bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of materials on rural middle school students' understanding of science concepts
Authors: Bradshaw Timothy , Cunningham Jessica , Bradley Kelly , Shafer Sally , Straley Joseph , Osborn Jeffrey
Published: June 2021

Newton's Universe (NU) is a professional development (PD) program utilizing hands‐on inquiry based distance learning to improve science content knowledge of rural middle school science teachers. This study examined the impact of NU PD and kits containing laboratory materials and inquiry based activities on student understanding of temperature and heat concepts. Experimental teachers (n=34) received PD and a kit, while control teachers (n=17) received neither PD nor a kit. To account for the impact of laboratory materials independent of PD, kit teachers (n=19) received a kit but no PD. Experimental teachers scored significantly higher than control or kit teachers on a 25 multiple choice question post PD assessment (16.97 + 3.66 vs. 12.71 + 2.71 and 13.00 + 3.72 correct responses, respectively). On 39 multiple choice questions, pre and post instruction assessments, experimental teachers' students' scores significantly increased from pre to post (14.41 + 4.07 vs. 17.95 + 5.67; n = 1091) while control teachers' students' scores did not change (14.26 + 4.10 vs. 14.54 + 0.71; n = 310). Pre and post instruction scores of kit teachers' students (n = 1714) are being evaluated. These data show that NU PD improves student performance in physical science. Thus, using distance learning to increase rural teacher access to effective PD will improve student achievement in middle school science. (Supported by NSF Grant #0437768).
Renin Immunofluorescence in the metestrus rat uterus
Authors: Hurley Mary Eder , Critelli Marta , Johnson Alexandria , DenHaese Jason , Jones Craig , Olivieri Marion P.
Published: June 2021

Renin, produced by juxtaglomerular cells of the kidney to activate the renin‐angiotensin‐aldosterone pathway in response to low blood volume and/or pressure, also is a hox gene product that is important in the development of abdominal structures. When linked to green fluorescent protein, renin is expressed by adrenal, renal, and gonadal vessels in the mouse embryo. The ovarian/uterine artery continues to express renin after birth, although the testicular artery does not, and the quantity of expression varies with stage of estrus cycle and during pregnancy. Renin is also expressed in the giant trophoblast cells of the placenta. In the rat, pericytes of the ovarian/uterine artery are immunofluorescent for renin. This project specifically examines the rat uterus in the metestrus stage of the estrus cycle. Using anti‐mouse renin antibody stain, renin is localized to the tunica media of spiral arteries in the uterus and a larger vessel coursing through the serosa. With anti‐rat renin antibody stain, renin also localized to the tunica media of the same vessels. These results help define a baseline in the amount of renin expression in the uterus with which to compare other stages of the estrus cycle and pregnancy to possibly link renin expression with human conditions of premenstrual syndrome and toxemia of pregnancy. Support by NSF grants #0132823 & 0726185, and D'Youville faculty council research fund
Dental precocity and secondary pneumatization
Authors: Smith Timothy D , Rossie James B , Cooper Gregory M , Carmody Kelly A , Schmieg Robin M , Mooney Mark P , Siegel Michael I
Published: June 2021

Paranasal sinus expansion integrates skeletal elements of the nasal capsule, orbit, and dentition. Computer modeling suggests that sinus cavities appear in spatial "gaps" within the craniofacial complex. Anthropoid primates provide excellent natural experiments for testing this model, since not all possess a full complement of paranasal sinuses. Using histological and microCT methods, we studied an ontogenetic sample of Saguinus spp., which possesses maxillary sinuses (MS), and Saimiri sciureus, which does not form a MS. In Saguinus, secondary pneumatization begins perinatally. From infant to juvenile stages, the MS cavity progressively overlaps all of the deciduous molars (dm). The MS overlays all permanent molars in the adult. In Saimiri, the primordial space of the MS (maxillary recess) extends no farther posteriorly than dm2 at birth and no farther than the last premolar in the adult. Three‐dimensional reconstructions of newborns reveal critical differences that may account for these differences, including relatively larger dm teeth in Saimiri. Radiographic density of dm teeth is more than 20% greater in Saimiri compared to Saguinus, indicating more precocious development. These findings support the hypothesis that secondary pneumatization is a novel, opportunistic growth mechanism that may be constrained by adjacent elements such as the maxillary dentition. Grant support: NSF BCS‐0820751.
Single molecule studies of spliceosomal RNAs U2 and U6
Authors: Guo Zhuojun , Karunathilaka Krishanthi , Rueda David
Published: June 2021

Splicing is an essential step in the maturation reaction of eukaryotic pre‐mRNA, in which intervening sequences (introns) are removed from the coding sequences (exons). The spliceosome is a dynamic assembly of five snRNAs and a large number of proteins that catalyzes splicing. U2 and U6 are the only two spliceosomal snRNAs strictly required for both steps of splicing, and they form RNA complex using base pairing. Major conformational changes are expected to take place during the assembly and catalysis of the spliceosome.

Here, we have used Single Molecule Fluorescence to study the structural dynamics of a protein free U2‐U6 complex from S. cerevisiae. Our data clearly shows a Mg2+ induced large amplitude conformation change of the U2‐U6 complex. In the absence of Mg2+, helix III and the U6‐ISL are in close proximity, while in the presence of Mg2+, these two helices are far from each other. The conformational change consists of a two‐step process, with an obligatory folding intermediate. We hypothesize that the observed conformational change is essential for spliceosomal activation between the first and second steps of splicing. We have tested this hypothesis by measuring the structural dynamics of a point mutant known to block the second step of splicing and the compensatory mutant. Our data strongly supports this hypothesis. We have also studied the role of the highly conserved base U80 in the folding dynamics of the U2‐U6 complex. The data shows that U80 plays an important role in stabilizing the interaction between helix III and the U6‐ISL and without it, helix III and the U6‐ISL cannot come close to each other. We hypothesize that in the absence of Mg2+, U80 flips out of the U6‐ISL and interacts with helix III to bring them in close proximity.

Financial support from Wayne State University (Start‐up funds), the National Science Foundation (CAREER 0747285 to DR), and the NIH (GM085116)
Structure and Function of a sno‐like RNA in Haloferax volcanii
Authors: Harris Julia Louise , Nist Richard , Ginn‐Pease Margaret E. , Hemmingsen Jens , Daniels Charles J
Published: June 2021

Small nucleolar RNA (snoRNA) is known to guide modification of RNA in eukaryotes and archaea. In this study, the structure and function of a conserved halophile sno‐like RNA (sRNA) from the moderately halophilic archaeon, Haloferax volcanii (Hvo.), has been characterized using genetic and biochemical methods. The sRNA is predicted to guide 2′‐O‐ ribose methylation of cytidine‐34 (C34) in elongator methionine tRNA (met tRNA). Northern analysis, RT‐PCR walking and RT‐PCR of cyclized sRNA show that the sRNA is likely to be cotranscibed with an adjoining gene and processed from a larger transcript. A mutant strain of Hvo. (δsRNA) has been constructed in which the sRNA gene has been deleted. The mutant exhibits normal growth, indicating that this gene is not essential. An RNase H cleavage assay has been used to characterize the methylation of met tRNA isolated from wild‐type and δsRNA strains. Preliminary results suggest that the mutant may be lacking in methylation at C34. This work furthers our understanding of the mechanisms of tRNA maturation in the archaea. This work was supported by a NSF grant to CJD and JH (MCB‐0620005).
Urokinase plasminogen activator's affect on cancer progression in non‐small cell lung cancer
Authors: Kutz Craig , Canine Jenny , Provost Joseph J , Wallert Mark
Published: June 2021

The Na+/H+ exchanger isoform 1 (NHE1) plays a key role in cytoskeletal anchoring and regulation of intracellular pH, at the leading edge of migrating cells. This distribution and activity of NHE1 in metastatic cancer cells shows a similarity to the localization of the urokinase plasminogen activator receptor (uPAR) resulting in the stimulation of growth factor pathways and degradation of the extracellular matrix. In this study, the mechanism of uPAR activation was examined for its impact in proliferation, stress fiber formation, and tumor growth. Additionally, the amino terminal fragment of uPA (uPA‐ATF) was also used to activate the uPA response. In H1299, uPA increased ERK activation but no significant change occurred in H460 and H358. In the presence of 10 nM uPA or uPA‐ATF, cellular proliferation increased by 20‐30% in H358 and H460, whereas H1299 showed no significant changes. To investigate whether uPAR activation stimulated cell migration, stress fiber formation was measured. All three cell lines showed a significant increase in stress fibers with uPA stimulation which was blocked by inhibition of NHE1. Finally, to determine whether migration and proliferation could combine to support new tumor formation, a soft agar assay was performed. H460 cells showed a significant increase in large tumor formation. Supported by NIH Grant 1R15CA135616‐01, And NSF Grant MCB‐0817784.
The Regulation of Malate Dehydrogenase by Citrate
Authors: Marion Jimmy , Bell Ellis
Published: June 2021

Malate Dehydrogenase (MDH) catalyzes the oxidation of malate to oxaloacetate through the reduction of NAD+ to NADH and is vital in a variety of cell processes important for metabolic and energy pathways. Glyoxasomal MDH (gMDH) is a homodimer with each subunit contains a dinucleotide binding domain and a malate binding domain. Citrate has long been suggested as an allosteric regulator of the enzyme but crystal structures show only citrate binding to the active site. To define the effects of citrate both kinetic and biophysical approaches have been used. The ability of citrate to inhibit gMDH is shown to be pH dependent in the range from pH 6‐8. At pH 7 & 8 Citrate only inhibits about 60% of the activity at apparently saturating concentrations suggesting subunit interactions are involved in the citrate effect. The extent of inhibition is also dependent upon substrate inhibition by oxaloacetate. Isothermal titration calorimetry (ITC) studies are also done to investigate the affinity of citrate for the active site of the dimeric gMDH. The effects of citrate on local flexibility and stability of subunit interactions [studied by limited proteolysis and dynamic light scattering] are correlated with the kinetic effects.

This research is supported by a Grant from the National Science Foundation: MCB 0448905 to EB
The mechanism of Zinc Inhibition of Glutamate Dehydrogenase Involves Disruption of Subunit Interactions
Authors: Sinanan Leander , Bell Ellis
Published: June 2021

Zinc is a potent inhibitor of higher eukaryotic glutamate dehydrogenase but the mechanism of inhibition has not been established. The goal of the current study is to correlate zinc binding under various conditions with changes in activity and conformational flexibility. Using Isothermal Titration Calorimetry it is shown that zinc binds to glutamate dehydrogenase with a 1:1 stoichiometry in the presence or absence of amino acid substrates. Using tryptophan fluorescence spectroscopy it appears that zinc affects the most hydrophobic of the three tryptophans in the protein. Examination of the three dimensional structure of the enzyme suggests that zinc may bind at the subunit interface in the "twisty tie" region of the molecule. This is consistent with both the location of the most hydrophobic tryptophan and the fact that zinc appears to inhibit by disrupting subunit interactions necessary for full activity with glutamate as substrate. Differential Scanning Calorimetry and Limited Proteolysis approaches demonstrate that zinc binding induces changes in the conformational flexibility of glutamate dehydrogenase which could be responsible for this effect.

This research is supported by a Grant from the National Science Foundation: MCB 0448905 to EB
Regulation of signaling, in human lung cancer cell lines, that controls cellular proliferation and migration: the effect of lysophosphatidic acid and phenylephrine.
Authors: Canine Jenny , Kutz Craig , Wallert Mark , Provost Joseph J
Published: June 2021

One of the hallmarks of cancer is the ability of cells to spread from a tumor of origin to other areas. Metastasis occurs through a coordinated series of events. The Na+‐H+ exchanger (NHE) plays a role in pH regulation and cytoskeletal attachment to the plasma membrane. In this study, we investigated the role of stress fiber formation in cellular migration and tumor formation. To do this, we screened cellular responses to, lysophosphatidic acid (LPA) and phenylephrine (PE) in the presence of absence of the NHE inhibitor, ethyl‐isoproplyamiloride (EIPA) to examine stress fiber formation, cellular proliferation, and tumor formation in three human non‐small lung cancer cell lines. Stimulation with LPA and PE lead to no significant increase in cellular proliferation but lead to an increase in stress by formation in the H460, H358, and H1299 cell lines. Inhibition of NHE by EIPA decreased proliferation by 30‐50% in all cell lines and returned stress fibers formation to basal levels. Using a soft agar assay to examine migration and tumor growth, LPA increased the total number of small tumor formed in H358 and H460 cells. In H358 both PE and LPA increased large tumor formations. These studies indicate that NHE plays a vital role in stress fiber formation and cellular migration and that NHE transport activity is necessary to both functions. Supported by NIH Grant 1R15CA135616‐01 and NSF Grant MCB‐0817784
Investigation of a conserved tyrosine residue in a yeast casein kinase 1 protein kinase
Authors: Day Jordan , Robinson Lucy C , Brame Cynthia J
Published: June 2021

Yck1/2 are essential and redundant plasma membrane‐associated casein kinase 1 (CK1) proteins in budding yeast. In addition to sequence features common to protein kinases, CK1s share features unique to this subfamily. We identified the peptide HIPYRE as such a feature and investigated its role in the function of Yck2 (H233‐E238). CK1s from S. pombe were shown to exhibit dual specificity and to be affected by Tyr phosphorylation. We find that Yck2 is Tyr phosphorylated, and we investigated the possibility that Yck2 activity is regulated by phosphorylation of Y236. We generated mutant YCK2 alleles to investigate growth phenotypes and in vitro kinase activity. Cells expressing only Yck2 deleted for Y236 show defects in common with the low activity yckts mutant, and the mutant protein shows decreased activity on casein in vitro. Y236S and Y236F substitutions do not appear to impair Yck2 activity in vitro or in vivo, but the Y236E protein neither sustains normal growth of yeast nor phosphorylates casein at normal levels in vitro. We conclude that Y236 is important for activity, and that Yck2 activity could be negatively regulated by phosphorylation at this site. Work supported by NSF grant MCB‐0517204.
Gastrointestinal activity of saponins from soy and tomato
Authors: Carlson Emily M. , Chitchumroonchokchai C. , Vodovotz Y. , Schwartz S. J. , Gunn J. S. , Bhatiya A. , Kerem Z. , Berhow M. , Bomser J. A. , Failla M. L.
Published: June 2021

Saponins, a structurally diverse family of secondary plant metabolites, have been suggested to possess anti‐carcinogenic, hypolipemic, hypocholesterolemic, and immune‐enhancing activities. We have initiated studies to compare the effects of saponins extracted from soy (soyasaponin A and B), tomato, and a commercial mixture of phytosterols (positive control) on GI absorption and metabolism of cholesterol and on interactions between microflora and the gut epithelium. During simulated gastric and small intestinal digestion, saponins from tomato and the phytosterol mix significantly inhibited micellarization of cholesterol (14 µM) from a food matrix (ratio of saponins/phytosterols to cholesterol was approximately 1:6.5). Saponins from soy and tomato, as well as phytosterols, also significantly impaired the transfer of cholesterol from Tween micelles to differentiated cultures of human intestinal Caco‐2 cells. Additionally, as saponins share structural similarity with bile salts that repress the transcription of Salmonella enterica invasion gene regulator sirC, studies examining the influence of dietary saponins on the transcription of sirC and on the influence of the bacterium with Caco‐2 cells are ongoing and will be reported Supported by OARDC and USDA BARD grant.
Regulation of LPS‐induced tissue factor expression in human monocytic THP‐1 cells by curcumin
Authors: Guo Weimin , Ratnasari Anita , Li Jing , Meydani Mohsen
Published: June 2021

Tissue factor (TF) is a transmembrane receptor, which initiates thrombotic episodes associated with various diseases. In addition to membrane‐bound TF, we have reported alternatively spliced form of human TF mRNA which was later confirmed that this form of TF mRNA expresses a soluble protein circulating in blood. Curcumin, a natural polyphenol, has been shown to inhibit the induction of TF activity in cultured endothelial cell. Since monocytes are the only circulating blood cells that can be induced to express TF, we tested the effect of curcumin on TF expression in human monocytic THP‐1 cell. We found that curcumin dose‐dependently reduced LPS‐induced procoagulant activity in intact THP‐1 cells, which indicates decreased membrane‐bound TF. However, curcumin at low concentration (10 µM) enhanced LPS‐induced TF protein in the whole cell lysate, and majority of the increased TF protein is a soluble form as indicated in Western blotting. TF mRNA level was also increased at 10 µM curcumin treatment; whereas high concentration of curcumin (40 µM) completely inhibited LPS‐induced TF mRNA and protein expression. Overall, our data show that curcumin could suppress the LPS‐induced procoagulant activity in THP‐1 monocytic cells, but it requires further investigation to understand how the two forms of TF are regulated by curcumin. (Supported by USDA agreement #58‐1950‐7‐707)
English walnuts (Juglans regia L.) improve antioxidant capacity in humans
Authors: McKay Diane L. , Chen Chung‐Yen , Steffen Lyn M. , Blumberg Jeffrey B.
Published: June 2021

The consumption of English walnuts (Juglans regia L.) promotes cardiovascular health, in part due to its favorable effect on serum lipids. Walnut polyphenols, including ellagitannins (ET) may also contribute to this benefit. To determine whether consuming walnuts in amounts readily incorporated into the diet (0.75 and 1.50 oz) has a dose‐response effect on plasma antioxidant capacity, we conducted a randomized, crossover study with a 1 wk run‐in period, 6 wk interventions with each walnut dose, and a 6 wk washout period between these tests in 20 healthy, non‐smoking adults ≥50 y. Subjects followed a low ET diet during the entire 19 wk study, and a low polyphenol diet for 2 d prior to each visit. Walnut intake tended to increase selected measures of plasma antioxidant capacity in a dose‐dependent manner with a higher proportion of responders in the 1.50 vs. 0.75 oz group, particularly in women. These data suggest that walnuts may modestly improve antioxidant capacity in humans, revealing another potential mechanism to further support the FDA qualified health claim that the regular consumption of walnuts decreases the risk of heart disease. (Supported by USDA and California Walnut Commission)
Green tea extract (GTE) protects against nonalcoholic fatty liver disease (NAFLD) in obese mice by decreasing liver injury and improving enzymatic antioxidant defenses
Authors: DiNatale Dana Anne , Park Hea Jin , Chung Min‐Yu , Koo Sung , Bruno Richard
Published: June 2021

GTE contains catechins and may protect against oxidative stress implicated in NAFLD. We hypothesized that GTE would protect against NAFLD by regulating hepatic enzymatic antioxidant (AOX) defenses. We fed 5‐wk old obese mice (ob/ob; n = 38) a diet containing 0, 0.5, or 1% GTE and 0% GTE to lean mice (n = 12) for 6‐wk. Then, serum alanine aminotransferase (ALT) and hepatic AOX defenses were measured. Obese compared to lean controls had 7‐fold greater ALT (p < 0.05) and GTE at 0.5 or 1% decreased ALT by 19‐25%. Mn and CuZn superoxide dismutase (SOD) activities were 25‐29% lower in obese mice and GTE increased Mn‐ and CuZn‐SOD by 29‐42% and 26‐28%, respectively. Catalase (CAT) activity was 40% lower in obese mice and GTE at 0.5 or 1% increased it 26‐42%. Glutathione peroxidase (GPx) activity of obese mice was 30% lower than lean controls and GTE increased it 14‐15%. Glutathione S‐transferase activity was greater in obese mice and further increased by GTE at 1% only. Glutathione reductase activity was higher in obese mice and unaffected by GTE. ALT was inversely related (p < 0.05) to CAT (r = ‐0.395) and GPx (r = ‐0.562) suggesting that these enzymatic AOX activities protect against hepatic injury. Collectively, these data suggest that GTE, in part, restores those AOX defenses impaired by obesity and that GTE may protect against hepatic injury associated with NAFLD. Supported by a grant from the USDA‐NRI (2007‐02303) to RSB.
Characterization, extraction efficiency, stability and antioxidant activity of phytochemicals in Angelica keiskei
Authors: Correa C R , Li L , Chen CYO , Chun H‐K , Cho S‐M , Park K‐M , Aldini G , Carini M , Blumberg J B , Russell R M , Yeum K‐J
Published: June 2021

Angelica keiskei (Ak) is a dark green leafy vegetable associated in traditional Asian diets with longevity. Phytochemicals in Ak and their extraction efficiency were characterized in ethanol/water (70/30, 40/60, 0/100, V/V) and stability at different temperatures were determined. Ak grown in three geographical areas were lyophilized, milled to a powder, and stored at 22ºC, 4ºC or ‐80ºC for 12 months (mo). Carotenoids and ascorbic acid were analyzed by HPLC systems, total phenols by spectrophotometry, polyphenols by LC‐ESI‐MS/MS, and antioxidant activity by fluorometry. Lutein was a principal carotenoid and chlorogenic acid, chalcones, and glucosides of luteolin and quercetin were prevalent phenolic compounds in Ak. Only 46% of lutein was extracted with 70% ethanol and no carotenoids were detected in 40% ethanol or 100% water extracts. The major Ak phytochemicals were stable at ‐80ºC for 12 mo while β‐carotene degraded at 22ºC and 4ºC in ≤2 mo and lutein at 22ºC by 12 mo. Thus, phytochemicals in lyophilized Ak are stable up to 12 mo, except for some carotenoids. Whole dried Ak power is a richer source of most of these phytochemicals than Ak extracts due to their low extraction efficiency in ethanol/water. [Supported by USDA ARS #58‐1950‐7‐707; USA & BioGreen 21 Program Code #20070301034009 RDA, Korea]
Effects of Resistant Maltodextrin on Fecal Microbiota Composition
Authors: Mai Volker , Ukhanova Maria , Baer David , Okuma Kazuhiro , Tagami Hiroyuki , Kanahori Sumiko , Henderson Theresa , Gordon Dennis T
Published: June 2021

Resistant maltodextrin (RM) is a soluble dietary fiber that although resistant to digestion by human enzymes is a substrate for fermentation by the commensal microbiota of the large intestine. We determined the effects of RM on gut microbiota composition in a randomized, controlled dose‐response feeding study in 15 healthy males (14 completed). Subjects were assigned to a treatment sequence that consisted of three 28 day treatments periods with controlled diets: a placebo diet (0 g/d RM + 50 g/d maltodextrin) and two levels of dietary RM (25 g/d RM + 25 g/d maltodextrin and 50 g/d RM + 0 g/d maltodextrin). Fecal samples for the microbiota studies were collected on Days 1, 13 and 24 of each period. Microbiota composition was determined using 16S rRNA based methods that included denaturing gradient gel electrophoresis (DGGE), fluorescent in situ hybridization (FISH), qPCR and 454 based pyrosequencing. Total numbers of bacteria/g of feces increased by 22% in subjects consuming 50 g/d RM (P =0.02). While microbiota diversity appeared unaffected, Bifidobacteria (FISH and qPCR) as well as Rumninococci and Prevotella (454 sequencing) were increased during RM periods. Differences in microbiota composition likely contributed to the variations in energy gain from RM intake observed for study subjects.

Supported by USDA, ARS and Matsutani Chemical Industry Co., Ltd.
Phytosterol ester substrate specificity of pancreatic cholesterol esterase
Authors: Brown Andrew William , Hang Jiliang , Dussault Patrick H , Carr Timothy P
Published: June 2021

Consuming phytosterols and their esters results in decreased LDL cholesterol, an atherosclerotic risk factor. To more completely characterize how phytosterols impart their effects, the present study aimed to determine if pancreatic cholesterol esterase (PCE; EC 3.1.1.13), the enzyme primarily responsible for cholesterol ester hydrolysis in the duodenum, is capable of hydrolyzing various phytosterol esters, and to compare their rates of hydrolysis in vitro. We found that 1) PCE hydrolyzes palmitate, oleate, and stearate esters of cholesterol, stigmasterol, stigmastanol, and sitosterol; 2) specificity was dependent on both the sterol and the fatty acid moieties in the following order of rates of hydrolysis: cholesterol > (sitosterol = stigmastanol) > stigmasterol, oleate > (palmitate = stearate); 3) addition of free phytosterols did not change hydrolytic activity of PCE, while addition of palmitate, oleate, or stearate increased hydrolytic activity. Thus, PCE may play an important but discriminatory role in vivo in the liberation of free phytosterols to compete with cholesterol for micellar solubilization and absorption.

Grant Funding Source

USDA‐NRI competitive grant no. 2007‐35200‐18298
Homeostasis of biotin in human lymphoma and liver cells
Authors: Mall Gaganpreet Kaur , Chew Yap Ching , Zempleni Janos
Published: June 2021

Biotin is covalently attached to carboxylases and histones in a reaction catalysed by holocarboxylase synthetase (HCS). Here we determined whether biotin concentrations in culture media affect the expression of genes maintaining biotin homeostasis in human lymphoid (Jurkat) cells. As a secondary objective we determined whether mechanisms of biotin homeostasis are distinct in cells with great metabolic activities, i.e., human hepatocarcinoma (HepG2) cells. Jurkat cells were cultured in media containing deficient, physiological, or high concentrations of biotin for 36 d. While the abundances of biotinylated carboxylases responded rapidly (<3 d) to altered biotin supply, changes in histone biotinylation were not apparent until day 36 when biotin supplementation caused increased biotinylation of histones in nuclear extracts. The expression of genes linked with biotin homeostasis depended on biotin in Jurkat cells. The expression of biotin transporters SMVT and MCT1 increased in biotin‐deficient cells, whereas the expression of HCS increased in biotin‐supplemented cells. In ongoing studies we investigate the effects of biotin supply on histone biotinylation at distinct genomic loci by ChIP assays, and we compare biotin homeostasis in Jurkat cells to HepG2 liver cells. (Supported by UNL ARD Hatch Act funds, NIH DK063945, DK077816 and ES015206, USDA CSREES 2006‐35200‐17138, and NSF EPSCoR EPS‐0701892.)
Oxidative stress in cardiac mitochondria caused by copper deficiency may be insufficient to damage mitochondrial proteins
Authors: Johnson William T
Published: June 2021

Copper (Cu) deficiency may promote the generation of reactive oxygen species (ROS) by the mitochondrial electron transport chain through inhibition of cytochrome c oxidase (CCO) and increased reduction of respiratory complexes upstream from CCO. In the present study, respiration, H2O2 production and aconitase activity were measured in isolated heart mitochondria from weanling rats fed diets that were either deficient (CuD, <1ppm Cu), marginally deficient (CuM, 1.5 ppm Cu) or adequate (CuA, 6 ppm Cu) in Cu for 8 weeks. State 4 respiration (μmole O2 ·min−1 ·mg−1) in CuD rats (2.81±0.71) was lower (P<0.05) than in CuM (3.74±0.88) or CuA rats (3.72±0.78). H2O2 production (nmol · min−1 ·mg−1) in the presence of rotenone was marginally higher (p=0.07) in CuD rats (11.2±4.1) than in CuM rats (8.5±2.4) or CuA rats (8.2±2.6). However, aconitase activity (mU ·mg−1) was higher (P<0.05) in CuD rats (58.5±12.5) than in CuM (25.8±7.0) or CuA rats (28.4±9.3). Aconitase is readily inactivated by ROS and these findings suggest that even though severe Cu deficiency impairs mitochondrial function, mitochondrial oxidative stress is not elevated to a level that leads to oxidative inactivation of susceptible mitochondrial enzymes. Enhancement of aconitase activity may reflect enhanced TCA cycle activity for maintenance of cardiac energy production during Cu deficiency.

Supported by USDA‐ARS
Bioavailability of purple carrot anthocyanins is influenced by acylation but not plant matrix effects
Authors: Charron Craig S. , Kurilich Anne C. , Clevidence Beverly A. , Simon Philipp W. , Harrison Dawn J. , Britz Steven J. , Baer David J. , Novotny Janet A.
Published: June 2021

Bioavailability of anthocyanins from juiced purple carrots was investigated through a human feeding study. Ten healthy adults consumed three doses of purple carrot juice, and bioavailability was assessed by appearance of anthocyanins in plasma for 8 h after the dose. Doses were 50 mL, 150 mL, and 250 mL of purple carrot juice, containing 76.1 µmol (64.5 mg), 228.1 µmol (193.6 mg), and 380.2 µmol (322.7 mg) total anthocyanins, respectively. The peak plasma anthocyanin concentration increased significantly with increasing dose, though this increase was smaller in magnitude than the increase in dose size. Acylated anthocyanins comprised 76% of total anthocyanins in the juice, yet their bioavailability was found to be significantly less than that of nonacylated anthocyanins. Peak plasma concentration of nonacylated anthocyanins was 4‐fold higher than that for acylated anthocyanins. Because the treatments were consumed as juice, it could be discerned that the difference in bioavailability of acylated vs. nonacylated anthocyanins was not related to interaction of the anthocyanins with the plant matrix. Funded by USDA/ARS and USDA/CSREES.
Renal 25‐hydroxycholecalciferol reabsorption is modulated in a type II diabetic rat model.
Authors: Anderson Rachel L , Rowling Matthew J
Published: June 2021

Alterations of serum levels of the major circulating form of vitamin D (25‐hydroxycholecalciferol, 25D3) and its active hormone derivative (1,25‐dihydroxycholecalciferol, 1,25D3) have long been associated with type II diabetes, although a mechanistic basis for this occurrence has not been fully elucidated. Renal reabsorption of vitamin D occurs by receptor‐mediated endocytosis of the 25D3‐vitamin D binding protein (DBP) complex via megalin‐, cubilin‐, and disabled‐2 (Dab2)‐mediated endocytosis. Because renal dysfunction can lead to decreased expression of megalin and proteinuria , changes in serum vitamin D status in the uncontrolled diabetic may be due to increased urinary excretion of protein‐complexed 25D3. In the present study, we used a type II diabetic rat model [Zucker Diabetic Fatty Rats (ZDF)] to characterize the relationship between renal reuptake of 25D3‐DBP and vitamin D homeostasis. Using immunoblotting and real‐time PCR, we analyzed transcript and protein levels of renal megalin, cubilin, and Dab2 and serum and urinary 25D3,1,25D3 and DBP levels in 16 wk old ZDF rats. Our preliminary data suggest that renal megalin and cubilin mRNA and protein expression is reduced in diabetic animals when compared to their lean non‐diabetic controls. In addition, preliminary results indicate that urinary excretion of DBP is markedly elevated in diabetic rats. Taken together, these data suggest that vitamin D status is compromised in diabetics due to poor vitamin D reabsorption in the kidney. Supported by USDA (Iowa State University Nutrition and Wellness Center SRPG to MR) and the Iowa State University Experiment Station.
The growth performance, carcass traits, meat pH and color, and CO2 production in pigs supplementated arginine with conjugated linoleic acid
Authors: go gwang‐woong , Wu Guoyao , Smith Stephen B.
Published: June 2021

This experiment tested the hypothesis that the combination of dietary conjugated linoleic acid (CLA) plus arginine will increase the growth performance, carcass traits, meat quality, and CO2 production. Eight pigs (80kg) were assigned to treatments in a 2 x 2 factorial design with two pigs per treatment (control: 2.05% alanine + 1% canola oil; CLA: 2.05% alanine + 1% CLA (mixed isomers); arginine: 1% arginine + 1% canola oil; arginine + CLA: 1% arginine + 1% CLA). Preliminary tests showed up to 2% arginine plus 1% CLA supplementation can be acceptable without interfering lysine absorption. There was no significant difference in growth performance including average daily feed, average daily gain over the five week treatment period. Dietary arginine and CLA did not affect either carcass or meat quality traits including loin area, back fat thickness, pH45min, pH24hr, meat color (L*, a*, and b*), and drip loss. Rates of glucose and palmitate conversion to CO2 in vitro in liver, longissimus dorsi muscle, abdominal and subcutaneous fat, and enterocytes were not affected by CLA. However, arginine increased CO2 production in all tissue except muscle. Also, CO2 production in both adipose tissue deposits were increased by CLA and arginine (P < 0.05), (This project was supported by National Research Initiative Competitive Grant no. 2008‐35206‐18762 from the USDA Cooperative State Research, Education, and Extension Service).
Calcium intake in African American and Caucasian female college students and its relationship with weight
Authors: Douglas Crystal C , Shin Hyehyung , Liu Pei‐Yang , Ilich Jasminka Z
Published: June 2021

National surveys indicate that women in the United States do not consume enough dairy products, and as a result, are not meeting the recommendations for dietary calcium (Ca). This might result in negative consequences not just for bone health, but for body weight as well, in view of studies showing a negative relationship between overweight/obesity and Ca. The purpose of this study is to assess dietary Ca intake among college females and identify whether it is related to body weight. Volunteers on college campuses were asked to complete a validated food frequency questionnaire assessing usual Ca intake from food. Volunteers include 50 African American and 54 Caucasian females, 18‐37y and BMI (kg/m2) of 26.8±6.6 and 23.1±4.0, respectively (mean±SD). Race, height, and weight were self‐reported. Ca intake was similar between African Americans and Caucasians (1291±761 vs. 1465±934, respectively; p= 0.30), but Caucasians consumed significantly more Ca per kg body weight than African Americans (23.7±16.1 vs. 18.6±10.1; p=0.05). There was a nearly significant difference in Ca intake between normal weight and overweight/obese volunteers such that those with a BMI=25 kg/m2 consumed more Ca per kg of body weight (23.1±14.1 vs. 17.8±12.4; p=0.06). These results show that surveyed college females are meeting recommendations for Ca and suggest that Ca intake may be related to markers of body weight. Funded by USDA/CSREES/NRI #2004‐05287
Summer doesn't reverse freshmen weight gain
Authors: Gropper Sareen S , Gaines Alisha , Saunders Desiree , Clary Kayla , Connell Lenda Jo , Simmons Karla , Ulrich Pamela
Published: June 2021

The "freshman 15" phenomenon, referring to the popular belief that freshmen gain 15 lbs their first year of college, was examined in 240 college freshmen. Body weight (wt), percent (%) body fat, and lean body mass (lbm) were measured using standard anthropometric techniques thrice during the 2007‐08 academic year (acad yr) and at the beginning of fall semester 2008 (BF08). About 66% of the freshmen gained wt over the acad yr; only 5% gained the notorious 15 lbs. Acad yr wt gain was 2.6 + 5.3 lbs for the entire group (n=205, 132 females, 73 males) and 6.0 + 5.3 lbs for the wt gain group (n=135, 82 females, 53 males). Percent body fat, but not lbm, significantly increased 1.4 + 2.2 % for the entire group and 2.0 + 2.2% for the wt gain group over the acad yr. About 47% of the students who had gained wt over the acad yr lost wt (‐3.7 lbs) over the summer, while 51% gained more wt (3.0 lbs). Summer wt gain for the entire group (n=167) averaged < 1/4 lb; however, 16% gained > 5 lbs. Wt and % body fat, but not lbm, were significantly greater BF08 than 1 year earlier for the entire group, but did not significantly differ from the end of the acad yr. These findings show that wt gained by college freshmen during the acad yr was not lost over the summer. Moreover, the "freshman 15" appears to apply only to a small percentage of college freshmen. This research was funded by USDA AL Agricultural Experiment Station (AAES) projects 013‐020 and 07‐020, and an AAES Initiative Grant.
Differences in variety of vegetable consumption in low income women
Authors: Lyly Marika , Keim Nancy L. , Casey Shavawn M , Aaron Grant J , Townsend Marilyn S
Published: June 2021

MyPyramid includes recommendations for quantity and variety of vegetable intake. In previous studies, increased variety of vegetable intake was inversely related to BMI. We examined this association in low‐income women in California. Participants (n=112) completed survey instruments to assess dietary intake, behaviors and attitudes related to food intake. Three groups were formed based on variety of vegetables eaten weekly: low variety (LV) ≤ 5 different vegetables/wk, moderate (MV) 6‐9 vegetables/wk, high (HV) ≥ 10 vegetables/wk. BMI did not differ between groups, but descriptors of diet quality were different. Daily consumption of all vegetables averaged 1.0 cups in LV, and 2.8 cups in HV (p<0.001). The Healthy Eating Index score was lower and energy density of the diet was higher in LV compared to MV and HV (p<0.001). Top 5 vegetables most commonly consumed by LV included 3 starchy vegetables of high energy density, but only nutrient dense vegetables were in the list of top 5 for HV. More women in HV thought it was important that foods they ate were healthy (p<0.03). Although variety of vegetables was not related to BMI in this low‐income sample, greater vegetable variety was linked to better diet quality and higher quantities of vegetables consumed. Interventions designed to encourage and support variety in vegetable intake for this population are needed. Supported by USDA CSREES NRI grant #2004‐35215‐14441.
Alterations in the Mitochondrial Proteome in Broilers fed a Lysine‐Deficient Diet
Authors: Pomeroy Stephanie Katherine , Williams Patricia M , Webb Kenny E , Blemings Kenneth P
Published: June 2021

In liver, the primary site of lysine degradation is the mitochondrial matrix, but the protein(s) responsible for lysine uptake to the matrix is(are) unknown. Changes in inner mitochondrial membrane protein abundances were observed in broilers fed lysine‐adequate (LA) or lysine‐deficient (LD) diets for 14 days. Two transport proteins were affected by the LD diet. Broilers consuming the LD diet expressed only 50% of a voltage‐gated anion‐selective and a voltage‐gated potassium channel compared to those consuming a LA diet, although presumably lysine is not transported via either of these proteins. The consumption of the LD diet impacted many aspects of metabolism. Glutamine synthase expression was decreased over 95% in the chickens consuming the LD diet. Electron transport chain (ETC) components were also altered. Broilers consuming the LD diet had over a 30% reduction in NADH dehydrogenase expression and almost a 90% decrease in the expression of ATP sythase. Proteins involved in fatty acid oxidation were also affected by the LD diet, as Acyl CoA dehydrogenase was reduced about 75%. These data indicate that a LD diet affects many aspects of metabolism that may contribute to the reduction of growth typically seen with a lysine deficiency. (Support USDA‐NRI 2004‐35206‐14160)
(‐)Huperzine A, replacement for pyridostigmine bromide as nerve agent pretreatment, measured in Guinea Pig plasma by a new ultrahigh‐pressure liquid chromatography (UHPLC)‐MS method.
Authors: Garcia Gregory E , Vernon Alison , Moorad‐Doctor Deborah , Ratcliffe Ruthie H
Published: June 2021

Huperzines are a class a of compounds extractable alkaloids from the club moss Huperzia serretia. Moss extract, as a tea, has been used in China to improve cognitive abilities. (‐) Huperzine A (HUP) is a potent reversible acetylcholinesterase inhibitor that can cross the blood‐brain barrier. It is under investigation as an Alzheimer's treatment. These properties may also be useful for a protection against organophosphate (OP) nerve agent poisoning of peripheral and CNS acetylcholinesterase. The guinea pig is the rodent model of choice for OP studies. It has low circulating levels of OP hydrolyzing carboxylesterase, like humans, but unlike rats or mice with high levels that can complicate protection studies. For HUP guinea pig studies we developed an ultrahigh‐pressure liquid chromatography (UHPLC)‐MS method with solid phase extraction (SPE) clean up. HUP recovery from SPE was > 90% for range 0.5 to 20 ng/mL. The UHPLC‐MS was sensitive with an LOD limit of 1 pg. The limits LOQ were 0.32 to 1000 ng/mL (highest tested) with the LLOQ 5 x S/N. Here we demonstrate assay utility in pilot ADME studies in guinea pig plasma from single I.M. doses of 3, 9, and 18 mg/kg. This work was supported by the Defense Threat Reduction Agency.
The physiological plasticity of salinity tolerance in Fundulus species
Authors: Galvez Fernando , Whitehead Andrew , Zhang Shujun
Published: June 2021

Despite their renowned euryhaline lifestyle, considerable variation in salinity tolerance appears to exist amongst different species of Fundulus, and amongst different populations of a single Fundulus species. This presentation will describe the physiological plasticity and the genomic underpinnings of abrupt salinity transfers in different populations of F. heteroclitus, F. majalis, F. diaphanus, and F. grandis collected along the Atlantic or Gulf of Mexico coasts of the U.S. Gills have been archived for transcriptomic profiling, and many other tissues have been analyzed for comprehensive physiological profiling. Physiology data processed so far (plasma osmolality and gill epithelial structure) indicate significant differences in plasticity between populations and species of Fundulus. Current work is also investigating the adaptive role of claudins, a constitutive component of epithelial tight junctions, in regulating the paracellular movement of solutes during osmotic stress. We have recently cloned several claudin genes from the gills of Fundulus, and are assessing their regulation during abrupt salinity transfers. This work was funded by an NSF grant to FG and AW (Award no. 0723771).
Direct and indirect signaling through the epidermal growth factor receptor (EGFr) exerts distinct effects on surface expression of Na+, K+, 2 Cl− cotransporter‐1 (NKCC1) in colonic epithelial cells
Authors: Chappell Alfred E , Barrett Kim E
Published: June 2021

NKCC1 participates in ion transport in many tissues, including chloride secretion by the intestinal epithelium. EGF regulates intestinal chloride secretion and EGFr can be differentially activated to elicit divergent, stimulus‐specific signaling. Using surface biotinylation, immunoprecipitaion, and western blot analysis in human T84 colonic epithelial cells, we now report that while NKCC1 is internalized in response to carbachol (CCh) through an EGFr‐dependent mechanism, it is not internalized in response to EGF. Thus, after 60 min. basolateral stimulation with 100µM CCh or 100ng/mL EGF, CCh induced significant internalization of NKCC1 (202.3 ± 15.8% vs. control, p<0.001, mean ± SEM, n=11) while EGF did not (n=11). Further, the effect of CCh was blocked by the EGFr inhibitor, AG1478 (1µM, n=6). NKCC1 was ubiquitinated in response to CCh (200.7 ± 41.6% vs. control, p<0.05, n=5) but not EGF (n=3). While it was seemingly not degraded, internalization was proteasome‐dependent and inhibited by MG132 (10µM, n=7). CCh‐stimulated internalization of NKCC1 was also inhibited by PD98059 (50µM) or SB203580 (10µM), indicating involvement of ERK and p38 mitogen‐activated protein kinases. We conclude that the differential effect of direct and indirect activation of EGFr on NKCC1 localization and perhaps activity may have implications for chloride secretion in the colon and in other tissues.

Supported by NIH and NSF.
Waveform distortion in TGF‐mediated limit‐cycle oscillations: Effects of TAL flow
Authors: Layton Anita T , Moore Leon C , Layton Harold E
Published: June 2021

Previously, we used a mathematical model to show that nonlinearities in TGF signal transduction produces a nonsinusoidal [Cl−] waveform at the macula densa (MD) when TAL inflow is sinusoidal. The distorted [Cl−] waveform exhibits slope asymmetry and a broad trough relative to the peak. These characteristic features are evident in many, but not all, experimental records of TGF limit‐cycle oscillations (LCO). In the present study, we used a mathematical model of the TAL with compliant walls to examine the effect of TAL flow perturbations on waveform distortion. Step‐like changes in TAL inflow pressure were examined, as were sinusoidal oscillations in inflow input pressure at differing baseline pressures. The results show that mean TAL flow strongly influences the [Cl−] waveform at the MD. This phenomenon is a consequence of the inverse relationship between flow and transit time, which produces asymmetry between up‐ and down‐slopes of the oscillation, and the nonlinearity of TAL transport at low flow rates, which broadens the trough of the oscillation relative to the peak. These results provide an explanation for the range of waveform distortion found in experimental recordings of TGF‐mediated LCO. The degree of distortion may provide insight into TAL function, including, e.g., fractional Na+ and Cl− reabsorption in the TAL. This research was supported in part by NIH grant DK‐42091 and NSF grant DMS‐0715021.
Search and repair of bona fide base‐pair mismatches in DNA
Authors: Hingorani Manju M , Zhai Jie
Published: June 2021

The DNA mismatch repair system (MMR) helps maintain genome integrity by correcting base‐pair mismatches and insertion/deletion loops in DNA. MMR proteins are thus critical for limiting mutagenesis, and their malfunction is linked to sporadic cancers and the HNPCC syndrome. MutS proteins perform the challenging task of finding a few errors against a background of millions of correct base pairs in DNA, and initiate repair. In order to understand the mechanism by which MutS proteins locate these errors, we measured the movement of S. cerevisiae MutS (Msh2‐6), between near Watson‐Crick (2‐Aminopurine:T) and mismatched (G:T) base‐pair sites on DNA. We found that Msh2‐6 binds both sites at equally high rates (2x107 M−1s−1), but it leaves the matched site ~50‐fold faster than the mismatched site (t1/2 ~ 0.4 versus 18 s, respv). These data suggest that while Msh2‐6 makes similar initial contacts with various base pairs, it recognizes mismatches by forming stable contacts specifically at these sites. Notably, Msh2‐6 leaves the matched site and arrives at the mismatched site without dissociating from DNA, which suggests that the protein slides on DNA, probing base pairs via transient contacts until it is trapped at a bona fide mismatch. A sliding mechanism likely enables MutS proteins to scan long stretches of DNA rapidly, thereby ensuring efficient repair of infrequent aberrations in the double helix. Research support: NSF.