bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence of RNA looping by PTB using Fluorescence Resonance Energy Transfer and NMR spectroscopy
Authors: Rueda David , Lamichhane Rajan , Auweter Sigrid D , Manatchal Cristina , Austin Keyunna S , Valniuk Oksana , Allain Frederic
Published: June 2021

In eukaryotes, alternative splicing plays an important role in proteome diversity and sound protein expression. Polypyrimidine tract binding protein (PTB) is a major alternative splicing factor that has been involved mostly with splicing repression. It has been proposed that the mechanism of action of PTB involves a looping out of exons flanked by pyrimidine tracts. Here, we present Fluorescence, NMR and single molecule data that directly support this mechanism. We show that the RNA binding domains 3 and 4 of PTB (RBD34) bind two distant pyrimidine‐tracts and can bring their 5' and 3' ends in close proximity, thus looping the RNA. Looping efficiency depends on the length of the intervening sequence with preference for a 15‐nucleotide spacer between the pyrimidine‐tracts. NMR chemical shifts perturbation show a specific directionality of the PTB inter‐action with RNA, RRM3 and RRM4 binding the 5' and the 3' pyrimidine‐tract, respectively. A PTB mutant reveals a synergistic effect between RBD3 and 4 for efficient looping in vitro and repress splicing in vivo.
Association of mRNA Export Factor Yra1 with the C‐terminal Domain of RNA Polymerase II
Authors: MacKellar April L , Greenleaf Arno L
Published: June 2021

The unique C‐terminal domain (CTD) of RNA polymerase II (Pol II) is composed of tandem heptad repeats of the consensus sequence YSPTSPS. This domain is subject to differential phosphorylation throughout the transcription cycle, mainly on serines 2 and 5 of the repeats. Proteins involved in transcription and RNA processing bind the CTD to localize to nascent pre‐mRNA during transcription. In S. cerevisiae, the mRNA export protein Yra1 (ALY/REF in metazoa) cotranscriptionally associates with mRNA and delivers it to the nuclear pore complex for export to the cytoplasm. Here we report that Yra1 binds in vitro to the elongating or hyperphosphorylated form of the CTD as judged by both Far Western and immobilized peptide binding assays. Using truncated versions of Yra1, we have determined that the C‐terminal segment of the protein is dispensable for phosphoCTD binding. PhosphoCTD binding also appears to involve non‐RRM residues of Yra1. In addition, co‐immunoprecipitation experiments with cell extracts demonstrate that the Yra1‐phosphoCTD interaction likely occurs in vivo. A model is proposed in which recruitment of Yra1 to the nacent mRNA is mediated by binding to and release from the phosphorylated forms of the CTD.
Glycogen Protects Clear‐Cell Carcinoma: dependence on the hypoxia inducible pathway and a potential therapeutic role for glycogenolysis inhibitors.
Authors: Parker Glendon J
Published: June 2021

Compromised oxygen delivery is a major feature of solid tumor biology. Carcinoma metabolism up regulates glycolysis to produce ATP in an oxygen independent manner. As a result, carcinoma cells are highly dependent on glucose delivery and availability. Clear‐cell carcinomas, a histologic variant present in many cancer types, have distinctive, large intracellular reserves of glycogen, the polymeric form of glucose. We hypothesized that glucose mobilized from glycogen reserves would protect renal cell carcinoma (RCC) from nutrient and oxygen deprivation. We induced glycogenosis in Caki‐2 human renal cell carcinoma cells with desferrioxamine (DFO), an activator of the hypoxia‐inducible factor (HIF) pathway. The induced glycogenosis was shown to be HIF‐dependent using RNAi targeted against HIF‐1alpha (p < 0.01). The accumulated glycogen reserves were responsive to metabolic status (p < 0.05). Mobilization of glycogen reserves protect Caki‐2 cells incubated with azide in glucose‐free media, whereas non‐glycogenotic Caki‐2 cells started to lose viability after 4 h (p < 0.05). Inhibition of glycogen phosphorylase removed the protective effect of glycogenosis (p < 0.05). This demonstrates that glycogen protects RCC and that inhibition of glycogenolysis would complement anti‐angiogenic compounds, which restrict oxygen delivery and increase dependence on glycogen metabolism.
Mice with targeted inactivation of nBmp2 exhibit increased daytime activity
Authors: Adam Michael Max , Nichols Caitlin A. , Schmidt Alina D. , Logonathan Sampath , Thomson David , Capecchi Mario R , Barrow Jeffery R. , Bridgewater Laura C.
Published: June 2021

Bone morphogenetic protein 2 (Bmp2) is a secreted signaling protein known to induce bone and cartilage formation, and has been implicated in several disease and developmental processes. Recently, a nuclear form of Bmp2 (nBmp2) was discovered in our lab. To determine the function of nBmp2, mice have been engineered in which the conventional secreted form of Bmp2 is produced normally, but nBmp2 cannot be translocated to the nucleus. Previous studies, including flexed arm‐hang strength tests, swim tests, and gait analysis, have been performed on the mutant and wild type mice and have revealed that mutant mice have significantly reduced strength. The objective of the current study was to characterize the running patterns in the mutant. Age‐matched wild type and mutant mice were allowed to run voluntarily on running wheels in their cages, and the distance covered was recorded at 30 minute intervals using the Lafayette Instrument Activity Wheel System. The results revealed little difference in total distance run during nighttime cycles, but male mutant mice ran approximately twice as much during the daytime as wild type mice. In addition, western blot analyses of muscle tissue from the WT and mutant males showed more cytochrome C in mutants. These results suggest that nBmp2 may play a role in regulating locomotor activity patterns by affecting muscle function. Supported by NIH grant AR48839 and by the Fulton Family Foundation.
Interaction of Abcb6 with Cytochrome P450s
Authors: Oruganti Mahitha , Krishnamurthy Partha
Published: June 2021

Modification of the enzymatic activity of cytochrome P450 (CYP450) enzymes can alter the metabolism of the drug substrate for that enzyme, and thereby alter the biological activity of the drug leading to potentially harmful drug‐drug interactions. The activity and in some cases the induction of these CYP450 family members is mediated by haem, an essential prosthetic group of these detoxifying enzymes. In the present study we investigated whether the mitochondrial half transporter Abcb6, which regulates cellular haem biosynthesis and cellular haemoprotein pool, plays a role in the expression and/or function of CYP450s. We found that over‐expression of Abcb6 in HepG2 cells lead to an increase in CYP3A4 mRNA expression. In contrast over‐expression of Abcb6 in K562 cells resulted in decreased expression of CYP3A4, CYP2E1 and CYP2D6 mRNA. We tested whether prototypical inducers of CYP450 expression regulated Abcb6 expression and found that both pregnenolone‐16α carbonitrile (PCN) and 1,4‐bis[2‐(3,5‐dichloropyridyloxy) benzene (TCPOBOP), two well known inducers of CYP450, also induced Abcb6 expression both in vitro and in vivo. Cumulatively these findings suggest that Abcb6 might play a role in the metabolism and disposition of drugs and environmental chemicals, and may affect drug clearance and drug toxicity.
Transcriptional Regulation of the Yeast PAH1 Gene
Authors: Pascual Florencia , Carman George M.
Published: June 2021

The PAH1 gene in Saccharomyces cerevisiae encodes the Mg2+‐dependent phosphatidate (PA) phosphatase. PA phosphatase catalyzes the dephosphorylation of PA yielding diacylglycerol and Pi. This enzyme generates the diacylglycerol used for the synthesis of triacylglycerol, and may generate the diacylglycerol used for the synthesis of phospholipids via the Kennedy pathway. The PAH1‐encoded enzyme also controls the cellular concentration of its substrate PA, which is the precursor for phospholipids that are synthesized via the CDP‐diacylglycerol pathway. Previous work utilizing a PPAH1‐lacZ reporter gene plasmid expressed in wild type cells showed that PAH1 expression is 5‐fold higher in stationary phase cells than that found in exponential phase cells, and inositol supplementation resulted in a further 2‐fold increase in β‐galactosidase activity in stationary phase cells. Preliminary studies indicated that regulation of PAH1 expression is mediated by the Gis1/Rph1 transcription factors. In this work, the cis‐ and trans‐acting elements involved in this regulation are examined. Supported by NIH grant GM 28140.
Phosphorylation of Yeast Phosphatidylserine Synthase by Protein Kinase A
Authors: Choi Hyeon‐Son , Carman George M.
Published: June 2021

Phosphatidylserine (PS) synthase catalyzes the committed step of phosphatidylcholine synthesis via the CDP‐diacylglycerol pathway in the yeast Saccharomyces cerevisiae. It is one of the most highly regulated phospholipid synthesis enzymes in yeast. Previous work showed that PS synthase is phosphorylated by protein kinase A, and that phosphorylation inhibited enzyme activity. In this work, the sites of protein kinase A phosphorylation were examined. Upon SDS‐PAGE, the PS synthase migrates as a doublet of two protein bands. Protein kinase A phosphorylation of the native enzyme resulted in the disappearance of the lower protein band, whereas treatment with alkaline phosphatase or protein phosphatase resulted in the disappearance of the upper protein band. A site‐directed mutagenesis analysis of putative protein kinase A sites (Ser46, Ser47, Ser140, Ser141) revealed that Ser46 and Ser47 were the major phosphorylation sites in the enzyme. A double S46A S47A mutation resulted in an enzyme that migrated on SDS‐gels as a single protein band, whereas single S46A and S47A mutant proteins migrated as doublets with a decrease in the abundance of the upper band. PS synthase was also subject to proteolytic degradation, and the phosphorylation of the enzyme at Ser46 and Ser47 protected the enzyme from proteolysis. Supported by NIH grant GM 50679.
Effects of short‐term cyclic tensile strain and TGF‐β1 on smad pathway in human mesenchymal stem cells
Authors: CHEN HUA HUA , DE ISLA NATALIA , OUYANG JINGPING , STOLTZ JEAN‐FRANCOIS
Published: June 2021

Transforming growth factor‐β (TGF‐β) is a key factor for chondrogenic differentiation of human mesenchymal stem cells (hMSCs). Mechanical loading has also been shown to be important in MSC differentiation, but mechanisms by which mechanotransduction occurs remain largely elusive. In this study, MSCs were obtained from bone marrow samples of healthy donors. MSCs at passage 4 were seeded on an elastic silicone membrane coated with collagen I (BioFlex plate). At 80% confluence, cells were pretreated with serum‐free medium for 24 h. Then chondrogenic medium with or without TGF‐β1was added and cyclic tensile strain (CTS, 0.5 Hz, 5% equibiaxial strain) was applied for 10, 30, 60 and 180 minutes using a Flexercell Tension Plus system (FX‐4000T, Flexcell International). As a control, cells cultured on the same type of plates, without stretch, with or without TGF‐β1 were also used. Using specific antibodies, Smad2/3 and Smad3 phosphorylation (pSmad3) were quantified by western blotting and localization of pSmad3 was observed by confocal microscopy. We have found that, the level of Smad2/3 is not significantly different in all conditions. Both the CTS and TGF‐β1 increased pSmad3 at all times tested. And CTS plus TGF‐β1 exhibited higher level of psmad3 than CTS and TGF‐β1 respectively. However, the increase was maximal at 1 hour of stimulation and decreased after 3 hours of stimulation. Smad3p translocation to the nucleus was observed submitted to CTS and TGF‐β1. Our results show that CTS affected smad3 phosphorylation and that the effect was time dependent. A synergy effect between CTS and TGF‐β1 was observed. CTS could stimulate MSC differentiation by the activation of endogenous TGF‐β signaling pathway.
Study of spinal cord cells interactions in a tissue‐engineered three‐dimensional in vitro model
Authors: Beaulieu Marie‐Michèle , Grenier Myriam , Berthod François
Published: June 2021

Amyotrophic lateral sclerosis and other motor neuron diseases are very complex as their development and progression involve many cellular processes and many cell types. To better understand motor neuron physiology in defined culture conditions and in a physiological environment that reflects the in vivo situation, we developed in vitro a three‐dimensional tissue‐engineered spinal cord model. Our aim was to use this model to study the pathogenesis of neurodegenerative diseases affecting motor neurons. Purified mouse spinal cord motor neurons, astrocytes and microglia were seeded on a biomaterial populated with fibroblasts that mimicked the connective tissue

through which motor axons have to elongate in vivo. Motor neuron axon elongation and the interactions with the glial cells were assessed by immunohistochemistry after 14 days of culture. Our reconstructed tissue promoted the survival, neurite elongation and maturation of motor neurons. This tissue‐engineered spinal cord model is well‐suited to study motor neurons in vitro in a physiological three‐dimensional environment to better understand neurodegenerative diseases.
Molecular Characterization of Antibiotic‐Producing Bacteria from Brooklyn Soils.
Authors: Fadael Rudgie , Bolnet Carolle
Published: June 2021

In our previous studies, physiological and morphological methods were used in an attempt to screen out new potent antibiotic producers from different locations in Brooklyn, NY: Manhattan Beach (MB), Coney Island Beach (CIB), Prospect Park (PP) and Downstate (DS). Cultures from CIB soil not only displayed the most bacterial diversity but also revealed colonies showing signs of antibiosis. Among these cultures, we were able to identify bacteria of the genus Streptomyces whereas the others could not be identified accurately by those two methods. In this study, we used molecular tools to characterize unidentified bacteria from CIB. DNA was extracted from pure cultures and amplified using a nested PCR designed for the 16S rRNA gene that significantly reduces the amount of non specific amplification. The cleaned PCR products were sequenced by using Big‐Dye chemistry and the ABI3730 automated sequencer. Using BLAST, the obtained sequences were compared to bacterial 16S rDNA sequences in GenBank. Analyses of the results revealed a 99% nucleotide similarity to the 16S rDNA sequence of Streptomyces hygroscopicus very well known as a producer of antimicrobial agents such as rapamycin, ascomycin as well as the immunosuppressant FK506.
Effects of puerarin on differentiation of 3T3‐L1 preadipocytes
Authors: Lee Ok‐Hwan , Seo Dong‐Ho , Park Cheon‐Seok , Kim Young‐Cheul
Published: June 2021

Puerarin (daidzein 8‐C‐glucoside), a major isoflavone glycoside derived from Kudzu root (Pueraria lobata), has been shown to favorably affect glucose and lipid metabolism associated with insulin resistance, diabetes, and cardiovascular diseases. Here, we examined the effects of puerarin on the changes of key gene expression associated with adipocyte differentiation and insulin sensitivity using 3T3‐L1 cell, a cell line that undergoes differentiation into adipocytes in the presence of adipogenic hormonal stimuli. 3T3‐L1 preadipocytes were differentiated in the presence of various concentrations of Puerarin for up to 6 days, followed by Oil Red‐O staining of lipids and gene expression using semi‐quantitative RT‐PCR. Puerarin treatment significantly enhanced differentiation of 3T3‐L1 preadipocytes in a dose‐dependent manner. At day 6 after the differentiation, 100µM puerarin increased expression of PPARγ mRNA (~1.7‐fold) and its target gene adipocyte‐specific fatty acid binding protein (aP2) mRNA (~1.2‐fold). Puerarin treatment also up‐regulated the expression of GLUT4 and adiponectin mRNAs (~2.0‐fold and 1.7‐fold, respectively), two important genes for insulin sensitivity. These data indicate that the insulin‐sensitizing effects of puerarin may involve activation of PPARγ and stimulation of GLUT4 and adiponectin gene expression, leading to overall increased glucose utilization
Marginal Vitamin B6 Deficiency Affects Fatty Acid Profiles in Healthy Men and Women
Authors: Zhao Mei , Lamers Yvonne , Ralat Maria A , Gilbert Lesa R , Keeling Christine E , Stacpoole Peter W , Gregory Jesse F
Published: June 2021

Vitamin B6 (VB6) deficiency alters fatty acid (FA) profiles of rat tissue lipids. The effects of VB6 deficiency on FA profiles in humans are not well understood. The aim of our study was to determine effects of VB6 deficiency on FA profiles of various blood fractions including plasma, red cells and lymphocytes. Healthy subjects (n=13) received a 2‐d adequate diet followed by a 28‐d VB6 restricted diet (0.5 mg /d) to induce a marginal VB6 deficiency (plasma pyridoxal‐5′‐phosphate 20‐30 nM). Plasma total FA, red cell and lymphocyte membrane FA were analyzed by gas chromatography/flame ionization. After 28d of VB6 restriction, plasma arachidonate (C20:4 n‐6) and docosahexaenoic acid (C22:6 n‐3) concentration decreased significantly from (mean ± SD) 533 ± 72 to 479 ± 59 µM (p=0.006) and from 111 ± 25 to 98 ± 20 µM (p=0.044). The ratio of arachidonate/linoleate, (C20:4 n‐6)/(C18:2 n‐6), in plasma also significantly decreased from 0.23 ± 0.04 to 0.20 ± 0.04 (p<0.001). For red cell membrane lipids, the percentage of oleate (C18:1 n‐9) and eicosapentaenoic acid (C20:5 n‐3) significantly decreased from 18.9 ± 1.3 % to 18.6 ± 1.1% (p=0.023) and from 0.45 ± 0.12% to 0.41 ± 0.12% (p=0.042). There were no significant changes in FA profiles of membrane lipids in lymphocytes. In conclusion, marginal VB6 deficiency has subtle but significant effects on FA profiles in humans. Supported by NIH grant DK072398 and GCRC grant M01‐RR00082.
Proteomic analysis of pancreas in a polygenic mouse model of type 2 diabetes
Authors: Wyatt Brantley , Hall Lorin , Kim Jung Han
Published: June 2021

TALLYHO/JngJ (TH) mice are a polygenic model for type 2 diabetes. We conducted a proteomic analysis in pancreas using 2D‐DIGE and mass spectrometry to identify molecules pathologically relevant to the diabetes in TH mice. Total protein was extracted from pancreas of TH and C57BL/6J mice and labeled with CyDye. Isoelectric focusing in the first dimension was conducted at pH 3‐10 to separate proteins based on pI, and in the second dimension in 8‐14% gradient SDS‐PAGE to separate proteins based on size. Differentially expressed protein spots were excised, digested with trypsin and subjected to MALDI‐TOF mass spectrometry. We observed changes in protein amounts or evidence of modifications in antioxidant defense proteins and heat shock proteins in TH mice. Proteins involved in growth and survival of pancreatic beta‐cells were also identified. In summary, the proteins identified in this study may provide links of molecular processes to the development of diabetes in TH mice, and their respective genes may serve as candidates for susceptibility genes in this polygenic model. Supported in part by NIH/NIDDK‐1R01DK077202‐01A2, AHA‐0855300E, & pilot and feasibility grant from the UT Obesity Research Center.
Prolactin regulates ZnT2 transcription through the JAK2‐STAT5 signaling pathway
Authors: Kelleher Shannon , Qian Linxi , Lopez Veronica , Seo Young Ah
Published: June 2021

ZnT2 plays an important role in zinc (Zn) secretion from the mammary gland and it is regulated by prolactin (PRL) during lactation. Herein, the objective was to explore the mechanisms through which PRL regulates ZnT2 expression in mammary cells. We determined that PRL increases ZnT2 mRNA level in cultured mammary epithelial cells (HC11) which was inhibited by pre‐treatment with the Jak2 inhibitor AG490. A fragment of the ZnT2 promoter was subcloned into a luciferase reporter vector and transfected into HC11 cells. The activity of the ZnT2 promoter was increased by PRL treatment. Cells co‐transfected with a dominant‐negative STAT5 abrogated this effect implicating the Jak2/STAT5 signaling pathway in the transcriptional regulation of ZnT2. Two putative consensus STAT5 binding sequences (GAS1 and GAS2) were identified in the ZnT2 promoter. Gel shift assays indicated that PRL induced STAT5 binding to the proximal GAS2 element with greater efficiency as compared with the distal GAS1 element. Mutagenesis analysis of these two GAS elements suggests that they synergistically modulate ZnT2 transcription. Finally, chromosome immunoprecipition assays confirmed that PRL activates STAT5 binding to the ZnT2 promoter in vivo. Our results indicate that PRL regulates ZnT2 transcription through activation of the Jak2/STAT5 signaling pathway coordinating the regulation of mammary Zn secretion processes.
A Rodent Model to Evaluate the Effect of Dietary Protein on Intestinal Calcium Absorption
Authors: Gaffney‐Stomberg Erin , Cucchi Carrie E. , Sun Ben‐hua , Simpson Christine A. , Kerstetter Jane E. , Insogna Karl L.
Published: June 2021

In humans, increasing dietary protein increases intestinal Ca absorption. To further study this effect, rats were randomly assigned to consume a control (20%), low (5%) or high (40%) protein diet for 7 days. All diets were isocaloric and contained 0.45% Ca, 0.35% P. Twenty‐four hr urines, blood and feces were collected to assess Ca metabolism, Ca balance and bone turnover. Brush border membrane vesicles (BBMV) were prepared from duodenal mucosa to measure Ca transport.

Urine Ca rose during the high protein diet and fell during the low diet (p<0.01). There were no changes in serum Ca or vitamin D metabolites on any diet. Serum PTH trended higher in the low protein group (p=0.07) and serum osteocalcin was significantly higher compared to the other groups (p<0.03). Rats consuming the high protein diet absorbed and retained more Ca compared to the low protein group (48.5% vs. 34.1% and 45.8% vs. 33.7% respectively, p<0.01). Ca transport was significantly higher in the BBMV from animals consuming the high protein diet (p<0.001).

This animal model recapitulates the findings in humans in that changes in Ca absorption explain the changes in UCa. The higher osteocalcin and PTH during the low protein diet suggest that bone turnover was increased during low protein intake. The BBMV studies indicate that increased transcellular Ca uptake contributes to the increase in Ca absorption during the high protein diet.
γ‐tocopherol supplementation improves postprandial vascular endothelial function in lean and obese men
Authors: Mah Eunice , Diaz Manuel Matos , Kawiecki Diana , Volek Jeff S , Bruno Richard
Published: June 2021

Acute hyperglycemia is associated with oxidative stress and impaired vascular endothelial function (VEF). Thus, we hypothesized that postprandial hyperglycemia would impair VEF by affecting plasma antioxidants. In a randomized, crossover study, lean and obese men (n=8/group) followed their usual diet or were provided γ‐tocopherol (γ‐T)‐rich capsules (500 mg/d, 5 d) prior to a fasting 75 g oral glucose challenge. Flow‐mediated dilation (FMD), plasma glucose, insulin, and antioxidant capacity were measured before and during the 3 h postprandial period. Insulin resistance (HOMA) was greater in obese (6.7±3.5) than lean men (3.9±2.0, p<0.05) and unaffected during the study. Postprandial FMD area under the curve (AUC) of obese men was 32% lower (p=0.02) than lean men. FMD AUC was 31% (p=0.006) higher in all men post‐γ ‐T supplementation relative to the control trial. Baseline oxygen radical absorbance capacity (ORAC), but not ferric reducing ability of plasma (FRAP), was lower (p=0.039) in obese compared to lean men. Postprandial FRAP, but not ORAC, was higher (p=0.027) with γ‐T supplementation regardless of group. These data suggest that obesity impaired VEF and that γ‐T‐rich vitamin E supplements improve VEF possibly by enhancing antioxidant capacity. Additional study is underway to define the protective effects of γ‐T on VEF. Support provided by a grant from International Life Sciences Institute to RSB.

Grant Funding Source

International Life Sciences Institute
Effect of different green tea formulations on diabetes in the Zucker Diabetic Rat
Authors: Myracle Angela Denise , Madian Ashraf , Burgess John R , Ferruzzi Mario , Regnier Fred , Janle Elsa
Published: June 2021

Chronic diseases such as Diabetes mellitus (DM) are associated with increased levels of oxidative stress which is thought to contribute to secondary complications. Oral consumption of GT has been shown to have modest effects on reducing body weight, food intake and blood glucose. Simulated in vitro digestion studies have demonstrated that GT catechins can be stabilized by ascorbate which may improve bioavailability. This study investigated the effects of formulated GT on blood glucose, insulin, body weight, food consumption, and oxidized proteins in the Zucker diabetic rat (ZDF). Four groups were dosed orally with water (control), ascorbic acid (17 mg/kg), GT (85 mg/kg) or GT and ascorbic acid (85 mg/kg + 17 mg/kg) for 6 weeks. Data collected included glucose tolerance tests (GTT), daily weight and food consumption. Plasma oxidized proteins were identified and quantified to determine the potential benefit of GT to alleviate oxidative stress. Both GT treated groups showed a trend toward lower basal glucose, peak glucose concentrations and area under the curve compared to the control and ascorbate treatment. Insulin levels were not affected by the treatments. There were differential responses in the levels of oxidized proteins which varied by treatment.

Supported by NIH, ODS and NCCAM Grant P50 AT 00477.
A Novel Method For Tissue Glutathione Redox Couple Analysis Using HPLC‐Boron Doped Diamond Detection (BDD)
Authors: Park Hea Jin , Courtemanche Kevin , Bruno Richard S.
Published: June 2021

Glutathione (GSH/GSSG) is a marker of oxidative stress. HPLC‐FL and ‐UV approaches have been used, but are challenging, time‐consuming, and column life is limited. Electrochemical (ECD) approaches have been used, but the high potential required for disulfide oxidation results in signal degradation. Thus, we aimed to measure hepatic GSH and GSSG simultaneously using HPLC‐BDD detection, a novel ECD approach having greater stability at high electrochemical potentials. Mice (n = 4/group) were injected ip with lipopolysaccaride (LPS, 0 mg or 4 mg/kg BW) and sacrificed at 24 h. Liver homogenates were mixed with perchloric acid and the isolated supernatant was used for analysis. The sample was separated isocratically at 32°C on C18 column using sodium dihydrogen phosphate, octanesulfonic acid, and acetonitrile as the mobile phase. Hydrodynamic voltamogram experiments indicated an optimal applied potential of 1400 mV for GSH and GSSG. GSH and GSSG retention times were 6.6 ± 0.02 and 16.6 ± 0.12 min, respectively, and standards were linear (r > 0.99) between 1‐8000 pmol injected for each. Within‐ and between‐day coefficient of variations were <2% and <7%, respectively. LPS decreased the GSH/GSSG ratio, reflective of decreased GSH and increased GSSG. These data indicated that hepatic glutathione can be measured reliably with minimal sample preparation and greater sample throughput using HPLC‐BDD.
Assessment of Sources and Dietary Intake of Isoflavone in the U.S. Diet
Authors: Haytowitz David B. , Bhagwat Seema
Published: June 2021

Soy products are a major source of dietary isoflavones, with weak estrogenic, and other biological properties that may contribute to the reduction of the risk of some chronic diseases, such as certain cancers, i.e. prostate and melanoma. This presentation will examine sources of isoflavones in the diet for different age and gender groups. Data for isoflavones taken from the USDA Database for the Isoflavone Content of Selected Foods, Release 2.0 were combined with food consumption data from NHANES. Using the information in the Food and Nutrient Database for Dietary Studies and the corresponding consumption data, weighted grams consumed for each food item reported consumed was calculated. By multiplying total isoflavone content by the grams consumed of the respective food items, the dietary intake of total isoflavones contributed by individual food items was determined. Soymilk was the leading source of total isoflavones, followed by meatless bacon bits and tofu. Many people do not consume soy foods directly, but soy‐based ingredients are commonly used in food manufacturing. Non‐soy products, such as frankfurters and various baked products which may contain soy‐based ingredients, such as soy flour or soy isolates, provide isoflavones to the diet. Knowing the quantities of isoflavones consumed and their relative contributions to dietary intake will assist in a range of studies relating to various diseases.
Dietary determinants of vitamin E status among a free‐living adult population
Authors: David Angeline B , Haddad Ella , Jaceldo‐Siegl Karen
Published: June 2021

Objective

The 2005 Dietary Guidelines for Americans identified vitamin E as one of six shortfall nutrients for adults, with a low probability of adequacy. We determined whether almonds in the diet contribute to plasma concentrations of alpha‐ and gamma‐tocopherol.

Methods

Eight‐one healthy subjects aged 25‐70 years were recruited for this 1‐year study. During the first 6 months subjects followed their habitual diets; in the second 6 months subjects added almonds equivalent to 15% of their total habitual energy intake. Intake was assessed using 14 random 24‐hour telephone recalls and plasma tocopherols by HPLC. Analysis of paired t‐tests and multivariate analysis were performed using Stata 8.0.

Results

Plasma alpha‐ and gamma‐tocopherol levels were significantly different between the habitual and almond‐supplemented diets (p<0.001 and p<0.05, respectively). In multivariate analysis including all food groups, vitamin E supplement use was the only significant predictor of alpha‐tocopherol during both study phases (p<0.001). When analyzed with age, sex, and adjusting for total energy intake, almond intake significantly predicted plasma alpha‐tocopherol during the habitual diet (p<0.05) but not during the almond‐supplemented diet (p=0.68).

Conclusion

Consuming vitamin E supplements is the only significant predictor of alpha‐tocopherol status in a free‐living adult population.
Prenatal Equol Treatment: Does Not Alter Newborn Genital Development but Alters Depressive‐like Behaviors in Pre‐pubertal Offspring.
Authors: Blake Crystal , Fabick Kim , Setchell Kenneth D , Lephart Edwin
Published: June 2021

In this study we examined equol, the intestinal metabolite of daidzein, and its effects on genital morphology and subsequent postnatal behavior. Pregnant rats were divided into six treatment groups before subcutaneous injection treatments: 1) Non‐injected Controls, 2) dimethyl sulfoxide (DMSO)‐injected controls, 3) Equol at 10.5 mg/Kg (in DMSO), 4) Equol at 21.0 mg/Kg (in DMSO), 5) Equol at 63.0 mg/Kg (in DMSO), and 6) Flutamide (90 mg/Kg, androgen‐receptor blocker). The mothers received daily treatment injections from gestational days 14‐20. After birth, body weight and anogenital distance (AGD) were measured and sex determined. Some of the pups and mothers (a subset at birth) were sacrificed and trunk blood collected for determining isoflavone and 5alpha‐dihydrotestosterone (5alpha‐DHT) levels. The remainder of the pups developed postnatally until day 29 where animals underwent analysis via the Porsolt forced swim test to determine depressive‐like behaviors. In summary, equol exposure during late pregnancy 1) does not alter 5alpha‐DHT levels in the males or females or alter male external genital development, 2) the high equol dosed mothers weighed the least before and after birth and their offspring also weighed the least at birth, and 3) after birth the high dosed equol treated animals that gained the most weight postnatally displayed the least depressive‐like behaviors. (USDA 2004‐01811)
School environment and overweight and obesity in Mexican student of primary and secondary school
Authors: Mundo Rosas Verónica , Shamah Levy Teresa , Rodríguez Ramírez Sonia
Published: June 2021

The prevalence of overweight and obesity in Mexican Student of primary and secondary school is elevated (31.3%).

Objective

To describe some key school environment items related to childhood obesity.

Methods

Information from 374 scholastic establishments from the National Health Survey of students (ENSE‐2008) was included in the analysis. The items have a documented relationship to childhood obesity and are likely contributors to diet quality and physical activity. Items included were: water available, sweetened beverage, foods with low nutritional value available in and out the school and physical activity at the school (minutes per day).

Results

In the school, 58.2% there was water available to drink, 85% sold candies, snacks, sodas and sweetened drinks; 49.3% sold fresh fruits and only 23.7% vegetables. Out side of the school, 48.2% sold candies, snacks, sodas and sweetened drinks; 29.9% sold fresh fruits and only 9% vegetables. The average of physical activity was 20 min/day).

Conclusion

Interventions that promote healthy eating and physical activity behaviors during childhood and adolescence are necessary in Mexican schools, since not only they may prevent some of the causes of illness but also may decrease direct health‐care costs and improve quality of life.

Supported by the Rios Aronte Foundation, and Fondo de Apoyo al Programa Intersectorial de Educación Saludable.
Anemia in Mexican students
Authors: Gómez Luz María , Shamah Teresa , Villalpando Salvador , Mundo Verónica
Published: June 2021

The objective of the study was to describe the prevalence and distribution of anemia in Mexican students from Elementary and Secondary Public Schools. Information was gathered from 55,034 students from the National Student Health Survey, 2008 (ENSE, 2008). A portable photometer was used to determine hemoglobin concentration by the capillary puncture method. The cut values used to classify anemia are those designated by WHO. Results showed that female students aged 12 years and over, had lower concentrations of hemoglobin (14.00 ± 1.50 in comparison with male students 14.21 ± 1.54 mg/dL; P <0.001). However, male students aged 12 years and over, had a higher prevalence of anemia (20.99 vs.11.72%; P<0.001), than female students. Once disaggregated by educational level, elementary school students showed a higher prevalence of anemia, than secondary school students (21.2 vs.14.4%; P<0.001). In a logistic regression model, the highest risk of anemia was associated with being a man (OR 1.26, CI95% 1.17, 1.36) and belong to secondary school (OR 1.15, CI95% 1.01, 1.30) and the probability of having anemia decreases as age increases (OR 0.87, CI95% 0.86, 0.89). Prevalence of anemia is still of high magnitude in Mexican students, so big efforts have to be done to undertake measures for prevention and control.

Supported by the Rio Aronte Foundation, and Fondo de Apoyo al Programa Intersectorial de Educacion Saludable.
Nutrition education and supplementary foods make a big difference in maternal and child nutritional health
Authors: Park Haeryun , Kim Jinyoung , Uhm Soomhee
Published: June 2021

A community‐based nutrition intervention, benchmarking the WIC program was launched with the support of a local government in Korea. Supplementary foods rich in protein, vit A, Fe, Ca and vit A, which are typically deficient in the Korean diet, were delivered along with nutrition education for low income families for 4 months. The mandatory nutrition education was provided at least once a month at the health center and subsequent individual nutrition counseling was provided at home. Pre and posttest were performed to test any changes in nutrition knowledge and attitude, height and weight, and iron status for the 405 subjects and 30 subjects in the control group. The nutrition knowledge and attitude scores were improved while those of the control group were unchanged. Hb and Hct levels also improved in all of the subject groups of pregnant women, infants, children, and postpartum mothers. Percentile values of weight for age compared to the Korean standard also improved. This evidence‐based nutrition intervention program should be expanded nationwide for better nutrition for high‐risk families.
Effect of acculturation on food label use among immigrants from the former Soviet Union
Authors: Ludman Nadia , Jasti Sunitha
Published: June 2021

Objective

To assess the effect of acculturation on use of food labels and food label reading skills among former Soviet Union (FSU) immigrants living in New York city.

Methods

A cross‐sectional survey using self‐administered questionnaire assessed acculturation, food label use, label reading skills, nutrition knowledge and socio‐economic characteristics of a convenience sample of 200 FSU immigrants.

Results

The study sample consisted of 63% females. Mean age was 43± 5.5 y, and mean length of US residency was 11.7±6.5 y. Annual household income was >$50,000 for 50%. The mean degree of acculturation was 2.3 ± 0.7 on a scale of 1 ‐ 5. Only 35% of the participants were acculturated scoring > 2.5 on the acculturation scale. 55% of the participants reported using food labels often/always, and only 32% had good label reading skills. Nutrition knowledge was low (<2 on 0‐7 scale) for 30% of the sample. Acculturation was not associated with food label use, but was significantly associated with label reading skills (p<0.001). In multivariate logistic regression analysis of label reading skills, acculturation lost its significance while age, length of US residency and nutrition knowledge were strong predictors of label reading skills.

Conclusion

Food label use and reading skills may be improved by education programs that target new and younger FSU immigrants.
Employed parents' satisfaction with food choice coping strategies: influence of gender and household structure
Authors: Blake Christine E , Devine Carol M. , Wethington Elaine , Jastran Margaret , Farrell Tracy J. , Bisogni Carole A.
Published: June 2021

The need to integrate work and family demands has implications for the food choices of employed parents. This study aimed to understand parents' satisfaction with the ways they integrated work‐family demands to manage food and eating. Employed, low/moderate‐wage, urban, U.S., Black, White, and Latino mothers (35) and fathers (34) participated in qualitative interviews exploring work and family conditions and spillover, food roles, and food‐choice coping and family‐adaptive strategies. Ten evaluative categories emerged, based on parents' satisfaction with their ability to enact preferred food roles as they attempted to integrate work and family demands. Conditions that contributed to parents' evaluations differed by gender. Mothers based their evaluations on their ability to achieve balance between work and family, while managing traditionally maternal food roles. Fathers based their evaluations on their ability to achieve stability in routines and schedules and participate in family meals, while meeting expectations to contribute to food preparation. The findings support intersecting economic and gender explanations for differences in household‐level strategies and adaptations used to integrate work and family demands. These findings have implications for understanding other aspects of work‐family spillover and for workplace and family policies affecting a vulnerable population.
Conversations: The commitment to breastfeed among low‐income women in East Tennessee
Authors: Joyce Stephanie , Granger Lauren A , Tait Stephanie D , Kavanagh Katie F
Published: June 2021

The purpose of this qualitative study was to explore the experiences of breastfeeding women in East Tennessee. Participants were recruited from the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) and from a birth registry housed at the university. Eligibility criteria included women who were currently breastfeeding infants less than 9 months of age and were income‐eligible for WIC. Data were collected through audio‐recorded in‐depth telephone interviews. Eighteen interviews were conducted. Recording error led to only 16 interviews being available for analysis. However, saturation was still achieved. Transcripts were coded by a coding team, using standard coding techniques. Using grounded theory and cluster analysis, major themes emerged including aversions to formula‐feeding ("...that was bad for him, with like chemicals and stuff..."), perception of inconvenience in learning to use formula, WIC group classes offering significant support for breastfeeding, commitment to overcome the challenges of breastfeeding, and assessing their abilities through a comparison of others' experiences. In conclusion, this qualitative work indicates that low‐income women who choose to breastfeed recognize the differences of others breastfeeding experiences and are aware of difficulties that can arise with either breastfeeding or formula‐feeding. Funding: Internal university funds.
Despite a large first‐pass extraction for urea synthesis, the systemic bioavailability of meal arginine is high and dose‐dependent in men
Authors: Mariotti François , Petzke Klaus J , Huneau Jean François , Szezepanski Isabelle , Tomé Daniel , Bos Cécile , Bonnet Damien
Published: June 2021

Dietary Arginine is supposed to be largely extracted by the liver for ureagenesis but favourable effects of dietary Arginine are mainly ascribed to a systemic utilization for NO synthesis.

Using [15N2‐(guanido)]‐Arginine, we traced the fate of L‐Arginine at two nutritional levels in a complete amino acid meal given to healthy men. We monitored the postprandial appearance of meal arginine (mArg) in the plasma, body and urinary urea derived from mArg, and plasma citrulline and urinary nitrate derived from mArg[FM1].

The relative urea production from mArg was high but similar whatever the mArg level (49±2% of mArg) and completed early after ingestion. With the lower level of mArg, intact mArg accounted for 56±4% of the marked postprandial increase in plasma arginine. In contrast, plasma concentrations of mArg‐derived citrulline were very low. After the 2.7‐fold higher mArg intake, plasma mArg AUC was 3.2±0.2‐fold higher, and plasma mArg‐derived citrulline AUC and urinary mArg‐derived nitrate excretion were 1.8±0.3 and 2.2±0.3‐fold higher, respectively.

Because its relative splanchnic extraction for urea synthesis stays steady, mArg exhibits a high incremental systemic bioavailability. However, our data suggests that the utilization of mArg for NO synthesis, although modulated by the systemic availability of mArg, stays very low in these healthy men.

Supported by a grant of the French ministry of research

[FM1] OU, pour gagner un peu de place," mArg‐derived body and urinary urea, and mArg‐derived plasma citrulline and urinary nitrate"
Dietary Glutamic Acid Increased Postprandial Leucine Availability in Protein Malnourished Rats
Authors: Sakai Ryosei , Kawamata Yasuko , Yoneda Junya , Torii Kunio , Kuwahara Tomomi
Published: June 2021

Glutamic acid (Glu), the most abundant amino acid in food proteins, is major intestinal energy source. Recently we have found that Glu removal from foods reduced dietary leucine (Leu) availability by reducing its oxidation. The present study investigated roles of dietary Glu on Leu metabolism in details using protein malnourished rats.

Fischer strain male rats were fed normal amino acid (AA) defined diet containing 4% Glu or low AA diet (50% reduction) for 1 week. Then, the rats in each dietary group were divided into two groups and given daily amount of experimental diets with or without Glu in which all the Leu was replaced with [1‐13C]Leu. After 24h collection of expired air samples, blood and tissue samples were harvested. Breath 13CO2 and [1‐13C]Leu incorporation in tissue proteins were analyzed by IRMS and GCMS, respectively.

Feeding of low AA diet reduced dietary Leu oxidation markedly. Glu removal from low AA diet significantly enhanced dietary Leu oxidation and reduced its incorporation in liver protein and plasma albumin but not kidney or muscle proteins, indicating decrease in dietary Leu availability for hepatic protein synthesis in these animals presumably due to increase in its intestinal oxidation. Besides, the effects of Glu removal from normal AA diet were not apparent. These results suggest Leu‐sparing effects of dietary Glu by reducing postprandial Leu oxidation especially in protein malnutrition.
Whole body amino acids are candidate precursors of postprandial hepatic neoglycogenogenesis in high protein fed rats
Authors: Fromentin Claire , Azzout‐Marniche Dalila , Stepien Magdalena , Even Patrick , Fromentin Gilles , Tomé Daniel , Gaudichon Claire
Published: June 2021

We previously proposed that under high protein feeding, dietary amino acids (AA) were partly involved in liver glycogen synthesis. We hypothesized that the entry of dietary AA into glycogen occurred early during the postprandial phase. Two groups of 24 rats were fed a normal (NP) or a high (HP) protein diet for 2 weeks and then sacrificed 1h, 2h, 3h or 4h after the ingestion of a 4 g meal containing U‐ 13C AA. Blood was sampled in the portal vein, vena cava and aorta, and the liver was collected. Hepatic glycogen content increased within the first h after the meal (18.6±8.1 mg/g) and then remained stable in NP rats, whereas in HP rats, the maximum was obtained at 3 h (13±6.7 mg/g). The apparent overall synthesis of glycogen was 170mg in NP vs. 92 mg in HP rats. Both hepatic glucose uptake and plasma insulin to glucagon ratio were significantly lower in HP than in NP rats, whereas the net liver uptake of alanine and proline was significantly increased in HP rats. The 13C enrichment measured in liver glycogen indicated a low uptake of dietary AA carbon skeletons. Considering that under HP feeding, dietary carbohydrate is entirely oxidized within the 4 postprandial hours (as previously obtained from a calorimetry study), we postulate that most of the liver glycogen in HP rats is synthesized from whole body alanine and proline.
Mitochondrial oxygen consumption and ATP content in control and pyrene‐exposed frogs
Authors: Reed Loren K , Carroll Jennifer R , McGirk Megan E , Stabenau Erich K
Published: June 2021

Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous environmental contaminants. Exposure to pyrene, a PAH, elicits significant decreases in exercise performance, muscle contractile strength, and mitochondrial membrane potential and O2 consumption in R. pipiens. This study quantified mitochondrial O2 consumption and ATP content in R. pipiens and R. catesbeiana liver, muscle, and skin following a 7 day in‐water pyrene exposure. Mitochondria were isolated by differential centrifugation, and complex I (CI) and complex II (CII)‐initiated mitochondrial O2 consumption was measured using a Clark‐type O2 electrode. CI ‐ initiated O2 consumption was stimulated by addition of malate/glutamate while CII was stimulated by succinate. Mitochondrial ATP content was quantified via a luciferin‐luciferase chemiluminescent assay. CI and CII ‐ initiated mitochondrial O2 consumption significantly decreased 15‐50% in the three tissues following pyrene exposure. ATP content significantly decreased after pyrene exposure. Decreased ATP could disrupt numerous ATP‐dependent processes and offer an explanation for impaired muscle performance following pyrene exposure. Funding was provided by the National Tri‐Beta Research Scholarship Foundation, Bjorklund Endowed Research Fund, NSF‐REU, Biotechnology Research and Development Corporation, and Bradley University Research Excellence Grant.