bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondria Targeted Cytochrome P450 2E1: Potential Role in Alcohol Mediated Mitochondrial Oxidative Stress
Authors: Bansal Seema , Liu Chuan‐Peng , Sepuri Naresh BV , Anandatheerthavarada Hindupur K , Milne Ginger L , Guengerich Frederich P , Avadhani Narayan G
Published: June 2021

Studies suggest a role for cytochrome P450 (CYP2E1) in oxidative stress. Here we investigated if the mitochondrial (mtCYP2E1) or microsomal (mcCYP2E1) counterpart is responsible for inducing alcohol mediated oxidative stress. Using an algorithm we altered the SRP binding affinity which also altered the mitochondrial targeting efficiencies of the protein. We also generated stable cell lines predominantly expressing the mtCYP2E1 (MT++, MT+) and predominantly the mcCYP2E1 (ER+). GSH depletion, carbonyl formation and formation of F2‐Isoprostanes as measures of oxidative stress were significantly higher in Mt++ and Mt+ cells compared to ER+ cells. Also ethanol mediated oxidative stress was higher in cells expressing Mt+ and Mt++ proteins compared to ER+ and WT protein. The toxicity was blocked by antioxidants (Mito Q, NAC) and inhibitor of CYP2E1 (DAS) suggesting the role of mitochondria targeted CYP2E1 in alcohol mediated cell injury. Furthermore, in yeast cell expression system, MT++ CYP 2E1 expressing cells, but not ER+ CYP2E1 cells exhibited respiratory deficiency and severe mitochondrial DNA damage similar to the Rho0 cells. These results suggests that mtCYP 2E1 play a direct role in inducing oxidative cell damage and augments alcohol‐mediated cell injury. (Grant support: GM‐034883‐23, AA‐017749 to NGA and R37 CA090426 to FPG).
Superoxide formation at the Q‐binding site of mitochondrial complex I does not arise from a semiquinone that has direct access to the bulk membrane QH2/Q pool
Authors: Treberg Jason R , Quinlan Casey L , Brand Martin D
Published: June 2021

Complex I (NADH:Q oxidoreductase) can form superoxide during either forward (NADH oxidizing) electron flux or, under conditions of sufficiently high proton motive force (PMF), reverse electron transport (NAD+ reducing). In the isolated enzyme, the fully reduced flavin produces superoxide (site IF). With mitochondria, maximal superoxide production from site IF (with rotenone) is far less than during reverse electron transport driven by succinate. The marked discrepancy between these two ‘maximal’ rates is consistent with a 2‐site model of superoxide generation. The reduction state of this second site (site IQ) of superoxide production is unknown, but is often considered a semiquinone. In this study, we determine if superoxide production during complex I reverse electron transport arises from a partially or fully reduced site. We designed the following system to titrate the reduction state of the superoxide generating site: a constant PMF generated by ATP, succinate to reduce the Q pool and inhibitors of complex III isolate the QH2/Q from oxidation via the Q‐cycle. Under these conditions, a stepwise oxidation of the QH2/Q by malonate titration showed no evidence for the formation a semiquinone, or other partially reduced species, that directly interacts with the bulk Q pool. These titrations suggest, unexpectedly, that superoxide production by site IQ is also by a fully reduced species, although we cannot distinguish if this is a fully reduced single or double electron carrier. Funded by NIH (ARRA) grant number: NIH R01 AG033542
The role of TMAP/CKAP2 in the regenerating liver
Authors: Kang Du‐Seock , Park Chi‐Hu , Park Joobae , Bae Chang‐Dae
Published: June 2021

TMAP/CKAP2 is frequently upregulated in various human primary cancers. Previously, we showed TMAP/CKAP2 was related to chromosome segregation in several established cell lines including C2C12 cells (Mol Cell Biol, 2007). The physiologic role of TMAP/CKAP2 in normal diploid cells, however, has never been explored. Here, we investigated the role of TMAP/CKAP2 in regenerating liver after partial hepatectomy. TMAP/CKAP2 was induced and reached maximum level at 48 h after partial hepatectomy. Targeting TMAP/CKAP2 using hydrodynamic injection of TMAP/CKAP2 shRNA before partial hepatectomy blocked regaining liver mass. Primary cultured hepatocytes from regenerating liver with prior injection of TMAP/CKAP2 shRNA showed multiple centrosomes, multipolar spindle formation. Hepatocytes with multiple centrosomes eventually underwent multipolar cell division and subsequent cell death. Overall mitotic cell death was increased more than 4 folds in TMAP/CKAP2 knock‐down hepatocytes compared to control luciferase shRNA injected hepatocytes. Taken together, we concluded that TMAP/CKAP2 is essential in cell cycle progression of normal diploid cells possibly through centrosome duplication process.
Tissue injury by microbeam irradiation depends on the stage of vascular maturation
Authors: Sabatasso Sara , Laissue Jean Albert , Hlushchuk Ruslan , Bräuer‐Krisch Elke , Bravin Alberto , Blattmann Hans , Djonov Valentin
Published: June 2021

Purpose

The aim of this study is to explore the effects of Microbeam Radiation (MR) on vascular biology using the chick chorio‐allantoic membrane (CAM).

Methods

CAMs were irradiated with multiple planar synchrotron X‐ray beams at doses between 100 and 300 Gy and were evaluated morphologically and in vivo at days 8 and 12.

Results

In vivo monitoring and morphological investigations of day 8 CAM immature vasculature 6 hr after MR revealed a near total destruction of the capillary plexus within the beams’ width. Surprisingly, arteries and veins were not affected. Conversely, at day 12, only well defined lesions in the microvasculature (mature ones, covered by pericytes) were observed.

After 300 Gy MR, TEM revealed enlargement of the interendothelial cell junctions which could explain the oedema, the microvessels along the beam path showed signs of disruption and apoptosis. The remaining vasculature recovered rapidly and CAM regained its normal thickness. Between 1 hr and 6 hr no additional morphological changes were observed.

Conclusions

The effects of MR are most likely mediated by capillary damage.

The vascular toxicity of MR depends on the stage of capillary maturation: the immature vessels are much more vulnerable than the mature ones.

The physiological effects of MR appear within a short time, the most important structural alterations being present between 15 and 60 minutes after irradiation.
TRP14 attenuates osteoclast differentiation by inhibiting NF‐κB and MAPK signaling
Authors: Hong Sohyun , Kim Hojin , Jeong Woojin
Published: June 2021

TRP14 is a disulfide reductase containing the CXXC motif which regulates NF‐κB and mitogen‐activated protein kinases (MAPKs) stimulated by TNFα. Given that NF‐κB and MAPK signaling play an important role in osteoclastogenesis, it was examined whether TRP14 can inhibit osteoclast differentiation. Osteoclast differentiation of RAW264.7 macrophages by receptor activator of NF‐κB ligand (RANKL) was enhanced by depletion of TRP14, while it was inhibited by TRP14 overexpression, as revealed by tartrate‐resistant acid phosphatase (TRAP) staining and TRAP activity assay. When RAW264.7 cells was exposed to RANKL, depletion of TRP14 augmented phosphorylation and degradation of IκBα, and activation of c‐Jun NH2‐terminal kinase (JNK) and p38 as well as consequent induction of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) that is a pivotal determinant of osteoclastogenesis. In addition depletion of TRP14 strongly expanded actin ring formation that is a prerequisite for osteoclast bone resorption, and significantly increased RANKL‐mediated ROS production. Taken together TRP14 attenuates osteoclast differentiation by inhibiting NF‐κB, JNK and p38 activated by RANKL.
Signalling pathway of prostate‐specific membrane antigen implicates different types of detergent‐resistant membranes
Authors: Schmidt Sonja , Heine Martin , Naim Hassan Y.
Published: June 2021

Prostate‐specific membrane antigen (PSMA) is a surface protein overexpressed in prostate cancer and neovasculature of further tumours. It is therefore one of the most promising biomarkers in diagnosis and treatment of prostate cancer.

PSMA is associated with detergent‐resistant membranes (DRMs). The mature form of PSMA is mainly insoluble in Lubrol WX, but does not associate with Triton X‐100 DRMs.

Recently we could demonstrate that cross‐linking of cell surface PSMA with specific antibodies activates different signalling cascades like the MAPK‐pathway. Here we show that internalization of PSMA is increased after antibody binding and this leads to redistribution of PSMA to Triton X‐100 DRMs in a time‐dependent manner.

Our results propose an essential role for different DRM types in the signalling pathways involving PSMA.
Ligand Induced Conformational Changes within the Metal‐Dependent PI‐PLC from Streptomyces antibioticus
Authors: Selby Thomas Lee
Published: June 2021

The objective of this study focused on understanding the conformational changes associated with calcium and inositol binding for the metal dependent phosphatidylinositol‐specific phospholipase C (PI‐PLC) from Streptomyces antibioticus (hereafter referred to as SaPLC). By monitoring fluorescence quenching using both iodide and acrylamide—in the presence and absence of metal and/or inositol—we were able to deduce that one or more tryptophan residues became solvent exposed when either ligand was present. We then performed site directed mutagenesis studies and systematically changed each tryptophan residue to phenylalanine to understand which tryptophan amino acids are involved in the structural transition. These conformation changes were corroborated using 15N‐HSQC NMR studies in an attempt to understand the global conformational changes associated with substrate and cofactor binding. Based on the recently solved x‐ray crystal structure completed by our lab, and this study, we propose that SaPLC undergoes significant active site rearrangement during substrate binding, which may help explain its novel stereochemical mechanism. Future studies include additional NMR experiments and fluorescent studies using terbium as an acceptor for the tryptophan fluorescence emission to better understand these active site conformational changes.
Characterization of two synthetic ligands of peroxisome proliferator‐activated receptor γ (PPARγ) by cofactor recruitment, site‐directed mutagenesis and structure analysis
Authors: Crestani Maurizio , Mitro Nico , Gilardi Federica , Scotti Elena , Pochetti Giorgio , Lavecchia Antonio , Novellino Ettore , Fracchiolla Giuseppe , Laghezza Antonio , Tortorella Paolo , Aschi Massimiliano , Mazza Fernando , Montanari Roberta , Loiodice Fulvio
Published: June 2021

In this study we report the behavior of two PPAR enantiomeric ligands (R‐1 and S‐1). Cell‐based reporter assays indicate that both enantiomers are dual PPARα/γ ligands, being R‐1 a full agonist of PPARα and γ and S‐1 a partial agonist of PPARγ. 3D‐structure analysis of the PPARγ ligand binding domain in the complex with the two ligands shows that the suboptimal conformation of helix 12 in the PPARγ/S‐1 complex is the consequence of a steric hindrance between the ethyl group of the ligand and the Q286 in helix 3. Site‐directed mutagenesis confirms that Q286 is a key residue in determining the activity of different ligands. By using FRET assays, we found that the coactivators SRC‐1, PGC‐1α, RIP140, TIF‐2 are recruited by R‐1, S‐1 and rosiglitazone with similar EC50, whereas CBP affinity is higher in the presence of rosiglitazone. Conversely, only S‐1 allows the association of the corepressor N‐CoR to PPARγ as opposed to rosiglitazone and R‐1, providing a functional explanation to the partial agonist behavior of S‐1. ChIP assays confirmed that S‐1 enhances the association of N‐CoR to PPARγ target promoters in the native chromatin context, whereas rosiglitazone and R‐1 recruit less N‐CoR. Altogether, our results provide a rationale for the distinct behavior of different PPAR ligands.

Supported by CARIPLO Foundation, Harvard‐Armenise Foundation
Thioflavin S inhibits hepatitis C virus RNA replication and the viral helicase with a novel mechanism
Authors: Frick David N , Belon Craig A
Published: June 2021

The NCI mechanistic diversity set (827 compounds) was screened a with a molecular beacon helicase assay to identify inhibitors of the helicase encoded by the hepatitis C virus (HCV). All of the small molecule inhibitors identified also interacted with the DNA substrate in a direct binding assay except one, Thioflavin S. When administered to cells that have been stably transfected with a subgenomic HCV replicon Thioflavin S was not toxic, and it decreased the amount of positive sense HCV RNA by 21%. Thioflavin S was more effective when it was administered with RNA upon transfection, decreasing both positive and negative sense RNA by 42% and 85% respectively in 6 days when present at 50 μM. These decreases in HCV RNA after transfection were concentration dependent and similar to those seen when interferon was administered in the same manner to the same cells. Thioflavin S inhibited NS3 catalyzed DNA unwinding with an IC50 of 13 μM, ATP hydrolysis with and IC50 of 39 μM, and peptide cleavage by 40% when present at 15 μM, but unlike other well‐characterized helicase inhibitors, the compound did not compete with either RNA or ATP for known binding sites. The compound inhibits the NS3 protein from various HCV genotypes, Japanese encephalitis virus, and Dengue Virus, but has no discernable effects on related proteins. This study was supported by NIH grants AI052395, MH085690, & AI088001.
Studying the Efficacy of Antidotes for Metabolic Cyanide Poisoning
Authors: Harrison M. , Haines D. C. , Petrikovics I.
Published: June 2021

Cyanide is found in a variety of environmental and industrial sources. Cyanide inhibits mitochondrial cyctochrome c oxidase, the terminal electron acceptor in the electron transport chain, resulting in decreased oxidative metabolism and decreased oxygen utilization. The goal of our study was to test novel prophylactic and therapeutic treatments for their ability to protect against cyanide toxicity. Mice were treated with cyanide and various antidotes, and then brain mitochondria were isolated and the cytochrome c oxidase activity in the brain mitochondria determined. The antidotes tested were sulfur donors including dimethyltrisulfide, thiotaurate, and thiosulfate administered with exogenous rhodonase, an enzyme that uses sulfur donors to convert cyanide to much less toxic thiocyanate. Sodium nitrite, which oxidizes hemoglobin to methemoglobin which can sequester the cyanide, was also administered in the presence and absence of the novel therapeutics. The cytochrome c oxidase activity data complemented survival data in the determination of antidote efficacy.
Primary cilia regulates endothelial cell directional migration through HSP‐27 dependent modulation of actin cytoskeleton and focal adhesions
Authors: Jones Thomas Joseph , Adapala Ravi K , Bursley Christopher , Nauli Surya M , Thodeti Charles K
Published: June 2021

Cilia are mechano/chemo‐sensing organelles that communicate extracellular mechanical signals to intracellular responses. Altered function of primary cilia plays a key role in the development of various diseases including polycystic kidney disease. Here, we show that endothelial cells from the oak ridge polycystic kidney (ORPK) mouse with impaired assembly of cilia, exhibits a reduced formation of actin stress fibers and focal adhesions compared to wild type endothelial cells. Further, these cells showed decreased directional migration in scratch‐wound assays. Interestingly, we found that expression of heat shock protein‐27 (hsp‐27), which is required for the organization of the actin cytoskeleton, is down regulated in these cells. However, the phosphorylation of focal adhesion kinase (FAK), a known downstream effector of hsp‐27, is unchanged. Taken together, these results demonstrate that disruption of primary cilia formation compromises endothelial cell directional migration through the suppression of hsp‐27 dependant F‐actin organization and focal adhesion formation which may lead to vascular dysfunction in ciliopathies.
Pheromone‐ and Rsp5‐Dependent Ubiquitinaton of G Beta Subunit Ste4 in Yeast
Authors: Zhu Ming , Torres Mathew , Dohlman Henrik , Wang Yuqi
Published: June 2021

Ste4 is the beta subunit of the heterotrimeric G proteins that mediates mating signaling in budding yeast Saccharomyces cerevisiae. In this study, we show that Ste4 undergoes ubiquitination and this was largely promoted by mating pheromone. Ubiquitination of Ste4 requires a loop region, composed of the residues from 310 to 346 and critical for Ste4 phosphorylation. Interestingly, Ste4 phosphorylation‐defective mutants fail to undergo ubiquitination in response to pheromone stimulation. We found that the residue K340 within the loop region serves as a primary ubiquitination site, since Ste4K340R mutant significantly diminishes its ubiquitination in response to pheromone stimulation. In addition, our study also identified Rsp5 as an E3‐ligase mediating Ste4 ubiquitination. Ste4 ubiquitination is completely blocked in Rsp5‐disruptive mutant. Rsp5 physically interacts with Ste4 in vivo and is capable of catalyzing Ste4 ubiquitination in vitro. Altogether, our findings revealed the novel ubiquitination of the Gβ, Ste4, catalyzed by the E3 enzyme Rsp5 on K340. The ubiquitination‐defective mutants of Ste4 generated in this study would provide a powerful and specific tool for examing the functional consequences of Ste4 ubiquitination on mating signaling.
Structure and Function of a Novel Bi‐Functional Xylanase‐Esterase
Authors: Bae Brian , Dodd Dylan , Mackie Roderick , Cann Isaac , Nair Satish
Published: June 2021

While plant cell wall material is an abundant renewable polymer and has great potential for bio‐fuel production, it is very recalcitrant to hydrolysis due to insolubility and heterogeneity. As a result, invention and optimization of enzymatic processes that process cellulosic biomass to produce monomeric sugar as a fermentable source has been a global focus. Here, we show structural and functional characterization of a novel bi‐functional enzyme that was identified from the rumen of herbivores. This enzyme is unique because it is shown to harbor high xylanase and ferulic acid esterase (FAE) activity within a single polypeptide. The determined crystal structure of the apo‐enzyme and binary complex structure of protein and synthetic substrate provide the basis for rational design and engineering of munti‐functional plant cell wall enzymes. The structures reveal an (á/â)8‐barrel xylanase domain and á/â hydrolase fold ferulic acid esterase domain, linked by carbohydrate binding module (CBM) ‐like folded linker region. Based on the sequence alignment and the conformation of active site, we can postulate a mechanism for plant cell wall hydrolysis.
ELEMENTAL IMAGING OF HUMAN EMBRYONIC STEM CELLS
Authors: Finney Lydia , Wolford Janet , Jin Qiaoling , Chishti Yasmin , Ward Jesse , Vogt Stefan , Chen Liaohai
Published: June 2021

A critical aspect of understanding human embryonic stem cell (hESC) differentiation is regulation of protein activity. After all, it is by selectively activating and inhibiting various proteins that cells are able to proliferate and differentiate. One way Nature achieves this is by changing the availability of the metal ions that activate or inhibit existing proteins. An estimated one third of all proteins bind metal ions which play numerous critical cellular roles including as cofactors for enzymatic reactions, structural nuclei for protein folding motifs, and various signaling pathways.

We are elucidating the role of metals in the dynamic cellular process of hESC differentiation at a systems level and developing analytical tools applicable to any cellular system in which metal ions play such roles. Combining existing molecular and cell biology approaches with cutting‐edge x‐ray fluorescent capabilities at the APS, we are currently investigating dynamics in metal ion distribution across stem cell colonies upon induction of differentiation, seeking a paradigm for the role of metal ions in developmental biology. We find that cellular zinc levels may serve as a biomarker of differentiation, indirectly correlating with the expression of pluripotency markers.
Antioxidative Effects of Native Berries, Empetrum nigrum var. japonicum K. Koch and Rubus buergeri Miq., of Jeju Korea
Authors: Lee Sunjoo , Park Shin Young , Lee Eun Suk , Han Sang Heon , Lee Hyo Yeon , Baek Seungjun , Bu Eunju
Published: June 2021

Native berries of Jeju in Korea, Empetrum nigrum var. japonicum and Rubus buergeri Miq., were extracted each fruit or leaves with stem in 80% ethanol to investigate the antioxidant activities. Total contents of flavonoids in leaves and fruit of Empetrum nigrum were 7.33 ± 0.02 mg and 0.44 ± 0.01 mg in 1 g of each plant part, respectively. Total amounts of phenolic compounds in leaves were higher than the fruit for both berries. In 1,1‐diphenyl‐2‐pycryl‐hydrazyl (DPPH) free radical scavenging and superoxide dismutase(SOD)‐like enzyme assay, all extracts showed strong antioxidative effects. Protective effects against H2O2‐induced cow pulmonary artery endothelium(CPAE) cell cytotoxicity were analyzed. CPAE viability was greatly reduced and the survival rate was about 37% when the cells were treated with 1mM H2O2 for 24h. The viability of these H2O2‐treated cells was dramatically improved by preincubation with leaf extracts of both berries. The extracts also inhibited lipid peroxidation in human LDL, measured as the reduction in malondialdehyde(MDA) production. When human LDL was incubated with 100 μg/mL leaf extracts of winter strawberry or crowberry, the value was 1 μM and 1.63 μM of MDA, respectively. This is about 26 times lower than the untreated sample (27.5 μM). Results suggested that Korean native species E. nigrum could be used as a functional material for drinks because of its property of natural antioxidant.
If you can't be with the one you love – Can you make a model of it?
Authors: Dunbrack Roland
Published: June 2021

One of the many techniques used in the Protein Structure Initiative to obtain structures of target proteins has been to express a large number of orthologues and attempt crystallization on all of them. Thus, it is often the case that the PSI may solve the structure of a protein that is a relative of the one under study in the laboratory. At the Fox Chase Cancer Center, we have developed and used methods for protein structure prediction on large number of targets under study by investigators. The purposes of molecular modeling are highly varied – from understanding sequence/structure/function relationships to ligand docking for lead drug design. Often, molecular modeling is used to design specific mutations to disrupt particular protein‐protein interactions while leaving others intact. We have developed databases and software to model proteins in biologically relevant states, including homooligomers and complexes of different proteins. This requires the ability to accurately identify biologically relevant states within protein crystals. We have found that the public databases of “biological units” are inconsistent, and that comparison of protein‐protein interfaces in all available template structure crystals is often required for identifying the best template oligomeric structure. This work was funded by the NIH, grants P20 GM76222 (PSI2) and R01 GM73784.
Targeting protein‐protein interaction networks: Structural genomics of FeS cluster assembly and human cancer biology
Authors: Montelione Gaetano T
Published: June 2021

The NESG is one of four Large‐Scale Centers of the NIGMS Protein Structure Initiative (PSI). A key goal of the PSI is to generate 3D structures for some 5,000 proteins selected using broad biological, genomic, and bioinformatics criteria, together with targets selected from specific biological theme projects, so as to provide significant structural coverage of a large number of protein sequences in nature. The PSI also develops new methods for protein structure analysis, and proactively disseminates these technologies to the broader biological community. Our efforts span three classes of proteins: (i) selected to provide course‐grained coverage of large protein domain families; (ii) nominated in collaborations with the biomedical research community; and (iii) NESG Biomedical Theme of ‘Networks of Proteins Associated with Human Cancer and Developmental Biology’. NESG technology development emphasizes: (i) new technologies for production of proteins, (ii) new methods for protein NMR; (iii) hybrid approaches exploiting synergies of crystallography, SAX, NMR, and computational prediction, and (iv) improved methods for crystallizing proteins. This technology development is driven by our Biomedical Theme focus on human proteins, including protein complexes associated with cancer biology. The many structures, reagents, methods and technologies developed in this project provide starting points for traditional hypothesis‐driven biological research, and provide powerful, broad‐impact infrastructure for biological science and engineering.

Supported by Natl. Inst. of General Medical Sciences
In vitro digestion/Caco‐2 cell model for assessing transport of dietary folate across the intestinal mucosa
Authors: Yarbaeva Shakhlo N. , Lee Ji‐Young , Albrecht Julie A
Published: June 2021

An in vitro digestion coupled to Caco‐2 cell culture model was used to assess and evaluate the permeability of dietary 5‐methyl‐tetrahydrofolate (5‐methyl‐THF) from pinto beans. Folate‐depleted and folate‐sufficient Caco‐2 cell models were used. Caco‐2 cell confluence and integrity of monolayers were validated with transepithelial electrical resistance measurements and red phenol exclusion assay. Samples were 5‐methyl‐THF, 5‐methyl‐THF+2.5% v/v legume extract, 5‐methyl‐THF+5% v/v legume extract, 2.5% and 5% v/v legume extract. Pinto bean extracts at 5% (v/v) were not detrimental to Caco‐2 cells. Permeability coefficients (Papp) were determined in the apical‐to‐basolateral direction. Transport experiments were performed under sink‐conditions. Cumulative fractions of transported 5‐methyl‐THF increased linearly within 80 min of incubation for both folate‐depleted and folate‐sufficient cells. 5‐methyl‐THF was transported across the intestinal mucosa with Papp of 1.03–1.79x10−5cm/s for samples in folate‐depleted and with Papp of 0.99–1.32x10−5cm/s for samples in folate‐sufficient cells. This study demonstrates that 5‐methyl‐THF is bioaccessible from bean extracts in Caco‐2 cells. Experimental conditions for use of in vitro digestion/Caco‐2 cell culture model to assess intestinal transport of 5‐methyl‐THF from beans were established.
Localization of Retinoic Acid Receptor RARα in Adult Mouse Hippocampal Dentate Gyrus Neurons
Authors: Chen Wei‐Ping , Libien Jenny
Published: June 2021

Retinoids (Vitamin A) play an important role in differentiation and patterning in the developing nervous system. Retinoids bind to specific nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), in order to mediate cellular effects. Three subtypes of RAR receptor, RARα, β and γ, have been identified. Evidence has emerged that retinoid signaling required for synaptic plasticity, learning and memory behaviors, and for adult neurogenesis. In this study, we investigated the expression pattern of RARα in adult mouse hippocampus. We hypothesized that the receptor may have a developmental‐dependent expression in the new neurons of the dentate gyrus. Co‐labeling experiments were performed with markers for immature and mature neurons (Doublecortin, Calretinin, NeuN) and for astrocytes/stem cells (nestin, GFAP). We found that RARα expression was located at several different areas in the mouse hippocampus, CA1‐CA3, the granule cell layer, hillus and granule cell layer of DG. RARα immunohistochemical stain was clearly visualized in cell cytoplasm, but not in nucleus. There was colocalization of RARα in DG with NeuN, but not with astrocyte/stem cell markers GFAP or Nestin. RARα immunohistochemical stain was not colocalized with immature granule cell markers doublecortin and calretinin. Our results provide the first evidence for identify RARα localized only in mature granular neurons of adult mouse dentate gyrus.
The Impact of d‐δ‐Tocotrienol and Geranylgeraniol on Cell Cycle Progression and Apoptosis in Human and Murine Melanoma Cells
Authors: Fernandes Nicolle V , Katuru Rajasekhar , Dutta Dibyendu , Mills Nathaniel , Mo Huanbiao
Published: June 2021

The mevalonate pathway provides essential intermediates for the prenylation or dolichylation of growth‐related proteins including nuclear lamins, Ras and growth factor receptors. d‐δ‐Tocotrienol, a vitamin E isomer with farnesyl side chain, and geranylgeraniol, a diterpene, are suppressors of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase, the rate‐limiting activity of the mevalonate pathway. We evaluated the impact of d‐δ‐tocotrienol and geranylgeraniol on cell cycle progression and apoptosis in human A375 and A2058 melanoma cells and murine B16 melanoma cells. d‐δ‐Tocotrienol (11–38 μmol/L) and geranylgeraniol (30–90 μmol/L) induced concentration‐dependent cell cycle arrest at the G1 phase and apoptosis as detected by flow cytometry and fluorescence microscopy; the impacts were accompanied by decreased levels of cyclin‐dependent kinase 4 and cyclin D1 proteins and the activation of caspases‐3. d‐δ‐Tocotrienol also suppressed the level of Ras protein in A375 and A2058 cells. Blends of d‐δ‐tocotrienol and geranylgeraniol suppressed the growth of murine B16 melanoma cells to greater extents than the sum of individual impacts, suggesting a synergistic effect. The tocotrienols, geranylgeraniol and other mevalonate suppressors may have potential application in melanoma prevention and therapy. TDA & TWU REP.
Multiple retinol dehydrogenases (short‐chain dehydrogenases) and retinal dehydrogenases synthesize retinoic acid in hippocampus astrocytes
Authors: Wang Chao , Napoli Joseph
Published: June 2021

Little direct evidence identifies hippocampus cell types and enzymes that biosynthesize all‐trans retinoic acid (atRA). We show that primary rat astrocytes, but not neurons, biosynthesize atRA using retinol dehydrogenases (RDH) of the short‐chain dehydrogenase/reductase (SDR) gene family, and retinaldehyde dehydrogenases (RALDH). Astrocytes secrete atRA into their medium; neurons sequester atRA. Neurons and astrocytes both synthesize retinyl esters (RE). atRA autoregulates its concentrations by inducing RE synthesis (lecithin:retinol acyltransferase, LRAT) and stimulating its catabolism (cytochrome P450 CYP26B1). Knockdown by RNAi indicates that RDH10, RDH2, RALDH1, 2 and 3 contribute to atRA production. Knockdown of the SDR DHRS9 increases RA synthesis ~50% thru increasing RALDH1 expression. Culture‐time dependent expression of RALDH1 and 2, RDH10, DHRS9, LRAT and CYP26B1 correlate with atRA and RE synthesis, suggesting intrinsic adaption of retinoid homeostasis. These data show that adult hippocampus astrocytes rely on multiple RDHs and RALDHs to provide a paracrine source of atRA to neurons. Observation of cross talk between DHRS9 and RALDH1 provides novel insight into regulation of RA biosynthesis. Supported by NIH grant DK36870.
Cadmium downregulates ZnT‐4 and induces mammary gland involution in lactating mice
Authors: Tallkvist Jonas , Huang Liping , Öhrvik Helena
Published: June 2021

Numerous studies in various species have shown that cadmium (Cd) is retained in the lactating mammary gland (LMG) and is only poorly transferred to milk suggesting that the LMG may act as a barrier to protect the suckling offspring from exposure to Cd. The mechanism for the Cd retention is unknown but may involve interaction with metallothionein (MT) or members of the family of zinc transporters (ZnTs). The aim of the present study was to examine the morphology of the LMG and protein expressions of MT, ZnT‐2, ZnT‐4 or ZnT‐7 in Cd treated mice. A total of 12 primiparous NMRI dams with normalized litter sizes (n=8) were given 17.8 umol Cd/kg/day or vehicle on postnatal days (PND) 8, 9 and 10. Left inguinal mammary glands (4 and 5) were dissected on PND 11 for morphological examination, Zn‐analyses and immunoblotting. Livers were taken from the dams for MT westerns. The histopathology demonstrated increased fat content in the LMG of the Cd treated dams and a less active feature of the alveolar epithelium as compared to the controls, indicating a Cd induced involution. Total Zn‐levels in the LMG were unaffected by Cd. ZnT‐4 protein was significantly downregulated as a result of the Cd treatment whereas MT, ZnT‐2 and ZnT‐7expressions were unaffected. Liver MT was upregulated in the Cd treated dams. Our results indicate that Cd induces involution of the LMG and that ZnT‐4 is involved in this process.
Processing and matrix effects on the antioxidant capacity of fruit‐based beverages
Authors: Tadapaneni Ravi , Boettcher Kenneth , Patazca Eduardo , Edirisinghe Indika , Burton‐Freeman Britt
Published: June 2021

Fruits contain a variety of polyphenolic compounds with antioxidant properties. These compounds are associated with lower risk of chronic disease. Fruits are consumed as raw and in processed forms (e.g. beverages). The perishable nature of fruits/fruit‐based products demands processing strategies to enhance shelf‐life and assure food safety, while maintaining nutritional quality. Therefore, we proposed to develop a system to test standard and novel processing strategies on the antioxidant capacity (AC) of fruit‐based beverages containing appreciable amounts of polyphenolic compounds. In this study, strawberry (Str) formulated dairy‐(D) and non‐ dairy (ND) beverages were used to investigate the effect of high temperature‐short time (HTS‐72°C‐20 sec) and high pressure (HPP‐600 MPa‐1 min) processing on AC as measured by Oxygen Radical Scavenging Capacity (ORAC). Two dairy‐based beverages contained 0 or 10 g, freeze‐dried CA Str (D‐Ctrl, D‐Str), and 1 beverage contained Str (10 g) and water only (ND‐Str). HPP and HTS reduced ORAC of D‐Str and D‐Ctrl compared to their respective unprocessed beverages (D‐Str: 6.9±1.1 & 3.1±0.3 vs. 12.3±0.9 μMTE/mL and D‐Ctrl: 1.2±0.1 & 1.8±0.2 vs. 2.4±0.2 μMTE/mL, respectively, P<0.01). HPP did not affect ORAC of ND‐Str compared to unprocessed ND‐Str (11.1±1.0 vs. 10.6±1.1 μMTE/mL); however, HTS significantly reduced ORAC of ND‐Str (4.7±0.6 μMTE/mL) compared to unprocessed ND‐Str (P<0.01). These findings indicate a significant effect of processing and matrix on AC of fruit‐based beverages and support further work to determine the impact of these findings in in vivo systems.
Pantesin, an Active Derivative of Vitamin B5, Used as a Nutritional Supplement, Significantly Lowers Cardiovascular (CV) Disease Markers in Low to Intermediate CV Risk North American Subjects: A Triple‐Blind Placebo/Diet Controlled Study
Authors: Rumberger John Arthur , Evans Mal , Napolitano Joseph J , Azumano Isao , Kamiya Toshikazu
Published: June 2021

Serum cholesterol (C), LDL‐C, non‐HDL‐C, and apolipoprotein B (apo‐B) are well established risk markers for CV disease. The effect (and safety) of Pantesin (Daiichi Fine Chemicals) versus placebo on these risk markers was studied in 120 subjects (n=60/group) who were low to intermediate CV disease risk (NCEP ATP III) at screening. All subjects completed a 4 week diet lead‐in (TLC – Therapeutic Lifestyle Change) prior to randomization (baseline). Results for LDL‐C:

LDL‐C (mg/dL)
Screening
Baseline
4‐weeks
8‐weeks
16‐weeks

Placebo
115±26
108±25
112±28
114±29
115±27

Pantesin
112±24 (NS)
112±31 (NS)
104±27*
104±28*
108±30*

= p≤0.005

NS = non‐significant; data are mean±SD

Conclusion

Pantesin supplementation for 16 weeks (600 mg/d for 8 weeks then 900 mg/d for 8 weeks) significantly lowered LDL‐C as well as apo‐B (p=0.010) and non‐HDL‐C (p<0.001) over and above the effect of TLC diet alone. Pantesin was well tolerated with no differences between groups in adverse events. These results are noteworthy as prior studies have shown each 1 mg/dL reduction in LDL‐C lowers CV risk by 1%.
Pcynogenol Inhibits Adipogenesis and Simulate Antilipogenic Effect in 3T3‐L1 Adipocytes
Authors: Yang Jiwon , Jang Ja‐Young , Nam Da‐Eun , Lee Jeongmin , Hwang Kwon‐Tack , Jun Woo‐Jin , Lee You‐Yeon
Published: June 2021

Pycnogenol(Pyc), standardized extracts from the bark of French maritime pine (Pinus pinaster Ait.), has a wild range of phamacological effects. To explore the possibility that Pyc might influence an adipocyte differentiation and lipolysis, this study investigated the effects of Pyc on the expression of adipokines and PPARγ, the effects of Pyc on the regulation of fatty acid metabolism, and the induction of apoptosis in the differentiated 3T3‐L1 cells. With Oil‐Red O staining, treatment of Pyc at concentration of 10–200 μg/ml significantly inhibited the cytoplasmic lipid accumulation and glucose uptake at day 9 after induction of differentiation. In addition, we measured the protein expression of adipokines and PPARγ during differentiation. As result, Pyc suppresses the adipogenesis at post stage during differentiation by inhibiting PPARγ and adiponectin expression, but not leptin. We demonstrated that Pyc blocks adipogenesis by suppression on the expression of adipogenic transcription factors and adipokines. Anti‐lipogenic effects of Pyc on differentiated 3T3‐L1 adipocytes were investigated. Pyc inhibited the cytoplasmic lipid accumulation and has synergistically increased the glucose uptake by 32.6% in the differentiated 3T3‐L1. Pyc increased glycerol release from differentiated 3T3‐L1 and also up‐regulated LPL and HSL mRNA expression to degrade cellular fatty acid. With 72hr treatment, Pyc induced the apoptosis by modulating Bax/Bcl‐2 ratio. Apoptosis may, at least in part, occur through caspase‐3 signaling pathway.
Zinc increased the sensitivity of HepG2 and AML12 cells towards retinoic acid‐mediated growth inhibition
Authors: Ibbitson Deanna , Xu Zhaoming
Published: June 2021

Retinoic acid has been shown to reduce cell growth by interrupting cell cycle. This effect of retinoic acid involves retinoic acid receptor and retinoid X receptor, two zinc‐finger proteins. We hypothesized that the sensitivity of cells towards retinoic acid‐mediated growth inhibition can be modulated by zinc status. To test this hypothesis, hepatoma HepG2 cells were cultured in a low‐zinc media supplemented with 0 (low‐zinc group), 5 (adequate‐zinc group), and 10 (zinc‐supplemented group) μM of zinc followed by treating the cells with retinoic acid at 0 and 35 μM. Culturing the cells in the low‐zinc media depleted the labile intracellular pool of zinc by 86% while the total cellular zinc concentration was reduced by 28%. Treating the cells with retinoic acid reduced cell proliferation in the low‐zinc, adequate‐zinc, and zinc‐supplemented group by 36, 49, and 55%, respectively. Cell cycle analysis showed that retinoic acid treatment reduced the number of cells in the S‐phase in the adequate‐zinc and zinc‐supplemented groups by 27% compared to the low‐zinc group. Similar effects were obtained in normal hepatocyte AML12 cells using the same treatment regime. Collectively, these results suggested that zinc supplementation sensitized HepG2 and AML12 cells towards retinoic acid‐mediated growth inhibition. Supported by the Vitamin Research Fund.
Identification of Peptides Derived from Azuki Bean (Vigna angularis) which Stimulate Cholecystokinin Secretion from the Enteroendocrine STC‐1 Cells
Authors: Sufian Md. Kaosar Niaz Bin , Hira Tohru , Hara Hiroshi
Published: June 2021

Objectives

We found appetite‐suppressive peptides through cholecystokinin (CCK) secretion in vivo from a peptic digest of soybean β‐conglycinin. Later we found that peptic digest of dolicholin, a newly identified protein in country beans (Dolichos lablab), similar to β‐conglycinin, more potently stimulate CCK secretion from enteroendocrine cells. Our present study aimed to screen new CCK‐releasing peptides from beans that exist with homologous protein to dolicholin and to identify the active peptides therein.

Methods

Five kinds of beans were hydrolyzed with pepsin and were examined for CCK‐releasing activities in the enteroendocrine cell line STC‐1.

Results

Peptones prepared from azuki bean (ABP), mung bean (Vigna radiata) and country bean containing closely homologous protein were potent to stimulate CCK secretion in the STC‐1 cells, with the highest activity by ABP. Acetonitrile soluble peptides in ABP stimulated CCK secretion, and pronase treatment abolished its activity. Average molecular size of ABP was estimated to be ~3 kDa, and fractions containing 3–5 kDa peptides showed highest CCK‐releasing activities.

Conclusions

These results indicate that peptides in ABP are potent to stimulate CCK secretion and also suggest that the CCK‐releasing peptides are derived from protein similar to dolicholin.

(This research was supported by Japan Society for the Promotion of Science).
Postexercise appetite and hunger do not alter in parallel
Authors: Morishita Ai , Yamada Masako , Endo Masako Y , Miura Akira , Fukuba Yoshiyuki
Published: June 2021

It is generally recognized that moderate exercise induces to increase appetite/hunger in parallel. However, it is not still clear the effect of exercise intensity on these relationships. Therefore, the purpose of this study was to examine as to whether the exercise‐induced alternation of appetite/huger depend on exercise intensity. 12 healthy young male subjects performed cycling exercise at constant work rate (% of VO2max) of 30 % for 20 min (L‐ex), 50 % for 14 min (M‐ex), or 70 % for 8 min (H‐ex), in random order. The total amounts of three works were matched. The subjects were asked the feeling of appetite and hunger at prior‐ and post‐exercise (at 1 and 20‐min) using visual analog scale. The salivary flow and its alpha‐amylase activity were also measured. In H‐ex, appetite and hunger were suppressed immediately after exercise and returned to prior‐level at 20‐min. In L‐ex, both were unaffected by exercise and increased at 20 min. In M‐ex, however, the appetite for 20 min after exercise was close to those in L‐ex, while the hunger did show a similar change to those in not L‐ex, but H‐ex. Salivary flow significantly decreased after exercise in H‐ex. The results indicate that, for the intensity of prior‐exercise, there seem to be two different affected thresholds to appetite and hunger during the following short‐term recovery; i.e., below M‐ex for hunger, but above M‐ex for appetite. (Support: JSPS‐KAKENHI, 21500689 to AM)
Development and validation of a behavioral acclimation protocol for cats to respiration chambers used for indirect calorimetry studies
Authors: Shoveller Anna Kate , Duncan Ian J.H. , Atkinson Jim L. , Gooding Margaret A.
Published: June 2021

Cats exposed to stressful and novel environments initiate stress responses as evidenced by behavioural and physiological changes which, in turn, modify energy requirement and metabolism leading to the collection of unreliable results from nutritional trials conducted on stressed cats. 14 cats (10 ± 2 months) were subjected to an 11 week acclimation procedure to adapt cats to restriction within a chamber used for calorimetry studies. Cats were acclimated to the chambers on weeks 1–3, the study room on weeks 4–6 and increasing restriction periods of 1 to 20 hrs within the chamber on weeks 7–11. Ten additional cats (11 ± 1 month), used as controls, were subjected to 5 hrs of restriction without prior chamber or study room exposure to compare responses. Stress level, feed intake, fearfulness and eliminations were recorded. Cat‐Stress‐Scores (CSS) and latencies to approach a novel object peaked on weeks 4 and 7 (P<0.05). CSS declined with exposure to a novel experience and on week 11, stress levels were low and consistent (P<0.05). CSS was greater in un‐acclimated as compared to acclimated cats (P<0.05). In conclusion, acclimation protocols prepare cats for repeated, temporary restriction, whereas short acclimations do not. A step up acclimation procedure with behavioral indices of stress should be utilized to prepare cats for research that necessitates restriction. Supported by: The Procter and Gamble Co., Pet Care, Ohio, USA.
Mice bearing a targeted mutation of nBmp2 display decreased memory capabilities
Authors: Cordner Ryan Daniel , Ventura Julia S. , Blickenstaff Jacob W. , Walther Curtis K. , Mayo Jaime L. , Felin Jenny E. , Andreasen Brandalyn , Wallace Nicholas B. , Barrow Jeffrey R. , Edwards Jeffrey G. , Capecchi Mario R. , Bridgewater Laura C.
Published: June 2021

Bone morphogenetic protein 2 (Bmp2) is a growth factor involved in many important developmental processes including cartilage development, induction of osteoblast differentiation, and neurogenesis. Recently our lab discovered a nuclear form of Bmp2 (nBmp2). In an effort to determine the function of this nuclear variant, mice were generated which bear a targeted mutation that inactivates the nuclear localization of nBmp2 while still allowing the normal production of secreted Bmp2. Previous studies have shown that these mice have decreased muscle strength due to a calcium handling defect in the skeletal muscle. Our present study was intended to determine whether neurological function might also be affected by mutation of nBmp2. Age matched mutant and wild type mice were subjected to two different types of behavioral tests, the Morris water maze and the novel object recognition test. In both of these tests, the mutant mice displayed significantly impaired memory capabilities compared to the wild type mice. Synaptic plasticity of hippocampal slices is currently being tested by investigating long‐term potentiation in mutant and wild type mice. Our data suggest that in addition to intracellular calcium transport in muscle, nBmp2 also plays an important role in the processes of memory and learning, perhaps by affecting calcium transport in neural tissue. Supported by NIH grant AR48839.
Effect of natural elicitors on saponin content of soybean varieties
Authors: Smith James D , Salyer Joseph , Eswaranandam Satchithanandam , Gray Caroline P , Shakiba Ehsanollah , Chen Pengyin , Lee Sun‐Ok
Published: June 2021

Soy saponins (ssp) have shown to possess the health‐promoting properties. Finding soy varieties with high ssp content and increasing ssp content in a variety will increase their beneficial effects. The objective of this study is to find the effect of elicitors sprayed on Ozark, R95‐1705, and UA4805 on ssp content. Elicitors, salicylic acid (SA), ethyl acetate (EA), and methyl salicylate (MS), were sprayed at 10−3 and 10−1 M concentrations. Seeds were ground (60 mesh), defatted, and ssp was extracted. Variety had significant effect on ssp I, II, III, αg, βg, and βa of soybeans (P<0.0001). Ozark had higher total ssp content than UA4805 and R95‐1705 (4.2, 2.8, and 2.8 μmol/g soybean, respectively). Elicitor and variety had significant interaction on ssp II content of soybeans (P=0.0190). Elicitor had significant effect on total and ssp III contents (P=0.0058 and 0.035, respectively) of soybeans. Ozark had higher βg and βa contents than R95‐1705, followed by UA4805 (βg 2.70, 1.90, and 0.90; βa 0.96, 0.46, and 0.17 μmol/g soybean, respectively). The findings of this study will provide valuable data on the application of natural elicitors to increase ssp content. Higher content of ssp in soybean will improve health beneficial effects. Funding provided by Arkansas Biosciences Institute is greatly appreciated.