bluuebunny/crossref_metadata_2025 huggingface dataset:
Repressive effect of polyunsaturated fatty acids on serine dehydratase expression in rat hepatocytes
Authors: Contreras Alejandra , Ortiz Victor , Torres Nimbe , Tovar Armando R
Published: June 2021

Peroxisome proliferator‐activated receptor alpha (PPARα) is activated by ligands such as polyunsaturated fatty acids (PUFAs) and regulates expression of enzymes involved in fatty acid oxidation. Recently, it was found that PPARα also regulates the metabolism of amino acids in liver, however, the knowledge of molecular mechanisms of amino acid regulation in liver is limited. To assess the effect of natural (linoleic acid, LA) and synthetic PPARα ligands (Wy14643) on the expression of the amino acid degrading enzyme serine dehydratase (SHD) and the enzyme involved in fatty acid oxidation carnitine palmitoyl transferase‐1 (CPT‐1), rat hepatocytes were incubated with the agonist Wy14643, palmitic acid (saturated fatty acid) or linoleic acid (PUFA), and gene expression was measured. Addition of forskolin to the medium increased SDH gene expression as expected but did not altered CPT‐1 expression. However, addition of linoleic acid and palmitic acid to the medium increased CPT‐1 by 100% and 40%, but repressed SDH gene expression by 53% and 23% respectively. These data indicated that the insaturated linoleic acid reduced amino acid catabolism and stimulated fatty acid oxidation through PPARα in a higher extent than the saturated palmitic acid.

Supported by CONACYT and INCMNSZ.
The actin binding protein cortactin is overexpressed in metastatic cancer cells
Authors: Click Sarah , Kruchten Anne E
Published: June 2021

Cortactin is a Src substrate protein that interacts with actin and is involved in several cancer metastases. Previous studies indicate that cortactin protein is upregulated in several head, neck, and breast tumors. Cortactin phosphorylation has also been shown to enhance breast cancer cell metastasis to bone. While cortactin is present in many types of cells, we hypothesize that cortactin is upregulated in cell types that are highly motile such as metastatic cancers arising from a variety of tissue types. To determine if this hypothesis is true, we examined cortactin levels in three cell lines: normal green monkey kidney cells (Vero), non‐metastatic breast cancer cells (NM2C5), and metastatic breast cancer cells (M4A4). Using western blotting and immunofluorescence, we found that cortactin protein levels increase with the metastatic potential of the cell line. Future studies will examine additional metastatic cell types and further characterize cortactin in these cells. This work was supported by a Faculty Start Up Grant to A.E. Kruchten by The Murdock Charitable Trust and by a Linfield College Faculty Student Collaborative Research Award to A.E. Kruchten and S. Click.
Tetraspanin CD151 is Required for Both α3 and α6 Integrin Function
Authors: Zevian Shannin , Winterwood Nicole , Meland Marit , Stipp Chris
Published: June 2021

A benchmark characteristic of malignancy is an invasive phenotype often associated with tumor cell motility. We, and others, have described a critical role for tetraspanin CD151 in α3β1‐dependent tumor cell responses to laminin‐332. When CD151 is silenced, carcinoma cells exhibit impaired adhesion and migration on laminin‐332, phenotypes that can be reversed upon re‐expression of CD151. We have now begun to use this silencing and reconstitution system to explore the domains in CD151 required for its ability to promote α3β1 function. Mutation of a QRD motif in the EC2 domain of CD151 abolished CD151 association with α3β1 under harsh detergent conditions, as previously described. Surprisingly, however, the QRD mutation did not abolish CD151 association with α3β1 under moderate, Brij detergent conditions. Moreover, in experiments to date, the CD151 QRD mutant appears fully capable of rescuing α3β1‐dependent adhesion and migration. More severe EC2 mutations, which abolish α3β1 association even under mild conditions, cannot rescue the phenotypes of CD151‐silenced cells. In addition, CD151 palmitoylation sites are critical for CD151’s ability to link α3β1 to other tetraspanins, and for its ability to reverse the phenotypes of CD151‐silenced cells. Interestingly, palmitoylation mutants, but not severe EC2 mutants, are able to resist detachment when allowed to grow and spread on plates for at least 24 hours. Collectively, these data show that CD151’s ability to associate with its integrin partners and with other tetraspanins are both critical for its functions in adhesion and motility, and support a model in which one of CD151’s most important functions is to act as linker to target its integrin partners to tetraspanin microdomains. This work was supported by an American Cancer Society Research Scholar Award.
Anatomical Deviations Caused by a Monopartite Geminivirus Infection in Tissues of Tomato Plants
Authors: Montasser Magdy S.
Published: June 2021

Tomato yellow leaf curl virus (TYLCV) causing major yield losses in tomato (Lycopersicon esculentum L.) crops was isolated in Kuwait. The virus was found to produce characteristic external symptoms of yellowing and curly leaves on tomato test plants. Electron Microscopy of ultra‐thin sections in infected leaves showed several anatomical deviations in the ultrastructure of some organelles such as chloroplasts associated with alteration and severe damage in thylakoids and grana that contain photosynthetic pigments of chlorophyll and carotenoids. Abnormal building up of oily inclusions and empty vacuoles in the chloroplast stromas were observed. Uneven thickenings in the cell walls of the phloem tissues were found in all samples showed the characteristic external symptoms compared with healthy plant tissues. The virus identity was confirmed using Polymerase Chain Reaction technology, Southern Blot Hybridization and Dot Blot analysis. It was revealed that the causal virus was characterized as a monopartite Geminivirus. It has been found that uneven thickening in the cell walls resulted in developing the curly symptoms in tomato leaves. It was also concluded from this study that in response to TYLCV infection, severe damage in chloroplasts including thylakoids in grana resulted in 44 ‐79% reduction in chlorophyll 'a and 'b causing the yellowing symptoms and crop losses.
Vesicle trafficking of a RhoA guanine exchange factor regulates VEGF‐driven directional migration
Authors: Horowitz Arie , Agrawal Sudesh , Wu Chuanshen
Published: June 2021

Our objective is to test whether trafficking of the RhoA guanine exchange factor (GEF) Syx/Plekhg5 between tight junctions and the plasma membrane regulates VEGF‐driven directional migration of endothelial cells (ECs) and vessel sprouting. We found that unlike wt ECs, syx−/− ECs do not migrate in the direction of a VEGF‐A gradient, but randomly. We previously reported that syx knockdown zebrafish and syx−/− mice have similar vessel sprouting defects (Circ Res 103:710, 2008). Syx binds the PDZ adaptor protein synectin/GIPC1, which is involved in the docking of its ligand to uncoated vesicles (PNAS 103:12735, 2006). It is known that synectin−/− ECs migrate poorly in response to VEGF (Dev. Cell 10:783, 2006), indicating that synectin‐dependent trafficking is required for migration. Syx is present in tight junctions and at the leading edge of ECs migrating in response to VEGF‐A. We used endocytic markers in order to identify the trafficking pattern of Syx, and found that it collocates with Rab5 and Rab4. These observations indicate that Syx undergoes rapid recycling to the plasma membrane via early endosomes. Most of the cellular Syx population colocalized with angiomotin, a tight junction protein involved in EC migration. These results suggest that trafficking of Syx to the leading edge of cells sensing a chemoattractant gradient is involved in polarizing these cells along the direction of the chemoattractant gradient.
Metabolic imprinting effects in grass‐fed Wagyu (Japanese Black) beef production
Authors: Gotoh Takafumi
Published: June 2021

We aim at producing a high‐quality safe beef product while maximising the use of domestic grass resources. Alterations in foetal and early postnatal nutrition and endocrine status may result in developmental adaptations that permanently change the structure, physiology, and metabolism in the adult life of rats, mice, domestic species and humans. This phenomenon is referred to as “metabolic imprinting” based on the concept of “the developmental origins of health and disease (DOHaD)”. In this study, we investigate whether the metabolic imprinting effect of differences in feeding during an early growth influences on the meat quality of Japanese Black (Wagyu) steers or not. The high energy group (HE: n=12) underwent intense nursing till 3 months of age and was fed a high‐concentrate diet for 4 to 10 months of age. On the contrary, the Roughage group (R: n=11) underwent normal nursing and was fed only roughage ad libitum from 3 to 10 months of age. Both groups were fed only roughage ad libitum and grazed from 10 to 30 months of age. Samples of tissues from the longissimus muscles in all animals were collected and biopsied at 3, 10, 14, 20 and 30 months of age. Gene expressions related to adipogenesis, fatty acid synthase etc. in muscle were measured by real‐time PCR. We also investigated meat quality after slaughter. We could show the metabolic imprinting effects on meat quality and quantity of grass‐fed Wagyu.
Tropomyosin Phosphorylation in Healthy and Diseased Hearts
Authors: Sheikh Hajer N , Wieczorek David F
Published: June 2021

Tropomyosin (Tm) is an alpha‐helical coiled coil dimer involved in regulating striated muscle contraction. Although sarcomeric Tm is known to be phosphorylated at the penultimate amino acid Ser‐283, little is known regarding its phosphorylation levels in healthy and cardiac diseased states. The objective of this study was to study Tm phosphorylation levels with respect to age in wild type mice and in mice with familial hypertrophic cardiomyopathy (FHC). Phosphorylation levels were determined by western blotting of 2‐D gel electrophoresis of myofibrillar protein preparations. Results show that cardiac Tm phosphorylation is high during the perinatal period and decreases throughout adulthood in wild type mice. We also found that walls of the different cardiac chambers exhibit differential Tm phosphorylation.

Two transgenic mouse models harboring FHC mutations in alpha‐Tm (Glu180Gly, and Asp175Asn) were studied to determine the effect of this disease on cardiac Tm phosphorylation. Results show that Tm phosphorylation is altered in both FHC models. Unlike in wild type mice, Tm phosphorylation levels do not decrease after birth, but rather remain constant. Depending on the role of Tm phosphorylation, modulation of phosphorylation levels in FHC patients may be potentially of therapeutic value.

This project was funded by NHLBI HL81680 grant to DFW. HNS was supported by a fellowship from Dubai Medical College.
Quantitative Proteomics for Analyzing the Histone Code
Authors: Garcia Benjamin A.
Published: June 2021

Several single histone modifications are associated with both gene activation and silencing, but what type of effect distinct combinations of simultaneously occurring histone modifications (Histone Codes or patterns) have upon cellular events is poorly understood. Additionally, most recent research describes histone modifications as static events. The main reason for this lack of knowledge is that techniques for quantitative characterization or even qualitative identification of combinatorial Histone Codes in dynamic fashion by any method do not readily exist. We have specifically addressed this deficiency by developing novel mass spectrometry based methods for rapid comparison of dynamically changing histone modifications from multiple cellular states including quantitative tracking of hundreds of combinatorial Histone Codes in a single experiment. These studies in combination with biological experiments will help provide a systems‐wide outlook that will lay down the basic scientific foundation to advance several areas of chromatin biology.
Using a combination of chemical and systems biological approaches for defining a mechanism by which a novel anti‐aging compound greatly extends yeast longevity
Authors: Titorenko Vladimir , Goldberg Alexander , Bourque Simon , Beach Adam , Burstein Michelle , Richard Vincent , Kyryakov Pavlo , Gregg Christopher , Boukh‐Viner Tatiana , Juneau Mylène , Glebova Anastasia
Published: June 2021

We use the yeast Saccharomyces cerevisiae as a model to for unveiling the mechanisms of cellular aging in multicellular eukaryotes. Our findings imply that the chronological aging of yeast is a developmental program that progresses through two checkpoints. Implementing our understanding of longevity regulation, we used a custom‐designed screen of extensive compound libraries for identifying 25 novel anti‐aging compounds that extend yeast longevity. Using a combination of proteomic, lipidomic, metabolomic, and systems biological approaches, we established the molecular and cellular mechanisms underlying the life‐extending ability of one of these compounds, called LA. Our findings imply that LA extends yeast longevity by 1) activating mitochondrial fusion, thereby abolishing age‐related, mitochondria‐controlled apoptosis; 2) modulating mitochondrial production of reactive oxygen species, thereby activating expression of a compendium of stress response genes; 3) remodeling lipid metabolism in the endoplasmic reticulum, lipid bodies, and peroxisomes, thereby greatly delaying age‐related necrosis caused by the buildup of free fatty acids; and 4) modulating a non‐nuclear node of the protein kinase A‐governed glucose signaling network controlled by the protein kinase Rim15p. Importantly, LA has a similar effect on some of these longevity‐and disease‐related processes in cultured human cells.
Structure based design and structure activity relationship of new reversible peptides inhibitors of thrombin derived from D‐Phe‐Pro‐Arg substrate peptide
Authors: Clement Cristina Corina , Philipp Manfred
Published: June 2021

By employing in silico structure‐based design approaches, novel hexapeptides, pentapeptides and tetrapeptides were designed, new compounds that may be used as additives in the treatment of acute coronary diseases (ACD). Additionally, these new compounds were further tested for their ability to reversible inhibit alpha thrombin using kinetics, thermodynamics, and platelets aggregation assays. Initial molecular docking experiments generated a candidate group of compounds with both L‐ and D‐ amino acids, including two classes of sequence spaces: 1‐D‐Phe(P3)‐Pro(P2)‐Arg(P1)‐D‐Pro(P1′)‐P2′‐P3′‐CONH2 and 2‐D‐Phe(P3)‐Pro(P2)‐D‐Arg(P1)‐P1′‐P2′‐P3′‐CONH2. D‐Pro and D‐Arg replaced Arg in the P1′ and P1 positions, respectively, increasing the stability to proteolysis of the designed peptides. The newly‐designed peptides competitively inhibited the alpha‐thrombin induced cleavage of a chromogenic substrate, at 1,050‐0.8 uM. Additionally, some lead compounds inhibited the thrombin‐activated human platelets aggregation in the presence of its natural substrate, fibrinogen, effectively sustaining these peptides as powerful new anti‐coagulants. Molecular docking experiments suggest a model for achieving favorable hydrogen bonding interactions between the new peptides and thrombin, and provide a rationale for the development of a new series of anticoagulant drugs.
Physical models of enzymes engineered for biofuel cells: 2010 Pingry School S.M.A.R.T. Team Project
Authors: Hata Tommie Shintaro , Banta Scott , Campbell Elliot , Herman Tim , Ha Caryn , Kong Edward , Ma Florence , Ober Doug , Picard Mai‐Lee , Sukhin David , Sun Dylan , Vollbrechthausen Ricardo , Wang Connie , Xiao Ed , O'Mara Deirdre
Published: June 2021

The 2010 Pingry School S.M.A.R.T. Team (Students Modeling A Research Topic) has been working with the Banta laboratory at Columbia University to design and produce accurate, three‐dimensional physical models of alcohol dehydrogenase AdhD and other enzymes with applications for use in a biofuel cell. Features being engineered into these enzymes include (1) self‐assembly into hydrogels, (2) alternate cofactor use, and (3) broader substrate specificity. Discussions with the Banta laboratory allowed the students to use RP‐RasMol to design models of enzymes studied in the lab to highlight their structural and functional characteristics. These designs were used to direct rapid prototyping machines to build physical models of these enzymes. Along with Jmol tutorials created on the Team website (www.pingrysmartteam.com) and in Proteopedia (www.proteopedia.org), these physical models serve as “communication tools” used to enhance the understanding of these enzymes and their applications among the scientific and academic community. By contributing this new tool to the Banta laboratory research team, the students have the unique opportunity to experience and participate in the activities of a research laboratory. This work is supported by a grant awarded to Tim Herman by the NIH NCRR SEPA program and the HHMI Precollege Science Education Program.
Combinatorial design of a four‐helix bundle protein using high‐throughput thermal scanning
Authors: Magliery Thomas J. , Lavinder Jason J. , Hari Sanjay B. , Nguyen Chau , Sen Shila
Published: June 2021

Both the prediction and design of protein structure, using computational and rational approaches, remain significant challenges in protein chemistry. A major limitation to developing a comprehensive physicochemical model of the protein structure‐sequence relationship is the vastness of sequence space and the low‐throughput nature of biophysical studies. We are pursuing a combinatorial approach to understand better the sequence structure‐relationship: sorting large libraries of protein variants for structured proteins. We have developed a high‐throughput cell‐based screen for activity of the well‐studied four‐helix bundle protein Rop. To collect quantitative stability data for large numbers of variants, we have developed a method of high‐throughput hydrophobic dye binding called High‐Throughput Thermal Scanning (HTTS) which can be applied using automation and a real‐time PCR machine 96‐wells at a time. This system is being used to directly test the “rules” of protein design, taking those rules as hypotheses and sorting the resulting libraries for structure and stability. Here we will discuss the in‐depth analysis of a library of hydrophobic core variants, as well as initial results from surface libraries and rational design based on library statistics. This work was supported by a grant from the NIH (R01 GM083114) to TJM.
Investigation of class I integron presence and subsequent variable region characterization of 15 clinical isolates from Buenos Aires, Argentina
Authors: Kim Jun , Mercado Jeffrey , Centron Daniela , Tolmasky Marcelo
Published: June 2021

The aim of this study was to detect class I integron presence and characterize variable regions in 15 clinical isolates from Buenos Aires. Through PCR amplification of 15 isolates of various Enterobacteriaceae, 60% (9/15) were positive for the class I integron structure. NCBI Blast sequencing of 38 sequences revealed that 26% (10/38) contained a segment encoding the aac(6′)‐Ib and 21% (8/38) frequency of occurrence for the aadA1 gene cassette. Several studies have concluded that integron bearing isolates have a significant association with multi‐resistance and this conclusion is supported by our findings.
Interventions to improve breastfeeding among obese women
Authors: Rasmussen Kathleen M , Surber Christine D , Zelek Sarah T , Altabet Jamie D , Kjolhede Chris L
Published: June 2021

Maternal obesity is associated with poor breastfeeding (BF) outcomes, yet no intervention has been developed to improve them. To ascertain whether increased BF support or provision of a breast pump is a feasible, effective intervention to improve BF, we enrolled obese women who intended to breastfeed in 2 randomized trials. In Bassett Improving Breastfeeding Study (BIBS) 1, 40 women received targeted BF support in the hospital and via telephone or usual care. Information regarding BF was collected via telephone for 7 d after delivery and at 30 d and 3 mo postpartum. In BIBS 2, 34 obese mothers received a manual or electric breast pump to use for 10 d or no pump; data collection was similar. In BIBS 1, targeted care did not improve any measure of BF outcome. At 3 mo, fewer women in the targeted‐care group were still BF exclusively (P=0.03), and overall exclusive BF duration was 4.7 weeks shorter (P=0.03) than in the usual‐care group. In BIBS 2, provision of a breast pump did not improve any measure of BF outcome. At both 30 d and 3 mo, fewer women who received a pump were still BF (P<0.05) than those who did not. Execution of these studies was compromised by protocol violations, but regression analyses that accounted for these factors did not change the results. Further development of interventions of help obese women achieve optimal BF outcomes is required. (Funded in part by USDA/Hatch grant NYC‐399430; electric pumps donated by Medela.)
Informatics‐driven, fragment‐based drug discovery and refinement engine coupled with integrative pharmacology screens effectively prioritizes novel CNS experimental therapeutic selection
Authors: Chico Laura K , Watterson D. Martin
Published: June 2021

Adequate penetration of the blood brain barrier and avoidance of CYP2D6 substrates represents major challenges to success in the discovery of new molecular entities for CNS disorders. Thus, we sought to develop and validate a rapid, novel small molecule discovery engine for CNS experimental therapeutics characterized by front end designs that leverage trends from in‐house databases and informatics analyses and incorporate facile, high‐yield synthetic chemistry paradigms to sample diverse chemical space. The recursive nature of this platform permits the integration of pharmacological screens to focus on novel compounds with a high potential for success in later preclinical evaluation for investigational new drug (IND) status as a driving force for medicinal chemistry refinement. Additionally, the flexible nature of the discovery engine allowed for application to both function and single molecular target discovery campaigns. Commercial development of candidates emerging from this platform provides validation, as well as feedback for future optimization. Progress on preclinical and commercial development will be reported along with trends that have the potential for application to diverse chemotypes and targets.
Mutagenesis studies to investigate ligand binding to the carbon monoxide‐sensing heme protein, CooA, from Carboxydothermus hydrogenoformans
Authors: Wagoner Laura , Wagoner Josh , Goyne Thomas E. , Clark Robert W.
Published: June 2021

The work reported here is part of a larger effort to determine how amino acid residues that compose the heme pockets of gas‐sensing heme proteins impact the proteins' regulation mechanisms and gas specificity. In the present study, we have employed site‐directed mutagenesis to prepare protein variants of the carbon monoxide (CO)‐sensing heme protein, CooA, from Carboxydothermus hydrogenoformans. These mutants have been designed in an attempt to rationally alter CooA's effector specificity. We have prepared, isolated, and purified several protein variants to date, including C80T CooA. The C80T substitution is expected to increase the steric bulk on the proximal face of the CooA heme, but eliminates a potential site of S‐nitrosylation that may impact protein activation by nitric oxide (NO). Currently, we are investigating both C80T heme structure and this variant's ability to be activated for DNA‐binding by CO and NO. Our next goal will be to prepare mutants that allow us to study proposed cooperative interactions that take place between the monomers of the CooA homodimer. The authors wish to thank the Valparaiso University College of Arts and Sciences for financial support.
Spectroscopic and Functional Characterization of the Nitric Oxide Adducts of the Fe(III) and Fe(II) States of the CO‐Sensing Transcription Factor, CooA, from Carboxydothermus Hydrogenoformans
Authors: Clark Robert W. , Jani Edra , Francis Aarthi R. , LaRoche Elise , Malone Isabel
Published: June 2021

CooA is a carbon monoxide (CO) sensing transcription factor that is found in several bacteria and regulates CO metabolism. CO binding to CooA's heme groups initiates a conformation change that activates protein DNA binding. This study investigates the reaction of Fe(III) Carboxydothermus hydrogenoformans CooA (Ch CooA) with nitric oxide (NO). Previously, Clark and coworkers reported Fe(II) Ch CooA bound NO to form a 6‐coordinate (6‐C) Fe(II)‐NO adduct that was active for DNA binding in vivo and in vitro. This is in contrast to the best studied CooA homolog from R. rubrum (Rr) that was exquisitely specific for CO and formed an inactive 5‐coordinate (5‐C) Fe(II)‐NO adduct. In the current study, when Fe(III) Ch CooA was incubated with NO, a final 5‐C Fe(II)‐NO species resulted. This species had an electronic absorbance spectrum similar to Rr CooA; however, unlike Rr CooA, the 5‐C Fe(II)‐NO Ch CooA adduct retained some DNA‐binding activity. Results from mutagenesis studies suggest that S‐nitrosylation of a Cys residue located in the heme pocket may account for the unexpected reactivity differences of Fe(III) and Fe(II) Ch CooA with NO. Based on these data, it is proposed the ability of Ch CooA (and failure of Rr CooA) to be activated by NO is due to the binding of NO to the correct (i.e. distal) heme face. The authors wish to thank the Valparaiso University College of Arts and Sciences for financial support.
Bioorthogonal Ligation Using Site‐Specific Tetrazine‐Alkene Coupling On Proteins
Authors: Refakis Christian André , Blackman Melissa L , Fox Joseph M , Mehl Ryan A
Published: June 2021

The selective coupling of chemical labels to proteins is key to many applications in medicine and biotechnology. Chemical labeling strategies require two functional groups to react rapidly and selectively with one another under biological conditions to form a stable covalent bond. Despite their great utility, few effective bioorthogonal ligation reactions exist that are non‐toxic and react specifically to the programmed functionality. The objective of this study is to expand on the array of available bioorthogonal ligation reactions by using site‐specific unnatural amino acid incorporation to exploit the alkene‐tetrazine reaction. The amino acid p‐vinyl‐L‐phenylalanine (pVF) was synthesized and incorporated into proteins site‐specifically, in vivo, using an unnatural aminoacyl‐tRNA synthetase (RS) pair. The reactivity of a diverse family of tetrazines was explored with pVF‐protein. ESI‐MS confirmed the tetrzine‐alkene ligation reaction occurs with high fidelity, site‐specificity, and yield. We expect the results of this work will provide scientists with an additional venue for attaching small chemical labels and/or drugs to proteins.
Peroxisome Proliferator‐Activated Receptorγ Agonist Rosiglitazone Increases Expression of Lipin‐1 in Adipocytes
Authors: Kim Jina , Han Dong Cho , Kwon Byoung‐Mog
Published: June 2021

We investigate the possibility that lipin‐1 inhibits the phosphorylation of extracellular‐signal regulated kinases 1 and 2 (ERK1/2) and peroxisome proliferators‐activated receptorγ (PPARγ). Rosiglitazone (RGZ) is well known PPARγ agonist using for the type 2 diabetes mellitus therapy with side effect such as inducing obesity. RGZ enhances 3T3‐L1 adipocyte differentiation and lipin‐1 expression. Lipin‐1 is a novel family of Mg2+‐dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis. Phosphorylation of ERK1/2 and PPARγ are suppressed by RGZ in 3T3‐L1 cells. Phospho‐ERK1/2 is translocated into the nucleus and induces phosphorylation of PPARγ, which decreases PPARγ transcriptional activity and then inhibits adipocyte differentiation. In RGZ‐treated 3T3‐L1 preadipocyte, transfection of lipin‐1 small interfering RNA increased phosphorylation of ERK1/2 and PPARγ. These results suggest that RGZ induced adipogenesis through the expression of lipin‐1 to lead inhibition of ERK pathway and transcriptional activation of PPARγ. We conclude that rosiglitazone directly increases lipin‐1 expression, thereby inhibiting phosphorylation of ERK and PPARγ in adipocytes.
Small Molecule Activation of Apoptotic Caspases
Authors: Zorn Julie Anne , Wolan Dennis W. , Gray Daniel C. , Wells James A.
Published: June 2021

Caspases comprise a family of cysteine aspartyl proteases that are expressed as inactive zymogens, or procaspases. These enzymes require proteolytic processing by upstream proteases to form the active protein. Apoptotic caspases act via a two‐tiered signaling cascade whereby initiator caspases activate downstream executioner procaspases‐3, −6 and −7. Once activated, these executioner caspases cleave a plethora of substrates, resulting in apoptosis. The Wells lab has identified a small molecule (1541) from a high‐throughput screen that promotes the self‐activation of executioner procaspases‐3 and −6. This pan‐procaspase, small molecule activator has been validated in cellular assays to show rapid induction of apoptosis. We have synthesized a focused library of analogs around the initial hit to explore the mechanism of self‐activation and to generate selective activators for the individual procaspases. These activators are not only important as tools to explore caspase biology, but, as apoptotic inducers, they may also serve as potential chemotherapeutic agents.
Characterization of methyl group metabolism in the non‐obese diabetic (NOD) mouse
Authors: Neuman Joshua Charles , Nieman Kristin M , Schalinske Kevin L
Published: June 2021

Perturbed methyl group metabolism leads to elevated circulating levels of homocysteine, an independent risk factor for cardiovascular disease, as well as DNA hypomethylation. A streptozotocin‐induced type 1 diabetic rat model showed disrupted methyl group metabolism, characterized by increased levels of glycine N‐methyltransferase (GNMT), DNA hypomethylation, and hypohomocysteinemia. The present study examined similar measures of methyl group metabolism in the genetic non‐obese diabetic (NOD) mouse model. At 24 wk of age, NOD and ICR (control) mice were sacrificed and blood, liver, and kidney tissue were collected. NOD mice displayed 59% (P < 0.05) greater GNMT activity compared to the ICR mice. A correlation between both hepatic GNMT activity (r = 0.81, P < 0.002) and DNA hypomethylation (r = 0.59, P ≤ 0.05) with plasma glucose concentrations was also observed. Plasma homocysteine levels in the blood decreased by 53% in the NOD group (P < 0.005) compared to the controls. However, phosphatidylethanolamine N‐methyltransferase (PEMT) was down‐regulated in NOD mice, compared to its increase with streptozotocin treatment. This indicates that a genetic and chemically induced model for type 1 diabetes exhibit similar, but not entirely consistent, findings with respect to disrupted methyl group metabolism.

Grant Funding Source: NIH HD057315
BHMT deletion results in altered sulfur‐containing amino acids, disturbed one‐carbon metabolism, elevated hepatic fat deposition, and diminished white adipose tissue mass
Authors: Teng Ya‐Wen , Zeisel Steven H
Published: June 2021

Human single nucleotide polymorphisms (SNPs) in the betaine:homocysteine methyltransferase (BHMT) gene have been identified. Our laboratory generated a mouse with the gene encoding BHMT deleted (Bhmt−/−) to study the potential functional effects of such SNPs. Mice were fed a AIN76A diet. Homocysteine, cysteine, S‐adenosylhomocysteine were measured using HPLC; choline metabolites using LC/MS; triglyceride and phosphatidylcholine using GC. Bhmt−/− mice had hyperhomocysteinemia, hypocysteinemia, and elevated hepatic S‐adenosylhomocysteine (p<0.01) as compared to Bhmt+/+mice. Bhmt−/− mice had elevated betaine in various tissues, but reduced choline, phosphocholine, phosphatidylcholine, and glycerophosphocholine in the liver (p<0.05) as compared to Bhmt+/+mice. Starting at 5 weeks of age, Bhmt−/−mice had reduced body weight and fat mass with significant reductions in the gonadal, inguinal and retroperitoneal fat pads (p<0.01). Bhmt−/−mice had higher food intake (p<0.01), higher energy expenditure (p<0.05), but no difference in the activity level when compared to Bhmt+/+mice. Bhmt−/−mice also had elevated liver mass and hepatic triglyceride deposition, which were accompanied by reduced hepatic and plasma phosphatidylcholine, and reduced plasma cholesterol (p<0.01). We are currently studying the underlying mechanisms for these effects of BHMT deletion.
Effect of nitrous oxide (N2O) explosure during surgery on homocysteine (Hcy) concentrations of children
Authors: Pichardo Dubraiicka , Luginbuehl Igor , Shakur Yaseer , Wales Paul
Published: June 2021

Postoperative acute hyperhomocysteinemia has been associated with an increased risk of complications in adults. In this study we determined the effect of N2O on the Hcy concentrations of children and whether blood levels of folate vitamin B12, B6, methylmalonic acid (MMA) and methylenetetrahydrofolate reductase C6777T genotype affected this relationship. Total Hcy was measured before and after N2O. Vitamin levels, MMA and genotype were determined preoperatively. Median age of the 32 participants was 11mo. (3–126mo.). All children had folate and B6 levels above cut‐off values (7.4; 31nmol/L respectively). Five children had MMA levels above cut‐off (≥ 0.23μmol/L). Post‐exposure Hcy increased by 25% (P= <0.001). Length of exposure was strongly associated with the increase (r=0.696, P=<0.001). There was a trend between vitamin B12 and Hcy increase (r = − 0.450, P=0.080). Folate, vitamin B6 and genotype had no effect on Hcy. In conclusion, Hcy increases after exposure to N2O in children. Studies investigating clinical relevance of the increase and benefit of pre‐surgical vitamin B12 supplementation may prove worthwhile. Funding: Mead Johnson/Heinz/Weston Endowment for Nutrition/Metabolism Research; CONACYT.
Basal diet composition determines effects of supplemental selenium
Authors: Quiner Trevor E , Lindsay Heather S , Mason Brock A , Lephart Edwin D , Christensen Merrill J
Published: June 2021

The effects of supplemental selenium (Se) in animal models of prostate cancer and diabetes may depend upon the composition of the basal diet to which Se is added. In this study, we examined effects of dietary Se and isoflavones, alone and in combination, on body weight, fat mass, and serum T3 levels. Non‐transgenic male littermates of TRAMP mice, produced for a study of tumorigenesis, were weaned to one of two Se‐adequate stock diets, either low (Zeigler Bros. Phytoestrogen‐Red. Diet I) or high (Harlan Teklad 8604) in isoflavones, or the same diets supplemented with 3.0 ppm Se as Se‐methylselenocysteine. Mice were killed at 18 weeks of age. Serum levels of T3 were measured using ELISA kits. Hepatic expression of the iodothyronine deiodinase I gene was determined by real time RT‐PCR. Values in a column not sharing a common subscript are significantly different (p < 0.05). These data show a significant interaction between isoflavones and Se, and demonstrate opposite effects of supplemental Se based on the isoflavone content of the basal diet. (Supported by NIH CA122235 to MJC).

Final Body Wt (g)
Abdom. Fat (g/35 g BW)
T3 (pg/ml serum)
Relative DIO1 Exprsn.

Se − Iso
38.1 ± 4.9a
2.78 ± 0.29a
0.70 ± 0.13cd
1.0 ± 0.12b

+Se − Iso
35.2 ± 4.2ab
1.75 ± 0.51bc
0.93 ± 0.11bc
1.49 ± 0.12a

Se +Iso
31.4 ± 2.5b
1.23 ± 0.13c
1.99 ± 0.37a
1.47 ± 0.23a

+Se +Iso
35.5 ± 2.8a
1.90 ± 0.17b
1.31 ± 0.19b
1.11 ± 0.14b
tRNA (Ser)Sec coexpression affects SECp43 levels in HEK 293 cells
Authors: Gilman Christy Leinaala , Seale Lucia A , Seyedali Ali , Berry Marla J
Published: June 2021

Selenium is present in the active site(s) of selenoproteins in the form of selenocysteine (sec), the 21st amino acid. Sec is incorporated cotranslationally into proteins through recoding of the UGA codon. Key factors in this process include a unique tRNA for Sec (Sec‐tRNA), SECIS elements, SBP2 and EFsec. Additional factors include SLA/LP, SPS1 and SECp43. Previous studies in our laboratory have found that cotransfection of these three factors resulted in coprecipitation in nuclear and cytosolic fractions, indicating the formation of complexes. In GST pull downs, SECp43 and SPS1 coimmunoprecipitate in the cytosol in the absence but not in the presence of Sec‐tRNA. SECp43 is present in the nuclear fraction in the absence of other factors, in either the absence or presence of Sec‐tRNA. SECp43 pull‐down of SPS1 or vice versa is not seen when SLA/LP is added, suggesting mutually exclusive interactions. SECp43 in the presence of SLA/LP pellets with V5 beads independent of V5 tagged protein, suggesting it may be part of a large complex. This is not seen when SPS1 is also present, further supporting mutually exclusive interactions. We are currently following up on these findings to elucidate the mechanism by which Sec‐tRNA affects SECp43 levels and to understand the interactions of these factors.

Supported by NIH grant DK‐47320 to MJB.
Methylselenol, a selenium metabolite, plays common and different roles in colonic cancer and nontumorigenic colonic cell growth
Authors: Zeng Huawei , Briske‐Anderson Mary , Moyer Mary P.
Published: June 2021

There is increasing evidence for the efficacy of certain forms of selenium (Se) as cancer‐chemopreventive compounds, and methylselenol has been hypothesized to be a critical selenium metabolite for anticancer activity in vivo. To determine differential chemopreventive effects of methylselenol on colon cancer cells versus normal colon cells, colon‐cancer‐derived HCT116 cells and nontumorigenic colon NCM460 cells were exposed to methylselenol. We found that submicromolar methylselenol exposure inhibited cell growth and led to an increase in the G1 and G2 fractions with a concomitant drop in the S‐phase. In addition, methylselenol inhibited the extracellular‐regulated kinase 1/2 (ERK1/2) signaling and c‐Myc expression. Importantly, methylselenol showed greater inhibition of cell growth, cell cycle, ERK1/2 signal and c‐Myc expression in colon‐cancer‐derived HCT116 cells, when compared to NCM460 colon cells. Similarly, submicromolar methylselenol induced a higher apoptosis rate in HCT116 cells (up to 3.2 fold of the control) than that in NCM460 colon cells (up to 1.4 fold of the control). Taken together, our data demonstrate that methylselenol's stronger potential of inhibiting colon cancer cell proliferation when compared with normal colon cells, which may be a critical mechanism by which selenium exerts its anticancer activity. Supported by USDA/ARS
The mismatch repair protein hMLH1 regulates selenium‐induced DNA damage response in colorectal cancer cells
Authors: Qi Yongmei , Schoene Norberta W , Cheng Wen‐Hsing
Published: June 2021

Epidemiological studies indicate that selenium supplementation in humans suppresses risk of colorectal and other cancers. The majority of colorectal cancer is characterized by a defective DNA mismatch repair (MMR) system. The hMLH1‐hPMS2 heterodimer plays a critical role at the early stage of MMR repair. Here, we employed HCH116 colorectal cancer cells with intrinsic hMLH1 deficiency and the cell complemented with hMLH1, and treated them with sodium selenite (2–10 μM), methane seleninic acid (2–10 μM) and methyl‐seleno‐L‐cysteine (50–300 μM). We showed that complementation of hMLH1 sensitizes HCT116 cells to cell death after treatment with the selenium compounds. Interestingly, cellular exposure to organic forms of selenium compounds resulted in increased hMLH1 protein expression and hMLH1‐hPMS2 association. Furthermore, hMLH1 is involved in proper S and G2/M phase checkpoint responses and DNA‐PKcs phosphorylation at Thr‐2647, an early event of DNA damage response, after cellular exposure to selenite. Our results suggest that DNA damage checkpoint response, a barrier of tumorigenesis, is deficient in HCT116 cells after selenium treatment. Consequently, a lack of functional hMLH1‐hPMS2 complex may predispose colorectal cancer cells to genomic instability.

Grant Funding Source: University of Maryland
ATM is upstream of DNA‐PKcs in the activation of DNA damage response by sodium selenite
Authors: Rocourt Caroline , Cheng Wen‐Hsing
Published: June 2021

The role of selenium in mitigating tumorigenesis is not fully understood. Selenium in supranutritional doses can induce ROS, which indirectly causes DNA breaks. Previous reports indicate an interplay between Ataxia‐telangiectasia mutated (ATM) and DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in the early stages of DNA damage response. We recently found that ATM is required for a selenium‐induced senescence response in non‐cancerous cells at an early stage. Thus, we hypothesize that the selenium‐induced DNA damage response can modify this ATM‐DNA‐PKcs cross‐talk. Phospho‐specific antibodies against ATM and DNA‐PKcs were used to follow the phosphorylation events after selenium treatment in MRC‐5 normal fibroblasts and ATM shRNA and control shRNA U‐2 OS osteosarcoma cells. Results from immunofluorescence analysis showed that selenium treatment induces phosphorylation of DNA‐PKcs at T2647, an ATM‐dependent phosphorylation site, and phosphorylation of DNA‐PKcs at S2056, an autocatalytic site of DNA‐PKcs, in non‐cancerous MRC‐5 cells but not in U‐2 OS cancer cells. Further studies in MRC‐5 cells with an ATM kinase inhibitor, KU55933, showed attenuation of the selenium‐induced DNA‐PKcs phosphorylation at both foci, whereas pre‐treatment with a DNA‐PKcs kinase inhibitor, NU7026, does not prevent ATM phosphorylation at S1981, an event leading to ATM pathway activation. The selenium‐induced DNA‐PKcs phosphorylation at S2056 is decreased in S‐phase cells. Taken together, our results provide the first evidence of ATM and DNA‐PKcs activation as an early tumorigenesis barrier in response to selenium exposure in non‐cancerous cells, and places ATM upstream of DNA‐PKcs in the selenium‐induced DNA damage response process.

Grant Funding Source: University of Maryland
Possible rise in Ca:Mg ratio from food intake in U.S.A. adults
Authors: Rosanoff Andrea
Published: June 2021

A growing body of work suggests an importance for Ca:Mg ratio. We calculated the change of Ca:Mg intake from foods over time in the United States of America.

Method

U.S. Department of Agriculture Food Surveys from 1977, 1985, 1994, 2001–2, 2003–4, and 2005–6 provided mean Ca, Mg and Kcal intake per day in adult age‐gender groups. (USDA Surveys before 1977 do not include Mg; Data for adults age 50+ were not reported until 1994; Defined age/gender groups changed slightly for age from survey to survey.) Survey means were used to calculate % changes in Ca, Mg, Kcal, and Ca:Mg intakes from food for 1977 to 2006 (1994 – 2006 for age 50+ yrs).

Results

Mean Ca daily intake rose in each age/gender group: a low of 21% for elderly males and a high of 77% for adult females. Mean Mg daily intakes rose in each adult age‐gender group, but to a lesser extent than Ca, from 3% for elderly males and a high of 28% in adult females. Caloric intakes rose in each age/gender group comparable to the rise in Mg rather than to the rise in Ca: 7% for elderly males and 26% for adult females.

Conclusion

Large rises in Ca intakes between 1977 and 2005–6 result in an apparent rising Ca:Mg from food, observed from 1977 to 2005–6 for each adult age‐gender group; 20 – 38% rise for adult women and 18 – 32% rise for adult men. Supported by Center for Magnesium Education & Research.
Low vitamin D status among overweight and obese Puerto Rican adults
Authors: Gil Karen E , Palacios Cristina , Perez Cynthia M , Joshipura Kaumudi
Published: June 2021

Vitamin D deficiency is highly prevalent worldwide, even in locations where it was assumed that UV radiation was adequate to prevent vitamin D deficiency. The aims of this study were to measure vitamin D status in a convenience sample of overweight adults in Puerto Rico, which has plenty of sun exposure throughout the year, and relate it to vitamin D intake and sun exposure. Measurements included serum 25(OH)D, anthropometry, food frequency questionnaire and sun exposure questionnaire, obtaining a summary score ranging from 0 (no exposure, dark skin) to 38 (maximum exposure, light skin). Serum 25(OH)D was explained as a function of vitamin D intake, sun exposure, age, and % body fat using a multiple linear regression model. Complete data were obtained for 98 subjects (66% females), with mean age of 51.6±7.5 y, and BMI of 33.5±6.3 kg/m2. Mean serum 25(OH)D levels were 31.1±9.4 ng/ml. Vitamin D status was normal (>30 ng/ml) in 55%, insufficient (20–30 ng/ml) in 14% and deficient (<20 ng/ml) in 31% of the subjects. Mean vitamin D intake was 340±352 IU/d (58% were below the DRI) and sun exposure score was 21.5±6.7. The regression model explained only 7% of the variation of serum 25(OHD) levels, with only % body fat (p=0.07) and vitamin D intake (p=0.08) being marginally significant. In conclusion, in this group of overweight individuals, 45% had low vitamin D status, which was not explained by vitamin D intake or sun exposure.
Age related changes in CTX type bone biomarkers are differently affected by calcium status
Authors: Rio Maria Esther , Dupraz Hernán C , Zago Liliana B , Piazza Norma , Zeni Susana N
Published: June 2021

The influence of Calcium nutritional status (CaS) on biomarkers (BM) of normal bone development became recently recognized as a vacancy area in paediatric research. In this study a comparative analysis of the influence of CaS on the age‐linked changes of two different types of CTX markers was afforded. Basal urine samples of 160, both sex, healthy children 6 to 9 years old, normal according to body mass index (BMI by NCHS), were collected. CTXs α and βwere determined by ELISA (Nordic Bioscience Diagnostic, Denmark); calcium/creatinine ratio (Ca/C) was measured as estimator of Ca status: <0, 07 deficient (D); 0,07–0,15, adequate (A); >0,15 high (H). Analyzed as a function of age, and grouping data according CaS, neither significant influence of age nor of CaS was detected over βCTX along the frame of ages of the study. However, CaS modified α CTX evolution so that the although changes linked to age were not founded in D, the decreasing in values with age was similar in A and H groups. The slope of the curves αCTX vs. age was very similar for both (y= −1,6071x + 50,214) but the correlation coefficients(R2) were 0,5432 and 0,9643 respectively. Both CTX molecules,αα‐CTX and ββ‐CTX‐, reflect bone metabolic activity and bone remodelling but only the new biomarker &[alpha&[alpha]‐CTX responds to Ca status and appears appropriate for studies related to bone problems due to calcium inadequacy in paediatric patients.