bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning, Expression and Purification of Yeast Aminotransferase
Authors: Reyes Zoraya L , Karsten William E , Bobyk Kostyantyn D , Cook Paul F , Chooback Lilian
Published: June 2021

Lysine is synthesized in Saccharomyces cerevisiae via the α‐aminoadipate pathway.α‐aminoadipate is made from α‐ ketoadipate via the action of a PLP‐dependent aminotransferase that has not been identified. Sequence alignments and a homology search for conserved amino acid residues were used to identify six aminotransferase genes in S. cerevisiae. Based upon the homology, the identified genes code for two aromatic aminotransferases, two aspartate aminotransferases, one kynurenine aminotransferas and one putative aminotransferase of unkown function. The cloned genes were obtained from the Harvard Institute of Proteomics Plasmid Repository. The genes were subcloned into expression vector pET16b. The expressed his‐tagged proteins have been purified using a Ni‐NTA affinity column. The aromatic aminotransferase coded by the Aro 8 gene has Km values for L‐tyrosine and L‐phenylalanine of 0.5 and 1 mM respectively. The Kcat values are about 10 s‐1 for both substrates. The uv/vis spectra of the aromatic aminotransferases have been determined as a function of pH. Substrate titrations and initial rate studies have been done to determine the substrate specificity of these enzymes.
Biochemical characterization of the cellular biosynthesis and trafficking of Caspr2
Authors: Falivelli Giulia , Davide Comoletti , Dubi Noga , De Jaco Antonella , Wilson Jennifer , Taylor Palmer
Published: June 2021

Recent genetic studies have linked mutations of Contactin‐associated Protein‐like 2 (Caspr2), a gene product of CNTNAP2, in neurodevelopmental conditions such as autism and epilepsy. Although little information is currently available for the role of this protein in the human brain, recent lines of evidence suggest that Caspr2 is a key molecule in cell‐cell interactions important for normal neuronal function and cortical development. Here, we investigate the processing and trafficking of Caspr2 wild type and several mutants using biochemical and cell biological approaches. Through analysis of glycosylation processing in HEK293 cells, we show that some of the mutations impair Caspr2’s trafficking to its cellular location. Furthermore, analyses of confocal immunofluorescence co‐localization with chaperons like calreticulin demonstrate that certain mutants have increased retention in the endoplasmic reticulum and retarded transport of newly synthesized proteins to the cell surface. These observations promise to yield critical insights into congenital processes that give rise brain abnormalities in neuronal connectivity.

This work was supported by: USPHS Grant R37 GM‐18360 and NIEHS to PT, and Autism Speaks #2617 to DC
Extraction and evaluation of ganglioside‐like material in Lactobacillus bacterium
Authors: Sawdon Alicia J , Ettinger Anna C , Moore Kathleen H
Published: June 2021

Previous work has shown that many cultured dairy products are rich in gangliosides that may confer antimicrobial effects. It is unclear whether lactic acid bacteria (LAB) used to culture dairy products contain gangliosides; this effort seeks to isolate and characterize gangliosides from LAB membranes. LAB were grown in a ganglioside‐free thioglycolate medium. Cells were harvested by centrifugation and washed extensively with saline. After suspension in a 1:1 chloroform/methanol solution, cells were sonicated to disrupt cell walls and membranes. Subsequent Folch‐like fractions were separated from other lipid components by anion exchange chromatography into potential ganglioside pools. Sialic acid‐specific resorcinol reagent was used to screen chromatographic fractions for possible ganglioside content; additionally, the column fractions were analyzed by thin layer chromatography against ganglioside and sialic acid standards. Initial experiments with an LAB species have yielded a compound with ganglioside‐like mobility (RF = 0.34) and color response. Characterization studies include reactivity with N‐acetyl neuraminidase, as well as effect of exogenous sialic acid on production of ganglioside‐like material. The probiotic effect of LAB may relate to the presence of gangliosides. Supported by the Provost's Undergraduate Student Research Award.
Characterization of a Coenzyme Q biosynthetic complex in Saccharomyces cerevisiae
Authors: Allan Christopher Michael , Tran UyenPhuong , Gulmezian Melissa , Marbois Beth , Clarke Catherine
Published: June 2021

Coenzyme Q (Q) is a critical molecule for proper functioning of the electron transport chain in mitochondrial respiration. Q is synthesized by a series of enzymes in the mitochondria and the human Q biosynthetic proteins are well conserved in the yeast Saccharomyces cerevisiae, with several capable of functionally substituting for their yeast homologues. Yeast is thus a good model for understanding Q biosynthesis in higher eukaryotes. There is evidence that several of the Q biosynthetic proteins, the Coq proteins, are associated in a large complex in the mitochondria. All of the Coq proteins are peripheral membrane proteins on the matrix face of the inner mitochondrial membrane, except for Coq2p which is an integral membrane protein. It is thus possible that Coq2p serves as the membrane anchor for the other Coq polypeptides in the complex. Coq2p has also been demonstrated to exist in a high molecular mass complex via gel filtration chromatography and 2D Blue Native‐PAGE, although its partner proteins have not been identified. One of our aims is to characterize the Coq2p‐containing complex to determine its role in the organization and stabilization of the complex. BN‐PAGE will be used to determine whether other Coq proteins co‐migrate with Coq2p in a high molecular mass complex. Immunoprecipitation of Coq2p tagged with the HA epitope will identify interacting proteins.
Zhx2 protects the liver from high‐fat induced liver damage
Authors: Fleishaker Erica Leigh , Peterson Martha , Spear Brett
Published: June 2021

Obesity is a significant and growing health concern in the US due to its associated risks, including atherosclerosis and nonalcoholic fatty liver disease. A recent genetic study, identified a QTL on mouse chromosome 15, called hyplip2, associated with hyperlipidemia and increased atherosclerotic lesions (ATVB, 24:1928). We have found that the zinc‐finger and homeoboxes 2 (Zhx2) gene (PNAS, 102:396) is responsible for the hyplip2 phenotype. The objective of this study is to understand the role of Zhx2 in liver function during a high fat diet. BALB/c (wild‐type Zhx2) and BALB/cJ (mutant Zhx2) female mice were fed a normal or high fat diet. AFP mRNA and serum ALT levels were elevated in BALB/cJ on the high fat diet compared to all other groups. Hepatic TNF mRNA levels increased in response to the high fat diet but were not significantly different between the two strains. CYP8B1 (involved in bile acid synthesis) mRNA levels were higher in BALB/cJ than BALB/c on the high fat diet. This data suggests that Zhx2 may protect the liver from high fat‐induced damage and involved in regulating lipid metabolic enzymes.
A novel vision to the process of ERpahgy in yeast
Authors: Liu Ang‐Yu , Ho Kung‐Hsien , Huang Wei‐Pang
Published: June 2021

Under oxidative stresses, accumulation of misfolded protein in the endoplasmic reticulum (ER) activates downstream pathway ‐ the unfolded protein response (UPR) to protect cells. Autopahgy, on the other hand, is another protective response to cope with cellular stresses in eukaryotes, which involves in the degradation of cytosolic proteins and defective organelles. It is known that the ER‐selective autophagy, or ERphagy, will be induced during ER stresses and serve a protective role to maintain cell survival. However, the correlation of ERphagy with other type of autophagy is not clear. Here, we show that ERphagy is a selective process distinct from the cytoplasm‐to‐vacuole targeting (Cvt) pathway and the non‐selective macroautophagy. We found that under the dithiothreitol (DTT)‐induced ER stress, the ER‐containing autophagosomes (ERAs) would tend to accumulate in cytoplasm rather than fuse with vacuoles rapidly. However, soon after DTT is removed, ERAs will start to fuse with vacuoles and be degraded. We also discovered that the Cvt pathway would be partially inhibited at the vesicle forming stage during ERphagy, causing the delayed maturation phenotype of prApe1. Nevertheless, ERphagy seems to have limited effect on the process of the starvation–induced macroautophagy, and starvation treatment would not promote the clearance of accumulated ERAs in cytoplasm either. Taken these together, ERphagy as a selective process may compete with the Cvt pathway for utilizing the share of common regulatory machinery, but not preferable to the starvation‐induced macroautophagy.

NSC96‐2311‐B‐002‐012‐MY3
Possible immune functions of two mosquito multicopper oxidases
Authors: Lang Minglin , Gorman Maureen J , Gardner Stewart G , Yungeberg Sandi L , Kanost Michael R
Published: June 2021

The multicopper oxidase (MCO) family of enzymes has been implicated in a diverse set of physiological processes; however, little is known about their function in insects, including the malaria mosquito, Anopheles gambiae. Our goal is to characterize the function of two Anopheles MCOs: MCO1 and MCO3. We found that these two genes are upregulated in the midguts and Malpighian tubules of mosquitoes following a blood meal and in other tissues in response to infection with bacteria or malaria parasites. These results suggest that MCO1 and MCO3 may function in defense against pathogens. Preliminary data suggest that double silencing of MCO1 and MCO3 increases the susceptibility of mosquitoes to malaria infection. Overexpression of MCO1 or MCO3 in Drosophila increases their sensitivity to paraquat exposure, suggesting that MCO activity may lead to ROS production. If this is true, the MCOs may participate in an ROS killing mechanism against the parasites. Alternatively, the MCOs may restrict pathogen growth by acting as ferroxidases that reduce the amount of available iron. Finally, MCO1 and/or MCO3 may be required for protein crosslinking in the peritrophic matrix, which acts as a natural barrier against parasite invasion of the gut. We will present the results of experiments designed to test these three hypotheses. The work was supported by NIH Grants R03AI057816 and R01AI070864.
Involvement of protein kinase Cα in invadopodia formation in Src‐3T3 cells
Authors: O'Neill Audrey Kathleen , Gallegos Lisa Leon , Newton Alexandra C.
Published: June 2021

Dynamic structures termed podosomes or invadopodia take the form of actin puncta or rings and are involved in extracellular matrix remodeling, adhesion, and invasion. Though various protein kinase C (PKC) isoforms are known to affect these structures, the precise role of PKCα in signaling upstream of invadopodia in transformed cells remains unclear. We have begun to analyze the role of PKCα in NIH 3T3 fibroblasts transformed with active Src (Src‐3T3 cells). PKCα colocalizes with actin and the invadopodia marker Tks5 at rosettes, and inhibition of conventional PKC isoforms interferes with PKCα localization and rosette formation. The target of PKC that is responsible for invadopodia formation is unknown; however, one possible candidate is Discs Large 1 (DLG1), which we show is a substrate of PKC. DLG1 is present at numerous actin‐based structures including invadopodia and is involved in motility and invasion. Strikingly, coexpression of DLG1 and PKCα in COS‐7 epithelial cells causes formation of actin‐based rosettes (in a cPKC‐dependent manner), suggesting that phosphorylated DLG is sufficient for establishment of these structures.

This work was supported by NIH P01 DK54441 and NIH GM43154.
Identifying the essential arginine required for ELMOD1 GAP activity
Authors: Fritz Julia R. , East Michael P. , Kahn Richard A.
Published: June 2021

The Arf family of 20 kDa GTPases act in cells as molecular switches to regulate essential cellular processes, including energy metabolism, cell division, membrane traffic, and the cytoskeleton. Key regulators of these proteins include the GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). ELMOD1 and ELMOD2 are GAPs that have been shown to act on members of the Arf family, including Arfs and Arl2, though they lack the Arf GAP domain found in every other known ArfGAP. We sought to identify the essential residues for GAP activity to better define the yet unidentified Arl GAP and novel Arf GAP domain present in ELMOD1. By alignment of protein sequences from multiple species, we found three absolutely conserved arginine residues most likely to act as the “catalytic arginine” for this GAP domain. To test this, these residues were individually mutated to create three constructs which we used to transfect cells and compare to cells over expressing wild type ELMOD1. When wild type ELMOD1 is over expressed in HeLa cells, there is an obvious phenotype of golgi fragmentation. It was found that the golgi morphology differed from the wild type cells in only one mutant construct. These studies have identified the “catalytic arginine” that is involved in GTP hydrolysis on Arf and Arl2 proteins and serves as the basis for future experiments designed to determine the signature motif for this new family of cell regulators.
Allosteric modulation of Ras: a novel role for Glutamine 61 in catalysis
Authors: Mattos Carla , Buhrman Greg , Fetics Susan , Holzapfel Genevieve
Published: June 2021

The Ras GTPase and its effector Raf are key mediators of the Ras/Raf/MEK/Erk signal transduction pathway, which plays a central role in cell growth and malignant transformation. Mutants of residue Q61 impair the GTPase activity of Ras and are found prominently in cancers. Yet the mechanism through which Q61 contributes to catalysis has been elusive. We have found that Ras‐GppNHp in the crystal binds calcium acetate from the crystallization mother liquor at a site remote from the active site and most likely near the membrane, resulting in a shift in helix 3/loop 7 and a network of H‐bonding interactions that propagates across the molecule, culminating in complete ordering of switch II and placement of Q61 in the active site in a previously unobserved conformation for catalysis. In this structure Q61 interacts with a water molecule that bridges one of the γ‐phosphate oxygen atoms to the hydroxyl group of Y32. This active site arrangement suggests a novel role for Q61 where it interacts with the bridging water molecule to stabilize the transition state of the hydrolysis reaction. We propose that Raf together with the binding of Ca2+ and negatively charged group in the membrane at the allosteric site (mimicked in our structure by the acetate molecule), induce ordering of the switch I and switch II to complete the active site and bring intrinsic hydrolysis rates in Ras to biologically relevant levels. Funded by NIH grant CA096867.
Elg1 (Enhanced level of genome instability 1) has an essential role in early development and suppressing tumorigenesis
Authors: Sikdar Nilabja , Myung Kyungjae
Published: June 2021

Introduction

High levels of chromosome rearrangements known as gross chromosomal rearrangements (GCRs) such as translocations, deletion of chromosome arm, interstitial deletions, inversions and gene amplification, have been reported in many cancers. Multiple pathways, such as S‐phase checkpoints, DNA replication, chromatin remodeling, and telomere maintenance suppress the GCR formation. Elg1 was discovered during the genome wide screening of yeast S. cerevisiae as a suppressor of GCR formation. Elg1 is a suppressor of genomic instability and functions in DNA repair in yeast and human.

Objectives

To investigate whether the loss of Elg1 in mouse a in vivo model system causes any genomic disorder and null Elg1 MEF cells shows any Genomic instability phenotypes.

Results

Absence of Elg1 in mice caused early embryonic lethality and zebra fish Elg1 MO embryos also has developmental problem suggesting Elg1 has an essential role during early development. This embryonic developmental defect rescued in absence of p53 in zebrafish and developed normal. The heterozygous mice developed tumors including sarcoma, carcinoma, and adenoma in various organs from 11 month after birth. The Elg1+/mMEFs and Elg1+/− chicken DT 40 cells show high level of DN A damage sensitivity as well as chromosomal instability in respond to DNA damage. High levels of genomic rearrangements were observed in tumors developed in the Elg1+/mmice by comparative genome hybridization. The microarray and pathway analysis of tumors from the Elg1 heterozygous mice showed the upregulation of Smad independent Tgf‐β pathway.

Conclusions

Taken together, Elg1 has an essential role in early development that appears to have a close link to suppress genomic instability and ultimately tumorigenesis.
Multi‐analyte profiling reveals MCP‐1 and MMP‐9 as plasma biomarkers of cardiac aging
Authors: Chiao Ying Ann , Jin Yufang , Dai Qiuxia , Chou Youn‐Min , Lindsey Merry L
Published: June 2021

We have previously shown that cardiac sarcopenia occurs with age in C57/BL6J mice. However, underlying mechanisms and plasma biomarkers of cardiac aging have not been identified. Accordingly, the objective of this study is to evaluate plasma biomarkers that reflect cardiac aging phenotypes. Plasma from young (7.5 months, n=27) and senescent (31.8 months, n=25) C57/BL6J mice were collected and levels of 69 markers were measured by multi‐analyte profiling. Of these, 27 analytes were significantly higher in the senescent group. The majority were inflammatory markers associated with macrophage functions, including monocyte chemotactic protein‐1 (MCP‐1) and matrix metalloproteinase‐9 (MMP‐9). Immunoblotting showed higher MCP‐1 levels in left ventricle (LV) of senescent mice (p<0.05), and LV MCP‐1 levels correlated with plasma MCP‐1 levels (R=0.27, p<0.05). Both, immunoblotting of CD68 and immunohistochemistry for ER‐HR3 showed higher macrophage levels in the senescent LV. Further, the increased MCP‐1 and MMP‐9 levels correlated with the decrease in LV function in the senescent mice. In conclusion, this study suggests that MCP‐1 and MMP‐9 are potential plasma markers for cardiac aging and that augmented MCP‐1 and MMP‐9 levels and macrophage content in the LV could provide an underlying mechanism in cardiac aging. NSF 0649172 and NIH 1SC2 HL101430 to Y.J. and NIH R01 HL‐75360 and AHA 0855119F to M.L.L.
The effect of p66Shc on energy expenditure in mice
Authors: Stern Jennifer H , Hagopian Kevork , McDonald Roger B , Ramsey Jon J
Published: June 2021

Previous investigations have shown that the p66Shc protein has a significant effect on life span. While the mechanism underlying the impact that p66Shc has on life span remains to be elucidated, it has been proposed that p66Shc may interact with the IGF‐1/insulin signaling pathway to modulate metabolism, and possibly energy expenditure, under conditions of stress. As a first step in testing this possibility, oxygen consumption and carbon dioxide production were measured in 3‐month‐old male p66Shc knockout (p66Shc−/−) and wild type C57BL/6 mice for 24h at 23oC and for 5h at 12–13oC. Average 24h energy expenditure (EE) at 23oC did not differ (P > 0.10) between the p66Shc−/− and C57BL/6 groups. Dark vs. light phase EE (12h dark: 12h light) was greater in both groups, but did not differ among p66Shc−/− and C57BL/6 mice. Cold‐exposed EE also did not differ between the groups. RQ measured at 23oC was higher in the p66Shc−/− compared to C57BL/6 mice and this difference was most pronounced during the dark cycle. Cold‐exposed RQ was also greater in the p66Shc−/− vs. C57BL/6 mice (p66Shc−/−, 0.85 ± 0.004; C57BL/6, 0.83 ± 0.005). These preliminary data suggest that while EE does not differ in p66Shc−/− vs. C57BL/6 mice, the mix of substrate used for energy metabolism may be influenced by the p66Shc protein. The influence of the p66Shc protein on substrate utilization observed here is consistent with a role for this protein in the IGF‐1/insulin‐dependent intracellular signally pathway. (Supported by NIH PO1 AG025532)
Epigenetic marks of nuclear‐encoded mitochondrial genes by DNA methylation
Authors: Takasugi Masaki , Hirabayashi Keiji , Yagi Shintaro , Shiota Kunio
Published: June 2021

Mitochondria are semi‐self‐replicating organelles with it's intrinsic mitochondrial DNA (mtDNA). mtDNA is not under epigenetic regulation and, thus, unable to confer tissue or cell type dependent information in mammalian cells. However, of approximately 1,200 proteins in mitochondria, only 13 proteins are encoded by mtDNA. To investigate whether there are differences of epigenetic information of nuclear mitochondrial genes depending on tissues, we studied on tissue‐dependent and differentially methylated regions (T‐DMRs) in the promoters of nuclear mitochondrial genes. We analyzed methylation status of all known mitochondrial genes in mouse liver, brain, and heart. More than 200 mitochondrial genes were identified to have T‐DMRs which are specifically hypomethylated in one of the tissues. The mitochondrial genes with T‐DMRs were related to various mitochondrial functions. We also found that liver‐specific transcription factor binding site were enriched in the genes with liver‐hypomethylated T‐DMR. In conclusion, each tissue examined has inherent DNA methylation pattern of mitochondrial genes, and epigenetic regulation underlies the tissue‐dependent functions of mitochondria.

This work was supported by grant from NIBIO.
Dot1a‐AF9‐AF17‐mediated transcriptional changes in the epithelial sodium channel (ENaC) are translated into changes in ENaC‐mediated Na+ transport
Authors: Zhang Wenzheng
Published: June 2021

We previously reported that Dot1a‐AF9 complex inhibits the transcription of α‐ENaC by hypermethylating histone H3 K79 at its promoter. Aldosterone downregulates this complex through various mechanisms. AF17 upregulates transcription of all ENaC genes and their key regulators by competitively binding to and promoting nuclear export of Dot1a. However, the significance of these transcriptional mechanisms in regulating Na+ transport has not been fully defined. To address this question, mouse inner medullary collecting duct cell line‐3 (mIMCD3) and mouse cortical collecting duct M1 cells were used to determine ENaC activity as measured by benzamil‐sensitive intracellular Na+ concentration ([Na+]i) using fluorescence imaging or benzamil‐sensitive equivalent short circuit current (Isc) using an EVOM ohm/volt meter. In both cell lines, addition of benzamil significantly decreased the basal [Na+]i and Isc values. Benzamil‐sensitive [Na+]i or Isc values were significantly decreased by overexpression of Dot1a or AF9, but increased by AF17 overexpression. The effect of aldosterone on benzamil‐sensitive [Na+]i and Isc was impaired by blocking either transcription or translation. Our data suggest that ENaC is largely responsible for Na+ uptake in these cells, and that transcriptional changes in ENaC genes mediated by Dot1a, AF9, AF17 or aldosterone treatment are translated into changes in ENaC‐mediated Na+ transport.
Calcium Levulinate: Dietary Supplement and Pro‐drug of Abuse
Authors: Anderson Stephanie Rae , Zhang Guo‐Fang , Murphy Anne M. , Tomcik Kristyen A , Brunengraber Henri
Published: June 2021

The goal of the study was to investigate the metabolism of levulinate (4‐ketopentanoate, LEV) which as a calcium salt, is used as an oral or intravenous source of calcium. We hypothesized that (i) levulinate is converted to gamma‐hydroxypentanoate (GHP), a new drug of abuse, analog of gamma‐hydroxybutyrate, and (ii) the formation of GHP from LEV is enhanced by ethanol. We investigated the metabolism of LEV and GHP in perfused rat livers and live rats. In both models, LEV was converted to GHP, and GHP was converted to LEV. This interconversion involves a cytosolic NADP‐dehydrogenase. Ethanol decreases the uptake of LEV and increases the formation of GHP from LEV, without affecting the [GHP]/[LEV] ratio. Thus, ethanol appears to inhibit both LEV and GHP catabolism, presumably at the level of 3‐hydroxyacyl‐CoA dehydrogenase. In livers perfused with LEV there was substantial accumulation of LEV‐CoA, GHP‐CoA and 4‐phospho‐GHP‐CoA. In parallel, the concentrations of acetyl‐CoA, malonyl‐CoA, HMG‐CoA methylmalonyl‐CoA and succinyl‐CoA markedly decreased. Thus, the metabolism of LEV and GHP result in substantial CoA trapping which can affect a number of processes. The conversion of LEV to GHP, a drug of abuse, and the stimulation of GHP formation by ethanol is a public health concern since calcium‐LEV is freely available. Supported by NIDDK RoadMap and by NIEHS.
Selected beta‐apocarotenoids antagonize retinoic acid‐induced transactivation of retinoic acid receptors
Authors: Eroglu Abdulkerim , Hruszkewycz Damian , Curley Robert W. , Harrison Earl Howard
Published: June 2021

β‐carotene (BC) is the major dietary source of vitamin A and can undergo central or eccentric cleavage. Central cleavage of BC catalyzed by β‐carotene oxygenase (BCO) 1 yields two retinals, which can be oxidized to retinoic acid (RA). RA is the physiological ligand for the retinoic acid receptors (RARs). Eccentric cleavage of BC at double bonds other than the central double bond is catalyzed by BCO2 leading to the production of β‐apocarotenoids. This study sought to determine the effects of apocarotenoids on activation of RARs. All possible eccentric cleavage products of BC as the aldehydyes (or ketones) and acids were purified or synthesized. Reporter gene constructs containing the firefly luciferase gene driven by a DR5 retinoic acid response element along with vectors for each of the retinoic acid receptor subtypes were transiently transfected into COS‐1 cells, which were used in transactivation assays of these ligand‐dependent transcription factors. None of the β‐apocarotenoids activated any of the RARs. However, β‐apo‐10’‐carotenoic acid, β‐apo‐12’‐carotenoic acid, β‐apo‐14’‐carotenal, β‐apo‐14’‐carotenoic acid, and β‐apo‐13‐carotenone inhibited ATRA mediated activation of RARs. Molecular modeling shows that β‐apo‐13‐carotenone fits the RARβ ligand binding site. Some eccentric cleavage products of dietary β‐carotene may function as antagonists of retinoic acid signaling in cells. Funded by NIH
Selenium‐dependent expression of hematopoietic prostaglandin D synthase in macrophages
Authors: Gandhi Ujjawal H , Kaushal Naveen , Prabhu K. Sandeep
Published: June 2021

While playing a central role as effector cells inflammatory reactions and cell‐mediated immune responses, macrophages produce reactive oxygen species and other pro‐inflammatory substances. This is counter‐acted by a multitier antioxidant system, where the micronutrient selenium plays a pivotal role. We have shown that selenium increases production of 15‐deoxy‐D12,14‐prostaglandin J2 (15d‐PGJ2) in RAW264.7 and murine primary bone marrow‐derived macrophages. We have demonstrated a dose‐dependent increase in the expression of hematopoietic PGD synthase (H‐PGDS) in selenium‐supplemented macrophages, which sequentially leads to a corresponding increase in its downstream anti‐inflammatory products PGD2 and 15d‐PGJ2 upon stimulation with LPS. Furthermore, treatment with 15d‐PGJ2, a high‐affinity ligand of PPARgamma, as well as PPARbeta/delta agonists up‐regulated H‐PGDS, thus revealing a unique positive feedback loop in selenium‐supplemented macrophages. Studies with organic non‐bioavailable forms of selenium and the genetic manipulation of cellular selenium incorporation machinery indicated that selenoproteins were necessary for H‐PGDS expression and 15d‐PGJ2 production. Taken together, these results indicate interplay between selenoproteins and PPAR‐dependent mechanisms in expression of H‐PGDS in macrophages. NIH DK077152
Comparative analysis of genome‐scale metabolic models for Mycoplasma genitalium, Ureaplasma parvum, and Mycoplasma hominis reveals novel routes through the Pentose Phosphate Pathway
Authors: DeJongh Matthew , Poel Nathan B. , Best Aaron A. , Henry Christopher S.
Published: June 2021

We have recently developed a high‐throughput computational pipeline for generating genome‐scale metabolic models. To test this pipeline, we have generated models for three mollicutes: Mycoplasma genitalium G‐37 (Mg), Ureaplasma parvum serovar 3 (Up), and Mycoplasma hominis (Mh). All three genomes lack an annotated transaldolase (TAL) gene, resulting in incomplete pentose phosphate pathways (PPP). However, all three organisms require production of phospho‐ribose‐diphosphate (PRPP), a key product of the PPP. To meet this demand, all three models predict novel routes through the PPP. The route predicted in the Mg and Up models relies on secondary activities of the phosphofructokinase (PFK) and fructose‐bisphosphate aldolase (FBA) enzymes to cope with the accumulation of sedoheptulose 7‐phosphate. This PPP bypass was recently discovered in an E. coli mutant lacking the TAL gene. Because Mh lacks a gene for PFK, the Mh model predicts a different route, reversing the activity of FBA and postulating the presence of Sedoheptulose‐1,7‐bisphosphatase. There is no genomic evidence for the presence of known forms of this enzyme in Mh; in this case, the model reveals the necessity of clarifying the pathway of PRPP production in Mh. Our study highlights the potential for comparative analysis of metabolic models to predict novel biochemistry and suggest testable hypotheses in prokaryotic metabolism.

NSF MCB 0745100
Actomyosin contraction and adhesion control angiogenic sprouting and guidance
Authors: Fischer Robert S , Gardel Margaret , Ma Xufei , Adelstein Robert S , Waterman Clare M
Published: June 2021

Directional control of endothelial cell (ECs) migration through their surrounding extracellular matrix (ECM) drives angiogenesis & vascular patterning. In vivo, ECs at vessel tips migrate directionally via extension of subcellular pseudopodia. While it is clear these cells are responsive to VEGF signals, the molecular mechanisms of the cytoskeleton forming these protrusive structures are unknown. Using in vitro 3D model systems where migrating ECs display branched pseudopodia morphodynamics similar to those in living zebrafish, we have demonstrated that ROCK‐dependent myosin II activity is dynamically localized to the EC cortex, and is partially released under conditions that promote branching, such as decreased local ECM stiffness. Moreoever, local depletion of cortical myosin II precedes branch initiation, which can be mimicked by local inhibition of myosin II activity. Our research aims to translate these findings to the in vivo intersegmental vasculature of the zebrafish embryo where regulation of rac and rho guide tip cells, in response to both local mechanical cues as well as soluble guidance cues such as semaphorin/plexin signals.
Evaluation of rapid western blot incubation system
Authors: Lang Ewa Z. , Rupprecht Kevin R , Rae Tracey D , Fishpaugh Jeffrey R
Published: June 2021

Western Blot has been a powerful tool in life science labs for over thirty years. One of the encumbrances of this method is that it is labor and time intensive. In our lab, shortened incubation times are acceptable for identity testing, however long incubation times are still standard when detecting low amounts of antigen or when using low affinity antibodies. We evaluated the Millipore SNAP i.d™ for use in routine western blot analysis as well as for researching new proteins whose properties were not as well known. The SNAP i.d.™ uses vacuum assisted diffusion to greatly decrease incubation times. A variety of samples including recombinant proteins and antibody fragments were optimized for both methods. Proteins were transferred using the Invitrogen i‐Blot®. Direct dual‐development analysis, staining the blot with Sypro® Ruby prior to antibody exposure was used to differentiate closely migrating proteins from the protein of interest. Commercially available kits were utilized to develop the blots. This study indicated that the SNAP i.d.™ is suitable for a variety of applications of the Western blot technique, although substantial method development may be required for unfamiliar proteins and specific applications. The system was useful for quickly identifying epitopes in specific recombinant degradation products, aiding in the identification of the specific cleavage sites and detecting low level contaminants.
Biomarker discovery by stable isotope labeling and quantitative proteomics
Authors: Filiou Michaela D. , Zhang Yaoyang , Teplytska Larysa , Reckow Stefan , Frank Elisabeth , Kessler Melanie S. , Maccarrone Giuseppina , Hambsch Boris , Landgraf Rainer , Turck Christoph W.
Published: June 2021

In order to identify protein biomarkers for anxiety disorders we have compared the synaptosome proteomes of high (HAB), normal (NAB) and low (LAB) anxiety‐related behavior mice using 15N metabolic labeling and quantitative mass spectrometry.

For accurate mass spectrometric‐based relative quantification, mice were metabolically labeled with a 15N‐enriched diet achieving 92% 15N incorporation in brain tissue. To detect phenotype‐related differences unlabeled HAB and LAB synaptosomes were compared with synaptosomes from 15N‐labeled NAB mice. We have found differentially expressed proteins between HAB and LAB synaptosomes involved in energy metabolism, transport and phosphorylation processes. Selected candidate biomarkers were validated with immunochemical and enzymatic assays. The identified biomarkers provide important information on pathways involved in disease mechanisms and shed light on anxiety pathophysiology.

This work was supported by a BMBF QuantPro grant and the Max Planck Society.
Investigating the catalytic mechanism of the yeast palmitoyltransferase Akr1p
Authors: Guan Xiaomu , Roth Amy F , Davis Nicholas G , Fierke Carol A
Published: June 2021

Protein palmitoylation is a widespread posttranslational modification in which cysteine thiols on a substrate protein are modified with a palmitoyl group. Defining the active site and catalytic mechanism of palmitoyltransferases responsible for this modification represents a key step towards understanding its biological significance. Akr1p, one of the first identified protein palmitoyltransferases, is an 86 kDa yeast integral membrane protein. Using Akr1p purified from yeast, auto‐ and trans‐palmitoylation activity has been detected by the covalent modification of a substrate protein with a 3H‐labeled palmitoyl moiety in the presence of [3H]‐palmitoyl‐CoA and ATP. In addition to the previously identified substrate yeast casein kinase (Yck2), we have used mutagenesis to demonstrate that Akr1p catalyzes time‐dependent palmitoylation of two c‐terminal cysteines in the yeast protein, Ypl199c. We are examining the mechanism of Akr1p using mass spectrometry to identify covalent modifications and mutagenesis to examine the functional importance of the DHYC motif in a cysteine‐rich domain that is proposed as the active site of Akr1p.
Fluorescent intercalator displacement as a tool for RNA‐ligand discovery
Authors: Asare‐Okai Papa Nii , Chow Christine S
Published: June 2021

Fluorescent intercalator displacement (FID) is a useful tool for identifying new nucleic‐acid‐binding ligands. The success of this method is based on the fact that it can be fashioned into a versatile high‐throughput assay, which can then be used to assess relative binding affinities of compounds to nucleic acids in a simple and efficient manner. FID is becoming an increasingly attractive method because it is a tag‐less approach; neither the nucleic acid nor the small molecule under investigation has to be modified. In this study, a modified FID method for screening RNA‐binding ligands was established, using 3‐methyl‐2‐((1‐(3‐(trimethylammonio)propyl)‐4‐quinolinylidene)methyl)benzothiazolium (TO‐PRO) as the fluorescent intercalator. Electrospray ionization mass spectrometry (ESI‐MS) was employed to investigate the FID mechanism. Our results provide molecular evidence that correlates a reduction in fluorescence intensity in the FID assay with displacement of the TO‐PRO dye molecule from structured RNA. The FID assay was then used to screen a variety of RNA‐binding ligands. With FID, it is possible to differentiate ligands that bind to a variety of RNA constructs from the ribosome and HIV (A‐site, h31, H69, and TAR) with moderate selectivity.

This work is supported by the National Institute of Health (NIH)
Gene expression profile of selenoproteins in an obese pig model induced by a high‐fat diet
Authors: Li K. , Zhao H. , Xia X. , Lei Xin Gen
Published: June 2021

This study was to develop an obese porcine model and to determine effects of obesity on gene expression of the 25 selenoproteins. A total of 20 crossbred, castrated boars (20 kg body weight) were fed a corn‐soy basal diet (Se adequate, total fat < 0.82%) or the diet added with lard at 3% (20–50 kg), 5% (50–80 kg), or 7% (> 80 kg) for 6 months. In the end, pigs fed the high fat diet showed greater (P < 0.05) body weight (146 vs. 121 kg), back fat thickness (3.5 vs. 2.3 cm), and abdominal fat content (3.5 vs. 1.9 kg), along with hyperglycemia and hyperlipidemia, compared with the control pigs. The obese pigs had greater (P < 0.05) mRNA levels of gpx6, selV, and selW in skeletal muscle, selK and selO in thyroid, sps2 in perirenal fat tissue, selT in kidney, and gpx4 in hypothalamus than the control pigs. In contrast, the opposite was true for the mRNA levels of selI and selT in pituitary, selI in the skeletal muscle, gpx1, sel15, selW, and selX in perirenal fat tissue, selI in subcutaneous fat tissue, and selH, selO, and selV in heart. In conclusion, the high fat diet‐induced obesity up‐regulated the expression of 8 selenoproteins and down‐regulated that of 9 selenoproteins in various tissues of pigs, implying a potential link between the two events. (NSFC Projects 30628019, 30700585, and 30871844, and the Chang Jiang Scholars Program).
Phosphate‐dependent physiological changes during short‐term dietary phosphorus deficiency with or without a dietary phosphorus binder
Authors: Bobeck Elizabeth A , Cook Mark E
Published: June 2021

Patients with end‐stage renal failure are susceptible to increased plasma phosphate; hence, animal models to rapidly screen strategies to prevent dietary phosphate absorption are needed. The objective of this experiment was to evaluate a short‐term animal feeding model for estimating the effectiveness of a phosphate binder to limit dietary phosphate absorption. Nutrient‐adequate chicks (10 days of age) were either maintained on a phosphorus (P) adequate diet (0.70% total P, or 0.45% available P) or switched to a P deficient (0.35% total P) diet with or without 1% inclusion of the phosphate binder sevelamer hydrochloride (Sev) (n=15/trt). Body weight gain from 10–13d of age, plasma phosphate, and dry fat‐free tibiotarsus ash were determined on day 13. When chicks were placed on the P deficient diet, bone ash and plasma phosphate, but not body weight, were reduced (9% and 26%, respectively) within 3 days (p<0.05, main effect of P). The addition of Sev to the diet (main effect) had no effect on plasma phosphate or ash, but reduced body weight 22%. Since there was no interaction between P and Sev, these data would suggest, within the limits of this model, that Sev was unable to interfere with phosphate absorption even when animals were clearly deficient. These data also suggest body weight was reduced through a mechanism other than phosphate deficiency. This work was supported by the Wisconsin Alumni Research Foundation (WARF).

Grant Funding Source: WARF
Vitamin D status of elderly Mayan residents of the western highlands of Guatemala
Authors: Bermudez Odilia I. , Sud Sohil , Montenegro‐Bethancourt Gabriela , Heaney Robert , Armas Laura , Solomons Noel W.
Published: June 2021

Introduction

While the prevalence of various chronic diseases is known to be high among elderly Guatemalan Mayans, and research linking vitamin D (VitD) deficiency to such conditions continues to advance, little is known about vitD concentrations in older adults.

Objectives

To assess 25(OH)D in Mayans, 60y or older, from the highland province of Quetzaltenango, Guatemala.

Methods

Blood samples were collected and analyzed (for serum 25(OH)D concentrations) from 108 subjects (mean age=69y, 50% men, 78% rural).

Results

Mean 25(OH)D values were 53±15 nmol/L. As ages rose, 25(OH)D declined (r=−0.28, p=0.004). Values for 25(OH)D were optimal (>80 nmol/L) in 4%, suboptimal (50–80 nmol/L) in 50%, and deficient (<50 nmol/L) in 46% of subjects. Men had higher VitD than women (58±17 vs. 48±12 nmol/L, p=0.001). VitD was higher although non‐significant in urban vs. rural elders (55±15 vs 47±12 nmol/L, p=0.23). Traditional dress allows 6 to 13% solar exposure of skin.

Conclusions

Despite their optimal geographic location for adequate sunlight exposure, most elderly Guatemalan Mayans had suboptimal or deficient levels of VitD. Age, sex and skin exposure seemed to play a role on it.
Effect of peanut skin phenolics on pancreatic amylase activity and fructose‐mediated albumin glycation
Authors: Garner Pamela L. , Hargrove James L. , Hartle Diane K. , Pegg Ronald B , Greenspan Phillip
Published: June 2021

Peanut skins, a by‐product of the peanut industry, have not been incorporated into the human food supply in the United States. This high protein product contains a high concentration of polyphenolics; the majority of the phenolics are proanthocyanidins or condensed tannins, but lower molecular weight phenolics (catechin and epicatechin) are also present. This present study was designed to examine the bioactivity of peanut skin phenolics on two biological processes relevant to the development of diabetes and its complications. Extracts of peanut skins inhibited porcine pancreatic α‐amylase by 90% at approximately 2 μg peanut skin phenolics/ml. The 90% level of inhibition of fructose‐mediated glycation of albumin was observed at approximately 20 μg peanut phenolics/ml. The difference in the degree of inhibition between these two biological processes may well be due to the differences in the mechanism of inhibition. Peanut skin proanthocyanidins probably inhibit α‐amylase activity by binding directly to the enzyme. However, since there is 10 mg albumin/ml in the protein glycation assay, the inhibition observed with peanut skins probably reflects its intrinsic antioxidant and/or metal‐ion chelating activity.

Support provided by the State of Georgia Food Industry Partnership.
Combination of fenugreek and cayenne improves glucose handling of a type 2 diabetic mouse model
Authors: Hanlon Paul , Eshelman Bruce , Harriman William , Barnes David
Published: June 2021

Fenugreek and cayenne contain bioactive phytochemicals that have both been shown to affect glucose handling. The purpose of this study was to determine the effect of these two foods on glucose handling in a model of diabetes, as well as whether there would be a synergistic effect of these two foods combined. C57BL/KsJ‐db/db (leptin receptor deficient) diabetic mice were fed a control diet or experimental diets containing 1% ground fenugreek seed, 1% dried cayenne pepper powder or the combination of 1% fenugreek and 1% cayenne for two weeks. Inclusion of fenugreek and/or cayenne did not significantly reduce food consumption, nor did it affect the body weight of the animals. At the end of two weeks, glucose handling was measured by i.p. glucose tolerance test (IPGTT). Mice fed either the fenugreek or cayenne alone diets did not have significant improvement in IPGTT in comparison with control. However, the mice fed the combination of fenugreek and cayenne demonstrated significantly improved glucose handling (115% of baseline after 120 minutes) over control (blood glucose 167% of baseline after 120 minutes). Values for circulating adipokines and hemoglobin A1c are also reported.
Effects of fermented soy food containing natural S‐equol on tail skin temperature in ovariectomized rat.
Authors: Yoneda Takeshi , Ueno Tomomi , Iwashita Soh , Uchiyama Shigeto
Published: June 2021

Equol has been reported to have greater estrogenic activity, decreasing risks of osteoporosis, menopausal symptoms and hyperlipidemia in menopausal women. We succeeded in industrial production of a fermented soy food containing natural S‐equol (FSE) using equol‐producing lactic acid bacteria, Lactococcus 20–92. It was reported that ovariectomized (OVX) rat increase tail skin temperature (TST) like hot flashes in postmenopausal women. We examined the effects of FSE on TST in OVX rat, and compared with Premarin (conjugated estrogen drug).

The study groups were consisted of sham, control, Premarin and FSE groups. Sham and control groups were administered vehicle, Premarin group were administered Premarin 6 mg/kg BW and FSE group were administered FSE as equol 12 mg/kg BW for 4 weeks. TST was evaluated at 3rd and 4th week after OVX operation. Uterine weight was measured at 4th week.

TST was significantly higher in control group compared with sham group, and significantly lower in Premarin and FSE groups compared with control group at 3rd and 4th week. Premarin group significantly increased the uterine weight reduced after OVX operation, but FSE group did not.

These results suggest that FSE is capable to improve hot flashes without effect on reproductive organs in postmenopausal women. Therefore, natural S‐equol might be expected to have activity as a selective estrogen receptor modulators.
Abrogation of TCDD Induced Epigenetic Silencing of BRCA‐1 by Resveratrol
Authors: Papoutsis Andreas , Romagnolo Donato , Selmin Ornella
Published: June 2021

BRCA‐1 protein is involved in repair of DNA damage. Mutations of the BRCA‐1 gene confer a high risk of developing breast cancer. In sporadic breast cancers BRCA‐1 is silenced in the absence of mutations. 2,3,7,8 tetrachlorodibenzene(p)dioxin (TCDD) is a ligand of the aryl hydrocarbon receptor (AhR) that contributes to this etiology. Resveratrol is a dietary ligand of the AhR with potential protective effects. Activation of the AhR leads to its association with xenobiotic responsive elements (XRE) harbored in the BRCA‐1 promoter. The results of DNA binding studies in MCF‐7 breast cancer cells documented that TCDD induced the association of the AhR to the XRE which was reduced by cotreatment with resveratrol. Western blot analysis revealed that TCDD reduced BRCA‐1 protein expression and that this effect was counteracted by resveratrol. Epigenetic modifications investigated by chromatin immunoprecipitation assays (ChIP) indicated that TCDD facilitated, whereas resveratrol antagonized the recruitment of DNA methyltransferase 1 (DNMT1) and histone deaceytlase 1 (HDAC1). These findings support a model in which resveratrol prevents the assembly of a co‐repressive complex responsible for transcriptional inactivation of BRCA‐1. We are currently investigating changes in DNA methylation induced by TCDD at the BRCA‐1 promoter and the preventative effects of resveratrol.

Susan G. Komen Breast Cancer for the Cure