bluuebunny/crossref_metadata_2025 huggingface dataset:
A Pyruvic Acid Analog Primarily Targets GAPDH To Promote Cancer Cell Death
Authors: Ganapathy‐Kanniappan Shanmugasundaram , Geschwind Jean‐Francois , Kunjithapatham Rani , Buijs Manon , Vossen Josephina Anna , Tchernyshyov Irina , Torbenson Michael S , Cole Robert N , Syed Labiq H , Vali Mustafa
Published: June 2021

The objective of the current study is to elucidate the principal mechanism underlying the anti‐glycolytic effect of the pyruvic acid analog, 3‐bromopyruvic acid (3‐BrPA). Up regulation of glycolysis is a common event witnessed in majority of tumors. 3‐BrPA induces tumor cell death by depleting intracellular energy (ATP) through inhibition of glycolysis. Previous reports claimed 3‐BrPA as an energy blocker targeting the enzyme, type II hexokinase. However, substantial experimental evidences on the primary targets, essentially involved in the 3BrPA cytotoxicity, are lacking. Here, we used a combination of dose‐response studies coupled with proteomics in multiple cancer cell lines to identify the molecular targets of 3‐BrPA in an unbiased fashion. Radiolabeled 3BrPA studies showed glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) as the primary chemical and biological target at a dose that induced cell death. Loss of function studies through RNA interference (RNAi) confirmed the necessity of GAPDH for cell survival. Hence, we provide evidence for the first time that GAPDH is the primary molecular target of 3‐BrPA.

This study was supported by NIH/NCI grant RO1 CA 100883‐01 (J.F.G), Charles Wallace Pratt Research Fund and the Abdulrahman Abdulmalik Liver Cancer Research Fund.
Evolution of the Krebs cycle during the prepubertal‐pubertal transition in rat mammary gland
Authors: Piras Antonio , Vayalil Praveen , Wilson Landon , Kuo Hui‐Chien , Crowley Michael , Page Grier P. , Meleth Sreelatha , Lamartiniere Coral , Kim Helen , Barnes Stephen
Published: June 2021

In rat models of mammary carcinogenesis, the highest incidence of carcinomas occurs when the animals are exposed to carcinogens during puberty (typically postnatal day 50). Gene expression analysis of rat mammary gland in Sprague‐Dawley rats revealed a consistent down‐regulation of Krebs cycle genes in tissues from 50‐day old animals relative to 21‐day animals. This result was confirmed at the protein level by Western blot analysis and assessment of specific activity of selected Krebs cycle enzymes; succinate dehydrogenase (SDH)‐B, fumarase (FH), NAD+ isocitrate dehydrogenase (IDH3A) were significantly reduced at late puberty (day 50) compared with prepubertal (day 21) in the mammary gland. A nanoLC‐multiple reaction ion monitoring‐mass spectrometry method was developed that measures each of these proteins in a single assay in tissue and mitochondrial preparations. The results reported here support the gene expression data obtained by microarray analysis and suggest that the increased vulnerability of the rat mammary gland at late puberty to breast cancer‐inducing carcinogens is correlated with an overall down‐regulation of aerobic metabolism. Supported by NCI U54 CA100949.
The use of cinema as a tool in the classroom
Authors: Fischer Beth
Published: June 2021

Short vignettes or even entire films can be effective tools in the classroom. They can focus attention on an issue, provide depth to a discussion, demonstrate a point, or simply add variety to a lecture. A broad range of topics can be addressed. Films can be used to demonstrate a clinical condition or a behavioral test. They also are useful in teaching about professional skills, such as teaching, interview skills, and making oral presentations. And they can address ethical issues, such as research integrity and social responsibility. This session will focus on factors that influence the effectiveness of video use, provide tips for selecting and using films, and discuss copyright issues. Participants will receive a bibliography of recommended films and corresponding topics, and will be encouraged to discuss their own experiences.
Identification of a novel PPARα‐interacting protein
Authors: Bagattin Alessia , Hugendubler Lynne , Mueller Elisabetta
Published: June 2021

PPARα (peroxisome proliferator‐activated receptor α) is a ligand‐activated transcription factor involved in the regulation of fatty acid β‐oxidation. It is mainly expressed in tissues with elevated energy metabolism like liver, heart, kidney, skeletal muscle and brown fat. Upon ligand binding, PPARα heterodimerizes with the retinoid X receptor α (RXRα). The PPARα‐RXRα complex binds to specific PPAR response elements (PPRE) in the promoter regions of target genes enhancing their transcription. PPARα is activated by fibrates, synthetic compounds that induce peroxisome proliferation in rodents and are used as hypolipidemic drugs in humans. Many studies aim at the identification of novel agonists and cofactors that can modulate PPARα activity. We identified ACOX1 (Acyl Coenzyme A Oxidase) as a PPARα‐interacting protein by pull‐down using PPARα‐GST fusion proteins and 35S‐labeled‐in vitro translated ACOX1. ACOX1 is able to increase the transcriptional activity of PPARα in transient transfection experiments in U2OS (human osteosarcoma) cells. ACOX1 is the first and rate‐limiting enzyme of the peroxisomal fatty acid β‐oxidation and its expression is induced by PPARα. These results suggest the possibility of a novel regulatory loop between PPARα and a metabolic enzyme.

Supported by the Intramural Research Program of NIDDK, NIH.
Analysis of antibody‐antigen interactions using size‐exclusion HPLC and photodiode array detection
Authors: Sanny Charles G.
Published: June 2021

Analysis of antibody‐antigen interactions using size‐exclusion HPLC (SE‐HPLC) and detection at a single wavelength (e.g. 280 nm) have been described in the literature. The use of a diode array detector may provide additional information based on antibody and antigen component absorbance spectra. In this study, mixtures of an antivenin (Crotalidae Polyvalent Immune Fab (Ovine), Protherics, Inc.; FabO), and western diamondback rattlesnake (C. atrox) venom were analyzed by SE‐HPLC with photodiode array detection in the 210 to 320 nm range. Changes in elution profiles were consistent with antibody‐antigen complex formation with concomitant decreases in reactive antibody and antigen components. Spectral analysis indicated that the total absorbance of reaction mixtures are essentially proportional to the sum of the total absorbance of the reactants (FabO; C. atrox venom) at each wavelength. Based on spectral matching, there was greater dissimilarity between FabO and C. atrox venom total absorbance in the 240‐320 nm range compared to the 210‐320 nm range. A comparison between reaction mixture spectra and the sum of reactant spectra (FabO spectra + C. atrox venom spectra) indicated low dissimilarity in both the 210‐320 nm and 240‐320 nm range, suggesting no significant change occurs in the spectra of reactants during complex formation. Spectral matching was also used to help identify and compare regions of dissimilarity within the elution profiles of FabO, C. atrox venom, and reaction mixtures. Research support from the Department of Biochemistry and Microbiology, Oklahoma State University Center for Health Science.
Integrated structural and functional model of the human ESCRT‐II complex
Authors: Im Young Jun , Hurley James H
Published: June 2021

ESCRT‐II plays a pivotal role in receptor downregulation and multivesicular body biogenesis, and is conserved from yeast to humans. The crystal structures of two human ESCRT‐II complex structures have been determined at 2.6 and 2.9 Åresolution, respectively. The complex has three lobes and contains one copy each of VPS22 and VPS36, and two copies of VPS25. The structure reveals a dynamic helical domain to which both the VPS22 and VPS36 subunits contribute, which serves as a platform connecting the GLUE domain to the rest of the ESCRT‐II core. Hydrodynamic analysis shows that intact ESCRT‐II has a compact, closed conformation. ESCRT‐II binds to the ESCRT‐I VPS28 C‐terminal domain subunit through a helix immediately C‐terminal to the VPS36‐GLUE domain. ESCRT‐II is targeted to membranes by phosphatidylinositol 3‐phosphate binding to the GLUE domain and nonspecific polyanionic lipid binding by the first helix of VPS22. These data provide a unifying structural and functional framework for the ESCRT‐II complex.
Tetramer formation by Vps74p is required for proper localization of Golgi glycosylation enzymes
Authors: Schmitz Karl R , Setty Thanuja Gangi , Bessman Nicholas , Ferguson Kathryn M
Published: June 2021

The yeast protein, Vps74p, belongs to a family of Golgi‐associated cytosolic proteins, which are highly conserved throughout fungi and animals. We have found that Vps74p functions to maintain the steady state distribution of Golgi mannosyltransferases, and is thereby required for proper protein glycosylation in yeast. We have determined the X‐ray crystal structure of Vps74p, and observe a tetramer, the subunits of which adopt a novel alpha helical fold. Equilibrium between monomer and tetramer is observed in solution, and deletion of a key structural feature abolishes both tetramer formation and Vps74p function. We report details of the formation and regulation of tetramer formation in solution. Furthermore, we describe structural and functional similarities between Vps74p and its mammalian homolog. This research is supported by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund and by a US Army Breast Cancer Research Program Predoctoral Fellowship (BC051591). K.M.F is the Dennis and Marsha Dammerman Scholar of the Damon Runyon Cancer Research Foundation.
Selection of a brain‐seeking subline of breast cancer cells
Authors: Teixeira Kathryn , Galileo Deni S.
Published: June 2021

Our lab has shown that MDA‐MB‐231 human breast cancer cells can be isolated from the chick embryo brain after injection into the extra‐embryonic vasculature. Others have demonstrated with nude mice that re‐injection of these cells results in sublines with enhanced capability to metastasize to the brain. It remained unknown whether this was the result of cells specifically targeting the brain, or increased survival in the brain compared to other organs. We transfected MDA‐MB‐231 cells with neomycin resistance and lacZ genes to enable drug‐selection and visualization. This allowed us to quantitate cells which extravasated into the brain and grew as colonies after dissection, dissociation, and drug selection following injection. To determine the sensitivity of the chick embryo system for detecting brain metastases, cells were injected at high and low concentrations. Colonies were detected following injection of as few as 5,000 cells (average of 32.2 colonies/brain). Sublines of cells which had been through the brain up to five times were created. The number of colonies isolated from the brain and liver after re‐injection was compared to that of previously uninjected cells. Re‐injected cells produced a greater number of colonies, suggesting that these cells specifically target the brain. This project was supported by NIH grant 2 P20 RR016472‐07 under the INBRE Program of the National Center for Research Resources.
A role for MGA2, but not SPT23, in activation of transcription of ERG1 in Saccharomyces cerevisiae
Authors: Rice Caitlin , Stukey Joseph , McDonough Virginia
Published: June 2021

A major role of the Saccharomyces cerevisiae MGA2 gene product is regulation of unsaturated fatty acid production, by controlling transcription and mRNA stability of OLE1, the gene encoding the ?‐9 fatty acid desaturase. Lipid composition studies indicated that the mga2? strain contains elevated relative amounts of squalene when compared to wild‐type cells. The deletion of the MGA2 homologue, SPT23, does not similarly impact squalene amounts. To explore the role of MGA2 in the regulation of sterol synthesis, ERG1 transcription was studied using an ERG1 promoter‐lacZ reporter gene construct. We report here that in addition to Mga2p's role in regulation of unsaturated fatty acids, MGA2 is required for full basal expression of ERG1. Mga2p was found to differentially regulate activation of ERG1 and OLE1, as unsaturated fatty acids did not affect ERG1 expression levels as has previously been shown to control Mga2p's activation of OLE1. The spt23? strain shows similar ERG1 expression to wild‐type cells, while the mga2?/spt23? strain shows reduced ERG1 expression, comparable to the mga2? mutant alone, suggesting that the role of regulation of ERG1 transcription is unique to Mga2p. Finally, particular exogenous unsaturated fatty acid supplementation was shown to influence cellular relative squalene amounts, which was less dependent on genotype.

CR was supported by a Deans Research Award
Posttranslational Regulation of the Yeast PAH1‐encoded Phosphatidate Phosphatase
Authors: Soto‐Cardalda Anibal , Carman George M.
Published: June 2021

Phosphatidate (PA) phosphatase catalyzes the Mg2+‐dependent dephosphorylation of PA to form diacylglycerol. This enzyme catalyzes the penultimate step in the synthesis of triacylglycerols and the phospholipids phosphatidylcholine and phosphatidylethanolamine via the Kennedy pathway. The analyses of pah1 mutants in yeast have shown that the PAH1‐encoded PA phosphatase plays a major role in controlling cellular levels of PA and triacylglycerol, the transcriptional regulation of phospholipid synthesis genes, and the growth of the nuclear/ER membrane. Triacylglycerols, which accumulate in the stationary phase of growth, are synthesized during the later stages of the exponential phase. The expression of PA phosphatase activity correlated with the synthesis of triacylglycerols during growth. However, as cells entered the stationary phase, the levels of the PA phosphatase protein disappeared. Immunoblot analysis of ubc1 and ubc4 mutants, which are defective in the ubiquitin‐conjugating enzymes that are induced in stationary phase, indicated that PA phosphatase is subjected to ubiquitination and proteolysis. That PA phosphatase is ubiquitinated is being examined through immunoblot analyses with anti‐ubiquitin antibodies and with antibodies directed against myc‐tagged ubiquitin bound to the PA phosphatase enzyme. Supported by NIH Grants GM 28140 and GM 75378.
Glucosylceramide synthase regulates MDR1 gene expression through the association of globotriaosylceramide (Gb3) and histone acetylation
Authors: Liu Yong Yu , Gupta Vineet , Patwardhan Gauri Anand , Pinnoji Rajeswara , Hsia Victor S. , Jazwinski S. Michal
Published: June 2021

MDR1 gene is frequently overexpressed in drug‐resistant tumor, and encodes a large amount of P‐glycoprotein protecting cells from cytotoxins. Glucosylceramide synthase (GCS) converting ceramide into glucosylceramide initiates glycosphingolipid (GSL) synthesis and defaults drug‐induced apoptosis. It is poorly understood whether GCS has an epigenetic effect on MDR1 upregulation and contributes to drug resistance in cancer. In this study, we report that MDR1 and GCS are co‐overexpressed in drug‐resistant cells and GCS upregulates MDR1 expression. We found that drug‐resistant cells overexpress MDR1 and GCS; blocking GCS repressed MDR1 expression and sensitized these cells. Introduction of GCS gene into cells significantly enhanced MDR1 expression and paclitaxel efflux; suppression of GCS by using antisense oligo dramatically repressed MDR1 expression and cellular efflux. Promoter and Chip assays indicated GSLs modulated MDR1 transcription process. Further evidences on siRNA gene knockdown for Gb3 synthase and GD3 ganglioside synthase, and inhibition of histone deacetylase (HDAC) with trichostatin (TSA) pinpointed the epigenetic effect of GCS on MDR1 relied on Gb3 and histone acetylation (H3, H4). The present study demonstrated the role for ceramide glycosylation by GCS in regulating MDR1 expression. This work is supported by NIH grant (P20 RR16456).
Integration of Nanoscale and Macroscale cues in Bone Regeneration
Authors: Griffith Linda Gay , Platt Manu , Stockdale Linda , Serdy James , Alvarez Luis , Lauffenburger Douglas , Wells D.A. , Muschler G.F.
Published: June 2021

Marrow stromal cells (MSC) can act as a pluripotent source of reparative cells during injury. Many envisioned applications of MSC, such as treating large defects in bone, involve in vivo implantation of MSC attached to a scaffold, a process that creates an acute inflammatory environment that may be hostile to MSC survival and proliferation ‐ especially in large defects. We address ways of integrating nanoscale control of growth factor presentation with macroscale design of scaffold architecture with an eye toward influencing multiple biological processes, including growth, proliferation, migration, and differentiation of cells that contribute to bone formation. We have focused especially on the role of epidermal growth factor (EGF) receptor signalling. For some cell types, soluble EGF acts as a survival signal in the face of pro‐death stimuli, but MSC appear refractory toward survival when challenged with pro‐death stimuli in the presence of soluble EGF. In contrast, when EGF is covalently tethered to the scaffold, it can activate the EGFR signaling pathways and strongly promotes spreading and survival of MSC. Substrate‐tethered EGF also influences proliferation, colony formation, and differentiation of human MSC, hence holds potential for enhancing bone regeneration.
A novel approach for investigating glycan involvement in embryonic cellular interactions. II. lectin studies
Authors: Naminik Arjang , Oppenheimer Steven B.
Published: June 2021

Here we investigate the effects of 5 lectins at concentrations ranging from 0.0005 mg lectin ‐ 0.5 mg lectin per ml low calcium sea water on specific cellular interactions in the NIH model sea urchin embryo Lytechinus pictus using a microplate assay. 3 separate experiments were conducted with 12 microwells in each experiment at each lectin concentration, yielding a total of 36 wells with hundreds of embryos assessed for each lectin concentration. Lectins(or no lectin controls) were added at 24 hrs of development and the effects on archenteron/elongation/binding to the blastocoel roof were recorded after an additional 24 hrs. Phytolacca americana (specific for N‐acetyl‐beta‐D‐glucosamine residues) inhibited archenteron elongation/attachment to the blastocoel roof in a concentration dependent manner when controls and treated embryos were compared (p less than 0.0001 comparing complete archenterons in controls versus experimentals). Pisum sativum (specific for alpha‐D‐glucose and alpha‐D‐mannose) and Ulex europaeus UEA 1 (specific for L‐fucose) lectins both caused exogastrulation, where not only does the archenteron not attach but it everts from the embryo proper (p value of less than 0.0001 comparing exogastrulation in controls versus experimentals). Helix pomata (specific for N‐acetyl‐alpha‐D‐galactosamine) and Glycine max (specific for N‐acetyl‐alpha‐D‐galactosamine and D‐galactose) had no effects on the embryos, like the controls.

The results indicate that specific lectins that bind to specific saccharides influence a model cellular interaction. Most important, however, is that the microplate approach used here is able to quantitatively assess specific probed events in living embryos (Supported by NIH NIGMS SCORE (S0648680), RISE, MARC , the Joseph Drown Foundation and the Sidney Stern Memorial Trust).
Salt inhibition of immobilized lectin binding: a model for anti‐infection drug testing
Authors: Zem G. C. , Lim J. , Khurrum M. , Gabrielyan Z. , Gabrielyan A. , Khandakhchyan A. , Allen J. , Dayan M. , Petrosyan S. , Goldstein L. , Guiterrez M. , Jahanbakhsh A. , Abramian S. , Uber L. , Daco E. , Kojasaryan J. , Khademi T. , Basra J. , Aghakhanian S. , Oh P , Qazi U. , Oganyan S. , Yaghoubi A. , Margarian A. , Balazadeh H. , Dergrigorian L. , Norouzzadeh M. , Nguyen M. , Yaghoubi A. , Ghazarian A. , McNee Z. , Mendez F. , Oppenheimer S. B.
Published: June 2021

Past studies have examined the inhibition of binding of mannose‐rich yeast to immobilized concanavalin A (Con A) in the presence and absence of specific saccharides. This is a model system for testing potential drugs that could block pathogen binding to human cells. Here 2.0M, 0.2 M and 0.02 M NaCl and KCl were tested in a much more extensive study than in the past, for their ability to inhibit binding of yeast (Saccharomyces cerevisiae) to immobilized Con A over a 30 min time course. In about 15,000 replicate experiments, both salts inhibited binding in a nearly identical way, in a concentration dependent manner, ranging from about 20% to about 60% reduced binding over controls. The salt effects reached a plateau at 20‐30 minutes, with 2.0 M salt showing the greatest inhibitory effects. These results are similar to those obtained in studies with specific saccharides, suggesting that salt effectively can block lectin‐saccharide binding, with possible implications in pathogen binding to human cells (Supported by NIH NIGMS SCORE (S0648680), MARC, RISE, the Joseph Drown Foundation and Sidney Stern Memorial Trust.
O‐GlcNAc modification of proteins in mouse embryonic neural stem cells
Authors: Yanagisawa Makoto , Yu Robert K
Published: June 2021

In neural stem cells (NSCs), glycoconjugates and carbohydrate antigens are known to serve as excellent biomarkers for cellular differentiation and to play functional roles in determining cell fate. O‐Linked β‐N‐acetylglucosamine (O‐GlcNAc) modifying nuclear and cytoplasmic proteins on the serine/threonine residues is also expected to play the regulatory roles. It is not known, however, if O‐GlcNAc modification is expressed in NSCs and what the function of this expression is. In this study, we evaluated the patterns and possible functions O‐GlcNAcylation in mouse embryonic neuroepithelial cells (NECs) which are rich in NSCs. We found the expression of O‐GlcNAc transferase, O‐GlcNAcase, and a number of O‐GlcNAcylated proteins in NECs. Treatment of NECs with O‐GlcNAcase inhibitors induced robust O‐GlcNAc accumulation in NECs and reduction of the cell number. In O‐GlcNAcase inhibitor‐treated NECs, the Ras‐MAPK pathway and the PI3K‐Akt pathway, important for proliferation and survival, respectively, were intact, but caspase‐3, an executioner of cell death, was activated. These results suggest that O‐GlcNAc is involved in cell death signaling in NECs. Furthermore, we identified an O‐GlcNAc‐modified protein, Sp1 transcription factor. This work was supported by USPHS grants NS11853, AG027199 and a grant from the Childrens' Medical Research Foundation, Chicago, IL.
Expression of Sialyl Le(x), Sialyl Le(a), Le(x) and Le(y) Glycotopes in Secreted Human Ovarian Cyst Glycoproteins
Authors: Wu Albert M. , Yang Zhangung
Published: June 2021

Human blood group determinants expressed on ovarian cyst glycoproteins (gps) have been studied for over five decades. However, little is known about sialyl Lex and sialyl Lea glycotopes, which play essential roles in inflammation, and cancer cell metastasis. Furthermore, Lex and Ley were classified as glycotopes of unknown genes due to lacking specific antibodies. In this study, the occurrence of sialyl Lex, sialyl Lea, Lex and Ley reactivities in cyst glycoproteins was characterized by ELISA. The results indicated that most human ovarian cyst glycoproteins carried Lex (8/25) and/or Ley (17/25) glycotopes. The expression (epitopes) of the new genes described in previous reports are Lex and Ley glycotopes; the reactivities of sialyl Lex and sialyl Lea glycotopes in secreted cyst gps may be affected by the conditions of purification; the relationship between Ley and human blood group ABH was confirmed; recognition profiles of sialyl Lex, sialyl Lea, Lex and Ley present in the carbohydrate chains of water‐soluble cyst gps were illustrated; possible attachments of glycotopes to the internal carbohydrate complex of cyst gps have been reconstructed; proposed biosynthetic pathways for the formation of sialyl Lea, sialyl Lex, Lex, Ley, ALey and BLey determinant structures on Type I and Type II core structures of human ovarian cyst gps are also included in this study.
Phenylalanine 508 forms an intra‐domain contact crucial to CFTR folding and dynamics
Authors: Serohijos Adrian Wendil Ruba , Hegedus Tamas , Aleksandrov Andrei , He Lihua , Cui Liying , Riordan John R , Dokholyan Nikolay V
Published: June 2021

Cystic fibrosis is the most prevalent genetically inherited disease in the US. In 90% of CF cases, the disease is caused by the deletion of phenylalanine‐508 (Phe508) from the first nucleotide‐binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP‐binding cassette (ABC) transporter. Prior experimental studies suggest that Phe508 deletion cause the aberrant folding of NBD1 and the misassembly of various CFTR domains. To understand the role of Phe508 in channel function and of the impact of its deletion, we constructed a 3D structural model of CFTR using the structure of another ABC transporter, Sav1866. The CFTR model predicts, and we experimentally validated, that Phe508 mediates an interaction between NBD1 and the fourth cytoplasmic loop in the second membrane‐spanning domain. This explains the observed sensitivity of CFTR assembly to many disease‐associated mutations in CL4 and NBD1. Other cytoplasmic‐membrane domain contacts were also experimentally confirmed. Electrophysiological measurements and molecular dynamics simulations also show that the CFTR membrane‐cytoplasmic interfaces regulate channel gating and are highly dynamic. Aside from advancing our understanding of CFTR structure and function, this study identifies a focused region in CFTR that can be targeted in drug development.

Funding is provided by NIH (JRR), CFF (NVD), and AHA (AWRS).
VEGF Regulates Levels of PKC, PLC and eNOS mRNAs in the Cervix of Rodents During Remodeling
Authors: Irwin Whitney Wellborn , Mowa Chishimba
Published: June 2021

Cervical remodeling is an inflammatory response that is closely associated with microvascular changes. Mechanisms that underlie VEGF effects such as angiogenesis, vascular permeability and vasodilation are tissue‐dependent but not completely known. In some tissues they are mediated by signaling molecules, such as PKC, PLC and eNOS. The exact signaling pathway for VEGF effects in the cervix is not known. Our previous work has shown that levels of VEGF, VEGF receptors, and some key signaling molecules change during pregnancy. Here we verify our previous DNA microarray data using real time PCR. We show that VEGF agents (recombinant protein and blocker) in the cervix alter mRNAs of eNOS, PKC, and PLC‐γ dose‐dependently. Based on this data we conclude that the effects of VEGF on vascular events during cervical remodeling are likely mediated by PKC, PLC, and eNOS.
The Pathogenesis of Febrile Seizures: An Update
Authors: Wang Yuncui , Yu Fang , Peng Biwen , Liu Yanlan , He Xiaohua
Published: June 2021

Febrile seizures(FS)is a specific epilepsy syndrome induced by fervescence in childhood,and it has been suspected to have a genetic basis.KCNQ2 and KCNQ3 genetic mutations related to benign familial neonatal convulsions(BFNC) have been confirmed, and mutations associated with generalized epilepsy with febrile seizure plus(GEFS+)were identified in SCN1A?SCN2A?SCN1B and GABRG2 genes. But the penetrance of FS and GEFS+ are incomplete and the clinical expression are very heterogeneous, suggesting that modifying genes and/or environmental factors strongly affect the phenotype.So the effects of temperature on ion channels associated with GEFS+ and FS are attracting increasing attention. Previous study has suggested that interleukin‐1β, a pyrogenic proinflammatory cytokine,is involved in the generation of febrile seizures or enhanced seizure susceptibility in animals, also recent research has been shown a mutation in the GABAA receptor subunit GABRG2 gene causing a temperature‐dependent intracellular trafficking defect‐misfolding and impaired assembly and ER retention, increased degradation, or rapid endocytosis would be worsened by fever resulting in febrile seizures.Moreover,our previous simulation studies have been shown that there is an obvious conformational change of M channel at 40?compared with that at 37?, and the fact that considerable temperature sensitivity in KCNQ2/KCNQ3 potassium channel has been verified.The intracellular trafficking defect of channel proteins caused by elevated temperature provides a new insight into the molecular pathomechanism of FS .
Dynein motor contributes to stress granule dynamics in primary neurons
Authors: TSUI YAO‐CHEN , TSAI NIEN‐PEI , WEI LI‐NA
Published: June 2021

Mobilization and translation of mRNAs are essential for cellular response to environmental stress, and important to neuronal survive and activity. Studies have shown mRNAs accumulation in stress granules (SGs) in stressed cells. We previously reported the formation of SGs in embryonal carcinoma cells can be regulated by kinases such as focal adhesion kinase (FAK) and RNA binding proteins such as Grb7. However, the formation and disassembly of SGs in neurons remains unclear. In this study, we used arsenite to induce cellular oxidative stress in primary neuronal cultures, and found SGs formation in both cell bodies and neurites of stressed primary spinal cord neurons. By employing siRNA dockdown, we discovered that dynein motor subunit localizes in SG, and is important for SG assembly in neurons. Under stress, dynein motor subunit also facilitates translational repression and enhances the formation and integrity of SG in neurons. By blocking the energy source of dynein motor, both the formation and disassembly of SG are attenuated. These findings demonstrate, for the first time, that dynein motor complex plays a critical role in the formation of neuronal SGs, as well as translation of certain mRNAs. This work was supported by DA11190, DA11806, DK54733, DK60521, K02‐DA13926 to L.‐N. W.
Pasteurella multocida toxin‐induced Gαq‐dependent mTOR‐mediated ribosomal S6 protein phosphorylation
Authors: Oubrahim Hammou , Chock P. Boon
Published: June 2021

Pasteurella multocida toxin (PMT) is a potent mitogen. We investigated the effect of PMT treatment on the mammalian target of rapamycin (mTOR), a protein kinase believed to play a key role in cell growth, proliferation, and apoptosis. Western blot analysis revealed that PMT was able to stimulate mTOR in wild type mouse embryonic fibroblasts (MEF) and in Swiss 3T3 cells as judged by a hefty increase in the phosphorylation of its downstream target, 40S ribosomal S6 protein at Ser235/236 and Ser240/244. This observation was validated by results showing that PMT was capable of inducing mTOR phosphorylation at serine 2448 and S6K1 phosphorylation at serine 389 in 3T3 cells. However, in MEF cells lacking only Gα11, PMT was able to stimulate S6 phosphorylation at Ser240/244 but not Ser235/236. In MEF cells lacking both Gαq and Gα11, we observed a decrease in S6 phosphorylation at Ser235/236 and Ser240/244 following PMT treatment. To confirm that PMT‐induced S6 phosphorylation is directly mediated through mTOR, we showed that rapamycin, a specific inhibitor of TORC1, inhibits PMT‐induced S6 phosphorylation in wild type MEF and in 3T3 cells. Our results reveal for the first time that PMT induces a Gαq‐dependent activation of mTOR which in turn activates S6k1 and leads to S6 phosphorylation. This research was supported by the Intramural Research Program of the NIH, NHLBI.
Enhanced ROS production and redox signaling with combined arsenite and UVA exposures
Authors: Cooper Karen L , Liu Ke Jian , Hudson Laurie G
Published: June 2021

Solar ultraviolet radiation (UVR) is the major etiological factor in skin carcinogenesis. In vivo studies demonstrate that mice exposed to arsenic and UVR exhibit significantly more tumors and oxidative DNA damage than animals treated with either agent alone. Interactions between arsenite and UVR in the production of reactive oxygen species (ROS) and stress‐associated signaling may provide a basis for the enhanced carcinogenicity. Here keratinocytes were pretreated with arsenite (3 µM) then exposed to UVA (10 kJ/m2). Exposure to UVA following arsenite pretreatment enhanced ROS production, p38 MAP kinase activation and induction of heme oxygenase‐1, when compared to either stimulus alone. Arsenite exposure resulted in pronounced and persistent NADPH oxidase (NOX) activation whereas UVA activation was rapid and transient. Inhibition of NOX decreased ROS production in arsenite treated cells but had little impact on UVA exposed cells. Furthermore, arsenite‐, but not UVA‐, induced p38 activation and HO‐1 expression were dependent upon NOX activity. These findings indicate differences in mechanisms of ROS production by arsenite and UVA that may provide an underlying basis for the observed enhancement of redox‐related cellular responses. Work supported by NIH R01 ES012938, NIH AR 42989, NIEHS P30‐ES‐012‐72, and EPA STAR Fellowship FP‐91650201‐1.
Identification and characterization of HePP1, a Heart protein phosphatase 1 binding protein
Authors: Chen Chun‐Yu , Lai Ning‐Sheng , Yang Jaw‐Ji , Huang Hsien‐bin
Published: June 2021

We have conducted the search for the new protein phosphatase 1 (PP1)‐binding proteins of heart cells by using a yeast two‐hybrid system and identified six candidate proteins. One of those was a novel protein, Heart protein phosphatase 1 binding protein (HePP1). HePP1 contains 186 amino acids and is encoded by FLJ23654 . We have demonstrated that HePP1 was associated with PP1 in GST pull‐down assay, co‐immunoprecipitation, and far‐western blot. Northern blot analysis revealed that HePP1 mRNA was mainly distributed in heart and testis, but not in other tissues. The recombinant HePP1 cannot inhibit the PP1 activity, but antagonizes the inhibition of PP1 by phospho‐inhibitor‐1.
Characterization of EGFR pathway using small molecule inhibitors and In‐Cell ELISA technology
Authors: Narahari Janaki , Hughes Douglas E , Webb Brian
Published: June 2021

In‐Cell ELISA technology represents an emerging new technique for studying intracellular signaling proteins in context of the whole cell using post‐translational modification (PTM)‐specific antibodies. It offers a convenient and fast cell‐based assay methodology for analyzing signal transduction pathways. Typically, Western Blots are used to monitor post‐translational protein modifications (PTMs), using specific antibodies. However, western blotting involves time‐consuming steps such as lysate preparation, gel loading and electrophoresis, membrane transfer and probing of the membrane with the appropriate antibody. Western blots are also only semi‐quantitative and have very low throughput. PTMs associated with protein activation can be easily monitored by In‐Cell Elisa method in a 96‐ or 384‐well microplate format, which allows for assay scalability and automation while conserving valuable reagents like cell culture regents and stimulators/inhibitors.

Development of reagents for In‐Cell Elisa, compatible with either Colorimetric or Fluorescent detection of proteins, are described here. We demonstrate the utility of In‐Cell Elisa technology for simultaneous analysis of different proteins involved in Epidermal Growth Factor signaling using various small molecule activators and inhibitors. Results comparing our In‐Cell Elisa technology to more commonly used western blots technology will be demonstrated.
Keyhole limpet hemocyanin, an immunological adjuvant, inhibits locomotion and egg laying in C. elegans
Authors: Huggins Luke Graham , Wolf Brian Christopher , Getz Dustin J. , Vona‐Davis Linda
Published: June 2021

Keyhole limpet hemocyanin (KLH) is the copper‐containing respiratory pigment of the keyhole limpet mollusk. It has been used as an adjuvant in many vaccines. It is not known how KLH stimulates the immune system. KLH can also induce apoptosis and necrosis in Barrett's esophageal adenocarcinoma cells (McFadden et al. 2003). To understand the mechanism of KLH function, we tested its effects on C. elegans, a small, free‐living nematode worm frequently used as a genetic and developmental model system. In C. elegans treated with 300 mg KLH/mL, there was decreased feeding behavior, locomotion, and increased egg‐retention. All three of these behaviors are regulated by serotonin signaling. Using locomotion and egg laying assays, we screened for the effect of KLH on 13 mutant lines that modulate serotonin signaling in C. elegans. Loss of function mutants in the SER‐4 pathway showed a significant decrease in egg laying, while loss of function mutants in the SER‐1 pathway did not. SER‐1 mutants showed a significant decrease in locomotion indicating that SER‐1 is not affected by KLH. These results suggests that KLH functions primarily through the activating SER‐1 signaling pathway downstream of SER‐1. Supported by NIH Grant 5P20RR016477 to the West Virginia IDeA Network for Biomedical Research Excellence
Inhibition of castration‐resistant prostate cancer by the cancer/testis antigen PAGE4, a novel corepressor of the androgen receptor
Authors: Sampson Natalie , Dirnhofer Stephan , Berger Peter
Published: June 2021

Cellular homeostasis and function of the prostate is regulated by androgens and mediated via the androgen receptor (AR), whose activity is regulated by coactivator and corepressor proteins that enhance or silence AR, respectively. Aberrant reactivation of AR signaling is a pivotal mechanism by which tumors escape androgen‐ablation therapy, the current mainstay systemic treatment for prostate cancer (PCa), which is the second leading cause of cancer death in the US. Prostate‐associated gene 4 (PAGE4) is a member of GAGE‐related cancer/testis antigens, with expression in males confined to prostate and testis. PAGE4 overexpression in LNCaP cells (an AR+ PCa cell line) attenuates AR activity in luciferase assays, reduces androgenic proliferative response and expression of the androgen‐regulated gene Prostate Specific Antigen (PSA). Consistently siRNA‐mediated PAGE4 knockdown potentiates nuclear receptor activity and increases endogenous gene expression. Collectively these data indicate that PAGE4 is a novel AR corepressor. Using LNCaP xenografts to study tumor progression following castration‐mediated androgen depletion, loss of PAGE4 was observed, which temporally correlated with tumor regrowth, AR reactivation and resumed PSA expression. Moreover, tumor incidence and growth was significantly impaired in xenografts overexpressing PAGE4 under androgen‐depleted conditions. Thus by inhibiting AR activity PAGE4 prevents AR reactivation following androgen depletion and thereby prevents tumor progression. Targeting PAGE4 may thus represent a novel therapeutic strategy for advanced PCa.
Biological activity of some common lichens
Authors: Brennan Jarryd , Vaden Matthew , Lester Christen , Crixell Sylvia , Vattem Dhiraj
Published: June 2021

Lichens, are symbiotic organisms of fungal and an algal and/or cyanobacterial partner and are considered edible or used for their medicinal properties by many cultures. They produce a variety of secondary compounds unique to lichens, mostly from fungal metabolism. In spite of the diversity few studies have evaluated the biological activity of secondary metabolites from lichens. Our objective was to evaluate the antioxidant, antimicrobial and anti‐adhesive activity of common lichens in Central Texas against Pseudomonas aeruginosa. Lichens were harvested from trees and rocks using standard techniques and identified by morphological, biochemical and microscopic analysis. The low molecular weight fraction (LMWF) and high molecular weight fraction (HMWF) were isolated using standard protocols. Antioxidant activity was determined by Malondialdehyde (MDA) assay and radical quenching assay. Antimicrobial and anti‐adhesive activity of sterile extracts against P. aeruginosa was determined by Kirby‐Bauer (KB) and ex vivo skin assay respectively. Lichens harvested were identified as Ramalina celastri (RA), R. stenospora (RS), R. Americana (RA), T. chrysopthalmus (TC), Parmotrema austrosinese (PA) and P. perforatum (PP). The HMWF had higher antioxidant activity and neutralized 60‐75% radicals compared to LMWF which only neutralized 20‐40% of the radicals except PA whose activity was 79%. All extracts were able to reduce MDA formation by 30‐88% except LMWF of RA and PP. None of the extracts had antimicrobial activity in the KB assay. All the HMWF were able to reduce adhesion of P. aeruginosa by 30‐55% except PP. LMWF from RS, PP, PA and PP reduced bacterial adhesion by 41‐54%. Our results suggest that Lichens can be an important source for bioactive compounds with benefits against oxidative and infectious diseases and warrants further investigation.
Use of herbal supplements among rural African‐Americans participating in a cardiovascular disease prevention program
Authors: Dawkins Norma L , Phillips Beatrice W , Pace Ralphenia D
Published: June 2021

The use of alternative medicine (herbal products) among Americans has increased over the years. The increased use is associated with their proposed curative and preventive disease potentials and the promotion of health and longevity. The use of herbal supplement remains a concern for healthcare professionals. Many herbs can interact with prescription drugs, and may cause complications in certain disease conditions. This study assessed knowledge, use and safety of herbal supplement among African Americans participating in a cardiovascular disease prevention program. A questionnaire was designed for a rural low literacy population. It was distributed to 103 participants consisting of 88% women and 12% men. Frequencies and one‐way ANOVA was used to test the significance of selected variables. Eighty‐six percent of participants had knowledge of ginseng followed by St. Johns Wort (62%) and ginkgo biloba (61%). Only 27% used herbal supplements regularly while 28% used them sometimes and 41% not using them at all. Only 22% reported taking garlic pills. On safety issues 57% would stop taking herbal supplement. No difference (p>0.05) was observed between usefulness and safety. Based on the preliminary results no wide spread use of herbal supplement was observed this population group.
A Volatile Distillate By‐Product of Mint Oil Promotes Absorption of Co‐enzyme Q10 in Caco‐2 cells grown on semi‐permeable membranes.
Authors: Hintze Korry Joseph , Timbimboo Hunter , Finley John
Published: June 2021

Elevated serum cholesterol is a major risk factor for heart disease, and statin drugs are particularly useful for the reduction of cholesterol levels. Statins decrease body stores of Coenzyme Q10 (CoQ10), and many health care practitioners urge CoQ10 supplementation concomitant with statin use. However CoQ10 oral bioavailability is low, calling into question the efficacy of such supplementation. A natural mixture of volatile distillate compounds (VDC) derived from mint during the production of mint flavors was tested for the ability to promote the absorption of CoQ10. Absorption (cellular uptake and transcellullar transport) was studied in Caco‐2 cells (grown on 24 mm dia. semipermeable membranes). CoQ10 and VDC (at several ratios), BHT, bile salts and cell culture media were sonicated to induce micellular formation, applied to the apical surface of cells and incubated 4 or 6 hours. Control media without VDC did not induce absorption of CoQ10; media containing VDC induced a dose‐responsive increase in CoQ10 absorption with a maximum response at 1% VDC (maximum cellular and transcellullar transport were 10‐30‐fold that of controls). We conclude that VDC from mint oils may be a novel means of enhancing the low bioavailability of lipophillic compounds such as CoQ10.
The anti‐adipogenic effect of quercetin is associated with coordinated changes of antioxidant responses during adipogenic differentiation of 3T3‐L1 cells.
Authors: Lee Ok‐Hwan , Swick Jennifer , O'Fallon Kevin , Clarkson Priscilla M , Kim Young‐Cheul
Published: June 2021

Increased reactive oxygen species (ROS) from accumulated fat in obesity has been shown to elevate systemic oxidative stress, contributing to diabetes, hypertension, and coronary heart disease. Here, we investigated the effects of a flavonoid quercetin on the changes of gene expression associated with cellular antioxidant responses and adipogenesis during differentiation of 3T3‐L1 preadipocytes. The changes in levels of lipid, ROS, mRNA expression of PPARγ, glucose‐6‐phosphate dehydrogenase (G6PDH), glutathione reductase (GR), and catalase was determined. ROS production significantly increased in parallel with lipid accumulation in adipocytes, which correlated with increased mRNA expression of all of the genes. Exposure to 100µM quercetin inhibited PPARγ (~3‐fold) and G6PDH mRNAs (~4‐fold) as well as adipogenesis and ROS accumulation compared to control. Quercetin also markedly inhibited mRNA expression of GR and catalase (~5 and 3‐fold, respectively). The inhibition of PPARγ and G6PDH by quercetin likely prevented hexose monophosphate (HMP) pathway‐mediated NADPH generation required for lipid synthesis and GR activity. These observations suggest that, during adipogenesis, the antioxidant enzymes are positively regulated to manage oxidative stress from accumulating fat, and that the anti‐adipogenic action of quercetin may involve coordinated changes of cellular redox state in adipocytes
Effects of Lycium barbarum on Feelings of General Well‐being, Neurological/Psychological Traits and Plasma Levels of Catecholamines. A Randomized, Double‐blind, Placebo‐controlled Human Clinical Study
Authors: Amagase Harunobu , Nance Dwight M
Published: June 2021

We examined the effects of orally consumed Lycium barbarum, as a standardized juice (GoChi), in healthy adults (18‐60 years old). Subjects consumed 120 ml/day of GoChi or placebo for 30 days. Questionnaires, graded 0‐5 for general feelings of well‐being and neurological/psychological traits and plasma levels of catecholamines (epinephrine and norepinephrine) were assessed before and following the intervention, and analyzed for the changes between pre‐ and post‐intervention by the nonparametric Wilcoxon matched pairs tests and ANOVA, respectively. Significant improvements for overall effects, sleep quality, ease of waking and better circulation (p<0.05) were found in the GoChi group (N = 20). GoChi also significantly reduced feelings of impaired concentration, excess worry, unreasonable worry, memory loss and tiredness after exercise. Placebo group (N = 19) showed no significant changes, but there was a tendency to increased stress and depression. Plasma levels of epinephrine and norepinephrine in the placebo group, compared to pre‐intervention, were significantly increased by 20% and 31%, respectively, whereas the GoChi group showed no significant changes. These results suggest that daily consumption of GoChi increases subjective feelings of general well‐being and neurological/psychological traits, which may be related to the stabilization of catecholamine levels.
Raisins as a source of prebiotic compounds in the diet
Authors: Carughi Arianna
Published: June 2021

Fructans, oligo‐ and polysaccharides of fructose, include both frucooligosaccharides and inulins. They are not digested in the human small intestine and are therefore considered as dietary fiber. Fructans may provide a wide range of health benefits. Acting as prebiotic compounds, they promote the growth of beneficial intestinal flora and suppress the growth of potential pathogens in the colon. They may also increase stool‐bulking capacity; stimulate the gastrointestinal immune system and may reduce the risk of colon cancer. While fresh grapes have no detectable fructans, the dehydration process converts grape sugars into fructans. The objective of this study was to characterize the dietary composition of four different commercially important raisin types: sun‐dried raisins (conventional and dried on the vine); golden raisins and Zante currants. Total, soluble and insoluble fiber was determined by AOAC method 991.43, inulin by AOAC method 997.08 and fructan by AOAC method 999.03. Total dietary fiber values agreed with published results. Raisin inulin content ranged between 0.71% to 1.2% and fructan between 0.78% and 0.96%, thus having among the highest prebiotic content of commonly consumed fruits. Results suggest that the type of dehydration, storage and grape variety influence raisins fructan content.