperm samples caused a significant increase in CR (1.8 ± 0.40 times). This suggests that the CR of freshly diluted cocks sperm is highly associated with phosphorylation. We observed a drop in respiratory stimulation by 2,4 ‐ DNP (1,7±0,11 times) after thawing. This is an indication of an increase in free oxidation not associated with oxidative phosphorylation. The CR stimulation by 2,4‐DNP in the frozen samples ranged from 1.4 to 2.3, and the fertility of eggs ranged from 24 to 95%. The correlation between egg fertilization and stimulation of respiration by 2,4‐DNP was high (r = 0,76, P <0,01). Assessment of oxidative phosphorylation can serve as a marker of sperm viability after freezing and as criterion for predicting the fertilizing ability.Support or Funding InformationAuthors acknowledge financial support from Russian Science Foundation, Grant No: 18‐16‐00071This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NAG‐1/GDF15 inhibits HFD‐induced obesity and inflammation by modulating gut microbiota
Authors: Sang Tingting , Guo Chenjie , Guo Dandan , Na Kun , Wu Kaikai , Li Kang , Wang Xingya 
Published: June 2021

Nonsteroidal anti‐inflammatory drug‐activated gene (NAG‐1) or growth differentiation factor 15 (GDF15) is a divergent member of the transforming growth factor beta (TGF‐β) superfamily. Mice expressing NAG‐1/GDF15 have been shown to be resistant to high fat diet (HFD)‐induced obesity and inflammation. However, the exact mechanism underlying the anti‐obesity effects of NAG‐1/GDF15 remains unknown. The objective of this study was to examine whether the anti‐obesity effects of NAG‐1/GDF15 is associated with gut microbiota modulation and gut barrier function regulation. First, we confirmed that NAG‐1/GDF15 Tg mice are resistant to dietary induced obesity and inflammation. These mice also demonstrated improved insulin sensitivity compared to Wt littermates. We then found these phenotypic differences were correlated with differences in the composition of gut microbiota, gut barrier function and short chain fatty acids compositions in feces as determined by standard molecular biological methods. In addition, HFD reduced the expression of Cyp7a1, Cyp7b1, Cyp27a1, Ntcp, Mdr2, and Oatp that regulate bile acid metabolism in liver. However, the expression of these genes were increased in NAG‐1/GDF15 Tg mice. Furthermore, antibacterial peptide (Reg3g, Lyz‐1 and Defa) and MUC2 in small intestine were up‐regulated in NAG‐1/GDF15 Tg mice. We also found that NGA‐1/GDF15 down‐regulate the expression of TLR4 and its downstream signals in fat, liver and small intestines at both mRNA and protein levels as determined by Western blot and qRT‐PCR. Finally, we transplanted feces from NAG‐1/GDF15 mice into pseudo germ‐free mice and fed the mice with HFD and found that a shift in gut microbiota composition from NAG‐1/GDF15 mice could inhibit the development of obesity and inflammation. Taken together, our results indicate that the protection role of NAG‐1/GDF15 against HFD‐induced obesity and inflammation is mediated, at least in part, by the modulation of gut microbiota, function of gut barrier, and bile acid metabolism.Support or Funding InformationNational Natural Science Foundation of China Grant No. 81400822.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 Reassessment of Phosphatidylinositol Transfer Protein Alpha's Growth Factor Signaling Role
Authors: McDermott Mark I. , Diz Ramiro , Hur Seong , Lete Marta G. , Applebee Christopher J. , Grabon Aby , Tripathi Ashutosh , Wakelam Michael J. O. , Larijani Banafshe , Bankaitis Vytas A. 
Published: June 2021

In mammals, the START‐like phosphatidylinositol transfer proteins (PITPs) remain an understudied group of proteins implicated in human health and disease. Phosphatidylinositol transfer protein alpha (PITPa) is linked to human epidermal growth factor receptor (HER2)‐positive metastatic breast cancer drug resistance, and muscular dystrophy, but its precise mechanism of action is unclear. Current dogma states that PITPa is essential for growth factor signaling through the epidermal growth factor (EGFR) and netrin signaling pathways, but little data has emerged supporting these claims. Data from our laboratory, from multiple experimental systems, strongly dispute PITPa requirement in these pathways. Strikingly, mouse and human cell lines lacking PITPa sense and chemotax towards EGF, and signaling responses downstream of EGFR‐stimulation are unimpaired, including phosphatidylinositol (3,4,5)P3 generation, and phosphorylation of protein kinase B (Akt). Surprisingly, lack of PITPa in human cells results in potentiation of Akt phosphorylation (threonine 308) following EGFR stimulation, indicating that contrary to current thinking, PITPa may actually repress this pathway. The dispensability of PITPa for EGF‐signaling explains the remarkable absence of EGF‐signaling associated phenotypes in PITPa nullizygous (Pitpa0/0) mice. We have previously reported that Pitpa0/0 animals exhibit a complex phenotype including spinocerebellar neurodegeneration characterized by gliosis and neuronal apoptosis of cerebellum, and hindbrain, and that neuronal specific eviction of embryonic PITPa and PITPb results in progeny born lacking a forebrain. Axon guidance and mouse forebrain development require netrin‐1 signaling through the netrin receptor (DCC), a process reported to require PITPa. We demonstrate that while compromise of netrin‐signaling results in structural brain derangements, these defects do not result from absence of PITPa. Furthermore, we fail to reproduce the previously reported physical interaction between the netrin receptor (DCC) and PITPa. In light of our findings the ‘essential’ role of PITPa in growth factor signaling must be reevaluated.Support or Funding InformationThis work was supported by the Robert A. Welch Foundation (BE0017), and a National Institutes of Health grant (GM44530) awarded to Vytas A. BankaitisThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased hexokinase interactions with mitochondria protect against cell damage
Authors: Sampe David 
Published: June 2021

Deaths due to cancer and ischemic heart disease are the most common; dysfunction of mitochondrial energy (ATP) metabolism is key in these pathologies. Therefore, therapeutic strategies, which remain elusive, have focused on energetic metabolism. Cells use glucose and oxygen in glycolysis and oxidative phosphorylation to generate ATP. The cytosolic protein hexokinase (HK) plays a crucial role in glycolysis by catalyzing the breakdown of glucose to produce most of the ATP generated from mitochondria. For efficient glycolysis, HK must access the mitochondrial ATP, which flows out via the voltage dependent anion channel (VDAC), a 30‐kDa protein in the outer mitochondrial membrane (OMM). Cancer cells grow and proliferate by increasing access to ATP via increased HK‐VDAC associations. This strategy has potential implications for protection against cardiac ischemic injury. Ischemia promotes dissociation of HK from VDAC leading to cardiac damage, but cardio‐protective interventions can increase HK‐VDAC association, preserving function. The overall objective is to determine the biophysical and biochemical features that lead to HK‐VDAC association or dissociation. HK, a 100‐KDa dimeric protein, has 918 amino acids in two domains: an N‐terminus and C‐terminus joined by a linker a‐helix. HK interactions with mitochondria are not well known but, HK, with its non‐polar sequence of 15 amino acids on the N‐terminus, may insert in the OMM close to VDAC. Glucose and glucose‐6‐phospate have distinct binding sites on HK. Glucose binds at K173, D209, and E260 in the N‐terminus and at K621, D657, and E708 in the C‐terminus. The Brown Deer High School SMART Team designed a model of HK using 3‐D printing technology to investigate structure‐function relationships. Understanding HK‐VDAC binding mechanisms could provide better targeted approaches to manage pathologies, including cancer, cardiac and neurodegenerative diseases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.