bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Iron Metabolism Receptors, TfR1 and TfR2, Bind and Stabilize Transferrin Through Non‐Conserved Interactions
Authors: Kleven Mark Daniel , Enns Caroline 
Published: June 2021

Hereditary hemochromatosis, a disease marked by chronic iron overload from insufficient expression of the hormone hepcidin, is one of the most common genetic diseases in people of Northern European ancestry. One form of HH (Type III) results from mutations in the transferrin receptor‐2 (TfR2). How TfR2 functions to promote hepcidin expression remains unclear, though it appears to be an important part of a mechanism to modulate hepcidin expression based on systemic iron levels. This responsiveness may be achieved through TfR2's competition with its homolog, TfR1, for their ligand, the iron transport protein, transferrin (Tf). TfR1 is expected to be the preferred receptor for diferric, or holo‐, Tf and that TfR2 will bind holo‐Tf when Tf‐iron saturation levels are elevated. To better understand the nature of these protein‐protein interactions, a surface plasmon resonance binding study with the full‐length receptors was conducted for the first time. In agreement with previous studies, holo‐Tf binds to TfR1 significantly stronger than TfR2. However, the binding constant for Tf‐TfR2 is still far below that of physiological holo‐Tf levels, inconsistent with the hypothetical model and suggests that other factors mediate the interaction. Apo‐Tf only weakly binds TfR2 at serum pH, thus is not be able to effectively compete with holo‐Tf under these conditions. Like TfR1, TfR2 is able to bind apo‐Tf with higher affinity at the lower pH of endosomes, suggesting that it could function to take up iron, where stabilization of the apo‐Tf‐TfR complex is key. Tf‐binding Tf‐binding to a TfR2 chimera containing the TfR1‐helical domain indicates that the differences in the helical domain account for differences in on‐rate of Tf, but non‐conserved inter‐receptor interactions are necessary stabilize the Tf‐TfR1/TfR2 chimera complex. One possible site of stabilization, the apical arm junction, is not important for TfR1‐Tf complex stabilization. The junction is critical for the TfR2‐Tf interaction, however; highlighting one non‐conserved stabilization mechanism. Our results highlight the differences in Tf interactions with the two TfRs.Support or Funding InformationNIH T‐32 Training Grant GMMBI0231C; NIH R01DK072166This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LIMD2 is an intracellular activator of Integrin Linked Kinase(ILK) activity and GSK‐3/Akt/beta‐catenin signaling
Authors: Dedhar Shoukat , Awrey Shannon 
Published: June 2021

Integrin‐Linked Kinase (ILK) is an intracellular, focal adhesion, scaffold protein that couples beta 1‐integrins and growth factors to actin cytoskeletal organization and signal transduction via GSK‐3 beta and Akt. ILK has an atypical kinase catalytic domain, and has been considered to be a pseudokinase.Here, we demonstrate that a small LIM domain only protein, LIMD2, which is highly overexpressed in several types of cancers, and which interacts directly with ILK within the kinase domain ( Peng, H et al, Cancer Res, 2014), forms a complex with ILK in cancer cells and stimulates ILK kinase activity. LIMD2 overexpression results in the stimulation of the phosphorylation of GSK‐3 beta on Ser‐9, as well as of Akt on Ser‐473, in an ILK‐dependent manner. Furthermore we demonstrate a striking LIMD2 induced stimulation of phosphorylation of beta‐catenin on Ser‐552, shown previously to be a target of Akt, leading to 14.3.3‐dependent stablization of beta‐catenin and stimulation of Lef/TCF transcriptional activity that results in tumor cell invasion (Fang et al. J Biol Chem, 2007).Since LIMD2 upregulation in tumor cells is associated with increased cell invasion and metastasis, we examined the effect of inhibiting LIMD2 induced ILK activity on breast, thyroid and pancreatic cell invasion through collagen and matrigel. As expected, overexpression of LIMD2 induced tumor cell invasion, and this invasion was blocked by ILK siRNA or by a specific pharmacological inhibitor of ILK, QLT‐0267. Inhibition of ILK in these cells also resulted in dramatic suppression of Akt Ser‐473 and beta‐catenin Ser‐552 phosphorylation as well as of LEF‐TCF transcrptional activity.In summary, we describe a new paradigm for the intracellular activation of low activity protein kinases through protein‐protein interactions. Specifically we identify a new signaling axis through the activation of ILK by LIMD2, which results in the stimulation of a GSK‐3/Akt/Beta‐catenin pathway important in tumor cell invasion.Support or Funding InformationSupported by funds from the Cancer Research Society, Quebec, CanadaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐metastatic activity of capsaicin and natural capsaicin‐like compounds in human lung adenocarcinoma
Authors: Richbart Stephen D. , Nolan Nicholas A. , Akers Austin T. , Colclough Kate W. , Hurley John D. , Williams Cathryn D. , Rollyson William D. , Brown Kathleen C. , Denning Krista L. , Brown Linda G. , Egleton Richard D. , Dasgupta Piyali 
Published: June 2021

Lung adenocarcinoma (LAC) accounts for 65% of all non‐small cell lung cancer (NSCLC) cases. A substantial proportion of LAC present with extrapulmonary and distant metastasis at the time of their diagnosis. One of the earliest events of the metastatic process is the invasion of malignant cells through the surrounding extracellular basement membrane into the blood and lymph. The long‐term goal of our laboratory is to identify nutrition‐based agents which will suppress the growth and progression of human LACs. Capsaicin is the pungent ingredient of chili peppers. Published reports have revealed that capsaicin inhibits the invasion and metastasis of several types of human cancers including melanoma, prostate cancer, and cholangiosarcoma. However, the clinical application of capsaicin as an anti‐cancer drug is limited by its unpleasant side‐effect profile. This led us to compare the anti‐metastatic activity of capsaicin with natural non‐pungent capsaicin‐like compounds, namely capsiate and capsiconiate. The structure and bioactivity of capsaicin closely resembles capsiate. There are no published reports involving the biological activity of capsiconiate. We measured the anti‐invasive activity of these compounds by two independent invasion assays, namely the Boyden chamber assay and spherical invasion assay. We found that capsaicin and capsiate displayed anti‐invasive activity in three human LAC cell lines. In contrast, capsiconiate did not suppress the invasion of any LAC cell lines. Furthermore, we tested the anti‐metastatic activity of capsaicin in a syngeneic mouse model of metastasis. We observed that the daily dietary administration of capsaicin in AIN‐76A diet (with 5% lipid level) robustly decreased the area metastatic foci (in the lung) relative to vehicle‐treated mice. The results of our studies may foster the development of novel anti‐metastatic therapies for human LAC.Support or Funding InformationFunding for our study was supported by an NIH R15‐AREA Grant (2R15CA161491‐02) and a NASA Undergraduate Fellowship to NANThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of metabolic profiling of leelamine in vitro and in vivo by LC‐MS/MS
Authors: Shrestha Riya , Lee Taeho , Lee Sangkyu 
Published: June 2021

Leelamine is a diterpene compound found in the bark of pine trees that is generating considerable interest owing to its potent anticancer properties. The present study aimed to investigate the metabolic profile of leelamine in human liver microsomes (HLMs) and mice using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). We found that leelamine undergoes only Phase I metabolism, which generates one metabolite that is mono‐hydroxylated at the C9 carbon of the octahydrophenanthrene ring (M1) both in vitro and in vivo. The structure and metabolic pathway of M1 were determined from the MSn fragmentation obtained by collision‐induced dissociation using LC‐MS/MS in HLMs. Cytochrome p450 (CYP) 2D6 was found to be the dominant CYP enzyme involved in the biotransformation of leelamine to its hydroxylated metabolite while CYP2C19, CYP1A1, and CYP3A4 contributed to some extent. Moreover, further investigation of the in vivo metabolism of leelamine in mice using urinary and fecal analysis detected only one metabolite, M1, in the urine but none in the feces. In conclusion, leelamine was metabolized to mono‐hydroxyl metabolite by CYP2D6 and mainly excreted in the urine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Milk plus Carbohydrate Supplementation during Interval Walking Training Enhanced the Improvement of Blood Glucose and Blood Pressure Regulations in Older People
Authors: UCHIDA KOJI , MASUKI SHIZUE , MORIKAWA MAYUKO , FURIHATA MAYUKA , MANABE KAZUMASA , OGAWA YU , KATAOKA YUFUKO , AIDA TAKAMICHI , NAKANO SAKURA , NOSE HIROSHI 
Published: June 2021

AimsWe examined whether the milk + carbohydrate (CHO) supplementation during interval walking training improved the symptoms of lifestyle‐related diseases with enhanced methylation of the pro‐inflammatory gene, NFKB2.MethodsTwenty nine subjects (~70 yr) having performed interval walking training for >6 months but with >130 mmHg of systolic pressure and >110 mg/dl of blood glucose participated in the study. After the baseline measurements of peak aerobic capacity (VO2peak) by graded walking test, blood constituents, blood glucose by continuous method (iPro2R), and carotid arterial compliance with Doppler ultrasound (Vivid7R) and non‐invasive arterial pressure measurement (FinometerR), we randomly divided subjects into two groups: MILK (6 men, 9 women) and CNT (6 men, 8 women) consuming either milk (10 g protein and 9.5 g CHO) + CHO (8.8 g) or CHO (7 g) alone during 5‐month interval walking training, respectively. After the training, we measured the same variables as before.ResultsIn MILK, VO2peak and carotid arterial compliance increased and mean blood glucose decreased with a reduced fluctuation after standardized meal intake during the day (all, P<0.05); however, these changes were minimal in CNT (all, P>0.06). This was accompanied by enhanced methylation of NFKB2 in MILK than CNT (P=0.045).ConclusionsMilk + CHO supplementation during interval walking training enhanced the improvement of lifestyle‐related diseases with enhanced NFKB2 methylation.Support or Funding InformationThis study was supported by a grant from the Japan Society for the Promotion of Science (15H01830).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Sphingosine ‐1‐Phosphate on Expression of MAPK and Akt Signaling Pathways in Hypoxic Human Extravillous Trophoblasts
Authors: Swan Kenneth F. , Pridjian Gabriella , Swayze Thomas , Gagen Brennan R. , Intapad Suttira 
Published: June 2021

Preeclampsia (PE), is a serious pregnancy disorder characterized by placental ischemia, hypertension, and proteinuria. A successful pregnancy requires the migration and invasion of extravillous trophoblasts (EVTs), from the villous tips of the placenta into the maternal decidua and subsequently the spiral arteries. Failure of EVTs to adequately migrate and invade into the spiral arteries leads to insufficient blood flow to the developing placenta commonly found in PE. Sphingosine‐1‐phosphate (S1P) functions as a key regulator of cellular homeostasis in angiogenesis, inflammation, and endothelium permeability. S1P acts on its receptor to control cell migration through the PI3‐Akt and p38 MAPK signaling pathways. We have previously reported that expression of placenta ceramidase, sphingosine kinases 1, and sphingosine kinases 2 were altered in reduced uterine perfusion (RUP) mouse model. In the present study, we tested the hypothesis that serine 473 phosphorylated Akt (pAkt) and phosphorylated p38 MAPK expressions are decreased in hypoxic condition and treatment with S1PR 1 agonist and antagonist alter these expression levels.MethodsThe human extravillous trophoblasts (EVTs) cell line SGHPL‐4 was cultured in either normoxia 21% oxygen or hypoxia 2% oxygen conditions for 24 hours. The cultures were treated for one hour with vehicle control or with S1PR1 receptor agonists (SEW2871, 1uM) or S1PR antagonist (W146, 18 nM).ResultsBoth pAkt and p38 MAPK were significantly decreased in hypoxia EVTs compared to normoxia EVTs (p38 MAPK: 50% and pAkt: 25% decrease). SEW2871 increased p38 MAPK in hypoxia EVTs to a level similar to normaxia EVTs. Inhibition of S1PR1 by W146 decreased p38 MAPK in both hypoxia and normoxia. SEW2871 normalized the differences of pAkt expression between normoxia and hypoxia EVTs. While W146 did not change the pAkt in normoxia, it significantly increased pAkt in hypoxia EVTs cells. This data suggests that activation of S1P receptor type 1 increases the expression of angiogenic pMAPK in hypoxic SGHPL‐4 trophoblast cells.Support or Funding InformationDr. Intapad was supported by funding from the AHA 16SDG27770041, ASN Kidney Research Career Development grant, and Start‐up funds from Tulane University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AT1aR Dependent GABAa Inhibition in the MnPO Following Chronic Intermittent Hypoxia
Authors: Farmer George E. , Little Joel T. , Marciante Alexandria B. , Cunningham J. Thomas 
Published: June 2021

Chronic intermittent hypoxia (CIH) is an animal model that simulates the hypoxemia seen in obstructive sleep apnea (OSA). Rats exposed to CIH exhibit an increase in sympathetic tone and reactivity and a persistent elevation in blood pressure during periods of normoxia, similar to that observed in patients with OSA. The median preoptic nucleus (MnPO) exhibits increases in Angiotensin type 1a receptor (AT1aR) mRNA following CIH and blocking this increase in AT1aR mRNA prevents the sustained increase in blood pressure. Our lab has previously reported that AT1aR activation influences GABAa mediated inhibition within the MnPO. Here we investigate the role of Angiotensin II (Ang II) signaling via the AT1a receptor in the MnPO and its influence on excitatory/inhibitory balance in rats subjected to CIH. Under isoflurane (2–3%) anesthesia, male Sprague‐Dawley rats (250–350 g) received microinfusions (0.4 μL) of recombinant AAV construct containing GFP reporter and shRNA against AT1aR (AT1a KD) or an AAV construct containing the GFP reporter and a shRNA scramble (Scr) targeted to the MnPO. After recovery, rats were subjected to 7 days of CIH (0800–1600 hrs). The CIH protocol consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10×/hr for 8 consecutive hrs (during the normal inactive/sleep phase) on 7 consecutive days. After 7 days CIH, the rats were anesthetized with isoflurane (2–3%) and coronal slices (300 μm) containing the MnPO were cut using standard in vitro slice procedures. Loose patch recordings were obtained from GFP labeled neurons using borosilicate glass micropipettes containing aCSF as the internal solution (1–3 MΩ). Spontaneous action potential firing was recorded in response to focal application of Ang II (100 nM, 30s) or muscimol (100 uM, 30s). Brief focal application of Ang II produced a transient increase in spontaneous action potential frequency of MnPO neurons from both CIH/Scr (n = 11) and normoxic/Scr (n = 9) rats. The Ang II dependent increase in activity was blocked in neurons from AT1a KD rats from both CIH (n = 12) and normoxic (n = 10) groups. The GABAa agonist muscimol decreased action potential activity (n = 10) of neurons from normoxic/Scr rats. This GABAa mediated inhibition was blunted in neurons from CIH/Scr (n = 13) and normoxic/AT1a KD (n = 10) rats. However, GABAa activation from neurons in the CIH/AT1a KD (n = 15) group produced an increase in spontaneous activity. The current study shows AT1aR KD mediated reduction in GABAa inhibition is exacerbated to the point where GABAa activation is excitatory following CIH. Future studies will address the influence of reduced AT1a signaling combined with reduced GABAa mediated inhibition on downstream targets of the MnPO in AT1aKD from both normoxic and CIH rats.Support or Funding InformationSupported by P01 HL088052This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Hydrogen Sulfide (H2S) on Homocysteine Mediated Glutamate Excitotoxicity, Endoplasmic Reticulum Stress and Pyroptosis in Retina
Authors: George Akash K. , Singh Mahavir , Homme Rubens P. , Majumder Avisek , Tyagi Suresh 
Published: June 2021

High levels of circulating homocysteine (Hcy) in the blood i.e. hyperhomocysteinemia (HHcy) have been linked to several visual disorders and are associated with impairment of retinal functions. The precise molecular mechanisms of how HHcy affects the retinal biology are unknown. The main objective of this study was to investigate the effect of HHcy on retinal glutamatergic dysfunction, endoplasmic reticulum (ER) stress and inflammation and whether H2S; a potent anti‐oxidant, anti‐inflammatory, vasoactive gas can mitigate the Hcy effects. We used HHcy mouse model CBS+/− and the wild type (WT) treated with H2S donor GYY4137 (133 μM/kg‐1/d‐1). The animal groups were designated as follows: Group‐I: WT control + No treatment; Group‐II: CBS+/− +No treatment, Group‐II: CBS+/− + GYY4137 and Group‐II: WT control + GYY4137. Novel Object Recognition Test (NORT) and Light Dark Box Test (LDBT) were employed to check the visual functions. The bovine serum albumin (BSA) labeled fluorescein isothiocyanate (FITC‐BSA) was used to study the vascular permeability in the retinas of these mice from all the four groups. Western blotting and qPCR were carried out to analyze the protein and mRNA expression levels in the retinas from all the groups. Our NORT and LDBT data showed a significant impairment of the visual functions in the CBS+/− mice as compared to the WT control mice. We also found a significant amount of leakage in the blood vessels in the retina of the CBS+/− mice in comparison to the WT control mice and that was mitigated by GYY4137 treatment. We noticed oxidative stress, ER‐stress, Glutamate excitotoxicity and inflammation were prominent in the tissue samples collected from the CBS mice in comparison to WT controls. Interestingly, we found that GYY4137 treatment could mitigate the glutamatergic dysfunction, ER stress, neurovascular dysfunction and the pyroptosis along with improvement in the visual functions in the CBS+/− mice treated with H2S.Support or Funding InformationNIH Heart, Lung & Blood Institute (HL‐74815), Institute of Neurological Disorders & Stroke (NS‐084823).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Subcellular Ca2+ Puffs Mediated By Different Inositol Trisphosphate Receptor Isoforms
Authors: Lock Jeffrey T. , Alzayady Kamil J. , Yule David I. , Parker Ian 
Published: June 2021

The second messenger inositol trisphosphate (IP3) evokes Ca2+ liberation through IP3 receptor/channels (IP3Rs) in the ER membrane to generate a hierarchy of cytosolic signals, including local Ca2+ puffs that arise from concerted openings of clustered IP3Rs, and global Ca2+ waves that propagate through the cell. Mammalian cells express three isoforms of IP3Rs that are encoded by different genes, have ~ 60–80% sequence homology and are reported to display differing functional characteristics. Imaging Ca2+ puffs with single‐channel resolution provides important information on the localization and properties of IP3Rs in intact cells. However, interpretation has been complicated because studies have been limited to native cell types that typically express varying proportions of two or three IP3R isoforms, with additional complexity arising from hetero‐multimerization within the tetrameric IP3R. Here, we applied high‐resolution TIRF microscopy to image Ca2+ puffs in HEK‐293 cell lines in which IP3R isoforms were knocked‐out in pairs by CRISPR/Cas9 technology to generate cells expressing exclusively IP3R types 1, 2 or 3. Puffs were evoked by photoreleased i‐IP3 in all three cell lines, indicating that IP3Rs of each isoform are able to assemble into the clusters that underlie these local Ca2+ signals. Moreover, Ca2+ puffs mediated by all three isoforms displayed largely similar mean amplitudes, temporal characteristics and spatial extents, except that type 3 IP3R‐mediated puffs showed appreciably faster kinetics. A quantal analysis of fluorescence signals revealed the single‐channel Ca2+ flux to be equivalent between isoforms, further suggesting that clusters of different IP3R isoforms contain similar numbers of active channels. Finally, mapping the distances between puff centroid localizations suggested that clusters of type 1 IP3Rs are more dispersed than clusters of type 2 or 3 IP3Rs. Overall, our results indicate that, although there is substantial functional redundancy among the IP3R isoforms, each exhibit distinct properties and spatial arrangements that likely shape the spatial‐temporal patterning of cellular Ca2+ signals observed in native cells.Support or Funding InformationThis work was supported by NIH grants R01 DE019245 (D.I.Y) and R37 GM048071 (I.P)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.