bluuebunny/crossref_metadata_2025 huggingface dataset:
The design and characterization of an RNA nanoring for use in RNA interference drug delivery
Authors: Grabow Wade , Severcan Isil , Chworos Arkadiusz , Shapiro Bruce , Luc Jaeger
Published: June 2021

While RNA interference (RNAi) offers tremendous promise as a selective means to silence gene expression, novel packaging and delivery methods are needed to improve its overall therapeutic effectiveness. The following RNA nanoring was previously designed as a simple means to package and deliver RNAi. Characterization of the RNA nanoring and the contribution of the RNAI/RNAII inverse interaction responsible for its specific assembly were carried out through the rational design of monomers possessing helical stems with altered degrees of stability, twist, and rigidity. Experimental analysis of the different monomer assemblies confirms that the geometry of the loop‐loop interaction is the dominant factor in the formation of RNA nanorings. Subsequent studies on one specific RNA nanoring design have demonstrated a promising level of resistance toward ribonuclease degradation and confirm its potential as a platform for RNAi drug delivery. This work was funded by the National Institutes of Health (RO1 GM079604‐01) to Luc Jaeger.
PPARbeta and the translational status of PMA‐differentiated THP‐1 cells
Authors: Cambiaghi Tavane David , Luchessi Augusto Ducati , Takahashi Hilton Kenji , Curi Rui
Published: June 2021

The differentiation of THP‐1 cells in macrophages, induced by PMA, is associated to overexpression of peroxisome proliferator‐activated receptor b (PPARb). Previous studies have shown that the PPARb 5′ UTR negatively regulates its expression. In our study the translational status of PMA‐differentiated THP‐1 cells was investigated in association to overexpression of PPARb. Putative compatible Kosak initiation codons were identified in the PPARb uORFs and could be involved in the inhibitory effect of 5′ UTR. Decreased incorporation of L‐[U‐14C]Leucine in proteins revealed that the overproduction of PPARb in PMA‐differentiated THP‐1 cells coincides with a global decrease in the protein synthesis process. Translation impairment was confirmed by polysome profile assay. An intense dephosphorylation of 4E‐BP by PMA treatment was observed. Dephosphorylated 4E‐BP causes inhibition of eIF4E cap‐dependent translation initiation and favors IRES‐dependent translation. The PPARb 5′ UTR structure has some characteristics that resemble the one described for IRES. Therefore, the PPARb production may be controlled by IRES and the high production of PPARb occurs concomitantly with cap‐dependent translation inhibition.
Translational regulation of the stress response in a Saccharomyces cerevisiae cytokinesis mutant myo1Δ
Authors: Rivera‐Ruiz Marielis E , Rodríguez‐Medina José R.
Published: June 2021

Previous analysis of global mRNA expression in Saccharomyces cerevisiae myosin type II deficient strains (myo1Δ) revealed 547 genes that are regulated at the transcriptional level. The objective of this study is to explore the post‐transcriptional regulation of mRNAs in myo1Δ. To assess the mode of regulation of ribosomal protein (RP) mRNAs, we analyzed the polysomal distribution of three representative RP mRNAs in sucrose gradient fractions of myo1Δ and wild‐type (wt) cells. Our results showed that RPS8A, RPL7B and RPL3 mRNAs were recruited from heavy to lighter polyribosome fractions in myo1Δ. Furthermore, western blot analysis revealed accumulation of the phosphorylated form of eukaryotic initiation factor 2 (eIF2α‐P) in myo1Δ and RPS8A and RPL7B mRNAs were co‐precipitated with immunoprecipitated eIF2α‐P, suggesting a direct association between eIF2α‐P and translationally regulated RP mRNAs. Treatment of myo1Δ with rapamycin did not increase the levels of eIF2α‐P yet, a gcn2Δmyo1Δ strain exhibited a synthetic growth defect. Reduced steady state levels of the initiation factor eIF4G was also observed supporting regulation of translation by TOR pathway. These findings support the conclusion that post‐transcriptional control by translation inhibition is a key component of the stress response in yeast. Supported by NIGMS/NIAID (5SC1AI081658‐02), NCRR‐RCMI(G12RR03051) and MBRS‐RISE (R25GM061838).
Diabetes Mellitus: Clinical and Biochemical Perspectives
Authors: Merriam Kathryn , Stahelin Robert V
Published: June 2021

Diabetes mellitus is a disease in which the body cannot produce or properly use insulin, a hormone necessary for the uptake of glucose in cells. According to the American Diabetes Association, 23.6 million people in the United States (7.8% of the population) have diabetes. There are three distinct types of diabetes – type I, type II, and gestational – but this project will look specifically at types I and II which are characterized by failure to produce insulin and by insulin resistance respectively. Both type I and type II diabetes leave glucose free to circulate in the bloodstream since cells cannot take up this glucose without insulin present. High concentrations of glucose in the bloodstream have been shown to cause acute and long‐term health complications such as diabetic ketoacidosis, glycosuria, polyuria, nerve and tissue damage, retinopathy, stroke, and kidney disease, all of which we will present from a clinical perspective. We will also examine diabetes mellitus from the biomolecular level by investigating the relationship between hyperglycemia, PKCε activation, and β cell dysfunction. Additionally, we will research one particular diabetic complication – retinopathy – at the biomolecular level. Results will be discussed in terms of abnormal renal vasodilatation and vascular permeability through PKC‐dependent mechanisms.
Changing the binding mode to peroxisome proliferator activated receptor (PPAR) alpha/gamma: a new ligand with improved antidiabetic and antiobesity properties
Authors: Mitro Nico , Gilardi Federica , Giudici Marco , Loiodice Fulvio , Fracchiolla Giuseppe , Laghezza Antonio , Pochetti Giorgio , Lavecchia Antonio , Guerrini Uliano , Tremoli Elena , Rando Gianpaolo , Maggi Adriana , Novellino Ettore , Crestani Maurizio
Published: June 2021

PPARs are ligand activated transcription factors playing a key role in lipid, glucose and energy metabolism. PPAR agonists are used as hypolipidemic and antidiabetic agents, however since these ligands have shown side effects, the hunt for selective PPAR modulators avoiding adverse reactions is still open.

By a transfection based screening we identified LT175 that binds and activates both PPARα and γ subtypes. The 3D structures of PPARγ/LT175 complex reveals a new binding mode with a distinct coactivator recruitment profile. In transcription assays with PPRE‐Luc reporter mice, LT175 activates PPAR program in target tissues. In high fat fed mice, LT175 ameliorates the metabolic profile and reduces visceral adiposity. Circulating levels of adiponectin increases while plasma insulin decreases. LT175 enhances the expression of target genes in the liver and adipose tissue. We also observed increased brown fat mass that contributes to decrease body weight via fat burning an effect secondary to an increased FGF‐21 expression. Altogether, we demonstrate that LT175 is a lead compound with favorable effects on glucose and lipid metabolism that could be developed to treat diabetes and obesity

Funded by FP6 LSHM‐CT‐2006‐037498 from the European Community to MC and The Giovanni Armenise‐Harvard Foundation career development grant to NM
Effects of Intensities Treadmill Exercise on GLUT‐4 and MCT‐1 of Gastrocnimus Muscle and UCP‐3 Expression of Brown Adipose Tissue in Diabetic GK‐Rats
Authors: Il Na Chan , Kim Donghee , Lee Hayan , Kim Hyeok , Jung Jaekwan , Yang Meehyun , Kwon Soyoung , Moon Hyunwoong , Baek Changyei , Kim Daeyeol
Published: June 2021

In the present study, the effect of different exercise intensities training on the GLUT‐4 and MCT‐1 of gastrocnimus muscle and UCP‐3 expression of brown adipose tissue in rats was investigated. The GK rats were classified into four groups, 10 rats each: the control group, the low intensity exercise group, the moderate intensity exercise group and the high intensity exercise group, Rats of the exercise groups were made to run on a motorized treadmill by different intensity to each group for 30minutes once a day during 8 weeks. GLUT‐4 and MCT‐1 and UCP‐3 expression in rats were also measured by western blotting. It was shown that GLUT‐4 and UCP‐3 expression were increased significantly following the low and the moderate exercise training compared to the high exercise group after 8 weeks. But MCT‐1 was increased significantly following the moderate and the high exercise training compared to the low exercise group after 8 weeks. It is concluded that low and moderate intensity exercise induces translocation of GLUT‐4 and UCP‐3 which result in increased blood inflow thus glucose tolerance is increased. Increased UCP‐3 expression may be mediated by cyclic AMP increase, which is activated the lipolysis during exercise and then to increase GLUT‐4 transcription. Further research needs to be done to investigate the MCT‐1 increase by high intensity exercise training.
Pyruvate Dehydrogenase Kinase (PDK) 3 Contributes to the Decreased Metabolic Flexibility of Vascular Smooth Muscle (VSM)
Authors: Ward Kathryn R , Roberts Tina M , Hardin Christopher D
Published: June 2021

Previous data show that VSM exhibits decreased metabolic flexibility during acute dyslipidemia as compared to other muscle that we propose is due to differing control mechanisms of the pyruvate dehydrogenase complex (PDC). We examined the effects of the pyruvate dehydrogenase phosphatase stimulator D‐Chiro‐inositol (DCI) on glucose utilization and oxidation along with protein analyses of hog skeletal muscle and VSM PDK isoforms. Hog carotid arteries were incubated in physiological saline solution containing 5 mM [1‐13C] glucose and 1 mM or 0.1 mM [1,2‐13C] acetate with or without 25 mU/ml insulin and 200 μM DCI. Metabolic assays and 13C‐NMR analyses were used to determine glucose utilization and oxidation, respectively. Glucose utilization (p=0.04) and oxidation (p=0.03) were increased in the presence of DCI, insulin, and 1 mM [1,2‐13C] acetate. Glucose utilization was increased in contracted (p=0.001) and non‐contracted (p=0.0001) arteries in the presence of DCI, insulin, and 0.1 mM [1,2‐13C] acetate. Western blot analyses revealed 2.8 fold more PDK3 and 3.2 less PDK2 in VSM compared to skeletal muscle. We conclude that the PDC in VSM is chronically inhibited by the increased presence of PDK3 and decreased presence of PDK2 which may contribute to the metabolic inflexibility of VSM during acute dyslipidemia, potentially making VSM susceptible to lipotoxicity and disease.
Acetylcarnitine treatment increases mitochondrial protein‐lysine acetylation and protein expression
Authors: Kerner Janos , Yohannes Elizabeth , Virmani Ashraf , Koverech Aleardo , Chance Marc , Hoppel Charles
Published: June 2021

Myocardial carnitine content of Fischer 344 rats decreases with age and proportionally affects total, free, acyl‐, and acetylcarnitine (ALC). In both adult and elderly hearts, ALC is the major acylcarnitine representing approximately 75% of the acylcarnitine fraction. The carnitine and ALC content of elderly hearts is restored by a single intraperitoneal (IP) injection of ALC (1.28mmol or 260mg/kg body weight) or by oral administration of ALC in drinking water (1.5%) over a two month period. In ALC‐treated rat hearts mitochondrial protein lysine acetylation is increased in both subsarcolemmal and interfibrillar mitochondria as assessed by immunoblotting. Moreover, there are qualitative differences in protein lysine acetylation between SSM and IFM. Furthermore, 2‐DIGE analysis showed that ALC induced changes in protein expression. Thirty one proteins showed a two‐fold or greater change, eight showed an increase and twenty three proteins a decrease. Identification of these proteins will direct our future studies into the mechanism of the physiological effects of acetylcarnitine.

Supported in part by POI AG15885 and Sigma‐Tau SpA.
Development of a Real‐time PCR Assay to Quantify Mitochondrial DNA Deletions Relative to a Housekeeping Gene
Authors: Pitarresi Jason , Pogozelski Wendy
Published: June 2021

Mitochondria contain DNA in the form of a double‐stranded circle. This DNA (mtDNA) is often found to contain large‐scale deletions of several hundred to several thousand base pairs. These deletions correlate with a variety of disorders in oxidative phosphorylation. Using a real‐time PCR assay based on an external cloned mtDNA quantification standard, we observed that a particular deletion increases in cells treated with gamma rays. However, the data have not always been consistent. We are examining the use of a parallel approach that involves real‐time PCR but measures the deletions and the total mtDNA relative to a nuclear DNA gene.

This research was sponsored by the Camille and Henry Dreyfus Foundation, the Geneseo Foundation and the Geneseo Student Association.
Effect of LXR on the Renal NaPi transporters
Authors: Caldas Yupanqui A , Giral Hector , Sutherland Eileen , Sorribas Victor , Blaine Judith , Okamura Kayo , Levi Moshe
Published: June 2021

The Liver X Receptor (LXR) agonists have shown to modulate the lipid composition of different tissues including the vascular tissue and kidney. Since dyslipidemia has been associated with chronic kidney disease (CKD) and hyperphosphatemia is a consequence of CKD, we used both an in vitro and in vivo system to study the effect of the LXR agonists on renal NaPi cotransport protein regulation given their important role in maintaining Pi homeostasis. C57BL/6 mice fed chronically with either of the LXR agonists TO901317 and DMHCA show a reduction in the abundance of renal brush border membrane (BBM) NaPi transport proteins: 57% for NaPi‐2a, 40% for NaPi‐2c, and 30% for PiT‐2. After incubation for 24 hours with TO901317 the same effect was also observed in opossum kidney cells (OK: a cell culture model of the renal proximal tubule) in which levels of NaPi4 protein (the main type II NaPi transporter in OK cells) were reduced by 55% and Pi transport by 49%. In this cell line we have demonstrated increased nuclear expression of the endogenous LXR receptor following treatment with the LXR agonists. In addition, LXR agonists cause changes in the lipid profile and lipid content of the OK cell membrane. Our studies therefore document a novel role for the LXR agonists in modulation of renal NaPi transporters.

This research was supported by NIH (3R01 AG026529) supplemental minority grant.
SIRT3 Promotes the Urea Cycle by Deacetylating Ornithine Transcarbamoylase
Authors: Yu Wei , Hallows William , Smith Brian , Guan Kun‐liang , Denu John
Published: June 2021

Identifying the substrates for SIRT3 is paramount to understanding how reversible acetylation can modulate mitochondrial metabolism. Here we utilize a SPOT peptide library containing ~250 unique 9‐mer peptide sequences of previously identified acetylated mitochondrial proteins. Within the nine residue peptides, a central thiotrifluoro‐acetyl‐lysine was used as a tight‐binding mimic of acetyl‐lysine. Library screening revealed a wide range in binding affinity, which was validated by measuring the catalytic efficiency of deacetylation of the corresponding free peptides. One particular peptide that conferred very strong binding affinity was lysine 88 of ornithine transcarbamoylase(OTC) previously identified to be regulated by acetylation. OTC converts ornithine and carbamoyl phosphate into citrulline, and is a key enzyme in urea cycle to detoxify ammonium produced from amino acid metabolism. Reversible acetylation of lysine 88 plays an important role in regulating OTC activity through responding to cellular metabolic status. SIRT3 interacts with and deacetylates OTC on lysine 88, upregulating its activity. Knockdown of SIRT3 in cultured cells elevates OTC acetylation decreasing activity. Utilizing SIRT3−/− mice, OTC displays increased acetylation and decreased activity. Furthermore, measurements of urea cycle metabolites reveal defects in the SIRT3−/− animals. These findings demonstrate that SIRT3 regulates OTC and the ability to adapt to low energy input, avoiding a toxic buildup of ammonium from amino acid catabolism.
Gene Profiles of Host Biological Processes Targeted by Yersinia pestis
Authors: Miller Stacy‐Ann , Muhie Seid , Juibitu Meskerem , Moyler Candace , Hammamieh Rasha , Jett Marti
Published: June 2021

Yesrsinia pestis is a gram negative bacterium and is the causative agent of the bacterial disease plague. The bacterium is transmitted to humans via the bite of an infected flea or by inhalation of aerosolized infective droplets. Y.pestis causes deadly acute infection and has the ability to genetically manipulate the host and can potentially be used as a biological warfare weapon. Due to this it has been placed on the category A select agent list. One of the most important steps that will allow us to diagnosis plague early and quickly is understanding the early host responses at the molecular level. Our research focuses on defining the molecular events/network systems involved in the host‐pathogen interactions to identify biomarkers and therapeutic targets for pathogenic agents.

We exposed non‐human primates by aerosol to CO92 Y. pestis/sham at different lengths of time. We have collected whole blood, plasma, Peripheral Blood Mononuclear Cells (PBMCs) and various tissues from infected and control NHPs. RNA is isolated using the TRIZOL method and overall RNA integrity was confirmed by spectrophotometery and the Agilent 2100 bioanalyzer. Using gene expression microarray, microRNA microarray and miRNA real time PCR, we have identified genes that were differentially expressed in reponse to the pathogen. Functional annotaion of these genes revelealed an enrichement in genes coding for the transcription HIF‐1 pathway, molecular transport and apoptosis in Y.pestis infected monkeys. These findings provide a better understanding of the systemic progression of illness and a more in‐depth characterization of the dynamics of regulatory networks that are vital in the host‐pathogen interactions. Our aim is to eventually identify markers that will predict the eventual course of impending illness.
Control and Function of Translesion DNA Polymerases
Authors: Walker Graham C. , Cohen Susan E. , Jarosz Daniel F. , Foti James J.
Published: June 2021

Not only is E. coli's translesion DNA polymerase DinB (DNA pol IV) a much better polymerase when copying over N2‐furfuryl‐dG than over ordinary dG, but it is also remarkably proficient at extension, suggesting a biological role of carrying out relatively accurate DNA synthesis over common class(es) of N2‐dG adducts that are common to all domains of life. The products of the umuD and recA genes control functions of DinB as well as those of UmuC (DNA pol V and pre‐steady state kinetic analyses are offering insights into the nature of this control. A mutant DinB generated by altering a evolutionarily conserved tyrosine is capable of insertion opposite the lesion, but cannot complete TLS, stalling three nucleotides after an N2‐furfuryl‐dG adduct. Expression of this variant transforms a bacteriostatic DNA damaging agent into a bactericidal drug, resulting in profound toxicity even in a dinB+ background. Our discovery of physical and genetic interactions between DinB and NusA, a component of elongating RNA polymerase, indicates a previously unrecognized interaction between transcription and TLS that affects several aspects of DNA repair and mutagenesis.
Timing and spacing of ubiquitin‐dependent DNA damage bypass
Authors: Ulrich Helle D , Daigaku Yasukazu
Published: June 2021

DNA damage tolerance, also called postreplication repair (PRR), is a pathway that enables cells to overcome replication problems and complete the duplication of their genomes in the presence of DNA lesions. In eukaryotes, the pathway is controlled by the ubiquitylation of the replication clamp protein PCNA. Monoubiquitylation of PCNA activates translesion synthesis by specialized damage‐tolerant DNA polymerases, whereas polyubiquitylation is required for an alternative, error‐free pathway. Ubiquitin‐dependent PRR substantially influences the accuracy of replication and the resistance towards genotoxic agents; however, the details of when during the cell cycle the pathway is most relevant remain a matter of debate. We have now designed an inducible system of damage bypass in budding yeast that has allowed us to analyze the effects of PRR in the course of the cell cycle and to separate its activity from bulk replicative DNA synthesis. We show that PRR can act not only during S phase, but similarly contributes to survival when activated after replication of the genome is largely complete. We find that PRR both during and after S phase is largely mediated by translesion synthesis, with error‐free damage avoidance acting as a back‐up pathway. Finally, we have directly visualized PRR tracts by fiber spreading.
The effect of pressure cycling on proteolytic cleavage efficiency, reaction time and protein sequence coverage
Authors: Biringer Roger , Hühmer Andreas F. R.
Published: June 2021

Over the years many methodologies have been advanced to aid in the enhancement of proteolysis cleavage efficiency, reaction time, and protein sequence coverage for LC‐MS/MS based analysis. The series of experiments presented here examine the effect pressure cycling on each of these factors. Several individual proteins and a simple protein mixture were digested with different proteases under high pressure using a Barocycler NEP2320 pressure cycler and compared to overnight at 1 atm and 37 °C proteolysis conditions. The resulting peptides were identified and quantified using an LC‐MS/MS workflow with a LTQ XL with ETD. The results indicate that digestions using pressure cycling produce more distinct peptides and equivalent or higher protein sequence coverage when compared to standard overnight digestions.
Proteomic Analysis of Propiconazole Responses in Mouse Liver‐Comparison of Genomic and Proteomic Profiles
Authors: Bruno Maribel Enid , Ortiz Pedro , Moore Tanya , Nesnow Stephen , Winnik Witold , Yue Ge
Published: June 2021

We have performed a comprehensive profiling of changes in protein expression of soluble proteins in mouse livers treated with the liver tumorigen, propiconazole, to uncover the pathways altered by this fungicide. Using two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry (MS), we identified 62 proteins altered by propiconazole treatment. These proteins were mapped using Ingenuity Pathway Analyses™ canonical pathways and Tox lists and results compared to genomics results. Seventeen pathways/lists were unique to proteomics, 21 to genomics and 27 were shared by both analyses. The protein network analysis produced interacting sub‐networks including hepatocyte nuclear factor 4α (HNF4α), MYC, and glutathione S‐transferase (GST). Five GST isoforms were identified by proteomic analysis. Hepatic GST activities were compared between mice treated with propiconazole and 2 other conazoles and higher GST activities were found in the tumorigenic conazoles. This study has shown alterations in nuclear receptor activation, metabolism of xenobiotics, biosynthesis of biochemical intermediates, and oxidative stress. It provides insights into toxic mechanisms and/or modes of action of propiconazole required for human health risk assessment. This abstract does not necessarily reflect EPA policy. Mention of trade names of commercial products does not constitute endorsement or recommendation for use.
Identification and characterization of a novel ferritin subunit from Aedes aegypti
Authors: Patel Naren , Geiser Dawn L. , Gucinski Ashley C , Wysocki Vicki H , Winzerling Joy J
Published: June 2021

Mosquitoes consume and absorb significant amount of iron from a blood meal. Ferritin is a protein that is used to store iron. Iron is necessary for egg development in mosquitoes. We wanted to examine the proteome of ovaries from Aedes aegypti (yellow fever mosquito) that were fed an artificial blood meal with or without hemoglobin. The number of proteins identified in ovaries from mosquitoes that were fed an artificial blood meal with or without hemoglobin was 48 and 13 respectively. A previously uncharacterized glycosylated ferritin light chain homologue (G‐LCH) was found in ovaries of mosquitoes that were fed hemoglobin rich diet. Primers were designed specifically for G‐LCH to perform PCR and real‐time PCR. G‐LCH ferritin was cloned and sequenced. Real‐time PCR showed that eggs, larvae and pupae have similar expression levels of G‐LCH. Males and 72h post blood fed (PBF) females have higher expression levels of this message. Real‐time PCR will be performed on tissues of 24h and 72h PBF mosquitoes. The 5’‐UTR region of G‐LCH mRNA will be examined using rapid amplification of cDNA ends to determine if stem loop moieties are present. Characterization of G‐LCH subunit will give further insights into the unique regulation of iron metabolism in mosquitoes. Research support was from NIH(GM056812), the Agricultural Experiment Station and CALS at the University of Arizona.
Gene network analysis during early infection of human coronary artery smooth muscle cells by Trypanosoma cruzi and its gp83 ligand
Authors: Johnson Candice Angella , Nde Pius , Pratap Siddharth , Cardenas Tatiana C , Kleshchenko Yulia , Furtak Vyachslav A , Lima Maria F , Villalta Fernando
Published: June 2021

Trypanosoma cruzi, the causative agent of Chagas disease, infects heart and muscle cells leading to cardiac arrest followed by death. The genetic architectures in the early T. cruzi infection process of human cells are unknown. To understand the genetic architectures of the early invasion process of T. cruzi, we conducted gene transcription microarray analysis followed by gene network construction of the host cell response in human coronary artery smooth muscle primary cells (HCASMC) infected with T. cruzi or exposed to T. cruzi gp83, a ligand used by the trypanosome to bind host cells. Using seven RT‐PCR verified up‐regulated genes (FOSB, ATF5, INPP1, CCND2, THBS1, LAMC1, and APLP2) as the seed for network construction, we built an interaction network of early T. cruzi infection process containing 165 genes, connected by 598 biological interactions. This interactome network is centered on the BCL6 gene as a hub. Silencing the expression of two seed genes (THBS1 and LAMC1) by RNAi reduced T. cruzi infection. Overall, our results elucidate the significant and complex process involved in T. cruzi infection of HCASMC at the transcriptome level. This is the first elucidation into the interactome network in human cells caused by T. cruzi and its gp83 ligand (Supported by NIH grants AI080580‐03 and AI007281‐21).
Comparative proteome analysis reveals flotillin‐1 as a H‐Ras‐interacting lipid raft protein critical for breast epithelial cell invasion
Authors: Moon Aree , Yong Hae‐Young , Hwang Jin‐Sun , Kim Myeong‐Ok , Sohn Hwa‐Jin , Kim Eun‐Sook , Choi Wahn Soo , Kim Hyeong‐Reh Choi , Kim Ki‐Bum
Published: June 2021

The objective of the present study was to identify the protein profile specific to the biological processes for breast cell invasion/migration. Complete proteome profiles of lipid raft proteins were provided from three cell lines of differing invasive abilities: non‐invasive MCF10A and N‐Ras MCF10A cells and invasive H‐Ras MCF10A cells. Among 24‐lipid raft proteins which were upregulated in an H‐Ras‐specific manner, we tested whether flotillin‐1 plays a causative role for H‐Ras‐mediated MCF10A cell invasion/migration. Expression of flotillin‐1 was correlated with the pathogenically diagnosed cancer tissues compared to normal tissues, providing the in vivo relevance for the tumorigenic potential of flotillin‐1 in human breast cancer. Importantly, flotillin‐1 colocalizes with H‐Ras in the plasma membrane, but not with N‐Ras. Disruption of lipid raft caused a marked inhibition of H‐Ras activation and invasion/migration, suggesting that the interaction of H‐Ras and flotillin‐1 is critical to the H‐Ras‐induced invasion/migration. Moreover, the knockdown of flotillin‐1 expression reduced the activation of H‐Ras, Rac1 and p38 as well as MMP‐2 and invasive/migratory phenotypes. Taken together, this study identified flotillin‐1 as an H‐Ras‐interacting lipid raft protein critical for the activation of H‐Ras and consequently for the H‐Ras ability to induce invasive potential in breast epithelial cells.
Causation for inherited equine night blindness
Authors: Scott Michelle Lynn , Forsyth George W , Sandmeyer Lynne S , Bellone Rebecca R , Loewen Matthew E , Grahn Bruce H
Published: June 2021

Mutations associated with equine night blindness (CSNB) have been mapped to the Transient Receptor Potential cation channel family Member 1 (TRPM1) gene on ECA1. Reduced TRPM1 mRNA expression has been reported in retina and melanocytes in CSNB horses. A single unique intronic snp has been found in all CSNB horses examined to date. We hypothesize that this unique snp creates a binding site for the Nova intronic splicing enhancer which promotes aberrant splicing of an 80 bp downstream acceptor, therefore targeting TRPM1 mRNA for degradation. Nova‐1 and TRPM1 mRNA levels were measured in retina and in primary melanocyte cell lines established from the choroid of normal and CSNB horses. TRPM1 mRNA level in retina from a CSNB horse was 0.2% of that in the control horse retina. Choroidal melanocyes from normal and CSNB horses both had low TRPM1 mRNA levels similar to those observed in CSNB retina. We are investigating whether similar levels of TRPM1 mRNA in CSNB and normal melanocytes are due to removal of the effect of some upstream enhancer element in tissue culture, or result from insufficient Nova‐1 expression to cause the destabilizing TRPM1 mRNA splice event in CSNB melanocytes.

Supported by the WCVM Canadian Vitamins Class Action Settlement Research Grant.
The origin and role of cardiomyocyte stem cells in regenerating hearts of Notophthalmus viridescens
Authors: Miller Dwayvania , Piefer Andrew John , Sessions Stanley
Published: June 2021

One of the leading causes of death among humans is heart disease. Unfortunately, humans have only limited ability to repair or regenerate their heart tissue after it has been damaged. Interestingly, organisms such as zebrafish, and newts have the ability to regenerate severely damaged heart tissue. The purpose of this study was to investigate the origin and role of cardiomyocyte stem cells in the regeneration of heart tissue in N. viridescens (Red Spotted Newt). Regenerating newt hearts contain a blood clot that becomes populated by dividing cells. This clot may be the source of stem cells for new cardiomyocytes. This idea will be tested by conducting a two part study. The first part of the study will determine whether the clot formed after the partial amputation of the heart ventricle is a source of cardiomyocyte stem cells for newly regenerated heart tissue. BrdU – labeled blood cells from a donor newt will be transfused into a recipient newt. Approximately, 20% of this recipient newt's heart will be surgically removed and allowed to regenerate. The newly regenerated heart tissue will then be tested for BrdU‐labeled cardiomyocytes indicating that the clot is the source of stem cells. The second part of this study will use a northern blot to determine whether cells in the clot express pre‐cardiomyocyte transcriptional factors such as tbx20, nkx2.5, and gata4. The project is funded by the Departments of Chemistry and Biology.
Sam68 directs NF‐kB for IL2RA gene transcription
Authors: Wan Fengyi , Weaver Amanda , Lenardo Michael J.
Published: June 2021

The inherent complexity of the recognition of kB sites by transcription factor NF‐kB was underscored by our recent study demonstrating ribosomal protein S3 (RPS3), an essential non‐Rel NF‐kB subunit cooperates with Rel subunits to achieve full binding and transcriptional activity of NF‐kB. RPS3 preferentially directs binding to kB sites of certain NF‐kB genes, thus functioning as a “specifier” subunit. To explore the RPS3‐like “specifier” molecules in the NF‐kB complexes with different gene activation specificities, we identified the Src‐Associated substrate during mitosis of 68kDa (Sam68) as the “specifier” subunit of NF‐kB conferring the specific transcription of IL2RA, a RPS3‐independent gene encoding IL2 receptor alpha chain with important function in hematopoietic cells and high expression in cancers. Sam68 was not detected in the cytoplasmic IkB‐p65 complex in resting T cells, but nuclearly complexed with p65 after stimulation, where it synergistically enhanced NF‐kB complex binding to IL2RA kB site. The significance of Sam68‐conferred recognition was highlighted by the observations that Sam68 knockdown dramatically impaired TCR engagement‐induced IL2Ra upregulation and killed IL2Ra highly expressed cancer cells. Our discovery thus sheds new light on the long‐standing question of NF‐kB regulatory specificity. This research was supported by the NIH, NIAID Intramural Research Program.
Pin1 facilitates cancer proliferation by stabilizing phospho‐Sp1
Authors: Wang Shao‐an , Chuang Jian‐Ying , Chang Wen‐Chang , Hung Jan‐Jong
Published: June 2021

Sp1 is important for many gene transcriptions. How Sp1 is preserved in cancer cells during mitosis remains unknown. Here we found that Sp1 is phosphorylated at Thr739 by CDK1 before the entry of mitosis. Further, during mitosis, Pin1, a peptidyl‐prolyl isomerase, shields phospho‐Sp1‐Thr739 from dephosphorylation by PP2A via an action involving the isomerase activity of Pin1. Phospho‐Sp1 loses its affinity for DNA and appears to exist as a congregated ring in the periphery of chromosomes. At the end of mitosis and the beginning of interphase however, Sp1 becomes dephosphorylated by PP2A and returns to the chromatin. The results indicate that cancer cells utilize Pin1 to stabilize Sp1 in phosphorylated form and recycle the latter between mitosis and interphase. Pin1 thus, by stabilizing phospho‐Sp1, serves to facilitate the quick initiation and execution of interphase in order to maintain the favorable proliferative nature of cancer cells.
The role of PLZF in the transcriptional repression of the p21CIP1 gene
Authors: Choi Won‐Il , Yoon Jae‐Hyeon , Hur Man‐Wook
Published: June 2021

The PLZF which is involved in rare cases of acute promyelocytic leukemia, is a POK family transcription factor containing nine Krüppel‐like C‐terminal Cys2–His2 zinc finger motifs and an N‐terminus BTB/POZ‐domain. PLZF plays important roles in myeloid cell growth, differentiation, and apoptosis. We investigated whether PLZF can regulate genes of ARF‐MDM2‐p53‐p21 regulatory pathway, which is important in cell cycle regulation and oncogenesis. We found that p21 gene, an ARF/p53 downstream cell cycle regulator gene, is the molecular target of PLZF. We dissected the molecular mechanism of transcription repression of p21 gene by PLZF. We found that PLZF directly bound onto the proximal Sp1 binding GC‐box elements 5/6 and the distal p53 binding elements of p21 promoter, and thereby it blocked transcriptional activation by Sp1 and p53. Our data also suggested that PLZF represses p21 gene by recruitment of corepressor‐HDAC complex recruited onto the binding sites via PLZF. PLZF caused cellular transformation, promoted cell cycle proliferation, and also increased the number of cells in S‐phase probably by affecting the gene transcription of p53 pathway.
Sumoylation activates Pax‐6, an important transcription factor for eye and brain development
Authors: Gong Lili , Yan Qin , Li David W
Published: June 2021

Pax‐6 is a master gene controlling eye and brain development. Four isoforms of Pax‐6, p46, p48, p43 and p32/33, have been detected in the vertebrate eye. Absence of the paired domain in the 32kDa Pax‐6 distinguishes it from the other three forms. How is the 32 kDa Pax‐6 acting as a transcription factor is unknown. In the present study, we demonstrated that activation of the 32 kDa Pax‐6 requires sumoylation. First, nuclear and cytoplasmic distributions of Pax‐6 were detected in human lens epithelial cell line (FHL‐124) by Western‐blot analysis. Three Pax‐6 isoforms (46, 43 and 32 kDa) were detected in the nuclear extracts but only one isoform (43 kDa) was found in the cytoplasm. Second, a P3 sequence specific for HD domain was used to test the binding activities of different forms of Pax‐6 in gel mobility shifting assays. Only the 43kDa Pax‐6 displayed binding ability to the P3 sequence. 32 and 46 kDa Pax‐6 either extracted from FHL‐124 or synthesized in vitro lack the binding ability to P3 sequence. However, after incubating with the Pax‐6‐depeleted nuclear extract, 32kDa but not the 46kDa Pax‐6 exhibits strong binding ability to P3 sequence. Finally, both in vitro and in vivo biochemical analysis revealed the 32kDa Pax‐6 is sumoylated before binding to P3 sequence. Take together, the 32kda and 46kda Pax‐6 display differential DNA binding activity to the P3 sequence and may have differential regulatory mechanism on downstream target genes. Moreover, sumoylation of the 32 kda Pax‐6 is necessary to activate its transcriptional activity.
Inactivation of Natriuretic Peptides by Human Insulin‐Degrading Enzyme Reveals New Insights on Substrate Recognition and Selectivity
Authors: Ralat Luis Abel , Funke Todd A. , Dickey Deborah M , Potter Lincoln , Tang Wei‐Jen
Published: June 2021

Atrial, brain, and C‐type natriuretic peptides (ANP, BNP, and CNP) are 3 physiological hormones that regulate blood pressure. Currently, all 3 natriuretic peptides are known to be cleared by 2 accepted processes: 1) receptor‐mediated internalization followed by lysosomal degradation; and 2) enzymatic degradation by neprilysin, a zinc‐dependent protease expressed on the plasma membrane. However, rat insulin‐degrading enzyme (IDE), also a zinc‐dependent protease, can also cleave rat natriuretic peptides. Here, we used human IDE to define the selectivity and sequence of cleavage events for the human natriuretic peptides and the role of IDE in their physiological clearance. Our studies reveal that ANP and CNP are preferred substrates over BNP. Analysis of the cleavage sites shows a preference for basic or hydrophobic residues on the N‐ or C‐terminal side of a peptide bond. In contrast to most of the known substrates of IDE, the cleavage events for natriuretic peptides suggest that their degradation is probabilistic and not sequential. For ANP and BNP, the 1st cleavage site by IDE occurs at the noncyclic portions of the peptides followed by cleavages in the 17‐member ring. Conversely, for CNP, the 1st cleavage by IDE can occur at the noncyclic portion or the 17‐member ring. Cellular studies using HEK‐293 cells show that the degradation of natriuretic peptides by IDE affect the natriuretic peptide‐induced cGMP levels.
Identification and characterization of novel bisphosphonate inhibitors of squalene synthase
Authors: Wasko Brian M. , Smits Jacqueline P. , Tong Huaxiang , Wiemer David F. , Hohl Raymond J.
Published: June 2021

At the branch point of the isoprenoid biosynthetic pathway, squalene synthase (SQS) is an enzyme that utilizes farnesyl diphosphate (FPP) to make squalene, which is first committed step in the biosynthesis of cholesterol. A recombinant form of human SQS has been purified and in vitro assays have been established to measure enzyme activity. We have evaluated structure activity relationships among more than 30 novel bisphosphonates and have identified 9‐biphenyl geranyl bisphosphonate (BPGB) as a potent inhibitor of SQS (IC50 = 10 nM). Western blot analysis of farnesylated and geranylgeranylated proteins from HepG2 cells indicates that treatment with BGBP is specific for the sterol branch of the pathway and not other isoprenoid enzymes (i.e., farnesyl diphosphate synthase or geranylgeranyl diphosphate synthase). The coadministration of lovastatin with BGBP can prevent lovastatin‐induced inhibition of protein prenylation. BGBP at 25 μM for 24 hours resulted in more than 20 fold accumulation of the substrate FPP. In conclusion, this data identifies a novel and specific inhibitor of SQS and evaluates some of the cellular effects of squalene synthase inhibition. Research support comes from the Roy J. Carver Charitable Trust and the Roland W. Holden Family Program for Experimental Cancer Therapeutics.
The Search for Human Alpha‐Amylase Inhibitors as Therapeutics for Diabetes and Obesity
Authors: Brayer Gary D. , Williams Leslie K. , Tarling Chris A. , Woods Kate , Li Chunmin , Zhang Ran , Mahpour AmirAli , Andersen Raymond J. , Withers Stephen G.
Published: June 2021

Diabetes and obesity lead to a significantly reduced quality of life, with an increased risk of serious complications including cardiovascular disease, hypertension, stroke, kidney failure and nerve damage. Human pancreatic alpha‐amylase (HPA) provides a unique opportunity for the development of potential therapeutic agents for the treatment of these conditions. This enzyme plays a vital role in the breakdown of starch in the diet, and its activity has been correlated to postprandial blood glucose levels, the control of which is essential for maintaining quality of life for diabetic patients. Nonetheless, the discovery of specific high affinity inhibitors for HPA has proven elusive and the currently available therapies that target this enzyme cause many deleterious side effects due to their activity on a wide range of glycosidases. In an attempt to identify new inhibitors of HPA, we have screened over 80,000 pure chemicals and crude biological extracts. This has resulted in the exciting discovery of montbretin A, a glycosylated acyl flavonol that acts as a competitive HPA inhibitor with a Ki of 8.1 nM. Structural characterization of the binding mode of fragments of the montbretin A molecule have been undertaken and a model of montbretin A binding proposed. This work is supported by the Canadian Institutes of Health Research.
Meprins Affect Epithelial Barrier Function by Cleaving Tight Junction Proteins
Authors: Bao Jialing , Cornely Stephanie , Matters Gail , Bradley S.Gaylen , Bond Judith
Published: June 2021

Meprins are metalloproteinases that cleave a range of biologically important substrates such as extracellular matrix proteins, adherens junction proteins, and cytokines. Previous studies in our laboratory showed that disruption of the meprin A gene leads to altered outcomes of mouse models of inflammatory bowel diseases and endotoxin‐induced inflammation. Because disruption of epithelial cell interaction can contribute to inflammatory responses, the influence of mouse homomeric meprin A on the function of epithelial barrier has been explored. Cultured Madin‐Darby canine kidney (MDCK) cells exposed to active homomeric meprin A show decreased immunostaining for ZO‐1 and occludin in the tight junction of the confluent cell layer. Monolayers of MDCK cells incubated with active meprin A, but not latent meprin A, display decreased transepithelial electrical resistance and increased permeability to fluorescein‐5‐isothiocyanate (FITC)‐labeled dextran. The tight junction protein occludin, but not claudin‐4 is cleaved by meprin A in both membrane‐enriched cell fractions and intact monolayers of MDCK cells. These results indicate that meprin A impairs the integrity of epithelial barriers and selectively cleaves tight junction proteins, thereby affecting leukocyte migration and/or cytokines accumulation at sites of inflammation.
Cleavage of Extracellular Matrix Molecules: a role for ADAM8 in tumour cell invasion
Authors: Ferdous Taheera , Golfi Panagiota , Schlomann Uwe , Zack Marc D , Bartsch Joerg W
Published: June 2021

Processing of extracellular matrix (ECM) components is attributed mainly to zinc‐dependent proteases such as MMPs, as they are associated with invasive behavior of tumor cells. Interestingly, ADAM8, a member of the membrane‐bound zinc‐dependent family of ADAM proteases, seems to adapt an invasive phenotype when overexpressed and has been described in highly invasive cancer pathologies. It is also involved in conditions such as allergy, asthma and neurodegeneration. We postulate that cellular processing of ADAM8 eventually leads to ‘MMP‐like’ activity which could modulate the tumor microenvironment.

We investigate the mechanism by which ADAM8 contributes to cancer cell invasion. We identified a soluble form of murine ADAM8 released from transfected cells by proteolytic processing, which cleaves CD23 fluorescence peptide suggesting proteolytic activity. In vitro assay shows that recombinant soluble human ADAM8 cleave fibronectin, collagen IV and collagen type I. Furthermore, in both matrigel invasion and FITC‐labeled fibronectin assays, we have seen migration and cleavage of COS‐7 cells transfected with wildtype ADAM8 compared to an inactive mutant. Cancer cell lines with upregulated ADAM8 expression, such as human osteosarcoma (HOS), degrade collagen type I.

We conclude that ADAM8 degrades ECM molecules, both in vitro and in vivo, enabling cancer cell invasion. Thus, ADAM8 may be a potential target for therapeutic intervention in metastatic cancer.
IL‐6 is a Newly‐Discovered Substrate for Meprin Metalloproteinases
Authors: Keiffer Timothy Robert , Bond Judith
Published: June 2021

Meprins are unique metalloproteinases that are highly expressed in intestinal, kidney, and skin epithelial cells, and also in certain leukocytes (eg., monocytes and natural killer cells). Meprins are secreted and membrane‐bound proteases comprised of alpha and/or beta subunits. These proteases have been implicated in the pathogenesis of several inflammatory diseases such as: Inflammatory Bowel Disease (IBD), Urinary Tract Infections (UTI), Acute Renal Failure (ARF), and cancer. Previous work has demonstrated, using experimental mouse models, that meprin knock‐out mice have altered cytokine profiles compared to their wild‐type counterparts in the IBD, UTI, and ARF models. IL‐6 was one of the cytokines significantly increased in meprin alpha and alpha/beta knock‐out mice with IBD, leading us to hypothesize that IL‐6 is a meprin substrate and that meprin plays a role in modulating inflammation by inactivating IL‐6. So far, we have determined through SDS‐PAGE gel‐shift analysis that meprins do cleave IL‐6, which is the first direct in vitro evidence that IL‐6 is a meprin substrate. Additionally, the homomeric meprin A isoform fragmented IL‐6 differently than the homomeric meprin B isoforms. Subsequent studies are being done to measure the affinity of IL‐6 as a meprin substrate and determine whether meprin‐generated IL‐6 products are bioactive. (Supported by NIH DK 19691).