bluuebunny/crossref_metadata_2025 huggingface dataset:
Examining the Influence of the Sulfur Assimilation Pathway on the Regulation of Yap1 Activity in Saccharomyces cerevisiae
Authors: Brown Haley A. , West James D
Published: June 2021

Multiple transcription factors confer resistance to the damage caused by reactive oxidants and electrophiles. In S. cerevisiae, these factors include Yap1, a regulator of detoxification genes, and Met4, a regulator of genes involved in sulfur amino acid metabolism. While Met4‐dependent transcriptional activity can be regulated inducibly in yeast, it interacts with other DNA binding proteins, including Cbf1, to enhance expression of genes that influence intracellular levels of cysteine and methionine. Yeast mutants that lack cbf1 and yap1 exhibit decreased viability after treatment with hydrogen peroxide when compared with a wild‐type strain. In addition, activity of a Yap1‐regulated reporter gene is elevated in cbf1 deletion mutants in the presence and absence of hydrogen peroxide and the electrophile, N‐ethylmaleimide, in comparison to wild‐type. However, the effective concentrations for activating the Yap1‐regulated reporter are similar between wild‐type and cbf1‐deficient yeast. Our results suggest that Yap1 activity is potentially elevated in yeast strains lacking other transcription factors that confer resistance to reactive molecules.
Engineering Human Paraoxonase‐1 Double Mutants to Hydrolyze Organophosphorus Compounds
Authors: Norris Joseph , Chung Myra , Smith J. Richard , Kirby Stephen
Published: June 2021

Variants of human paraoxonase‐1 (PON1) are being developed as catalytic bioscavengers for the chemical warfare agents tabun, soman, and VX. It is preferable that the new PON1 variants have broad spectrum OP hydrolase activities to hydrolyze both G‐ and V‐class agents. H115W PON1 has shown improvements over wild‐type PON1 in its capacity to hydrolyze some OPs. We improved upon these activities by either substituting a tryptophan (F347W) near the active site for enhanced substrate binding, or reducing a bulky group (Y71A) near the putative active site. When compared to H115W alone, we found that H115W/Y71A and H115W/F347W maintained VX catalytic efficiency but showed mixed results for the capacity to hydrolyze paraoxon. Testing our double mutants against racemic soman, we observed a reduced KM for H115W/F347W, which enhanced catalytic efficiency over wild‐type 10‐fold. Contrary to previous reports, H115W could hydrolyze soman, but the double mutant H115W/F347W was better. We observed hydrolysis of tabun with wild type and mutant PON1s. To our knowledge, this is the first demonstration of tabun hydrolysis with PON1. These data demonstrate enhancements made in PON1 for the purpose of developing an improved catalytic bioscavenger against chemical warfare agents.
Insights into the Function of the α‐Helical Tail of Haemophilus influenzae 3‐Deoxy‐D‐manno‐Octulosonate 8‐Phosphate Phosphatase
Authors: Hanuszkiewicz Anna Dorota , Briski Laurence , Woodard Ronald
Published: June 2021

The cell envelope of Gram‐negative bacteria contains, in addition to the inner membrane and the peptidoglycan layer, a bilayered and asymmetrically organized outer membrane. The most predominant component of the outer leaflet of the outer membrane is lipopolysaccharide (LPS). LPS is an amphiphilic molecule composed of a hydrophilic heteropolysaccharide and the membrane‐embedded lipid A. The innermost 8‐carbon sugar 3‐deoxy‐D‐manno‐oct‐2‐ulosonic acid (KDO) is the only conserved structural element found in all LPS investigated to date, linking the lipid A to the carbohydrate domain of the molecule. Inhibition of KDO biosynthesis is lethal to most bacteria, making this pathway an ideal target for the development of novel antimicrobials. The KDO 8‐phosphate phosphatase (KdsC) is one of the enzymes of the KDO biosynthesis pathway and is responsible for cleavage of the phosphate group from KDO 8‐phosphate to yield KDO. In Escherichia coli, KdsC has been characterized as a homotetrameric molecule with a random‐coiled C‐terminal tail in each subunit. This tail plays a potential role in the enzyme's catalytic cycle. KdsC of Haemophilus influenzae is also a tetramer containing an α‐helical tail. In order to investigate the role of the tails in the catalytic mechanism of H. influenzae KdsC, a truncated (tail‐less) H. influenzae KdsC derivative was constructed by removal of its last C‐terminal eighteen amino acids. Both the native and the truncated H. influenzae KdsC were cloned, overexpressed, purified to apparent homogeneity, and functionally characterized.
Engineering Platelet‐Activating Factor Acetylhydrolase as a Catalytic Bioscavenger for Organophosphorus Compounds
Authors: Kirby Stephen , Norris Joseph , Cerasoli Douglas , Bahnson Brian
Published: June 2021

Platelet‐activating factor acetylhydrolase (PAFAH) offers a unique platform on which to engineer a catalytic bioscavenger for organophosphorus compounds (OP). Liposome‐associated enzymes, like PAFAH and PON1, circulate within the plasma and interact with OPs before cholinergic disruption. Wild type PAFAH and butyrylcholinesterase (BuChE) were tested for their ability to interact with the OPs tabun (GA), sarin (GB), and soman (GD). BuChE could be completely inhibited with 10 uM of each OP while PAFAH was resistant, and maintained at least 55% of original activity. We investigated W298, F322 and L153 within human PAFAH as sites for a secondary nucleophile to improve reactivation of the OP‐inhibited enzyme. Mutants screened were less reactive than wild type to the soluble substrates p‐nitrophenyl butyrate (PNPB) and 2‐thio PAF. We tested mutants against VX, VR, V2 (an analog of VX), and echothiophate, but found no appreciable catalytic power against these OPs. We mutated residue Q352 to G, N, and W to determine its contribution to binding soluble substrates. For the smaller substrate PNPB, all mutants had higher KMs, but catalytic efficiencies were similar to wild type. For 2‐thio PAF, Q352N had low activity, whereas Q352W increased the catalytic efficiency by 20‐fold over Q352G and 80‐fold over wild type by reducing KM. Spontaneous reactivation rates with a variety of OPs will be discussed.
Asn362 Contributes to Zinc Binding by Leukotriene A4 Hydrolase
Authors: Thompson Michael W , Schaible Alexis , Seipelt Rebecca L
Published: June 2021

Leukotrine A4 hydrolase (LTA4H) is a pro‐inflammatory zinc metallopeptidase/epoxide hydrolase that contains an atom of zinc at the active site. This zinc ion is bound to two histidine residues and a glutamate residue at the active site center. In order to more fully understand how catalysis occurs in the peptidase mechanism of the enzyme, conserved residues that were identified as possibly influencing zinc binding in the yeast version of the enzyme were altered by site‐directed mutagenesis to study their functions. One of these residues, Asn362, was mutated to Gln (N362Q), Glu (N362E), and Leu (N362L). While the N362E and N362L mutants exhibited minimal catalytic activity and reduced zinc affinity, the N362Q mutation exhibited only a slight reduction in catalytic activity and a slightly reduced zinc affinity. Taken together, these data indicate that Asn362 likely influences the chemical environment of the nearby zinc coordinating residue Glu363 through a possible hydrogen bond interaction. Trp356, situated at the edge of the active site cleft, appears to help maintain the conformation of the enzyme, but does not appear to contribute significantly to zinc binding. A better understanding of catalysis by LTA4H will help further the design of drugs to target the inflammatory process.
Role of the Reactive Center Loop of Protein Z Dependent Protease Inhibitor in Determining Specificity of the Protease Reaction
Authors: Yang Likui , Rezaie Alireza R.
Published: June 2021

Antithrombin (AT) and protein Z‐dependent protease inhibitor (ZPI) are two serpin inhibitors in plasma that contribute to regulation of the clotting cascade. Unlike AT, which can inhibit the proteolytic activity of all coagulation proteases, ZPI has a narrower specificity, thus being capable of inhibiting only factors Xa (fXa) and XIa. To investigate the contribution of residues of the reactive center loop (RCL) to restricting the specificity of ZPI, we engineered two AT mutants in which the P1‐Arg of the RCL was replaced with P1‐Tyr of ZPI (AT‐R393Y) in one construct and the P12‐P3’ residues were replaced with the corresponding residues of ZPI in the other. The mutants were characterized with respect to their ability to react with coagulation proteases, chymotrypsin and a fXa mutant containing a Ser substitution for Asp‐189 at its primary specificity pocket (D189S). The reactivity of AT‐R393Y with all coagulation proteases including fXa was dramatically impaired, however, the serpin mutant inhibited chymotrypsin with >four orders of magnitude higher specificity. On the other hand, the P12‐P3’ mutant of AT did not exhibit improved reactivity with chymotrypsin, but reacted with a normal reactivity with fXa, though the reaction was not enhanced by heparin cofactors. The fXa‐D189S mutant exibited similar poor reactivity with both AT and AT‐R393Y, however, the protease mutant exhibited normal or improved reactivity with ZPI and AT‐P12‐P3’. These results suggest that P1‐Tyr as well as other unique structural features within the ZPI RCL are responsible for restricting the protease specificity of the serpin.
Processing of the Ubiquitin‐like Autophagic Protein LC3 by the 20S Proteasome
Authors: Jiang Xuejun , Gao Zhonghua , Gammoh Noor , Wong Pui‐Mun , Erdjument‐Bromage Hediye , Tempst Paul
Published: June 2021

Ubiquitin‐proteasome system and autophagy are the two major mechanisms for protein degradation in eukaryotic cells. LC3, a ubiquitin‐like protein, plays an essential role in autophagy through its ability to be conjugated to phosphatidylethanolamine. In this study, we discovered a novel LC3‐processing activity, and biochemically purified the 20S proteasome as the responsible enzyme. Processing of LC3 by the 20S proteasome is ATP‐ and ubiquitin‐independent, and requires both the N‐terminal helices and the ubiquitin fold of LC3. The 20S proteasome processes LC3 in a stepwise manner, it first cleaves LC3 within its ubiquitin fold and thus disrupt the conjugation function of LC3; subsequently and especially at high concentrations of the proteasome, LC3 is completely degraded. Intriguingly, proteolysis of LC3 by the 20S proteasome can be inhibited by p62, an LC3‐binding protein that mediates autophagic degradation of polyubiquitin aggregates in cells. Therefore, our study implicates a potential mechanism underlying interplay between the proteasomal and autophagic pathways. This study also provides biochemical evidence suggesting cellular relevance of the controversial ubiquitin‐independent proteolytic activity of the 20S proteasome.
Yeast Kinome Screen Reveals a Novel G‐protein Kinase
Authors: Torres Matthew Phillip , Westrick Sarah Therese , Dohlman Henrik Gunnar
Published: June 2021

As mediators between cell surface receptors and intracellular effectors, G proteins are well positioned to serve as targets of post‐translational modification including phosphorylation. A recent phospho‐proteomic experiment identified a single phosphorylation site (S200) on the yeast G protein alpha subunit (Gα/Gpa1) in vivo (Li et al. 2007, Proteome Res). The objective of this study is to identify the responsible kinase and to determine its role in yeast G protein signaling. We discovered that phosphorylation at S200 results in an electrophoretic mobility shift in Gα/Gpa1, and we used this phenotype to screen over 100 different gene‐deletion strains representing the non‐essential subset of the yeast kinome. The results of this screen identified a single gene, ELM1, necessary for Gα/Gpa1 phosphorylation. Previous studies have shown that Elm1 phosphorylates proteins required for cytokinesis and adaptation to glucose‐starvation. We show that Elm1 is necessary for complete phosphorylation of Gα/Gpa1 in vivo. We have further established that Gα/Gpa1 is directly phosphorylated by Elm1 at multiple sites in addition to S200 in vitro. Finally, yeast lacking Elm1 or containing a catalytically inactive form of Elm1 exhibit significantly diminished responsiveness to pheromone stimulation. We conclude that Elm1 is a G protein alpha subunit kinase with an essential role in yeast G protein signaling.
Effects of Ziram on Humman Natural Killer Cell MAP Kinases and Cytolytic Proteins
Authors: Taylor Thyneice Rochelle , Whalen Margaret M.
Published: June 2021

Human natural killer cells (NK) are lymphocytes that can recognize and kill virally infected cells, antibody coated cells, and tumor cells without any prior sensitization. Ziram is used as a fungicide in agriculture and as an accelerating agent in the production of latex. Previous studies have shown that ziram inhibits the ability human NK cells to lyse tumor target cells. Mitogen activated proteins kinases (MAPK) are important signaling enzymes in NK cells. These signaling enzymes play a significant role in regulating cytolytic protein expression. Exposure of NK cells to another environmental contaminant, which also blocks NK lytic function, tributyltin (TBT), induced phosphorylation (activation) of MAPK. Thus, disturbances in MAPK activation state could contribute to TBT (and possibly ziram) –induced loss of lytic function. The current study examines whether ziram causes MAPK activation. The MAPKs, p44/42, p38 and JNK were examined. A 10 minute exposure to 10 μM or 5 μM ziram caused a significant increase in phosphorylation of p44/42 (3 fold and 2 fold, respectively). A 10 minute exposure to 10 μM, 5 μM, or 2.5 μM ziram caused a significant increase in phosphorylation of p38 (4 fold, 3 fold, and 2 fold, respectively. These results suggest that MAPK activation may be part of the mechanism by which ziram inhibits NK lytic function, and that MAPK activation may be an effect that is shared by several environmental contaminants that have been shown to decrease NK lytic function. Additionally, NK cell cytolytic protein expression is decreased after various ziram exposure time points.

Supported by NIH grant S06 GM008092‐33
Effects of breastfeeding peer counseling for obese women on infant health outcomes
Authors: Chapman Donna J. , Wetzel Katherine , Bermudez‐Millan Angela , Young Sara , Damio Grace , Pérez‐Escamilla Rafael
Published: June 2021

We evaluated the efficacy of a specialized peer counseling (PC) intervention promoting exclusive breastfeeding (BF) to obese low‐income women. During pregnancy 206 obese women considering BF were randomized to receive a specialized PC intervention (3 prenatal, daily perinatal, 11 postpartum (pp) visits) or standard care. At delivery 154 (76 intervention, 78 control) infants continued to meet inclusion criteria. Demographic, biomedical and infant feeding data were collected at birth, 2 weeks, and monthly through 6 months pp. Medical records were reviewed. Bivariate and logistic regression analyses identified predictors infant health outcomes. Logistic regression analyses adjusted for baseline group differences in age and delivery mode showed control group infants were 3.5 times more likely to be readmitted to the hospital by 3 months of age than those in the intervention group (OR = 3.46, 95% CI 1.01 − 11.89). Main reasons for hospitalization were respiratory infections and fever. These findings may be partly explained by better BF rates in the intervention (vs control) group at 2 weeks (93.1 vs. 83.8% (p=0.08)) and 2 months pp (67.7 vs 54.0% (p=0.11)). These preliminary findings suggest that BF peer counseling reduced the re‐hospitalization rate of infants born to obese, low‐income women.

Funded by Donaghue Medical Research Foundation and NIH/NCMHD grant P20MD001765
Peer counselors improve breastfeeding technique among low‐income, obese women
Authors: Morel Katherine , Chapman Donna J , Kyer Nan , Bermudez‐Millan Angela , Young Sara , Perez‐Escamilla Rafael
Published: June 2021

Data included in these analyses are from a larger randomized controlled trial (RCT) (N=206) evaluating a specialized (SP) peer counseling intervention targeting obese women (3 prenatal, daily hospital and 11 postpartum (pp) visits). The control group received standard (std) care including access to a std peer counselor (PC). These analyses include 45 women from the RCT (Intervention n=22, Control n=23). The objective was to evaluate the impact of a SP breastfeeding (BF) PC on BF technique. Two BF sessions were taped on days 2, 4, and 7 pp. Tapes were edited to remove identifying characteristics and rated independently by 3 Lactation Consultants using the Infant BF Assessment Tool (IBFAT) and Via Christi BF Assessment Tool (VC). T‐tests revealed no significant difference on days 2 or 4 pp. On day 7 pp better BF technique was observed in the intervention group with significantly higher VC scores vs. controls (7.5±1.0 vs. 5.6±3.3, respectively p=.026). IBFAT scores tended to be higher in the intervention vs. controls (11.5±1.2 vs. 9.4±4.3, p=.068, respectively). Maternal report of PC contact during the first week pp was significantly higher in the intervention group vs. controls (143.8±101.6 vs. 39.8±37.1 mins respectively, p=.001). These preliminary findings suggest specialized BF PC provided at a high intensity during the first week pp improved BF technique by day 7 pp.

Funded by CDC/ATPR and NIH/NCMHD Grant P20MD001765
Consumption of either one egg or lutein‐enriched egg per day increases HDL cholesterol, reduces apolipoprotein B while increasing plasma carotenoids and macular pigment density in adult subjects
Authors: Ata Shymaa , Barona Jacqueline , Kopec Rachel , Jones Jenifer , Calle Mariana , Schwartz Steven , Fernandez Maria Luz
Published: June 2021

In this study, subjects were assigned to consume either one egg (n =11), one egg substitute (n = 12) or one egg fortified with 600 μg of lutein (n = 11) per day. We measured plasma lipids, LDL subfractions and circulating carotenoids, macular pigment density (MPOD) by use of a densitometer, and hand carotenoids by use of a biophotonic scanner at baseline and after 12 wk. No changes in plasma LDL cholesterol or triglycerides were observed over time. However, both egg groups presented increases in HDL cholesterol compared to baseline (P < 0.01). All subjects had decreases in apolipoprotein B (P < 0.05) associated with a lower number of small LDL particles (P < 0.01). Independent of source, egg intake increased MPOD (P < 0.01) while hand carotenoids did not change over time for any of the groups. Plasma lutein concentrations increased by 9% (P < 0.01) and by 35% (P < 0.001) in the egg and lutein‐enriched egg groups, respectively. In this study we have demonstrated that MPOD increases whether subjects are consuming regular eggs or lutein enriched eggs. However, circulating lutein concentrations were associated with higher intake of this carotenoid, which may increase antioxidant protection in plasma. These studies demonstrate that consuming one egg per day may protect the eye against macular degeneration while increasing HDL cholesterol and favoring the formation of less atherogenic particles. [Supported by the Egg Board]
Cytoprotective activity of leaf crude extract of Eugenia uniflora L. and quercetin in insulin‐producing cells
Authors: de Oliveira Emilio Henriette Rosa , Braatz Simoní Marinéia , Felipe Elise Tatiane , de Oliveira Márcia Regina Paes , Kanunfre Carla Cristine , Carpinelli Angelo Rafael , Curi Rui
Published: June 2021

Eugenia uniflora L. is used in popular medicine as an alternative to the treatment of this disease because they are rich in phenolic compounds. In this study was evaluated the cytoprotective activity of leaf crude extracts and quercetin by HE stain and by MTT assay. Acute and chronic assay were performed through pre‐incubation of RINm5f cells for 1h or 24 h, and posterior induction of stress by H2O2 (150μM/2h). Acute essay showed a reduction in the viability of the control group of 33%. Cytoprotection was partially achieved with aqueous extract (50μg/mL) and quercetin (6.25 > 3.13 μg/mL). Chronic essay showed a reduction in the viability of the control group of 45%. The cytoprotection achieved with aqueous and alcoholic extract was about 16.5% and 18%, respectively. The cytoprotection achieved with quercetin was 12% (6.25μg/mL) and 25% (3.13μg/mL). Through HE stain was verified loss of intercellular adhesion and membrane and shape cell alterations. Aqueous extract showed partial prevention in acute essay. There was a more intense prevention of damage by 3.13 μg/mL quercetin. At chronic essay, aqueous extract (75μg/mL) and quercetin (3.13μg/mL and 6.25 μg/mL) partially prevented morfologic damage. These effects suggest that the tested compounds reduce oxidative stress, probably by their direct (acute essay) and indirect (chronic essay) antioxidant activity. Supported by Araucaria Foundation.
Antioxidative properties of dark chocolate and bloomed dark chocolate
Authors: Hong Mee Young , Nemoseck Tricia
Published: June 2021

An increasing body of epidemiologic evidence supports that polyphenol‐rich diets promote health and attenuate or delay the onset of various diseases, including cardiovascular disease (CVD) by maintaining antioxidant status. Cocoa and dark chocolates have among the highest antioxidant concentrations compared to other antioxidant food sources. In the present study, total polyphenol levels and antioxidant values were evaluated between regular and lightly bloomed dark chocolates. Lightly bloomed chocolates showed slightly lower total antioxidant capacity (422 μmoleTE/g vs 546 μmoleTE/g) determined by Oxygen Radical Absorbance Capacity and total polyphenolics (57.03 ± 1.55 mgGAE/g vs 63.59 ± 0.33 mgGAE/g) than regular chocolate. Both regular and lightly bloomed chocolate groups increased total serum antioxidant capacity than control (no chocolate) (P=0.0001). Chocolate groups increased activities of super oxide dismutase (P<0.0001) and catalase (P=0.0706). Compared to control group, dark chocolate, regardless if bloomed or not, reduced serum C‐reactive protein (CRP) by 33% (P<0.0001). These results suggest that dark chocolate bloomed or not, may be beneficial for improvement of the risk factors of CVD by elevation of serum antioxidant capacity, antioxidant enzyme activities and CRP values. Chocolates were provided by Ghirardelli (San Leandro, CA). Supported by CHNR08‐810 and SDSU NUTR 302L class.
Effect of manganese exposure and antioxidant therapy on oxidative stress in the rat brain
Authors: Fordahl Steven , Cooney Paula , Milatovic Dejan , Aschner Michael , Erikson Keith
Published: June 2021

Manganese (Mn) is an essential element implicated in the etiologies of metal induced neurological disorders. Over‐exposure to Mn results in its accumulation in iron‐rich brain regions leading to symptoms analogous to Parkinson's disease. Mechanisms governing this response are largely undefined though it is hypothesized that Mn‐induced oxidative stress may play a role. In this study we examined weanling rats exposed to Mn (1 g/L d.i. water) for total glutathione (GSH), glutathione peroxidase (GPx) and catalase mRNA, and F2‐isoprostane (F2‐IsoP) production in the caudate putamen (CP) and hippocampus (HC). N‐acetyl‐cysteine (NAC; 200 mg/kg) or (−)‐epigallocatechin‐3‐gallate (EGCG; 5mg/kg) injections were administered to observe any protective effect against Mn‐induced oxidative stress. Mn accumulated significantly versus control in each region (p < 0.05) in Mn‐exposed groups. This did not correspond to alterations in total GSH or production of F2‐IsoPs. EGCG was also associated with significant Mn accumulation in the CP (p < 0.05). Significant up‐regulation of GPx gene expression (p < 0.05) observed in the HC correlated with increased Mn levels, and was augmented by EGCG. These data suggest a biological response in antioxidant gene expression to Mn exposure, even in the absence of identifiable oxidative stress. Additionally, antioxidant therapy may enhance this response.
Antioxidant capacities of seven flavonoid compounds isolated from pulp of acai fruit (Euterpe oleracea)
Authors: Kang Jie , Wu Tong , Li Zhimin , Jensen Gitte S , Schauss Alexander G , Wu Xianli
Published: June 2021

The pulp of açai fruit (Euterpe oleracea Mart.) has been demonstrated to exhibit extremely high antioxidant capacity. Seven major flavonoids were isolated from freeze‐dried acai pulp by various chromatographic methods. Their structures were elucidated as orientin (1), homoorientin (2), vitexin (3), luteolin(4), chrysoeriol (5), quercetin (6), and dihydrokaempferol (7) by NMR, MS and compared to authentic standards. Antioxidant capacities of these flavonoids were evaluated by the following assays: oxygen radical absorbance capacity (ORAC); cell‐based antioxidant protection in erythrocyte (CAP‐e); ROS formation in polymorphonuclear granulocytes (ROS PMN). ORAC values varied widely (1,423.6 – 14,817.4 μmol TE/g) among the seven compounds based on numbers and positions of hydroxyl groups and/or substitute groups. The ORAC values of aglycones were generally higher than that of glycosides. CAP‐e results indicated that three compounds (4, 6, 7) could enter the cytosol and significantly reduce oxidative damage within living cells. ROS PMN assay showed four compounds (2, 3, 7, 6) had exceptional anti‐inflammatory properties in reducing ROS formation in PMN cells, even when high amounts of ROS were produced upon hydrogen peroxide administration. Supported by MonaVie LLC.
Fast‐food consumption among college students and their attitudes toward healthier fast‐food options
Authors: Haines Caitlyn , O'Neil Carol E , Zanovec Michael
Published: June 2021

Fast‐food meals often provide energy‐dense, nutrient‐poor food; however, attitudes and practices regarding fast‐food consumption among college students is understudied. To determine these, an online survey was developed, piloted tested (n=55), and sent to all students enrolled in the College of Agriculture (n=1,594). A total of 344 students (22% of total; 79% female, 88% white, 68% junior/senior), ages 18–30 y, completed the survey. Eighty‐four percent of students consumed fast‐food with 54% eating it at least once a week. Low cost and good taste were the main reasons students consumed fast‐food, while high cost and preference for other restaurants were main reasons not to consume it. Fast‐food was consumed most often at lunch (46.4%); hamburgers and fried chicken were the top choices; 54.7% of students spent $5–6 per meal. Seventy‐seven percent reported it likely that healthier meals would cost more and of those students, 88% would pay more for healthier meal options; however, few students would opt for choices such as low‐fat milk or salad. Data suggest that college students frequently consumed fast‐food, and would pay more for, but would not select, nutrient‐dense options.

Funding: N/A
Peroxynitrite and Hydrogen Peroxide Increase Arginase Activity through the RhoA/Rho Kinase (RAK) Pathway
Authors: Chandra Surabhi , Romero Maritza J , Shatanawi Alia , Caldwell Ruth B , Caldwell Robert William
Published: June 2021

Nitric oxide (NO) produced from L‐arginine by NO synthase (NOS) is needed for normal vascular function. During diabetes, aging, and hypertension, arginase (ARG) competes with NOS for L‐arginine, reducing NO and increasing O2·− production, via NOS uncoupling. Elevated O2·− combines with NO to form peroxynitrite (ONOO−) further reducing NO. Oxidative radicals increase ARG activity, though mechanism(s) involved are not known. We hypothesized that oxidative species increase ARG activity through the RAK pathway. To test this, we determined effects of the ONOO− generator (SIN‐1) or H2O2 on ARG activity/expression in bovine aortic endothelial cells (EC). SIN‐1 (25 μM, 24 hr) increased ARG activity (35%) and expression (25%) which was prevented by pretreatment with ONOO− decomposition catalyst, FeTPPS (10 μM) or Rho kinase inhibitor, Y‐27632 (10 μM). Exposure to SIN‐1 (2 hr) increased membrane translocation of p115RhoGEF (20%), which activates RAK. Treatment with H2O2 (25 μM, 8 hr) increased O2·− production (71%), as well as ARG activity (50%) and expression (36%) which was prevented by pretreatment with the NADPH oxidase inhibitor apocynin (30 μM) or Y‐27632. Thus, our data indicate oxidative species ONOO− and H2O2 increase ARG activity/expression through the RAK pathway. Further, effect of H2O2 on ARG activity appears to be due to O2·− formation via NADPH oxidase (Support NIH grant HL70215).
Inhibition of reverse‐mode sodium‐calcium exchange by the anti‐anginal agent ranolazine.
Authors: Soliman Daniel , Wang Liango , Hamming Kevin SC , Matemisz Laura C , Clanachan Alexander S , Light Peter E
Published: June 2021

Myocardial calcium overload observed during ischemia‐reperfusion is largely mediated via reverse mode cardiac sodium‐calcium exchange (NCX1.1), resulting from intracellular sodium overload that occurs via sodium‐hydrogen exchange and induction of late sodium current (late INa). The anti‐anginal agent Ranolazine (Ranexa™) is thought to act by selective inhibition of late INa thus reducing the NCX1.1‐mediated intracellular calcium overload occurs. In preliminary studies, we observed that ranolazine reduces ouabain‐induced calcium overload in rat hearts perfused in working‐mode, that is likely independent of late INa. Therefore, we hypothesized that ranolazine may also directly inhibit reverse‐mode sodium‐calcium exchange. To test this notion, we investigated the effect of ranolazine on recombinant NCX1.1 currents using the inside‐out excised patch‐clamp technique. Interestingly, we observed that ranolazine is a selective and potent inhibitor of the inactivating reverse‐mode NCX1.1 currents (IC50 = 40.8 ± 3.9 nM), with no effect on the non‐inactivating forward‐mode NCX1.1 currents. To further probe the inactivation dependence of ranolazine, we examined the effects of ranolazine on reverse‐mode currents from the NCX1.1 mutants F258E and K264Q that display either no inactivation or accelerated inactivation. Furthermore, ranolazine inhibits reverse‐mode evoked NCX1.1 activity in intact neonatal rat cardiac myoyctes. These results strongly suggest that, at therapeutic concentrations in the micromolar range, ranolazine may exert some of its cardioprotective efficacy via direct inhibition of reverse‐mode NCX1.1 in addition to inhibition of late INa. Source of Research Support: Canadian Institutes for Health Research.
Up‐regulated phosphoinositide 3‐kinase γ (PI3Kγ ) promotes breast cancer metastasis
Authors: Xie Yan , Kirui Joseph K. , Jiang Haihong , Deng Caishu , Tu Yaping
Published: June 2021

We recently reported that dysregulation of Gi‐coupled receptors promotes breast cancer metastasis via Rac‐dependent pathways (Xie et al., Cancer Res. 69: 5743–5751). The PI3Kγ is selctively activated by Gi‐coupled receptors. The objective of this study was to define the role of PI3Kγ in breast cancer metastasis. Immunohistochemical staining and Western blot analysis showed that PI3Kγ expression was selectively up‐regulated in human metastatic breast cancer cell line and breast cancer specimens. Transwell chamber assays indicated that migration and invasion of metastatic breast cancer cells in response to chemoattractants of NIH‐3T3 conditioned media was inhibited by a selective inhibitor of PI3Kγ but not PI3Kα or β isoforms. Furthermore, blocking PI3Kγ not PI3Kα and β disrupted the Rac‐dependent lamellipodia formation induced by the activation of Gi‐coupledreceptors, such as protease‐activated receptor 1 and CXC chemokinereceptor 4. In contrast, the lamellipodia formation induced by epidermal growth factor (EGF), a process not dependent on G‐proteins, was blocked by inhibitors of PI3Kα and β not by the PI3Kγ Inhibitor. Our data demonstrated that PI3Kγ functions as a key molecule mediating signal flow from Gi‐coupled receptors to Rac‐dependent pathways and suggests that PI3Kγ may be a novel therapeutic target for the treatment of breast cancers. Supported by Nebraska State LB692 (YX).
Elevated glucose compromises myocardial mitochondrial DNA (mtDNA) integrity by activation of mitochondrial topoisomerase activity.
Authors: Edwards John G. , Medikayala Sushma , Piteo Brian , Labinskyy Nazar
Published: June 2021

Mitochondrial dysfunction has a significant role in the development and complications of diabetic cardiomyopathy. The goal of this study was to determine if chronically elevated glucose promoted a decline in mitochondrial DNA (mtDNA) integrity that contributed to mitochondrial dysfunction and identify the underlying basis for mtDNA damage. Functional tests of mitochondria (Complex I activity, cytochrome oxidase activity, and ATP generation) were significantly depressed by chronically elevated glucose, while AnnexinV binding (a marker of apoptosis) was significantly increased after 7 days. 13 days of elevated glucose significantly increased mtDNA damage both globally and across the region encoding for cytochrome oxidase. Using mitochondria isolated from H9c2 cells exposed to elevated glucose, we observed significant increases in mitochondrial topoisomerase‐linked DNA cleavage, and this was exacerbated by H2O2. We conclude that even mild increases in glucose presentation will compromise mitochondrial function as a function of mtDNA damage. Separate from a direct impact of oxidative stress on mtDNA, ROS‐induced alteration of mitochondrial topoisomerase activity appeared to propagate an increase in mtDNA damage. These findings indicate that the mitochondrial topoisomerase function may have a significant role in the development and complications of diabetic cardiomyopathy.
The Isolation of the Receptor for Advanced Glycation End‐Products from Avian Vasculature
Authors: Cousens Emily N. , Braun Eldon J.
Published: June 2021

Accumulation of glucose in the plasma results in the formation of Advanced Glycation End Products (AGEs) by a non‐enzymatic reaction with albumin. These products bind to Receptors for AGEs (RAGE) on endothelial cells. This alters the basement membrane structure leading to accumulation of fluid in tissues as seen in Type II Diabetes. Humans normally maintain plasma glucose concentrations of 4.4 to 5.5mM/L whereas birds maintain concentrations of 16.6mM/L or greater. If plasma glucose concentrations in humans exceed 6.6mM/L for 4–6 weeks, symptoms of diabetes will begin to develop. Birds can serve as an animal model for the study of Type II diabetes due to their high plasma glucose levels and that they are insulin insensitive resisting the effects of these levels. In the present study, SDS PAGE and Western Blot analysis were used to isolate RAGE from the vasculature of Mourning Doves. RAGE N‐16 goat polyclonal IgG (Santa Cruz) was used as the primary anti‐body which detects the presence of RAGE in mice, rats, humans and cows increasing the likelihood of detecting RAGE in birds. Vascular tissue isolated from rats was used as a control. We hypothesized that birds lack RAGE as they do not suffer Type II Diabetic pathologies. Our data indicate a band at 55kda suggesting the presence of a RAGE in birds. The density of these receptors in the vasculature is to be determined by immunohistochemistry.

E. Cousens was a recipient of an APS UGSRF.
Tissue Angiotensin II Type 1 Receptor Expression in Development of Hypertension in SHR/Brown Norway Congenic Rats
Authors: Collett J. A. , Ighodaro A. , Patterson E. , Osborn J. L.
Published: June 2021

Angiotensin II (AII) has been linked to hypertension in Okamoto Spontaneously Hypertensive Rats (SHR). The transmission and progeny expression of AII Type 1 receptors (AT1r) in SHR and the development of genetic hypertension remains unknown. It is hypothesized that tissue AT1r expression of genes derived from SHR are linked to elevated blood pressure in offspring of SHR crossed with normotensive, Brown Norway (BN) rats. Hypertensive female F1 progeny of SHR/BN matings were backcrossed with the founder BN male to generate F2 progeny. Arterial pressures (MAP) were measured by tail cuff plethysmography and offspring were sacrificed at 15–20 weeks of age for analysis of tissue AT1r. MAP of parent SHR was 151 mmHg and BN was 94 mmHg. Progeny were grouped according to normotension (NT<105mmHg; F1 n=0, F2 n=2), borderline hypertension (BHT 105 ≤ MAP < 122 mmHg; F1 n=18, F2 n=13) and hypertension (HT MAP ≥ 122 mmHg; F1 n=15, F2 n=13). AT1r expression in forebrain (0.918 ± 0.176), kidney (0.905 ± 0.050), and hypothalamus (1.28 ± 0.252) tissues in the F1 progeny of BHT and HT were similar (western blot analysis). These data suggest that tissue AT1r expression of genes transmitted to offspring from SHR females are correlated to elevated progeny blood pressure. Tissues of F3‐F6 SHR/BN congenic progeny will be analyzed to further evaluate the interactions of tissue AT1r expression and the genetic transmission of hypertension by female SHR.
Soluble Epoxide Hydrolase Is Involved in Angiotensin II‐induced Atherosclerosis
Authors: Wen Liang , Ai Ding , Hammock Bruce D. , Shyy John Y‐J , Zhu Yi
Published: June 2021

Angiotensin II may contribute to the development and progression of atherosclerotic lesions because of its growth and proinflammatory effects. Epoxyeicosatrienoic acids(EETs), hydrolyzed and degraded by soluble epoxide hydrolase (sEH), enhance arteries dilation and provide anti‐inflammation, thus are cardioprotective. In this study, we investigated the role of sEH in angiotensin II (Ang II)‐induced atherosclerosis in apoE‐deficient mice. AngII (1.44 mg/kg·d) was administered to apoE‐deficient mice via osmotic minipumps. The animals were placed on either standard chow or an atherogenic diet with or without sEH‐selective inhibitors. After 4‐week atherogenic diet, the mean atherosclerotic lesion area in ventricle efferent track of animals was increased compared to those on a standard chow diet. AngII treatment increased the lesion area, which was further increased by atherogenic diet. On the other hand, sEH‐inhibitor significantly reduced atherogenesis. Western blot shows that AngII increased the expression of sEH and ICAM‐1 in mouse aortas. Furthermore, sEH inhibitor abolished the ICAM‐1 induction by AngII. Our results suggest that sEH is upregulated by Ang II, which is atherogenic.
Nitric Oxide‐mediated Proteins' S‐nitrosation in Endothelial Cells under Shear Flow or Rosuvastatin Treatment
Authors: Wang Ling D. , Huang Bin
Published: June 2021

Atheroprotection of shear flow to endothelial cells (ECs) is partly contributed by the increased nitric oxide (NO) production. Cholesterol lowing drug, statin, is known to increase NO levels via the induction of eNOS expression and activity. NO‐mediated S‐nitrosation of Cys residue has emerged as a fundamental post‐translational modification of proteins in ECs. S‐nitrosation can alter protein's function and affects vascular physiology. In the present study, proteins' S‐nitrosation was examined in HUVECs after shear flow or statin treament. Increased eNOS activation was observed in ECs after rosuvastatin (10 μM) or shear flow (25 dynes/cm2) treatment. The enhanced S‐nitrosoproteins in rosuvastatin‐treated ECs were detected by biotin switch method coupled with Western blot‐based 2‐DE, while the shear flow‐enhanced S‐nitrosated proteins were detected by CyDye DIGE. There were 12 and 18 proteins showing increased S‐nitrosation in ECs under flow and statin treatments, respectively. S‐nitrosation in protein disulfide isomerase (PDI), mitochondrial heat shock protein 70 (mt‐HSP70), heat shock transcription factor, tropomyosin and vimentin were shown to be increased in both treatments. Further analysis by nLC‐MS/MS and MASCOT in‐house algorithm, the S‐nitrosated Cys residues on the mt‐HSP70, tropomyosin and vimentin were also determined. Our results indicating that statin or shear flow exerts similar atheroprotection to ECs is at least partly due to the NO‐mediated S‐nitrosation in proteins. Proteins' S‐nitrosation may be essential for maintaining endothelial integrity.

(This work ws supported by National Science Council and Academia Sinica, Taiwan)
Carbon Monoxide, a heme oxygenase metabolite, decreases ATP release by macula densa cells in vitro
Authors: Leung Pablo , Garvin Jeffrey L. , Liu Ruisheng , Carretero Oscar A.
Published: June 2021

Tubuloglomerular feedback (TGF) is the mechanism by which macula densa (MD) cells sense changes in luminal NaCl concentration and trigger signals that alter the vascular tone of afferent arterioles. TGF is mediated by ATP. We have shown that heme oxygenase (HO) metabolites blunt TGF, but the mechanism is unknown. We hypothesize that carbon monoxide (CO), released by HO, reduces ATP release by MD cells in response to increased NaCl thus blunting TGF. We measured: 1) expression of HO‐1 and 2 in laser capture microdissected MD cells from Sprague‐Dawley rats by real‐time PCR; and 2) the effect of a CO releasing molecule (CORM‐3) on NaCl‐induced ATP release by MD cells in culture. ATP release was quantified by an ATP luciferin‐luciferase bioluminescence assay. We found that MD cells from Sprague‐Dawley rats express both HO‐1 and HO‐2, but HO‐2 expression was higher (33.5 ± 0.5 vs. 31.4 ± 0.6 cycles at inflection; n=3, p<0.05). In the presence of 10 mM NaCl, ATP release was 3.2 ± 1.01 pmol/mg protein. In the presence of 75 mM NaCl it was 38.4 ± 6.4 pmol/mg protein, 11‐fold greater (n=6, p<0.002). CORM‐3 (50μM) reduced ATP release in 75 mM NaCl by 33% (25.8 ± 4.3; n=6, p<0.002), but had no effect on ATP release in 10 mM NaCl. Increasing chloride without increasing sodium by adding choline chloride to the media, had no significant effect on ATP release. We conclude that CO released by HO blunts TGF in part by reducing ATP release by MD cells.
The role of Nuclear Respiratory Factor‐1 in regulating GLUT4 expression
Authors: Mukwevho Emmanuel , Ojuka Edward
Published: June 2021

Overexpression of NRF‐1 in transgenic mice has a dramatic increase in GLUT4 and myocyte enhancing factor (MEF)‐2 (Baar et al., 2003). The increase in GLUT4 was surprising since NRF‐1 has no binding sites on the Glut4 gene; hence the question is how does NRF‐1 regulate GLUT4? Our hypothesis is that GLUT4 expression is a secondary effect of NRF‐1 on the Mef2a gene, which has NRF‐1 binding sites. The aim of this study was to investigate the molecular mechanism leading to GLUT4 upregulation by NRF‐1.

C2C12 cells were used to develop a tetracycline based gene expression system (Tet‐On) to form a double stable cell line which overexpresses NRF‐1 upon activation by Doxycycline.

Our results show that NRF‐1 overexpression increases the binding of NRF‐1 to the Mef2a and δAlas promoters and MEF2A to Glut4 promoter. Furthermore NRF‐1 overexpression resulted in increase expressions for MEF2A, GLUT4 and δALAS proteins. Using small interference RNA directed against MEF2A, overexpression of NRF‐1 resulted in downregulation of GLUT4 protein expression.

From these results we could conclude that NRF‐1 overexpression increases GLUT4 via Mef2a binding resulting in increased MEF2A which in turn increases its binding to the Glut4 gene resulting in GLUT4 expression. The parallel increases in mitochondrial and GLUT4 expression are essential in alleviating metabolic disease such as type II diabetes.
Differential vasodilatory responses to local thermal stimuli in facial, glabrous and hairy skin
Authors: Wilson Thad E , Ramos Lesley , Tomc Christa , Simpson Martha A , Simpson Christopher , Sammons Dawn
Published: June 2021

Local heating of the cutaneous vasculature traditionally produces a biphasic vasodilatory response related to axon reflex and nitric oxide mechanisms, respectively. This study tested skin blood flow (% of maximum cutaneous vascular conductance; %CVC) responses of 22 healthy subjects to local heating (42°C for 45 min) in the forehead (facial skin that does not often flush), cheek (facial skin that actively flushes), forearm (control hairy skin), and palm (control glabrous skin) to gain insight into facial flushing disorders. Baseline flow at 32°C was higher in the palm (65±11) compared to the forehead and cheek (15±1 and 17±2) and compared to the forearm (7±2%CVC; P<0.05). Palm skin did not demonstrate a biphasic local heating response; 1st peak was 20±9% greater than the 2nd peak and the nadir returned to baseline values (P>0.05). No differences were observed in the 1st peak between the forehead, cheek, and forearm (85±3, 92±2, and 91±6%CVC). In the check, nadirs were not observed in most subjects and nadir values (82±3) were greater than both the forehead and the cheek (69±3 and 73±4%CVC; P<0.05). These data suggest differing local regulation of facial and glabrous skin compared to hairy skin.
Impaired expression of prostaglandin E2 synthesis and degradation enzymes in urothelial cells from patients with interstitial cystitis.
Authors: McHowat Jane , Rickard Alice , Marentette John
Published: June 2021

The differentiated superficial cells of the urothelium restrict urine flow into the bladder wall. We have demonstrated that urothelial cells isolated from bladders of patients with interstitial cystitis (IC) fail to release PGE2 in response to tryptase. We measured protein expression of 15‐hydroxyprostaglandin dehydrogenase (PGDH), cyclooxygenase‐2 (COX‐2) and prostaglandin E2 synthase (PGES) in immortalized urothelial cells isolated from the bladders of IC patients (SR22A, SM28) or normal subjects (PD07i, PD08i) during differentiation produced by increased calcium and fetal bovine serum in the culture medium for 1, 3 and 7 days. PGES expression increased during differentiation in normal and IC urothelial cells. COX‐2 expression also increased in cells from normal patients following differentiation. Remarkably, no COX‐2 expression was detectable in IC cells. PGDH decreased in normal cells after 1 and 3 days of differentiation, but returned to normal after 7 days. PGDH expression was unchanged during differentiation at 1 and 3 days, but was more than 2‐fold higher at 7 days compared to day 0 in the IC cells. Urothelial cells isolated from IC patients demonstrated no PGE2 release in response to tryptase under any of the experimental conditions studied. Taken together, our results indicate that PGE2 release is compromised during differentiation in IC urothelium and may contribute to impaired barrier function.
P‐glycoprotein mediates cellular host‐defense response against Listeria monocytogenes invasion in HeLa cells
Authors: Lavrentyev Eduard N , Swanson Joseph M , Neudeck Brien L
Published: June 2021

Cellular host‐defense is a novel area of research, which may help in prevention of diseases caused by intercellular pathogens. Expression and function of intestinal P‐glycoprotein (Pgp) plays an important role in resistance to early Listeria invasion. However, the mechanism of Pgp‐related anti‐Listeria resistance is unclear and was the subject of the present study. We used HeLa cells expressing human Pgp cDNA in pRevTet‐Off vector. Cells were incubated with Listeria and the number of intercellular pathogens was counted via confocal microscopy. Cell lysates were subjected to RTQ‐PCR and Western Blot to analyze Pgp, β‐catenin and PI3Kα abundance. Our data suggests that Pgp expression does not affect the rate of the primary Listeria invasion, but rather inhibits the growth of intercellular microorganism via S100A8/S100A9 complex activation. This effect was mediated by β‐catenin phosphorylation at Ser‐552. In PgpON cells β‐catenin phosphorylation was higher and was observed 1½ h earlier than in PgpOFF cells. In PgpON cells, Listeria‐induced increase of S100A8/S100A9 expression was more prominent and appeared 2 h earlier than in PgpOFF cells. Moreover, Pgp expression diminished Listeria‐induced PI3Kα phosphorylaiton that, presumably, decreased Listeria invasion via intracellular gap‐junctions. Thus, Pgp plays an important role in cellular host‐defense against Listeria invasion.