bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxysterol‐Related Binding Proteins Regulate Glucocorticoid Production and Cholesterol Metabolism in H295R Human Adrenocortical Cells
Authors: Escajadillo Tamara , Li Donghui , Sewer Marion B
Published: June 2021

Oxysterol‐binding protein‐related binding proteins are a family of lipid binding proteins that serve as lipid transporters, signaling mediators, and sensors of cellular lipid concentrations. We have recently identified ORP2 and ORP10 as members of a protein complex that is implicated in the production of cortisol. Based on these findings, the aim of the current studies was to determine the functional significance of ORP2 and ORP10 in adrenocortical steroidogenesis. Using stable cells lines that express shRNAs targeted against either ORP2 or ORP10, we show silencing both family members resulted in an increase in the expression of multiple genes, including 3beta‐hydroxysteroid dehydrogenase, CYP11A1, CYP17A1, lipoprotein density receptor, the melanocortin 2 receptor, and steroidogenic acute regulatory protein. ORP2 and ORP10 suppression also led to an increase in the expression of genes required for de novo cholesterol biosynthesis, including HMG CoA synthase, lanosterol synthase, and 24‐dehydrocholesterol reductase. Interestingly, ORP2 knockdown reduced the cAMP‐stimulated secretion of cortisol, but resulted in an accumulation of cellular free cholesterol. We conclude that ORP2 and ORP10 modulate cortisol production by regulating the expression of genes required for cholesterol homeostasis and steroid hormone biosynthesis. These studies are supported by NIH DK094151.
Conserved Mediator subunit MDT‐15 assures metabolic homeostasis
Authors: Taubert Stefan
Published: June 2021

Metabolic regulation occurs to a significant extent at the level of gene expression. We study the Mediator complex, a conserved multiprotein transcriptional coregulator. Interestingly, several Mediator subunits regulate lipid biology. We previously showed that the Caenorhabditis elegans Mediator subunit MDT‐15 is required for normal fatty acid distribution and for ingestion‐associated stress responses. Here, we show that loss of mdt‐15 leads to increased ER stress, as shown by upregulation of HSP‐4/BiP and of spliced XBP‐1 mRNA. We observed similar changes in worms with depleted fat‐6/Stearoyl‐CoA desaturase, suggesting that lack of monounsaturated fatty acids causes ER stress. However, although dietary complementation with oleic acid rescued ER stress in fat‐6 worms, it only partially rescued the ER stress in mdt‐15 animals, suggesting that additional defects cause excess ER stress. Interestingly, we find that mdt‐15 is also required for oxidative stress responses, suggesting that disturbed redox balance contributes to ER stress in mdt‐15 worms. Our data provide evidence for broad cytoprotective actions of MDT‐15, which maintains normal lipid profiles and assures a favorable redox environment.
Nem1p‐Spo7p‐mediated dephosphorylation of yeast Pah1p phosphatidate phosphatase
Authors: Su Wen‐Min M. , Han Gil‐Soo , Carman George M
Published: June 2021

Yeast Pah1p phosphatidate phosphatase (PAP), which catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol and Pi, has emerged as one of the most important and highly regulated enzymes in lipid metabolism. The enzyme controls the cellular amounts of phosphatidate and diacylglycerol, which in turn regulate the synthesis of phospholipids and neutral lipids through genetic and biochemical mechanisms. Because Pah1p is principally located in the cytosol, its association with the nuclear/ER membrane where the substrate phosphatidate resides is essential to PAP function in vivo. The association of Pah1p with the membrane is regulated by the phosphorylation state of the enzyme; phosphorylated Pah1p is mainly found in the cytosol, whereas dephosphorylated Pah1p is mainly associated with the membrane. The phosphorylated enzyme is recruited to the membrane where it is dephosphorylated by the Nem1p‐Spo7p phosphatase complex. This dephosphorylation leads to anchoring of Pah1p to the membrane allowing for the PAP reaction and triacylglycerol synthesis. The enzymological properties (pH optimum, divalent cation dependence, kinetics) of the Nem1p‐Spo7p‐mediated dephosphorylation was examined in a well‐defined system using the purified phosphatase complex and purified Pah1p that was phosphorylated with the Pho85p‐Pho80p protein kinase‐cyclin complex. Supported by NIH grant GM‐50679.
Signalling and cholesterol metabolism
Authors: Luu Winnie , Sharpe Laura J. , Stevenson Julian , Brown Andrew J.
Published: June 2021

Cholesterol is a vital lipid and performs diverse functions on a whole body and cellular level. However, excess cellular cholesterol is toxic, and thus, elegant mechanisms have evolved to tightly regulate this important lipid. Cellular homeostasis is governed by two major transcriptional factors, the sterol regulatory element binding proteins (SREBPs) and liver X receptor (LXR). SREBP‐2 is up‐regulated when cholesterol levels are low, promoting genes that are involved in synthesis and uptake, whilst LXR up‐regulates efflux genes such as ATP‐binding cassette transporter A1 (ABCA1). While there is a large body of work elucidating these cholesterol‐related pathways, less is known about the role of signalling in these processes. We found that Akt activates SREBP‐2 and its target genes, whilst tyrosine phosphatase inhibition down‐regulates gene expression of ABCA1. These findings offer new insights into the regulation of cholesterol metabolism.

The Brown lab is funded by grants from the National Health and Medical Research Council and National Heart Foundation of Australia.
Yeast Pah1p phosphatidate phosphatase is subject to proteasome‐mediated degradation
Authors: Pascual Florencia , Soto‐Cardalda Anibal , Carman George M.
Published: June 2021

In yeast, Pah1p phosphatidate (PA) phosphatase catalyzes the penultimate step in the synthesis of triacylglycerol. The importance of this enzyme is exemplified by the severe phenotypes of the pah1Δ mutant that include deregulation of phospholipid synthesis, an aberrant expansion of the nuclear/ER membrane, a decrease in lipid droplet formation, a massive reduction in triacylglycerol content, defects in vacuole homeostasis and fusion, and increased sensitivity to lipotoxicity. On the other hand, the attenuation of Pah1p PA phosphatase function is also essential to normal cell physiology. Indeed, the overexpression of an unregulated form of PA phosphatase inhibits cell growth, and this is attributed to the depletion of PA needed for phospholipid synthesis via CDP‐diacylglycerol and the accumulation of diacylglycerol to a toxic level. Triacylglycerol accumulates in the stationary phase of growth, and the lipid analysis of the pah1Δ mutant established the essential role of Pah1p PA phosphatase in this process. While the PA phosphatase activity was elevated in stationary phase cells, the expression of PAH1 mRNA and Pah1p was maximal in the exponential phase, but declined as cells entered the stationary phase. The levels of Pah1p were stabilized in stationary phase cells treated with the proteasome inhibitor MG132 as well as in several mutants that exhibit a decrease in proteasome function. This work reveals a novel mechanism for the control of Pah1p PA phosphatase function in yeast. Supported by NIH grant GM 28140.
A regulator of complex and hybrid N‐glycan synthesis expressed mainly in testis
Authors: Batista Frank , Huang Hung‐Hsiang , Stanley Pamela
Published: June 2021

Complex and hybrid N‐glycan synthesis is initiated by N‐acetylglucosaminyltransferase I (GlcNAcT‐I, MGAT1). MGAT1 is inhibited by membrane‐bound GlcNAcT‐I Inhibitory Protein, GnT1IP‐L. The C‐terminal 39 amino acids of GnT1IP‐L are required to inhibit MGAT1, but not for co‐immunoprecipitation with MGAT1. To determine where GnT1IP‐L is active in the secretory pathway, a series of chimeric proteins were generated. The stem and luminal domain (St+L) of GnT1IP‐L was attached to the cytoplasmic and transmembrane domain (Cy+TM) of proteins known to reside in a specific compartment of the secretory pathway. GnT1IP‐L (St+L) targeted to the endoplasmic reticulum (ER) did not inhibit MGAT1 activity. GnT1IP‐L targeted to the medial Golgi, to the trans Golgi or to the Trans Golgi Network, also did not inhibit MGAT1. However, targeting of GnT1IP‐L to medial Golgi via the (Cy+TM) domain of MGAT1, inhibited MGAT1 activity, and the synthesis of complex N‐glycans. This suggests that GnT1IP‐L and MGAT1 may reside in the kin recognition complex. We are investigating whether GnT1IP‐L inhibits other activities of the kin recognition complex. The GnT1IP gene is highly expressed in spermatocytes but not spermatids, and is regulated during spermatogenesis. We have generated inactive and conditional GnT1IP alleles to investigate the consequences of deleting GnT1IP in spermatogonia and during embryonic development.
The role of O‐fucosylated glycans in Notch signaling
Authors: Shim Jeongsup , Petryniak Bronislawa , Lowe John B.
Published: June 2021

Signaling from the Notch receptor family is important in development, and its role in disease has assigned the Notch signaling pathway as a therapeutic target. Mammalian Notch receptors are single‐pass transmembrane glycoproteins containing 29–36 EGF‐like repeats. Notch receptors on “receiving” cells signal when they interact with Notch ligands expressed on “sending” cells. Glycosylation of some Notch EGF‐like repeats modulates Notch‐dependent signaling. EGF repeat glycosylation includes constitutive fucosylation of some EGF repeat‐localized serine/threonine residues, and elongation of fucose‐initiated glycans regulated by the Fringe family of glycosyltransferases.

To understand how glycosylation controls Notch signaling, we sought to determine how O‐fucosylated glycans modulate Notch receptor‐ligand interactions, using (1) surface plasmon resonance, (2) cell‐based binding assays, and (3) cell‐based Notch signaling assays. We find that lunatic Fringe modification of Notch1 receptors enhances Dll1 and Dll4 binding affinities and signaling, while it reduces Jag1‐mediated binding affinity and signaling. Fringe modification of Notch1 does not significantly alter Jag2‐mediated binding affinity and signaling. We also find that Dll1 binding and signaling is enhanced by lunatic Fringe primarily when Notch1 expression is low, and this enhancement is blunted when Notch1 expression is high.
Identification of Osteoblast Proteins O‐GlcNAc Modified During Osteogenesis
Authors: Nagel Alexis K , Schilling Michael , Berkaw Mary , Ball Lauren E
Published: June 2021

The nutrient responsive O‐GlcNAc post‐translational modification (PTM) modulates signaling and transcriptional pathways contributing to cellular development and survival. Supporting a role for this PTM in osteoblasts (OB), parathyroid hormone‐or forskolin‐induced RUNX2 activity is enhanced in murine MC3T3E1 preOB in the presence of the β‐N‐acetyl‐D‐glucosamindase inhibitor PUGNAc. In this study we employed lectin weak affinity chromatography (LWAC) to facilitate the identification of O‐GlcNAcylated OB proteins by C18 nLC‐MS/ MS with CID/ETD fragmentation (LTQ‐XL). Twenty O‐GlcNAc modified peptides from 18 total proteins were elucidated in in 7‐day differentiated MC3T3E1s. Seventeen sites of O‐GlcNAc modification were unambiguously mapped, including 5 novel sites of O‐GlcNAc modification on TAB2, SBNO1, UBAP2l, LPP, and CBP. Both expression and activity of alkaline phosphatase (ALP), an intermediate marker of extracellular matrix maturation, were significantly enhanced in bone marrow mesenchymal stem cells cultured under osteogenic conditions for 6 days in the presence of Thiamet G, a specific inhibitor of OGA. These data provide the first evidence that O‐GlcNAc signaling can modulate extracellular matrix maturation during osteogenesis. Future studies will focus on pathways regulated by O‐GlcNAc in OB. This work is supported by NIH/NIDCR grant nos. RO1‐DE020925–01A1 and T32‐DE017551–05.
Tropical Cellulolytic Bacteria Isolated From Hindgut Of A. Evuncifer Potential Candidates for Bioconversion of Lignocellulosic Waste Biomass
Authors: IGWO‐EZIKPE MIRIAM NWANNA , Nwahiri‐Ogbu Chika , Oyebamiji Olayemi , Ndukwe Nelly , Ilori Matthew O. , Ogbunugafor Henrietta A.
Published: June 2021

Studies into the use of lignocellulosic biomass as alternative low cost energy source are on the increase, this has thus necessitated the search for cellulolytic microorganisms to be employed in bioconversion of lignocellulosic materials. This study investigated the degradation of lignocellulosic materials and glucose production potential of bacteria isolated from the hindgut of wood‐eating worker termites Amitermes evuncifer collected from two locations in Lagos, Nigeria. The hindguts of A. evuncifer were inoculated into sterilized cellulolytic media containing wood pulp as sole carbon source and incubated at 37 0C for 14 days. Bacteria were isolated and identified as Bacillus subtilis, Bacillus cereus, Enterobacter cloacae, Enterobacter aerogene, Acinetobacter sp. and Chryseobacterium sp. There was variability in the presence and occurrence of the isolates between the localities. However, the isolates showed zones of clearance on wood pulp agar plates which suggested their utilization of cellulose as the sole carbon and energy source. In addition, growth of B. subtilis, B. cereus, E. cloacae, E. aerogene, Acinetobacter sp. and Chryseobacterium sp. on wood pulp yielded 77.89 ± 1.12, 54.83 ± 1.28, 29.40 ± 2.98, 38.94 ± 2.12, 22.25 ± 1.42 and 61.99 ± 2.52 (mgL‐1) glucose respectively. This study demonstrated the use of tropical cellulolytic bacteria isolates from hindgut of A. evuncifer as potential candidates for bioconversion of lignocellulosic waste biomass.
Natural products as powerful tools for drug discovery: insights from the phorbol esters
Authors: Blumberg Peter M.
Published: June 2021

Natural products identify critical biological targets, provide tools for their characterization, and afford lead structures for therapeutic development. The phorbol esters were the paradigm for tumor promotion, opened a window into diacylglycerol generation as a central pathway in cellular signaling, and have brought bryostatin and PEP005 into clinical trials for cancer therapy. A central emerging theme has been the diversity of targets and regulatory mechanisms downstream of diacylglycerol, with protein kinase C now only one of multiple families transducing diacylglycerol signaling. The phorbol ester derivative resiniferatoxin helped unlock a second pathway of great therapeutic importance, one mediating chronic and inflammatory pain. Resiniferatoxin proved to function as an analog of capsaicin, the hot ingredient in red pepper. It differed, however, in being extraordinarily potent – up to 4‐orders of magnitude that of capsaicin – and in partially dissecting the twin properties of capsaicin of acute irritation and subsequent desensitization; resiniferatoxin was much more effective at desensitization relative to its acute irritation. Once again, these natural products have proven key to revealing great regulatory complexity and its associated therapeutic opportunities.
Effects Of Dextromethorphan Mediated Bitter Taste Receptor Activation In Pulmonary Artery Smooth Muscle Cells
Authors: Upadhyaya Jasbir Deol , Singh Nisha , Sikarwar Anurag Singh , Bhullar Rajinder Pal , Dakshinamurti Shyamala , Chelikani Prashen
Published: June 2021

Activation of bitter taste receptors (T2Rs) in human airway smooth muscle cells lead to muscle relaxation and bronchodilation. However, the presence of T2Rs in human pulmonary artery smooth muscle cells (hPASMCs) has not been established. The previous finding in airway smooth muscle led to our hypothesis that T2Rs, if present in hPASMCs, might be involved in regulating the vascular tone. RT‐PCR analysis revealed the expression of multiple T2R transcripts in hPASMCs. Functional analysis showed that these cells responded to many bitter‐tasting compounds, by increasing intracellular calcium concentration, indicating that T2Rs in hPASMCs are functional. Since the response of T2R1 to dextromethorphan (DXM) has been characterized in our previous studies, T2R1 was selected for further analysis in this study. Knockdown with T2R1‐specific shRNA decreased mRNA levels and DXM‐induced calcium responses by up to 40%. ELISA data obtained from in vitro studies using hPASMC showed that DXM‐treatment led to increased levels of endothelin‐1, biomarker of systemic hypertension. To analyze if T2R1 is involved in regulating the pulmonary vascular tone, ex vivo studies using pulmonary arterial rings are being pursued. Furthermore, it remains to be analyzed if this effect of DXM in hPASMCs is T2R1 specific or due to the blockade of NMDA receptors.

Supported by MHRC, NSERC and a New Investigator Award from HSFC.
Lysophosphatidic acid‐mediated phosphorylation of protein kinase D2 is dependent on PKC delta
Authors: Lynch Christine M , Matheny Ronald W
Published: June 2021

Protein kinase D (PKD) is a serine/threonine kinase and an important regulator of skeletal muscle function and development. PKD can be activated by a number of stimuli, including G‐protein coupled receptors (GPCRs); however, the identities of the signaling molecules that mediate GPCR‐PKD activation are not well‐defined in myoblasts. To address this, C2C12 myoblasts were treated with the GPCR agonist lysophosphatidic acid (LPA), Gö6983, and (or) small interfering RNA (siRNA), and the phosphorylation of PKD at both Ser 916 and Ser 748 was analyzed. LPA stimulated PKD phosphorylation by ~6‐fold as compared to unstimulated cells (P < 0.01). This phosphorylation was found to be dependent on Protein kinase C δ (PKCδ) because treatment with Gö6983 or siRNA targeting PKCδ resulted in 100% inhibition of LPA‐stimulated PKD phosphorylation at Ser 916 and Ser 748 (P < 0.01). Real‐time PCR identified PKD2 and PKD3 as the dominant PKD isoforms expressed in myoblasts, and siRNA against PKD2 (but not PKD3) eliminated LPA‐stimulated PKD phosphorylation by 100% (P < 0.01). These findings demonstrate that LPA‐stimulated activation of PKD2 is dependent on PKCδ, thus identifying a GPCR‐PKCδ‐PKD2 signaling pathway in skeletal muscle myoblasts. This research was supported by an appointment to the ORAU Postgraduate Research Participation Program at the US Army Research Institute of Environmental Medicine (CML).
The involvement of prohibition in Akt and Raf co‐regulated apoptosis of HaCaT keratinocytes upon UVB irradiation
Authors: Wu Qiong , Wu Shiyong
Published: June 2021

Prohibitin (PHB) is involved in regulation of many cell functions, such as cell‐cycle control, cell proliferation, senescence, apoptosis, and tumor suppression. However, the roles of PHB in regulation of UVB‐induced apoptosis of keratinocytes are largely unknown. In this study, we evaluated the role of PHB in regulation of Akt and Raf activity in UVB‐irradiated HaCaT cells. Our data showed that PHB was localized to the membrane from the cytosol upon UVB irradiation. A co‐immunoprecipitation analysis indicated that UVB altered the interaction among PHB, Raf, and Akt. Using siRNA‐mediated knockdown as well as stable shRNA‐mediated knockdown, we demonstrated that upon UVB irradiation, the cells with reduced expression of PHB showed an increase in apoptosis, as well as an increased phosphorylation of Akt and a decreased phosphorylation of ERK. In addition, our data showed that the effect of L‐NAC on UVB‐induced apoptosis is independent of PHB knockdown, which suggests that the PHB‐knockdown‐mediated cell apoptosis is independent of UVB‐induced oxidative stress. Based on these results, we propose that PHB regulates UVB‐induced apoptosis of keratinocytes via Akt and Raf, not the oxidative‐stress signaling pathway.

This work is funded by NIH RO1 CA086928 (to S. W.)
The differential effects of carnosol in regulation of HaCaT cell apoptosis upon high or low dose of UVB irradiation
Authors: Tong Lingying , Wu Shiyong
Published: June 2021

Carnosol, a dietary diterpene extracted mainly from rosemary and sage, was previously reported to have an anti‐cancer effect on breast and prostate cancer by interacting with estrogen and androgen receptors. However, little is known of its effect on carcinogenesis and signaling pathways upon ultraviolet (UV) irradiation. In this report, we showed that the effect of carnosol on apoptosis of HaCaT cells was dependent on the dose of UVB irradiation. Upon a higher dose of UVB irradiation (50 mJ/cm2), carnosol protected the cells from apoptosis. Moreover, we showed that carnosol inhibited the UVB (50 mJ/cm2)‐induced elevation of nitric oxide (NO) and peroxynitrite (ONOO‐). The reduction of NO and ONOO‐was correlated with a partial inhibition of UVB‐induced reduction of IÎºB, activation and phosphorylation of NF‐ÎºB as well as phosphorylation of JNK. However, upon lower dose of UVB irradiation (6 mJ/cm2), carnosol promoted apoptosis and inhibited recovery of the irradiated cells. Meanwhile, there was no detectable effect on phosphorylation of NF‐ÎºB or JNK upon the lower dose of UVB irradiation and carnosol. Our results suggest that carnosol may be involved in different pathways in regulation of cell death induced by different doses of UVB irradiation. This work is funded by NIH RO1 CA086928 (to S.W.)
XPLN is a novel regulator of mTORC2
Authors: Khanna Nidhi , Fang Yimin , Yoon MeeSup , Chen Jie
Published: June 2021

mTORC2 (mammalian or mechanistic target of rapamycin complex 2) signaling controls a wide range of cellular and developmental processes, but the regulation of mTORC2 is incompletely understood. Here we report the identification of a novel regulator of mTORC2. In a yeast two‐hybrid screen using the C‐terminal 1188 amino acids of mTOR as bait, we identified XPLN (eXchange factor found in platelets and leukemic and neuronal tissues), a GEF (Guanine Exchange Factor) for RhoA and RhoB, as a positive hit. The physical interaction between XPLN and full‐length mTOR was validated by pulldown assays, and found to be dependent on rictor but not raptor suggesting that XPLN specifically interacts with mTORC2. Recombinant XPLN inhibited mTORC2 kinase activity toward Akt in vitro, and overexpression of XPLN suppressed Akt phosphorylation in both HEK293 cells and C2C12 myoblasts. Conversely, knockdown of XPLN increased AKT phosphorylation in those cells. Furthermore, we found that XPLN negatively regulated cell survival and myoblast differentiation through Akt. Surprisingly, these functions of XPLN were independent of its GEF activity. Taken together, our results reveal XPLN as a novel regulator of mTORC2 signaling to Akt in the regulation of cell survival and differentiation.

This work was supported by NIH grants to JC (AR048914 & GM089771).
Caveolin‐1 is a critical determinant of autophagy and oxidative stress
Authors: Romero Natalia , Shiroto Takashi , Kalwa Hermann , Sartoretto Juliano , Sugiyama Toru , Michel Thomas
Published: June 2021

Reactive oxygen species (ROS) are implicated in the pathogenesis of many disease states. Caveolin‐1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules. Caveolin‐1null mice have marked cardiovascular abnormalities, yet the molecular mechanisms are incompletely understood. We performed unbiased metabolomic analyses of lysates prepared from endothelial cells following siRNA‐mediated caveolin‐1 knockdown, and discovered striking (up to 30‐fold) increases in levels of cellular dipeptides, consistent with an increase in autophagy. We found that plasma 8‐isoprostanes levels were significantly higher in caveolin‐1null mice compared to wild‐type control mice (454 ± 44 vs. 319 ± 3 pg/ml, p<0.05). We injected caveolin‐1null and wild‐type mice with a recombinant lentivirus expressing the hydrogen peroxide (H2O2) biosensor HyPer2; quantitative fluorescence imaging studies revealed significant increases in H2O2 in the endothelium of arterial preparations isolated from caveolin‐1null mice (p<0.05 compared to wild type). Caveolin‐1 knockdown in endothelial cells led to significant (p<0.05) increases in H2O2 levels (measured using both the HyPer2 biosensor and the Amplex Red assay), associated with a marked decrease in the GSH/GSSG ratio. The mitochondrial inhibitor rotenone attenuated the increase in H2O2 levels seen after caveolin‐1 knockdown, whereas the NADPH oxidase inhibitor VAS‐7820 had no effect. Cellular imaging of mitochondrial ROS production (assayed with MitoSox Red) and of mitochondrial membrane potential revealed marked increases after caveolin‐1 knockdown, with no change in mitochondrial abundance. These findings establish that caveolin‐1 plays a key role in the regulation of cellular oxidative stress, and suggest that caveolin‐1‐modulated pathways may represent novel targets for the amelioration of oxidative stress in cardiovascular disease states.
Interaction between RGS7 binding protein and receptor protein tyrosine kinases
Authors: Fatora Christopher , Witherow D. Scott
Published: June 2021

The function of the dimer between the G protein beta subunit 5 and the regulator of G protein signaling 7 (Gβ5‐RGS7) in cell signaling is not well understood. However, it is known that the Gβ5‐RGS7 dimer reversibly binds the regulator of G protein signaling binding protein (R7BP). To determine potential binding partners that might be involved in regulation of Gβ5‐RGS7 and R7BP activity, we performed a yeast two‐hybrid screen with R7BP. Of numerous potential interacting proteins, we have studied the interaction of the two specific isoforms of protein receptor tyrosine phosphatases, delta and kappa. Using in vitro binding assays with recombinant proteins as well as cell‐based immunoprecipitation assays, the interaction of R7BP with the protein receptor tyrosine phosphatases was analyzed in the presence and absence of Gβ5‐RGS7. These data suggest new signaling pathways that might be regulated via the Gβ5‐RGS7 dimer.

This work was supported by a University of Tampa David Delo Research Professor Grant (DSW), a University of Tampa Dana Foundation Grant (DSW), and the University of Tampa Honors Program (CMF).
Prolactin receptors and signal pathways in the rat prostate following the increase of systemic prolactin
Authors: Hernandez Maria Elena , Pascual Luz Irene , Rodriguez Sara , Aranda‐Abreu Gonzalo E , Manzo Jorge
Published: June 2021

The prostate is a sexual gland in charge of semen production. Several hormones regulate its function, such as prolactin (PRL) that modulates the density of prolactin receptors, and promote the proliferation, differentiation, and survival of the prostatic epithelial cells. Systemic elevation of PRL induces the increase of the epithelial height and alveolus, and pathologies as dysplasia or neoplasia; and alterations of sexual behavior. Notwithstanding, it is still unknown whether short periods of systemic PRL increase have some effects on the expression of PRL messenger or signal pathways. Thus, in this work we evaluated the effect induced by short periods of PRL elevation in sexually expert male rats. Wistar males were used. PRL elevation was induced by an adenohypophysis transplant to the renal capsule or by the intramuscular injection of 50 μg of PRL (one injection every 12 h) for 15 days. The ventral prostate was obtained and submitted to analyze the expression of the short and long PRL receptor mRNA, and the density of the signal pathways STAT 3, STAT 5, and Mapk. Results showed that the transplant decreased the long PRL receptor mRNA and increased in the short one; also, it increased the three signal pathways. The injections of PRL increased the expression of both the short and long receptor messenger; and no signal pathway was modified. As expected, no treatment altered the sexual behavior. Hence, the increase of PRL induced modifications in the expression of PRL receptor messenger as well as signal pathways. It suggests that the changes are a direct response of the hormone and not as a consequence of the quality in the execution of sexual behavior. Results suggest that the morphological changes seen in the prostate tissue are directly related to the alteration in the receptor expression and the constant activation of signal pathways. Supported by CONACyT‐Mexico grant 106531 to MEH.
The NAADP/ TPC2/Ca2+ Signaling Antagonizes Autophagosome Maturation
Authors: Lu Yingying , Yue Jianbo
Published: June 2021

Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remains poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP) elicits Ca2+ release from lysosomes via two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in Hela cells, which lack the expression of endogenous TPC2, inhibited autophagosomal‐lysosomal fusion by raising lysosomal pH. Treatment of TPC2 overexpressing cells with a cell permeant NAADP agonist, NAADP‐AM, further inhibited the fusion, whereas Ned‐19, a NAADP antagonist, promoted the fusion. Moreover, intracellular acidification mitigated the fusion blockage in TPC2 overexpressing cells. Interestingly, autophagy is gradually induced during in vitro neural differentiation of mouse embryonic stem (ES) cells initiated by the monolayer culture, accompanying with the marked downregulation of endogenous TPC2. Consistently, TPC2 knockdown or overexpression in mouse ES cells promoted or inhibited the fusion between autophagosome and lysosome during early neural differentiation, respectively. Correspondingly, TPC2 knockdown accelerated mouse ES cells entry into neural lineages, whereas TPC2 overexpression in ES cells markedly inhibited it. The development of novel agonists or antagonists of NAADP should be a good approach to control the maturation processes of the autophagosome.
The effect of camptothecin on nuclear RanGTP levels in HeLa cells
Authors: Steinheiser Amy Marie , Resendes Karen K.
Published: June 2021

Nuclear transport is essential for the survival of all cells. The flow of molecules passing through the nuclear envelope via nuclear pore complexes is mediated by the karyopherin protein family, consisting of importins and exportins to which cargo can bind. The directionality of nuclear transport is regulated by a small GTPase, Ran. Most modern chemotherapeutic agents function by entering the cell nucleus via active transport and thus initiate apoptosis. Much time and research has been invested into the study of this exact mechanism by which certain cells are targeted for cell death. Using the apoptosis‐inducing drug, camptothecin, the goal of this project was to analyze the effect of camptothecin on the Ran gradient in HeLa cells and to establish a timetable during which delocalization of Ran no longer occurs and the breakdown of the nuclear envelope is initiated. In order to do so, two effective concentrations of camptothecin were administered to HeLa cells and fluorescence based assays were used to observe apoptosis, Ran localization, and nuclear pore integrity. Our results have shown that delocalization of RanGTP does not occur prior to nuclear envelope disassembly, suggesting that mechanism that is employed by camptothecin differs from other similar inducers of apoptosis, such as staurosporine and etoposide. Further research will determine whether this change is beneficial or a deficiency. Funding and supplies for this research were provided by Westminster College Biology Department and Westminster College Drinko Center for Experiential Learning Research Grant.
Andrographolide activates the Wnt pathway and modulates the APP processing by direct inhibiton of GSK3β
Authors: Tapia Cheril Cecilia , Mejías Cristóbal , Bustamante Hianara , Burgos Patricia , Inestrosa Nibaldo C
Published: June 2021

Objectives

We carry out a detailed analysis of the effect of Andrographolide (ANDRO), the major component of Andrographis paniculata, on the activation of Wnt signaling and APP processing.

Methods

Human H4 neuroglioma cells stably expressing APP695 and slices from wild‐type mice were incubated with ANDRO. The samples were used for biochemical analysis.

Results

ANDRO modulates the Wnt/β‐catenin signaling components increasing the levels of the β‐catenin by inactivation of GSK‐3β and inducing the transcription of Wnt target genes. ANDRO acts downstream from the ligand‐receptor binding, by direct inhibition of GSK3β. Finally, ANDRO seems to stimulate non‐amyloidogenic proteolytic cleavage of APP, leading to a significant increase in C83 levels, a product of α‐secretase.

Conclusions

We conclude that ANDRO activated Wnt pathway modulating the non‐amyloidogenic APP processing pathway by a mechanism that target inhibition of GSK3β.
The mechanism by which the TfR2/HFE complex affects iron homeostasis
Authors: Worthen Christal Anna , Chloupková Maja , Noguchi Ken , Enns Caroline A
Published: June 2021

Iron homeostasis is tightly controlled. Since there is no regulated way of excreting excess iron, levels are finely tuned through dietary uptake. This is mostly accomplished by the peptide hormone, hepcidin, which is produced in the liver and regulated by the BMP‐signaling pathway. The hereditary hemochromatosis protein (HFE) and transferrin receptor 2 (TfR2) form a complex and regulate the BMP‐signaling pathway in response to blood transferrin‐bound iron levels. Mutations in either protein result in a reduction in BMP signaling and hepcidin expression, leading to iron overload. How the TfR2/HFE complex affects BMP signaling is unknown. The purpose of this study was to determine the mechanism by which TfR2 and HFE affect BMP‐signaling and iron homeostasis. We found that TfR2 and the BMP co‐receptor, hemojuvelin, interact through immunoprecipitation, and that HFE stabilizes this interaction. This establishes an important link between the iron‐sensing TfR2/HFE complex and BMP signaling. To further understand how this complex affects BMP signaling, we created mutant forms of TfR2 and HFE and studied them in vitro, using minicircle DNA technology in primary hepatocytes, and in vivo, using adeno‐associated virus under the control of a liver‐specific promoter.

NIH RO1 DK054488

T32 5T32GM071338–08
Hyperthermia‐Induced NDRG2 Upregulation Inhibits the Invasive Potential of HepG2 cell via Suppressing ERK1/2 Signaling Pathway
Authors: Zhang Jing , Guo Yan , Li Xia , Zhang Jian , Liu Wenchao , Yao Libo
Published: June 2021

Hyperthermia (HT) has proven to be able to alter invasion ability of cancer cells. However, the detailed mechanisms responsible for the anti‐metastasis effects of HT have not been elucidated. N‐myc downstream‐regulated gene 2 (NDRG2) is a member of NDRG family, which has been suggested to be highly responsive to various stress and associated with tumor suppression. The present study aimed to investigate biological role of NDRG2 in invasion of HCC cells under hyperthermia. We found that NDRG2 could be induced by hyperthermia at 45□. Meanwhile, NDRG2 overexpression can inhibit expression of MMP2 and MMP9 as well as invasion of HCC cells. Whereas knocking‐down of NDRG2 increased invasion ability of HepG2 cells. Moreover, NDRG2 was proved to decline HT‐induced phosphorylation of ERK1/2 notably in HepG‐2 cells exposed to hyperthermia. Taken together, these data suggest that NDRG2 could be induced by hyperthermia and mediates hyperthermia‐caused inhibition of invasion in HCC cells via Suppressing ERK1/2 Signaling Pathway. This work was supported National Natural Science Foundation of China ((No.30973437; 81230043).
The selective growth inhibition of BRAF mutant glioma cell lines by a new selective BRAF kinase inhibitor UI‐152
Authors: Kim Na‐Yeon , Ahn Jun‐Ho , Lee Michael
Published: June 2021

Malignant gliomas are aggressive central nervous system tumors with poor prognosis. Activating mutation of BRAF (V600E) has been reported in a subset of these tumors. The aim of the present study was to understand the relationship between the activation state of RAF and the antiproliferation potential of a potent oncogenic BRAF kinase inhibitor UI‐152 in glioma cell lines with differing BRAF mutational status. We investigated the individual IC50 values of UI‐152 on the six malignant glioma cell lines. KG‐1‐C, NMC‐G1, and DBTRG‐05MG cell lines are heterozygous for BRAF‐V600E; A‐172, T98G, and U‐87 MG cell lines are homozygous for wild‐type (WT) BRAF. UI‐152 displayed the greatest level of selectivity for BRAF‐mutant cells, inhibiting their proliferation more potently than that of cells expressing BRAFWT. The IC50 of UI‐152 in B‐Raf mutant cells was less than 5 ìM, whereas the IC50 of cells expressing BRAF‐WT were not reached even at 20 ìM UI‐152. Corresponding immunoblot results showed that UI‐152 completely abolished MEK‐ERK phosphorylation in DBTRG‐05MG cells harboring BRAF‐V600E. However, in U‐87MG cells expressing BRAF‐WT, UI‐152 caused the paradoxical activation of the MAPK pathway. Taken together, our data suggest that UI‐152 may be an effective BRAF inhibitor and contribute to a potential therapeutic strategy for brain tumor patients with BRAF mutations.
Using Chemical Genetics to Define Zipper‐Interacting Protein Kinase Signalling Events
Authors: Al‐Ghabkari Abdulhameed Saleh , Sutherland Cindy , Walsh Michael P , MacDonald Justin A
Published: June 2021

Zipper‐interacting protein kinase (ZIPK) has emerged as an important regulator of apoptosis, cell motility and vascular smooth muscle (VSM) contraction. To identify the precise role of ZIPK in these processes, we set out to identify ZIPK substrates using a chemical‐genetic approach. Mutation of ZIPK at the conserved gatekeeper residue (L93G) within the ATP‐binding site was performed in order to develop cell‐based model systems for the analysis of ZIPK function. As proof of principle, kinetic analyses support the selectivity of analog‐sensitive kinase inhibitors (pyrazolo[3,5‐d]pyrimidine; PP1) for L93G‐ZIPK with minimal inhibitory potential observed for WT‐ZIPK or other VSM contractile kinases (ROK or MLCK). In this regard, the 1NM‐PP1 inhibitor (10 mM) provides maximal distinction of L93G‐ZIPK and WT‐ZIPK activities with essentially no off‐target effects. Moreover, the PP1 compounds have no effect on Ca2+‐dependent or Ca2+‐independent VSM contractions. These results are expected since the PP1 inhibitors have high specificity for the mutated kinase but minimal ‘off‐target’ effects on endogenous kinases. Although some novel off‐target effects of the analog‐sensitive kinase inhibitors were identified (i.e., able to inhibit ROK in vitro), we conclude that application of the chemical‐genetics approach with the L93G‐ZIPK and 1NM‐PP1 pairing will enable us to define the explicit actions of ZIPK.

This submission is sponsored by Justin A. MacDonald, society affiliation member of ASBMB, jmacdo@ucalgary.ca

Supported by the Heart & Stroke Foundation of Canada
Loss of Shp2 phosphatase favors alternative activation of macrophages, contributing to pulmonary fibrosis in mice
Authors: Tao bo , Zhang Xue , Jin Wei , Zhang Yun , Cheneg Hong qiang , Ke Yue hai
Published: June 2021

Alternative activation of macrophages (M2) in lungs has been implicated as driving pulmonary fibrosis. However, the mechanisms underlying M2 polarization are largely uncharacterized. Shp2, a ubiquitously expressed cytoplasmic tyrosine phosphatase, has previously been shown to be a critical signal transducer in various inflammatory diseases. In this report, we found that inactivation of Shp2 phosphatase augments IL‐4‐mediated M2 polarization in vitro, suggesting a potential negative regulation for this enzyme. To further unveil physiological roles of Shp2 in M2, we generated macrophage‐specific Shp2‐knockout mice under the control of LysM‐Cre promoter (Shp2flox/flox, Lysm‐Cre), Shp2‐deficient mice exhibit a marked shift of polarized balance in favor of IL‐4 mediated alternative activation. Additionally, there was a notable increase in arginase 1 activity when chitin was intraperitoneally administered to Shp2‐deficient mice. Moreover, these mice appeared to be more sensitive to bleomycin‐induced inflammation and pulmonary fibrosis. Mechanistically, Shp2 associates with IL‐4R¦Á and inhibits downstream JAK1‐STAT6 signaling by its catalytic domain in Shp2‐deficient mice, contributing to the observed phenotypic changes. Together, these findings identify Shp2 as a novel regulator of alveolar macrophage activation, and indicate that deregulation of Shp2 results in pulmonary fibrosis.
Pleiotropic effects of PI3K/Akt pathway in hippocampal neurons exposed to iron‐induced oxidative stress
Authors: Uranga Romina María , Giusto Norma María , Salvador Gabriela Alejandra
Published: June 2021

PI3K/Akt pathway is a key component in synaptic plasticity and neuronal survival. The aim of this work was to investigate the participation of PI3K/Akt pathway and its outcome on different molecular targets such as GSK3β and FoxO transcription factors during mild oxidative stress triggered by iron overload. The exposure of mouse hippocampal neurons (HT22) to different concentrations of Fe2+ (25–200 μM) for 24 h led us to define a mild oxidative injury status (50 μM Fe2+) in which cell morphology showed typical changes of neuronal damage, with increased lipid peroxidation and cellular oxidant levels but no alterations of cellular viability. Under these experimental conditions, SOD1, SOD2 and Trx expression was diminished, catalase showed no changes, and HO1 was increased, with respect to the control. Simultaneously, both Akt and GSK3β phosphorylation was increased. Levels of phospho‐FoxO3a (inactive form) increased in the cytosolic fraction of cells treated with iron in a PI3K‐dependent manner. Moreover, PI3K and Akt translocated to the nucleus in response to oxidative stress. Iron‐overloaded cells harboring a contitutively active form of Akt showed decreased oxidants levels. Our results show that the activation of PI3K/Akt pathway during iron‐induced neurotoxicity regulates multiple targets such as GSK3β, FoxO transcriptional activity and the generation of reactive oxygen species.
A picture is worth a thousand data points: An analysis of textbook visualizations in the molecular life sciences
Authors: Offerdahl Erika , Arneson Jessie
Published: June 2021

Calls to transform science instruction underscore the need to develop students’ understanding of the disciplinary practices of scientists. Visual thinking – the ability to interpret and make sense of visualizations (e.g. graphs, diagrams, figures) – is one such practice that is seldom an explicit learning outcome of undergraduate curricula.

Extracting meaning from visualizations requires translation across levels of organization (e.g. organismal level to the molecular) and levels of abstraction (e.g. from graphs to schematic models). The goal of this study was to determine the role of textbook figures in developing students’ visual thinking skills.

Figures from biology and biochemistry textbooks and from five high‐profile scientific journals were analyzed for level of organization, level of abstraction, and the nature of transitions between levels (e.g. implicit vs. explicit). We found that textbook figures generally display only a single level of organization, represent relationships between levels implicitly, and show little progression in complexity from intro to upper‐level. Further, figures from upper‐level textbooks are less complex and seldom represent quantitative data when compared to scientific figures. Our results suggest that visualizations used in traditional instruction lack the complexity necessary to explicitly support the development of visual thinking skills.
Two‐year food security transition and nutrient intake among homebound seniors
Authors: Sharkey Joseph R. , Xu Jin , Dean Wesley R.
Published: June 2021

Little is known about food security transition over time and the influence on nutrient intake among homebound seniors. Data are from 262 homebound older adults who received home‐delivered meals and completed in‐home assessments at two time‐points (1 year interval). Dietary intake was determined through 3 24‐hour dietary recalls at each time‐point. NDS‐R software was used to calculate nutrient intake; energy‐adjusted multiple regression models examined variables associated with nutrient intake at time 2. Food security stability was reported by 39% (n = 103), persistent risk for food insecurity by 26.3 %, and persistent food insecurity by 2.3%; 23% became less food secure; and 9% became more food secure. Nutrient intake at baseline predicted intake 1 year later. Food security transition was associated with time 2 intakes of energy, fiber, niacin, and phosphorus. Although food security transition is a predictor of energy‐adjusted dietary intake among homebound older adults, baseline dietary intake is the most significant predictor of dietary intake one year later. Thus, it is critical to monitor both food security status and dietary intake in vulnerable older populations.
Regulation and specificity of the Chd1 chromatin remodeler
Authors: Bowman Gregory , Patel Ashok , Chakravarthy Srinivas , Nodelman Ilana
Published: June 2021

Chromatin remodelers are ATP‐dependent enzymes required for the active reorganization of nucleosomes. Accompanying the conserved ATPase is a broad diversity of auxiliary domains that define different remodeler subfamilies, and presumably help guide the outcome of remodeling reactions. We discovered that the double chromodomain unit of the Chd1 remodeler regulates activation of the ATPase motor by blocking access to DNA. Disruption of the chromo‐ATPase interface results in a loss of specificity for nucleosomes over naked DNA, allowing for robust DNA‐dependent ATPase stimulation. Interestingly, this regulatory interface also appears to dampen the nucleosome sliding activity of the remodeler. We are investigating the stages at which chromodomain regulation impacts nucleosome sliding, and the potential impact of this regulation on substrate selection.
Protective Effect of Peanut Sprout Extract on Glutamate‐induced Neuronal Toxicity and Kainic acid‐induced Memory Loss
Authors: Kim Jong‐Sang , Kim Seong Soon , Kim Hyo Jung , Seo Ji Yeon , Park In Sil , Kang Hye‐Ryung , Kim Bo Ram , Zakharova Alexsandra
Published: June 2021

Glutamate, one of major excitatory neurotransmitter in central nervous system, has been known to induce oxidative stress such as reactive oxygen species (ROS) by overstimulation of neuron. In this context, we investigated the neuroprotective effect of peanut sprout extract (PSE) against glutamate/kainic acid‐induced oxidative stress in the mouse hippocampal HT22 cells and ICR mice. PSE significantly increased the cell viability and decreased the ROS generation under glutamate‐induced oxidative stress in HT22 cells. PSE induced heme oxygenase‐1(HO‐1) known as a phase 2 enzyme and, its protective effect was abolished by HO‐1 and PI3K inhibitors. Furthermore, to examine the effect of PSE on learning and memory abilities in mice, kainic acid was treated to induce learning and memory loss, an analogue of glutamate. Passive avoidance and Y‐maze tests were performed to examine abilities of learning memory and spatial cognition respectively. The results showed that PSE had protective effect on kainic acid‐induced memory loss in mice, suggesting that PSE protects neuronal cells from glutamate/kainic acid‐induced oxidative stress through induction of HO‐1.

Grant Funding Source: ASN
Relations between pregnancy risk, infant growth and cognition
Authors: Miner Jessica , Kennedy Tay , Colaizzi Janna , Tisdale Ashlee , Thomas David G.
Published: June 2021

Visual information processing, heart rate attention and growth were assessed in 132 infants at 3, 6 and 9 months of age. The infants’ mothers were predominately white, well‐educated and married; and all infants were predominately breastfed at 3 months of age. There were three groups of infants based on changes in attention termination and encoding speed over the study period. Pregnancy risk was assessed using a standardized questionnaire and risk scores were developed based on maternal response when the infants were 3 months of age. Performance did not differ between groups based on scores associated with pre‐pregnant health (p = .07); pregnancy health problems (p=.21) or late term pregnancy complications (p=.96). The three groups were significantly different in weight for length z‐ scores at 3 months of age (p= .006). In conclusion, cogitative performance on these tasks did not differ based on maternal health just prior to or during pregnancy but did differ based on early growth in this low‐risk sample.