bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the structure and function of a Forkhead Associated (FHA) domain containing gene, DAWDLE (DDL), in Arabidopsis thaliana
Authors: Narayanan Lakshmi Ayiloor , Chevalier David
Published: June 2021

DDL encodes a protein bearing an FHA domain in Arabidopsis thaliana. FHA is a phospho‐threonine binding domain in plant and animal proteins that function in DNA repair and cell cycle regulation. N‐terminal region of DDL displays sequence homology with proteins involved in RNA processing. DDL can bind to RNA and microRNA in vitro. SMAD nuclear interacting protein 1, a DDL ortholog in human, regulates RNA stability of Cyclin D1. We propose that DDL is involved in RNA processing and stability of genes regulating cell cycle in plant and aim to elucidate the potential role of DDL in RNA stability and its signaling pathway by a genetic approach. T‐DNA insertion knockout alleles of DDL exhibit pleiotropic developmental defects with short root and hypocotyl, reduced fertility and distorted organs. At the molecular level, ddl plants display reduced amount of microRNAs. Twelve point mutations spanning DDL are being studied to compare the severity of each point mutation allele and knockout alleles to understand the structure‐function of DDL. A genetic screen for identifying components of the DDL signaling pathway yielded one strong and two weak suppressors of ddl. Phenotypic characterization and molecular mapping are in progress for the suppressors. In conclusion, the potential role of DDL in RNA stability and its pleiotropic phenotype makes it a crucial gene to be studied. This research is funded by Mississippi State University.
Postnatal AFP Repression by Zhx2 through RNA Splicing Inhibition
Authors: Turcios Lilia , Spear Brett , Peterson Martha
Published: June 2021

The alpha‐fetoprotein (AFP) gene is highly expressed in the fetal liver and repressed at birth. This postnatal repression is controlled, in part, by Zhx2 (PNAS, 102:396). BALB/cJ mice have reduced Zhx2 expression and higher adult liver steady‐state AFP mRNA levels compared to other mouse strains. The AFP promoter is sufficient to confer Zhx2‐mediated regulation to a reporter gene in transgenic mice, suggesting that the promoter is required for repression. Despite differences in steady state AFP mRNA levels in the adult liver between BALB/cJ and wild‐type mice, transcription rates across this gene were similar. We found accumulated unspliced RNA across multiple AFP introns in wild‐type mice where mature AFP mRNA levels are low. In our analysis of mRNA from cytoplasmic and nuclear RNA fractions of BALB/cJ mouse liver with or without the Zhx2 transgene, we found unprocessed cytoplasmic AFP transcripts in the presence of Zhx2, consistent with a global repression of splicing. We are purifying Zhx2‐associated proteins from liver nuclear extracts; the identity of co‐purified factors will provide insight into the mechanism by which Zhx2 couples transcriptional/post‐transcriptional control of AFP repression.
Using Thermotolerance to Explore Differences in Caenorhabditis elegans Diet
Authors: Lee Laura , Gomez Fernando , Srinivasan Chandra , Clarke Catherine F
Published: June 2021

Previous studies indicate that aerobic respiratory metabolism in the Escherichia coli diet of Caenorhabditis elegans is important in reducing worm lifespan and compromises the worms’ ability to survive in high temperature environments for prolonged periods. We hypothesized that fermentative metabolism in the E. coli diet of C. elegans will also provide increased thermo tolerance and increased chemical tolerance. Wild type worms were raised at 22°C on one of two E. coli strains (OP50 vs. GD1 or AN180 vs. AN120) for 48 hours (first larval stage to L4 young adult). We then exposed the L4 worms to 35°C to induce oxidative stress and activate heat shock survival pathways and monitored their survival. In our chemical stress assay, we induced the production of superoxide radicals by exposing L4 worms fed either OP50 or GD1 on plates containing varying concentrations of paraquat (0 mM to 100 mM) for 20 hours. After four thermostress trials with OP50 vs. GD1 we found that 50% worms fed GD1 bacteria survive approximately 1.5 hrs longer than worms fed OP50 (p=0.005). The worms exposed to AN120 vs. AN180 E. coli showed no difference in survival between the two groups; time to 50% survival was 8–9 hours for both groups. Three paraquat trials showed that worms fed GD1 had a similar trend in survival between trials, however, there is a large amount of variation between the survival curves of the OP50 fed worms.
The role of methionine cycle metabolites in autoimmune neurodegenerative diseases
Authors: Gardner Lidia A , Hartzes Anastasia M , Clark Daniel , Gotuzzo Eduardo , Levin Michael C
Published: June 2021

Objective

To compare levels of Homosysteine (Hcy), S‐adenosyl‐methionine (SAM) and Methionine‐Adenosyltransferase‐II (MAT‐II) enzyme activity in patients with multiple sclerosis (MS) and human T‐lymphotrophic virus type 1 (HTLV‐1) associated myelopathy/tropical spastic paraparesis (HAM/TSP).

Four groups were evaluated: 1) HAM/TSP; 2) asymptomatic carriers of HTLV‐1; 3) MS and 4) normal age‐matched controls.

The mean plasma tHcy levels were higher in HAM/TSP (9.49±1.53μM/L), MS (9.85±1.77μM/L) and the asymptomatic group (12±1.4μMol/L) compared to controls (6.56±0.98μMol/L). The asymptomatic HTLV‐1 infected patients had statistically higher (P=0.0046) tHcy levels compared to the other groups (P=0.0277, Kruskal‐Wallis test). Also, when stratified by age, there was a trend towards significance in Hcy levels comparing diseased (HAM/TSP, MS) to normal controls. Next, we compared MAT‐II activity between MS patients and age‐matched controls. MAT‐II was higher in MS group (Km = 18.84 in MS vs. 12 in controls, P=0.0074). There was no significant difference in SAM (mean range from 10.92 to 12.97μMol) levels between any of the groups. These data underscore the complexity of the methionine cycle and suggest that high tHcy levels may be a marker for viral infection or cause damage to neurons by activation of oxidative injury and DNA damage pathways in autoimmune neurological disease.
Enhanced expression of Glia maturation factor in Alzheimer's disease
Authors: Zaheer Asgar , Zaheer Smita , Thangavel Ramasamy , Ramamoorthy Lavanya , Yang Xi , Sahu Shailendra K
Published: June 2021

We have previously demonstrated that Glia maturation factor (GMF), a brain‐specific protein isolated, sequenced and cloned in our laboratory, is a prominent mediator of inflammation in CNS leading to the death of neurons. In the present study we demonstrate, for the first time, a significant up regulation of the GMF in AD brain compared to age matched ND (non‐demented) controls. We analyzed brain regions by ELISA using specific anti‐GMF antibodies developed and characterized in our laboratory. We observed a significant increase in GMF concentration in entorhinal cortex, parietal cortex, frontal cortex, occipital cortex, perirhinal cortex, and temporal cortex of AD brain. The association between augmented expression of GMF and APs/NFTs in AD was also evaluated. Double immunofluorescence labeling for GMF and APs show strongly GMF‐positive cells with neuron and glia morphology are co‐localized with APs and NFTs. The astrocytes adjacent to the APs display ramified processes and express GMF and GFAP, indicative of an activated phenotype associated with inflammation. Our results provide strong evidence that under conditions of neuro degeneration in AD, the expression of GMF is significantly upregulated. These results imply that augmented expression of GMF is associated with AD pathology.

(Supported by VA Merit Review award and NIH/NINDS grant NS‐47145)
DYSREGULATION OF LIPOLYSIS AND LIPID METABOLISM IN VISCERAL AND SUBCUTANEOUS ADIPOCYTES BY HIGH‐FAT DIET
Authors: Ceddia Rolando B. , Anthony Nicole M. , Patel Prital , Noor Fawad , Hawke Thomas J , Gaidhu Mandeep P.
Published: June 2021

This study investigated the molecular mechanisms by which high‐fat diet (HFD) dysregulates lipolysis and lipid metabolism in mouse epididymal (visceral, VC) and inguinal (subcutaneous, SC) adipocytes. HFD feeding increased ATGL content and CGI‐58 expression, whereas HSL phosphorylation and perilipin content were reduced. This was accompanied by increased basal but blunted epinephrine‐stimulated lipolysis and increased DAG content in VC and SC adipocytes. In HFD VC adipocytes, activation of adenylyl cyclases by forskolin increased HSL phoshorylation and matched the lipolytic response of control VC cells. However, in HFD SC adipocytes, forskolin induced lipolysis without detectable HSL phosphorylation. This suggests activation of an alternative lipase in response to the HFD‐induced suppression of HSL phosphorylation/activation in VC and SC adipocytes. HFD also powerfully inhibited basal, epinephrine‐, and forskolin‐induced AMPK activation as well as the expression of PGC‐1α and PPARα, citrate synthase activity, and palmitate oxidation in both fat depots. In summary, defective adrenergic receptor signaling combined with up‐regulation of ATGL, and suppression of HSL and AMPK signaling mediate HFD‐induced dysregulation of lipolysis and lipid metabolism in VC and SC adipocytes.
Role of 4‐hydroxynonenal in oxidative stress induced signaling
Authors: Chaudhary Pankaj , Sharma Rajendra , Sharma Abha , Vatsyayan Rit , Yadav Sushma , Hadley Marcus , Awasthi Sanjay , Awasthi Yogesh Chandra
Published: June 2021

Objectives

We have shown that 4‐HNE induces signaling for apoptosis in cells via death receptor (Fas) mediated extrinsic and p53‐mediated intrinsic pathways. However, the nature of interactions of 4‐HNE with the components of these signaling pathways are not understood. During present studies, we have elucidated the pathways of 4‐HNE induced signaling for apoptosis in HepG2 cells.

Results

Our results showed that 4‐HNE promoted apoptosis in HepG2 cells through activation of p53, Bax, p21, JNK, and caspase3. Our results also showed that exposure of HepG2 cells to 4‐HNE leads to the activation of Fas, Daxx, ASK1, JNK, and caspase3 and these effects of 4‐HNE could be inhibited by the over expression of hGSTA4‐4. 4‐HNE also induced translocation of Daxx from nucleus to cytoplasm which caused inhibition of apoptosis and the activation and the translocation of HSF1 to nucleus resulting in the up‐regulation of HSP70. We also demonstrate that 4‐HNE does indeed bind to Fas, Daxx, and p53 to affect Fas and p53 mediated signaling.

Conclusion

These findings suggest that 4‐HNE generated during oxidative stress promotes apoptosis through multiple pathways, but upon sustained oxidative stress conditions, 4‐HNE may provide protection to cells by up regulation of heat shock proteins and by Daxx mediated inhibition of apoptosis.( NIH grants ES 012171, EY04396 (YCA) and CA 077495 (SA))
Investigating Mechanisms that Mediate Cyclooxygenase‐2 Signaling in Colon Cancer Development
Authors: Bean Ronald R. , Harpster Kelsey L. , Hessel Kara M. , Jakobi Rolf , Chang Yu‐Wen E.
Published: June 2021

During early development of colon cancer, cyclooxygenase 2 (COX‐2) is constitutively induced. Constitutive expression of COX‐2 promotes colon carcinogenesis, however the underlying mechanisms are not totally understood. Western blot analysis showed reduced protein levels of an adhesion molecule α‐catenin in HCA‐7 colon cancer cells that endogenously express COX‐2 and HCT‐15 colon cancer cells that stably express recombinant COX‐2 (HCT‐15‐COX‐2). High levels of phosphorylated active ERKs were also detected in HCA‐7 and HCT‐15‐COX‐2 cells. Inhibition of COX‐2 stimulated α‐catenin levels and prevented activation of ERKs. Scratch wound gap assays were used to monitor cell motility and proliferation. HCT‐15‐COX‐2 cells closed the gap faster than parental HCT‐15 cells. Inhibition of COX‐2 or ERKs in HCA‐7 and HCT‐15‐COX‐2 cells decreased the gap closing. The Human Cancer RT2‐miRNA PCR array screening showed that 39 microRNAs (miRNAs) are modulated by COX‐2 signaling. Quantitative RT‐PCR confirmed that expression levels of 6 miRNAs were significantly lower in HCA‐7 or HCT‐15‐COX‐2 than in HCA‐7 cells that were treated with the COX‐2‐selecetive inhibitor NS‐398 or HCT‐15 parental cells. Those 6 miRNAs were selected due to their relevance to cancer cell migration. In summary, we have identified that activation of ERKs and modulation of miRNAs mediate COX‐2 signaling to promote colon carcinogenesis.
Biodegradation of Cellulosic Wastes Using Aspergillus Niger: Prospect for Poultry Feed Formulation
Authors: Babalola Musa Oladayo , Okochi Ihuaku Veronica , Okafor Uzoma Anthony
Published: June 2021

Bioconversion of cellulosic biomass to commodity products and fuels has many potential benefits. This could alleviate food and energy shortages, diminish municipal pollution problems, and transform renewable organic matter into valuable resources. Two cellulosic materials; Aboria sawdust and sugarcane pulp pretreated by milling and ammonia steeping, then biodegraded with Aspergillus niger. On fermentation, the release of reducing sugar, change in pH, protein, and cellulosic hydrolases were monitored for a period of fifteen days. The fermented cellulosics were then used to formulate poultry feed. The formulated feed contained other ingredients in conventional feed except the carbon source. The formulated feed was fed to three‐weeks‐old broiler chicks along with convectional fed (control group). Their gain in body weights was monitored. The birds in the test group fed with the formulated feed showed slower body weight gain compared with control (birds on conventional feed). However, there was no statistical difference in the weights of both groups at p>0.01. Post sacrifice analyses reveal that the difference in weights was due to the difference in cholesterol deposition in the flesh of the birds, although a health advantage. Other organs were found not to be significantly different (p>0.01) from each other. By implication, the disposal of cellulosic wastes through biodegradation to value added products could lead to production of animal feed for healthier source of poultry birds and also to achieve wealth.
Investigation of Mutations of Sunflower Cotyledon Acetoacetyl CoA Thiolase
Authors: Cadet Melissa , Gomez Iris , Schiedel Anke C. , Oeljeklaus Silke , Dyer James H.
Published: June 2021

Acetoacetyl CoA thiolase is one of two thiolase activities found in sunflower cotyledons. This thiolase activity is involved in the oxidation of the short‐chain substrate acetoacetyl CoA to produce two acetyl CoAs and also has been characterized as a synthetic thiolase via the formation of acetoacetyl CoA from two acetyl CoAs. Crystal structures of thiolase from other organisms have identified conserved cysteines and a histidine present in the active site of the enzyme indicating that these residues probably play a key role in the reaction mechanism and other residues have been implicated in the stabilization of the enolate intermediate in the synthetic reaction. Site‐directed mutagenesis of the various amino acid residues was done and the effect of these mutations on the catalytic activity was measured.
Endothelial Tip Cell Guidance and Mechanisms
Authors: Franco Claudio Areias , Jakobsson Lars , Rosewell Ian , Bentley Katie , Busse Marta , Gerhardt Holger
Published: June 2021

Blood vessel formation through sprouting angiogenesis is a highly dynamic process involving selection of leading tip cells that will guide extending vessels along attractive and repulsive cues. VEGFRs ‐1, ‐2 and 3 are differentially expressed in the endothelium of growing vessels. The importance of such restricted receptor expression in patterning of the blood vasculature has not been clarified. Using a stem cell based mosaic sprouting assay, we illustrate that differential VEGFRs expression determines the potential of individual endothelial cells to reach the tip cell position and therefore outcompeted neighbour cells adquire stalk fate. Interestingly, we show that Dll4‐mediated Notch signalling is involved in the establishment of the differential receptor expression. Blocking Notch signalling abrogates the advantage of wildtype cells over VEGFR2 heterozygous cells, suggesting that VEGFR levels act upstream of Dll4/Notch signalling in the competition process, through induction of Dll4 expression. Importantly, mosaic in vivo studies confirm these observations. Our findings illustrate the importance of regulated VEGFR levels and the tight integration with the Dll4‐Notch signalling mechanism for shaping the vasculature.
Stoichiometric Quantification of Akt Phosphorylation using LC‐MS/MS
Authors: Atrih Abdelmadjid , Turnock Dan , Sellar Grant , Thompson Alastair , Ferguson Michael , Huang Jeffrey
Published: June 2021

Quantification of phospho‐Akt is a standard way of assessing the activity of the Ptdlns‐3‐kinase signalling pathway. Here we report an LC‐MS method to measure the stoichiometry of Akt phosphorylation in biological samples with good precision. The procedure includes immunoprecipitation, gel electrophoresis, in‐gel digestion, addition of stable isotope labelled internal standards and LC‐MS/MS. Two proteolytic enzymes, chymotrypsin and trypsin, were used to generate suitable peptide fragments for measuring Thr308 and Ser473 phosphorylation, respectively. The inter‐day imprecision was estimated to be 3.8% and 2.3% for Thr308 and Ser473, respectively. This method has been tested on human T‐cells grown in presence and absence of pervanadate with or without a PI3‐K inhibitor. The results suggest that the levels of Akt phosphorylation in untreated cells were below 1% for both phosphorylation sites. Pervanadate treatment provoked a 39‐ and 18‐fold increase of phosphorylation at Thr308 and Ser473, respectively. A comparison between LC‐MS/MS and Western blotting suggests that the LC‐MS based method provides a more accurate phosphorylation stoichiometry and more in‐depth information.
Fluorescent Peptide Biosensors for Imaging Protein Kinases involved in Cell Proliferation and Cancer
Authors: Morris May C , Kurzawa Laetitia , Pellerano Morgan
Published: June 2021

Cyclin‐dependant kinases play a central role in coordinating cell growth and division and in sustaining proliferation in many pathological conditions, thereby constituting established biomarkers of proliferation and attractive molecular targets for the development of anticancer drugs. Faced with the lack of diagnostic approaches to evaluate the relative levels of these intracellular biomarkers within tumours in a standardized fashion, we have developed peptide biosensors that recognize molecular interfaces specific to cyclin‐dependent kinases. Efficient delivery of biosensors into living cells was achieved thanks to nanoparticle formulations of cell‐penetrating peptide carriers (PEP & CAD family), thereby allowing for direct visualization of endogenous CDK‐cyclin complexes. Moreover, we developed a strategy for ratiometric quantification of CDK‐cyclin levels in different healthy and cancer‐derived cell lines, providing a means to monitor alterations in cyclin‐dependent kinases associated with tumoral progression. This technology can be further extended to probe other intracellular targets, thereby offering promising perspectives for development of non‐invasive diagnostic approaches.
Defining the mechanisms by which palmitoylation regulates the localization and function of RGS4
Authors: Dissanayake Kaveesh , Bastin Guillaume , Heximer Scott P
Published: June 2021

Regulators of G‐Protein Signalling (RGS) proteins are strong candidates for modulating cardiovascular signalling based on their high expression in cardiac and sinoatrial node myocytes, and their ability to potently inhibit heterotrimeric G‐proteins. RGS4 regulates G‐protein function at the plasma membrane (PM) and it has been demonstrated that palmitoylation, the post‐translational addition of a 16 carbon palmitate group to cysteine, is important for RGS4 translocation to the PM. Confocal microscopy data demonstrated that mutation of cysteine residues 2 and 12 removed RGS4 localization to the PM. Calcium imaging confirmed that RGS4‐C2A,C12A elicited less inhibition of Gq‐dependent calcium release than wildtype in HEK293T cells. A library of 23 mammalian palmitoyl‐acyl transferase enzymes has been cloned and isolated, each sharing a conserved catalytic Asp‐His‐His‐Cys (DHHC) site [Fukata et al, 2006]. We set out to establish the DHHC isoforms that regulate the localization and function of RGS4 in HEK cells. We created dominant negative mutations of five DHHCs most highly expressed in HEK cells. Confocal microscopy data showed that co‐transfection of mutant DHHCs 3 and 7 with RGS4 removed localization of RGS4 to the PM. Further studies will examine changes in Gq‐dependent calcium release following transfection of RGS4 in HEK cells where target DHHCs are knocked down using siRNAs. This study was funded by the Heart and Stroke Foundation of Ontario Grant‐in‐Aid Program grants T6799 and the Heart and Stroke/Richard Lewar Centre of Excellence Studentship Award.
Simultaneous quantification of histone acetylation, methylation and ubiquitination at multiple sites by LC/MS/MS‐MRM
Authors: Zhang Kangling , Schrag Matthew , Darmanto Agus , Semotiuk Andrew , Xu Guoliang , Kirsch Wolff
Published: June 2021

Histone post‐translational modifications (PTMs) plays a vital role in directing the fate of gene silencing in the direction of either normal cell development and differentiation or aberrant permanent silencing/activation of cancer‐related genes. To accurately quantify histone modifications in one specific cell line or cancer tissue becomes essential for the study of epigenetic alteration in diseases. Currently, Western‐blot is widely used to assess global histone modifications while ChIP or ChIP‐on‐Chip is used for the analysis of histone modifications at a specific gene location. Mass spectrometry has been recently emerged as a new and amazing tool for qualitative and quantitative analysis of histone modifications. Here, we report an LC/MS/MS‐MRM method for simultaneous quantification of histone acetylation, methylation and ubiquitination at multiple sites using an Agilent triple quadrupole mass spectrometer. The limit of quantification of H2B ubiquitination is 7.3 ng/ml, or 0.15 ng on the column (20 μl of injection volume), for the tryptic ubiquitinated H2B peptide. The dynamic range of quantification is 4~5 order of magnitude. Using this method, we find that H2B ubiquitination in leukemia cell line‐U937 is at least two times of that in leukemia cell line‐HL60, and that the ubiquitination is regulated by AF10 protein. In brain tissue of a person with Amyloid‐positive Alzheimer Disease (AD), histone H3 acetylation and H3 K79 methylation is 40% higher than in the brain tissue of a person with Amyloid‐negative AD or a person without AD.
Hyperactive mice show elevated D2High receptors, a model for schizophrenia: calcium/calmodulin‐dependent kinase II alpha knockouts
Authors: Novak Gabriela , Seeman Philip
Published: June 2021

The cerebral frontal cortex of schizophrenia patients shows elevated levels of RNA for calcium/calmodulin‐dependent protein kinase II beta (CaMKIIβ). In addition, recent research shows that animal models for schizophrenia consistently show elevated levels of dopamine 2 receptors in their high‐affinity state (D2High), the major target for antipsychotic medication. The present study was done to examine whether an alteration in the levels of CaMKIIβ, in respect to CaMKIIα, could lead to altered levels of D2High receptors.

We used quantitative real‐time PCR to measure CaMKIIα and CaMKIIβ mRNA levels in heterozygous and wild type CaMKIIα knock‐out mice, which show reduced CaMKIIα levels, and a D2R binding assay to determine the levels of D2High.

The heterozygous CaMKIIα knock‐out mice, which show features analogous to schizophrenia, showed a 50% reduction in CaMKIIα mRNA, and no change in CaMKIIβ in the frontal cortex, as expected, but also a 29% upregulation of CaMKIIβ mRNA in the striatum and a 2.8‐fold increase in D2High receptors. In addition, we found that the CaMKII inhibitor, KN‐93, markedly reduced D2High states in rat striatum.

The elevated levels of CaMKIIβ mRNA in the striatum suggest that this enzyme may increase D2High in animals and possibly in schizophrenia itself.
A Screening Assay For Z α1‐Antitrypsin Aggregation
Authors: LeMieux Monique Jenaie , Rowe Erica , Berthelier Valerie
Published: June 2021

It is well established that improper folding of proteins often leads to the formation of aggregates whose consequences are cellular impairment and cell death. Therefore, finding small molecules that can be used as therapeutic agents to prevent the aggregation process would revolutionize the treatment of numerous conformational diseases including α1‐antitrypsin deficiency. Aggregation of the mutant Z α1‐antitrypsin protein results in blocking of its secretion, consequently forming inclusion bodies and contributing, not only to the development of chronic obstructive pulmonary disease but also to hepatitis, cirrhosis and hepatocellular carcinoma. Critical molecular mechanisms of the aggregation process have been established and some crystallographic structures solved, however, no specific potent drug‐like small molecule “breakers” of aggregation have been identified. We have designed a 96‐well high‐throughput screening assay that can identify small molecules capable of inhibiting or slowing down Z α1‐antitrypsin aggregation.
Validity of Maternal Assessment of Infant Breastfeeding Behavior: A Cross‐cultural Comparison
Authors: Matias Susana L , Nommsen‐Rivers Laurie A , Chantry Caroline J , Cohen Roberta J , Dewey Kathryn G
Published: June 2021

We evaluated the validity of two potential ‘screening’ questions asked of primiparous mothers about a breastfeeding episode on days 0 and 3 post‐partum (pp) in Peru (n=154) and the US (n=248). A dichotomous measure (suboptimal vs. optimal infant breastfeeding behavior) based on Infant Breastfeeding Assessment Tool scores recorded by trained observers was the reference. Sensitivity (Se) and Specificity (Sp) of maternal assessment of suckling behavior (suckled well continuously/intermittently vs. otherwise; MASB) and maternal satisfaction (very satisfied/satisfied vs. otherwise; MAS) with a breastfeeding episode were calculated for independent and parallel use. Depending on the day pp, Se of MASB was 36–63% in Peru and 44–57% in the US; Sp was 97% in Peru and 97–98% in the US. Se of MAS was 61–85% in Peru and 48–58% in the US; Sp was 64–73% in Peru and 89–92% in the US. Parallel use of both questions improved Se in the US (55–73%). Increasing the cut‐off of MASB improved Se on both days in both samples (85–99%), but reduced Sp (31–69%). The use of these screening questions may help identify breastfeeding dyads who would benefit from further lactation support, especially in settings where availability of specialized lactation evaluation is limited.

Supported by grant R40MC04294 from the Department of Health and Human Services, Maternal Child Health Branch and by the Gifford Center for Population Studies at UC Davis.
Activation of mice skeletal muscle 6‐phosphofructo‐1 kinase by serotonin is mediated by PLC‐ β PKC dependent pathway
Authors: Coelho Wagner Santos , Sola‐Penna Mauro
Published: June 2021

Serotonin (5‐HT) is implicated in regulation of many physiological and pathological events, presenting the ability to stimulate glucose uptake on skeletal muscle. We previously reported that 5‐HT activates 6‐phosphofructo‐1‐kinase (PFK) from mice skeletal muscle in a non insulin‐dependent pathway, through 5‐HT2A receptor subtype. This study aims to gain further insights into the mechanisms involved in glucose metabolism control by 5‐HT, through enzyme activity assays and western blotting analysis using mice muscle stimulated with 5‐HT and inhibitors of signaling cascade intermediates. Here we show that activation of PFK promoted by 5‐HT is prevented by the PLC‐β inhibitor U‐73122, with concomitant reduction in 5‐HT‐mediated tyrosine phosphorylation of PFK. Moreover, 5‐HT effects are mimicked by phorbol myristate acetate (PMA) and blocked by bisindolylmaleimide‐1 (BIS‐1), which respectively activates and inhibits protein kinase C (PKC). KN‐62, a calmodulin kinase II (CaMKII) inhibitor, abolishes serotonin‐induced PFK activation. Furthermore, 5‐HT promoted co‐immunoprecipitation of PFK and CaMKII, suggesting a modulation mechanism. Yet, 5‐HT fails to promote extracellular regulated kinase 1/2 (ERK 1/2) phosphorylation. Altogether, our results support evidences that glycolytic flux control promoted by 5‐HT occurs in a PLC‐β PKC dependent pathway.

Research Support: CNPq, FAPERJ, FAF/FECD
Oxidative inhibition of MAPKAP kinase 2 (MK2) is predominately mediated through the activation loop cysteine
Authors: Forsberg Brian J , Patel Rutvi , Schumacher Carol , Chrestensen Carol A
Published: June 2021

MAPKAP kinase 2 (MK2) is a member of the mitogen activated protein kinase activated protein kinase family. MK2 is activated by p38 MAP kinase and this signaling pathway is important in cellular responses to inflammatory cytokines and stress (e.g. UV, osmotic shock, oxidative stress). Mammalian MK2 has 7 conserved cysteines, that are not known to participate in disulfide bonds. Among these is a cysteine residue in the activation loop that is conserved in other kinases including protein kinase A (PKA) and protein kinase C (PKC). PKA and PKC are inhibited by oxidative modification at this conserved cysteine residue. A non‐radioactive fluorescent based peptide assay was used to assess MK2 activity after various treatments using either bacterially expressed active‐MK2 or myc tagged MK2 immunoprecipitated from transiently transfected cells. MK2 was inhibited by oxidized glutathione in vitro and in cells by treatment with diamide. Mutation of the activation loop cysteine residue produced an active kinase that was considerably less sensitive to oxidative inhibition. This work was supported by the Research Corporation (CC6942) and a Mentor Protégé grant from Kennesaw State University.
Neurosteroid binding domains of the NMDA receptor NR2B and NR2D subunits
Authors: Cameron Krasi , Borkowski Brian , Kim Joon , Rhoads Sarah , Cassada Gray , Rocha Monica , Gentile Lisa
Published: June 2021

AMPA, NMDA and kainate receptors belong to the ionotropic glutamate receptor (iGluR) family. As binding to glutamate, a major fast excitatory neurotransmitter, causes activation of these channels, they play an important role in synaptic plasticity, memory and learning. Our research focuses on understanding how these receptors are regulated, for potential applications in conditions such as Alzheimer's and Parkinson's disease.

The data presented here is aimed at understanding the differential regulation of NMDA receptors by the endogenous neurosteroids pregnenolone sulfate (PS) and 3‐alpha‐hydroxy‐5‐beta‐pregnan‐20‐one sulfate (PREGAS). PS potentiates the activity of NMDA receptors containing an NR2B subunit while it inhibits those containing an NR2D subunit. PREGAS negatively regulates all iGluRs. Intrinsic and extrinsic fluorescence studies will be presented that confirm the binding of the NMDA NR2B S1S2 and amino terminal domains (ATD) to both PS and PREGAS. Unlike the NR2B subunit, the NR2D S1S2 domain does not bind to PS and PREGAS, however the NR2D ATD does bind to both neurosteroids. Data from isothermal titration calorimetry and Stern‐Volmer analysis will be presented to help differentiate the binding site of each of these neurosteroids on both the NMDA NR2B and NR2D subunits.
Characterization of bivalent peptides that bind to X‐linked inhibitor of apoptosis protein
Authors: Speer Kelsey , Splan Kathryn
Published: June 2021

X‐linked inhibitor of apoptosis protein (XIAP) is a regulator of programmed cell death and an important target in the development of cancer therapeutics. Biochemical studies reveal that the homodimeric protein Smac, the natural binding partner of XIAP, binds to XIAP via simultaneous contact to the BIR2 and BIR3 domains. Furthermore, the interaction is mediated primarily via the first four N‐terminal amino acids Ala‐Val‐Pro‐Ile. We and others have shown that dimeric peptides and peptidomimetics based upon the Ala‐Val‐Pro‐Ile motif also bind to XIAP through both the BIR2 and BIR3 domains. Despite the high structural and sequence similarities between the BIR2 and BIR3 domains, Ala‐Val‐Pro‐Ile binds to BIR2 with substantially lower affinity than to BIR3. Herein we explore the potential for asymmetric bivalent Smac mimetics wherein one binding motif has been optimized for the BIR2 domain. We show that the tetrapeptide Ala‐Glu‐Ala‐Val has a higher affinity for BIR2 than Ala‐Val‐Pro‐Ile, and we have incorporated this motif into dimeric peptides with both BIR2 and BIR3 targeting motifs. We also will present mutagenesis experiments aimed at restoring the tight binding of the BIR3 domain to BIR2. The implications of these results in the design of XIAP antagonists will be discussed. We acknowledge the Research Corporation (Cottrell College award to K.E.S.) and the Beckman Scholars Program (undergraduate research fellowship to K.F.S.) for support of this research.
Impact of heme reconstitution on the spectroscopic properties and DNA binding activity of the H82A variant of the carbon monoxide‐sensing heme transcription factor, CooA
Authors: Troester Rachel E. , Clark Robert W.
Published: June 2021

CooA is a gas‐sensing heme protein that regulates the expression of genes responsible for growth by carbon monoxide (CO) in several bacteria. CO binding to the CooA heme groups results in an allosteric conformation change that initiates the protein's DNA binding function. All known CooA homologs have a conserved histidine residue that i) acts as the proximal heme ligand and ii) is trans to CO. Together with the results from mutagenesis studies, this observation suggests the presence of a proximal histidine ligand is required for CooA's DNA‐binding activity. However, since mutants lacking this His have been observed to lose heme upon purification, an alternative hypothesis is that the absence of heme, and not the presence of histidine per se, accounts for this loss of function. To test this hypothesis, we measured the spectroscopic properties and DNA‐binding activity of heme‐reconstituted H82A. Results obtained using electronic absorbance spectroscopy suggest heme‐reconstituted Fe(II)‐CO H82A has an iron environment similar to that of wild‐type CooA. Preliminary results from a fluorescence anisotropy assay also reveal that reconstituted H82A possesses modest DNA binding activity, which suggests the presence of His‐82 is not essential for this activity. The authors wish to thank the Valparaiso University College of Arts and Sciences for financial support.
Interconversions of plasma folate species in response to dosing with oral folic acid and 5‐methyltetrahydrofolate: an LC‐MS/MS pilot study
Authors: Fazili Zia , Bailey Steven W , Paladugula Neelima , Ayling June E , Pfeiffer Christine M
Published: June 2021

Information is sparse on changes in plasma folate species in response to high folic acid (PGA) intake. As part of a pharmacokinetics study, we investigated the conversion of oral PGA (1 and 2.5 mg) and 5‐methyltetrahydrofolate (5MTHF; 5 mg) into plasma folate species (5MTHF, PGA, 5‐formylTHF [5FTHF], THF, and 5,10‐methenylTHF) by LC‐MS/MS in 6 subjects/dose at five time points (0, 1.5, 3.0, 6.5 and 24 h) and compared results to a placebo group. 5,10‐methenyl‐THF was not detectable. At baseline, total folate (TFOL) consisted of 80–97% 5MTHF, 1–7% PGA, 1–14% 5FTHF, and 0–12% THF. In the placebo group, TFOL and folate species remained unchanged over time. In the two PGA dose groups, the highest increases were observed for PGA at 1.5 h; smaller increases (≤5‐fold) were observed for the other folate species. In the 5MTHF dose group, 20‐fold increases were observed for 5MTHF and 5FTHF and < 5‐fold increases for PGA and THF. Changes in folate species pattern generally returned to baseline by 24 h. Preliminary results from this sensitive LC‐MS/MS method show that there are small interconversions of folate species in response to dosing with PGA and 5MTHF. Supported by NIH grant HL068165 to JEA.

Folate concentration (nmol/L) at 0 h/x‐fold increase at 1.5 h

Placebo
1 mg PGA
2.5 mg PGA
5 mg 5MTHF

5MTHF
19.3/1.0
17.2/1.7
31.5/2.0
20.4/19

PGA
0.5/0.9
0.6/57
0.7/238
0.4/4.4

5FTHF
1.5/1.0
0.6/2.5
1.2/2.1
0.6/18

THF
0.9/0.3
0.3/5.3
0.4/4.4
1.1/2.8
Impact of vitamin B‐6 availability on gluconeogenesis from amino acid precursors in human hepatoma (HepG2) cells
Authors: da Silva Vanessa Rodrigues , Ralat Maria , Gregory Jesse F.
Published: June 2021

Endogenous glucose synthesis is crucial to the maintenance of adequate glucose supplies for the body. The conversions of glycine to serine and of serine and alanine to pyruvate are pyridoxal 5′‐phosphate (PLP)‐dependent, leading to a metabolic intermediate which can be used for gluconeogenesis. This study aims to assess the impact of vitamin B‐6 deficiency on gluconeogenesis from amino acid precursors in a human hepatoma cell line. HepG2 cells were cultured in vitamin B‐6 replete (2000 nM pyridoxal) or vitamin B‐6‐deficient Dulbecco's minimal essential medium containing 10, 20, or 50 nM pyridoxal. At steady‐state, intracellular PLP levels were 29.1, 37.6, 42.5, and 57.2 pmol/mg protein, respectively. Cells were cultured for 24h in media enriched with a [U‐13C3]serine tracer, and enrichment of intra‐ and extracellular serine, glycine, and glucose measured by GC/MS. Maximum glucose enrichment occurred as the singly‐labeled species at 4h. Investigation is underway evaluate the effect of vitamin B‐6 status on gluconeogenic rate.

Grant Funding Source: UFL
Effects of selenium source on blood selenium content, blood cell counts and peripheral blood mononuclear cell mRNA profiles in maturing beef heifers
Authors: Brennan Kristen M , Burris Walter R , Boling James A , Matthews James C
Published: June 2021

To determine if source of dietary selenium (Se) supplementation affects blood Se content and expression of biomarkers of immune status in blood cells of growing cattle, Angus heifers were assigned (n = 10) to 4 Se treatments (TRT) for 126 d. The basal diet contained 0.08 mg Se/d, whereas the mineral supplements provided no (control, C), or 3 mg Se/d as inorganic (sodium selenite, SS), organic (Sel‐Plex®, SP), or a 50/50 mix (SS/SP). Jugular blood was collected for blood cell counts, Se analysis and peripheral blood mononuclear cell (PBMC) isolation. Whole blood (WB), plasma, and red blood cell (RBC) Se content was greater (P < 0.01) for all TRT versus C. SS/SP and SP heifers had greater (P < 0.01) WB and RBC Se content than SS, while plasma Se tended (P = 0.06) to be higher in SS/SP heifers than SS. PBMC count was negatively correlated (P < 0.05) to plasma and WB Se content in SP heifers, and tended (P = 0.06) to be negatively correlated to WB Se in SS/SP heifers. Se TRT did not affect interleukins 1β, 8, and receptor 2α mRNA levels in PBMC, while selenoprotein S (SelS) mRNA content was decreased (P < 0.05) in SS/SP and SP versus C heifers. Because SelS plays an important role in the regulation of the inflammatory cytokine response, decreased SelS mRNA levels indicate supplementing with 3 mg/d SP or SS/SP may result in cattle with a less challenged immune state. This work is supported by the Alltech‐University of KY Animal Nutrigenomics Alliance.
Selenoprotein P reduces lipid hydroperoxides in human embryonic kidney cells following exposure to 15‐ hydroperoxyeicosatetraenoic acid
Authors: Rock Colleen , Moos Philip J
Published: June 2021

Reactive intermediates formed during cell membrane metabolism can contribute to carcinogenesis through oxidative damage to DNA. Potential chemopreventive effects of selenium supplementation have been attributed to the antioxidant activity of selenocysteine residues in selenoproteins. Selenoprotein P (SelP) is an extracellular glycoprotein that functions both in selenium distribution and as an antioxidant protein. The major objective of this study was to determine the effect of SelP on lipid hydroperoxides produced during the metabolism of arachidonic acid by 15‐lipoxygenase‐1. Enzymatic reduction of 15‐ hydroperoxyeicosatetraenoic acid (15‐HpETE) by SelP was observed in a NADPH‐coupled biochemical assay. Diphenylpyrenylphosphin was used to measure lipid hydroperoxides in SelP treated human embryonic kidney (HEK‐293) cells following exposure to 15‐HpETE. Lipid hydroperoxides increased with 15‐HpETE treatment of cells, and SelP reduced this effect, even when the protein and metabolite were added simultaneously. This shows that SelP can work as an antioxidant enzyme against reactive intermediates formed during inflammation. This effect may serve to protect cells against the oxidative damage induced by these lipid metabolites.

This work is supported by NCI grant CA115616
Intestinal Cell Calcium Uptake and the Targeted Knockout Of the 1,25D3‐MARRS Receptor/PDIA3/Erp57
Authors: Nemere Ilka , Garbi Natalio , Hammerling Gunter
Published: June 2021

We have crossed ERp57lfx/flx mice with mice expressing villin‐driven cre‐recombinase. Lysates of enterocytes from knockout (KO) mice and littermates (LM) were used in Western analyses with Ab099 against the N‐terminus of the 1,25D3‐MARRS receptor: LM mice exhibited one positive band, which was absent in preparations from KO mice. Saturation analyses of cell lysates with [3H]1,25D3 revealed negligible binding in either female or male KOs. Lysates from female and male LM mice had similar affinities but different numbers of binding sites. Isolated enterocytes were tested for steroid stimulated calcium uptake. Treatment of cells from female or male LM mice with 1,25D3 elicited enhanced within 5 min. Intestinal cells from KO mice exhibited a severely blunted or completely absent response to hormone. Confocal microscopy of intestinal cells revealed the presence of cell‐surface VDR. However, antibodies to the VDR failed to block 1,25D3‐ stimulated calcium uptake. In chick enterocytes we have found that the PKA pathway mediates calcium uptake. The time course for activation of PKA in mouse enterocytes paralleled that for enhanced calcium uptake. Enterocytes from female or male KO mice failed to exhibit 1,25D3 stimulated PKA activity, but did respond to forskolin with enhanced calcium uptake. We conclude that the 1,25D3‐MARRS receptor is of central importance to steroid hormone stimulated calcium uptake in mammalian intestinal cells.
Effects of tomato and broccoli on the bone status of growing female rats with different levels of calcium intake
Authors: Wang Changzheng , Huang Lingyu , Butler Cecil
Published: June 2021

A total of 130 weanling female SD rats were allowed one week of adaptation before they were assigned into 13 treatment groups. The baseline group was killed at the beginning of the experiment. Groups A, B, C and D were fed the AIN93G diet containing 0.1% calcium; groups E, F, G and H were fed the AIN93G diet containing 0.25% calcium; and groups I, J, K and L were fed the AIN93G diet containing 0.5% calcium. Corn starch (20% of the diet) was replaced by tomato powder for groups B, F, and J; by broccoli powder for groups C, G and K; and by a 50:50 mixture of tomato and broccoli powders for groups D, H and L. The rats were fed the assigned diets for 8 weeks. Bone length, ash, calcium content and breaking strength became higher as dietary calcium concentration increased. Substitution of the corn starch by tomato and/or broccoli powders did not significantly affect the bone status of rats fed diets containing 0.25% or more of calcium. However, tomato and/or broccoli improved the bone status of rats fed diets containing 0.1% calcium. These results indicate that vegetables such as tomato and broccoli can have beneficial effects on bone when calcium intake is very low.
Megalin‐ and Disabled‐2‐mediated uptake of vitamin D‐binding protein is modulated by all‐trans‐retinoic acid in prostate and colon epithelial cells
Authors: Ternes Shantel B , Rowling Matthew J
Published: June 2021

Disabled‐2 (Dab2) is an adapter protein, that along with the membrane receptors megalin and cubilin, is essential for uptake of protein‐complexed 25‐hydroxycholecalciferol (25D3) in the kidney and maintenance of serum vitamin D homeostasis. Previously, we showed that mammary cells (T‐47D) also express Dab2, megalin, and cubilin and internalize vitamin D‐binding protein (DBP) by receptor‐mediated endocytosis. Moreover, induction of Dab2 and megalin by the differentiating agent all‐trans‐retinoic acid (RA) correlated with DBP uptake in mammary cells. These findings suggest that expression of Dab2, megalin, and cubilin are critical for the uptake and anti‐tumorigenic effects of vitamin D in cancer‐prone tissues. In this present study, we have extended our work by characterizing the uptake of DBP and the expression of Dab2 and megalin in prostate cancer (PC‐3 and LNCaP) and colon cancer (Caco‐2) cells. Our results indicate that Dab2 and megalin mRNA and protein are abundant in PC‐3, LNCaP, and Caco‐2 cell lines. This expression was markedly enhanced when treated with 10 μM RA. Furthermore, DBP was readily internalized by PC‐3, LNCaP, and Caco‐2 cells. Taken together, these are the first studies to our knowledge that have characterized a potential role for Dab2 and megalin in the uptake of vitamin D in prostate and colon cells. Supported by NIH SR03CA128091‐03 and Iowa State University Experiment station to MJR.
Magnesium supplementation attenuates aldosterone‐induced hypertension, kidney damage and oxidative stress in genetically hypomagnesemic mice
Authors: Yogi Alvaro , Callera Glaucia E , O'Connor Sarah E , He Ying , Tostes Rita C , Mazur Andrzej , Touyz Rhian M
Published: June 2021

Hyperaldosteronsim is associated with hypertension, fibrosis and hypomagnesemia. Here we investigated the effect of Mg2+ supplementation on aldosterone‐induced hypertension and kidney damage in mice genetically bred to have normal (MgH) or low (MgL) intracellular Mg2+ level. Male MgH and MgL mice were infused with aldosterone. Basal systolic blood pressure (SBP) was higher in MgL vs MgH (p<0.05). Aldosterone infusion increased SBP, albuminuria and renal collagen deposition (P<0.05) in both strains. TRPM6 channels are key regulators of Mg2+ reabsorption. MgL mice had reduced basal TRPM6 expression (P<0.05). Aldosteone infusion increased renal TRPM6 expression in MgH (P<0.01) but not MgL mice. Aldosterone induced oxidative stress, NF‐êB activity and expression of pro‐inflammatory markers in both MgH (1‐fold) and MgL mice (2‐fold). Aldosterone failed to increase SBP in mice fed with high Mg2+ diet. In these animals, aldosterone‐induced deleterious effects were prevented. Our data show that Mg2+ supplementation attenuates aldosterone‐induced hypertension and renal damage through changes in oxidative stress and inflammatory pathways. These findings suggest that aberrations in Mg2+ status may be associated with elevated SBP and renal damage in chronic diseases associated with hyperaldosteronism. Supplementation with Mg2+ may be protective in this context.

Support: HSFC, CIHR.
Sprasis crispa mushroom extract affects adipogenesis and lipolysis in 3T3‐L1 cells
Authors: Park Jin‐kyung , Lee Me‐Ae , Choi You‐Youn , Kim Hye‐Ryeon , Park Yoo Kyoung
Published: June 2021

Sprasis crispa (SC, cauliflower mushroom) has high substance of ß‐(1,3)‐D‐Glucan which has various physiological effects on living organism. The purpose of this study was to investigate effects of SC water‐soluble extract in 3T3‐L1 cells. The cytotoxicity of SC in 3T3‐L1 preadipocytes were evaluated by MTT assay. SC treatment (0–1000μg/ml in the preadipocytes) did not show toxicity. Preadipocytes were treated with differentiation cocktail (MDI) in the absence or presence of various concentrations of SC (200,400,600μg/ml). Samples were analyzed on 2nd, 5th and the 8th day of MDI treatment in 24hr, 48hr and 72hr of mature adipocyte. SC had significantly inhibited lipid accumulation compared with control in a time‐and dose‐dependent manner by ORO staining in the adipocyte differentiation and in the mature adipocyte. We also observed increased glycerol release in a dose dependent manner. Furthermore, SC reduces the expression of PPARγ, C/EBPβ in the adipocyte differentiation and increases CPT‐1 in the mature adipocyte. These results indicate that SC suppresses adipogenic enzymes and stimulates lipolytic enzyme providing a basis for anti‐obesity effects of SC.