bluuebunny/crossref_metadata_2025 huggingface dataset:
Salmonella effector AvrA promotes cellular proliferation
Authors: Jones Rheinallt , Wu Huixia , Luo Liping , Neish Andrew
Published: June 2021

Certain enteric pathogens evade elimination by secreting proteins that have potent suppressive effects on apoptosis and innate immunity. For example, the intracellular pathogen Salmonella secretes AvrA which specifically inhibits host JNK pathway signaling. We created a transgenic Drosophila harboring avrA, allowing directed expression of AvrA in fly tissues. We previously showed AvrA is a potent inhibitor of pro‐apoptotic JNK signaling in Drosophila. Here, we report that AvrA expression in a background of constitutive Eiger expression (Drosophila TNF‐alpha) resulted in increased proliferation of Drosophila epithelial tissue and importantly of Drosophila phagocytes (hemocytes) whereas AvrA activity in a non Eiger expressing background did not. Reporter gene assays in mammalian cultured cells showed AvrA mediated inhibition of JNK signaling, together with TNF‐alpha stimulation resulted in increased levels of pro‐proliferative ERK signaling. Finally, a murine Salmonella infection model showed that wild type Salmonella caused increased tissue BrdU positive splenocytes, compared to infection with an AvrA null mutant where no BrdU positive cells were detected. Together, these studies show Salmonella has evolved a protein that inhibits the pro‐apoptotic JNK pathway and also stimulates epithelial tissue and phagocyte growth under pro‐inflammatory conditions.
Students and teachers reaching for the STARS: a cadaveric exploration of anatomy
Authors: Bruzzini Kristen Blake
Published: June 2021

Area high school students and teachers had the opportunity to learn and teach human gross anatomy during an intensive workshop entitled Students and Teachers Anatomical Research Series (STARS) at Maryville University. Science teachers attended week one, which consisted of focused lectures on the anatomy of the thorax, abdomen, back, and upper and lower extremities, "hands‐on" cadaveric dissection, and development of lesson plans for use at home institutions. Week two involved putting into practice their new knowledge and teaching techniques. High school students spent this week performing traditional cadaveric dissection, as well as participating in either instructor assisted or self‐directed small group workstations using handouts, models and computer programs. Real world applicability of human anatomy was also incorporated. Daily assessment forms, end of course evaluations, and content testing of fourteen area high school students and two participating teachers demonstrated enthusiasm for the topics covered, an increased appreciation and understanding of the human body, and retention of the material covered at the end of the course. All teachers indicated that they learned information they would apply in their own courses and strongly felt that the workshop was an excellent opportunity for the university to generate an appreciation of anatomy and an excitement for learning at the high school level.

Grant Funding Source

American Association of Anatomists
An anatomy course based on clinical reasoning
Authors: Elizondo Omaña Rodrigo Enrique , Guzmán López Santos
Published: June 2021

To describe a general view of how anatomy can contribute in an important way in the development of clinical reasoning.

Learning based on clinical reasoning focuses in reaching an anatomical diagnosis out of any anomaly or normality. The basic intellectual abilities consist in the identification, description, comparison, definition and classification. The superior intellectual abilities are the analysis, synthesis and evaluation. Its systematic use allows the student to construct a mental structure for problem solving.

An anatomical course based on clinical reasoning has two main objectives: favour anatomical knowledge acquisition and to make the student aware of the clinical reasoning process.
Neuroscience Teacher Training Workshops for Elementary School Teachers
Authors: Puder Barb
Published: June 2021

A teacher training workshop was designed to instruct elementary school teachers regarding basic neuroscience content and provide guidelines for planning anatomical life science units for the classroom. This introduction of neuroanatomical sciences allows teachers to receive appropriate training in neuroanatomy, comply with the National Science Education standards, and create classroom activities that will encourage children to investigate topics regarding neuroscience.

Two half day weekend workshops were implemented which included a neuroscience lecture. Following the lecture, teachers participated in a hands‐on laboratory session filled with activities and experiments that can be replicated in the elementary school classroom.

To measure the impact and effectiveness of the workshop, participants were administered a pre and post test regarding key ideas presented in the workshop. Participants also filled out a survey rating the workshop's overall effectiveness, and course content. There was a statistically significant improvement from pretest to posttest (p< 0.0001).

These workshops provided necessary teacher instruction in the anatomical sciences which promotes student interest in neuroscience.

Supported by AAA Education Outreach Grant
Use of peer behavior video in teaching peer evaluation in Team‐Based Learning anatomy curriculum.
Authors: Vasan Nagaswami , DeFouw David , Compton Scott
Published: June 2021

Peer evaluation is a contentious process among medical students especially if it involves grades. Often, students are inexperienced in evaluating peers, unsure of what they are evaluating and what reference to use. Hence, we created an instructional video and educational module addressing acceptable and unacceptable behaviors and attributes of a good team. Attitudes and behaviors were described and compared between male (n=121) and female (n=69) ratings using chi‐square and Fisher's exact tests, as appropriate. The overall percentages of students are presented that agreed the video helped them to: understand acceptable behavior (99.3%); evaluate team members fairly and honestly (93.5%); understand and accept constructive peer evaluation (87.3%); modify my interactive skills to become a productive team member (90.1%). While many students answered that they have prior experience with peer evaluation, none involved grading. Greater number of female than male students agreed that the video helped them to understand and accept constructive peer evaluation (92.8% vs 81.8%; p=0.038). We conclude that the peer‐interaction instructional video educational module was well received by both male and female students, and resulted in highly positive self‐reported attitudes and behaviors regarding TBL. Supported by the Department of Cell Biology and Molecular Medicine.
Temporal order of events in the assembly of a ternary clamp loader‐clamp‐DNA complex
Authors: Bloom Linda B. , Paschall Christopher O. , Chiraniya Ankita , Thompson Jennifer A.
Published: June 2021

Sliding clamps are ring‐shaped protein complexes that increase the processivity of DNA synthesis by tethering DNA polymerases to DNA. Clamp loaders catalyze the assembly of sliding clamps onto primed templates. Binding and hydrolysis of ATP promote conformational changes in the clamp loader that alter interactions with the clamp and DNA to facilitate the mechanical clamp loading reaction. Our laboratory developed two independent fluorescence‐based assays to measure clamp loader▸clamp binding and clamp opening for the Escherichia coli γ‐complex clamp loader and β‐sliding clamp. Real time binding measurements revealed that when the γ complex is equilibrated with ATP, clamp binding is rapid and followed by a slower clamp opening reaction. This result demonstrates that the clamp loader binds and opens the clamp rather than capturing clamps that are transiently open in solution. When the β‐clamp and ATP are added to the γ complex simultaneously, a slow ATP‐dependent conformational change in the γ complex precedes clamp opening but not clamp binding. Kinetic assays with wild‐type γ complex and experiments with Arg finger mutants of γ complex were done to define ATP requirements for clamp binding and opening. A model will be presented in which a defined temporal order of events in assembling a ternary clamp loader▸clamp▸DNA complex is established by the rates of a series of ATP binding events and conformational changes.
Eukaryotic elongation factor 2 kinase (eEF2K) downregulation correlated with de novo DNA methylation and decreased DNA accessibility in neonatal IUGR rat liver
Authors: Fu Qi , McKnight Robert A , Yu Xing , Callaway Christopher W , Lane Robert H
Published: June 2021

Uteroplacental insufficiency (UPI) alters protein synthesis in intrauterine growth restriction (IUGR). A key regulator of protein synthesis is eEF2K, which regulates protein synthesis by phosphorylating and inactivating eEF2. eEF2K is a classic CpG island gene. We hypothesized that UPI would affect hepatic eEF2K expression by altering eEF2K DNA methylation and possibly DNA accessibility in neonatal IUGR rats.

Methods

IUGR rats were generated by bilateral uterine artery ligation. Nucleosome position and DNA accessibility was established by M.SssI methyltransferase (MMT) footprinting. EEF2K CpG methylation, mRNA and protein levels were determined.

Results

IUGR animals had decreased hepatic eEF2K mRNA and protein levels. 45 CpG sites around the transcription start site (TSS) from ‐282 through +217 on the eEF2K gene were analyzed. Increased CpG methylation between ‐206 and ‐282 occurred in IUGR relative to control. The position of 2.5 nucleosomes around the TSS was mapped by MMT footprinting. The ‐1 nucleosome was highly accessible in both groups, while ‐2 and +1 nucleosomes had significantly decreased accessibility in IUGR relative to control.

Conclusions

UPI downregulates hepatic eEF2K expression in neonatal IUGR rats. This downregulation is correlated with de novo DNA methylation and reduced DNA accessibility. These results demonstrate IUGR induced alterations to nucleosome positioning.
Identification of Potential Enhancers in the RUNX1 Gene
Authors: Gutierrez Soraya Elisa , Rebolledo Boris , Stuardo Marcela , Martinez Milka , Lian Jane , Javed Amjad , Stein Gary , Stein Janet
Published: June 2021

Human RUNX1 gene is the most frequent target for chromosomal translocations associated with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). The highest prevalence in acute myeloid leukemia (12‐15% of all cases) is noted with (8;21) translocation. Interestingly, all the breakpoints mapped to date in t(8;21) are clustered in intron 5 of the RUNX1 gene and intron 1 of the partner ETO gene. Even though, no homologous sequences have been found at the t(8:21) recombination regions; DNaseI hypersensitive sites (DHS) and topoisomerase II cleavage sites have been mapped to these areas of the genes. Presence of DHS sites is commonly associated with regulatory elements such as promoters, enhancers and silencers. In this study we used a combination of comparative genomics and chromatin analysis to identify transcriptionally active regions within intron 5 of the RUNX1 gene. Our genomic analysis identified nine potential enhancer regions that are evolutionarily conserved. Analysis of histone acetylation status of these regions in myeloid cells demonstrate a high association of 9,14 diacetylated histone H3; a hallmark of enhancer modules . Our results suggest that some of the identified conserved non coding sequences in the RUNX1 gene locus may act as regulatory sequences in hematopoietic cells.

Grants: ACT 44; NIH R03 TW007170
Transcriptional Activation of Regulator of G Protein Signaling 4 (RGS4) by Inverted CCAAT Box Element (ICE) Binding Factor NF‐YA Attenuates G Protein Signaling
Authors: Yang Jianqi , Huang Jie , Chatterjee Tapan K. , Twait Erik , Fisher Rory A.
Published: June 2021

RGS4 is a member of a family of proteins that play critical roles in attenuating GPCR signaling. Enriched in brain, RGS4 is linked to several pathologies, including Parkinson's disease. However, the mechanisms underlying regulation of RGS4 expression have not been studied. Here, we elucidated these mechanisms in PC6 cells, in which RGS4 is induced markedly at cellular confluence. Using gene promoter analyses, we demonstrated that an ICE in the human RGS4 promoter was required for transcriptional activation of RGS4. Phylogenetic analysis revealed that the ICE was one of four conserved transcription factor binding sites. Functionally, the ICE interacted with NF‐YA, an ICE binding transcription factor, in EMSA and biotin‐labeled DNA oligo pull‐down assays. Exogenous expression of NF‐YA or C/EBPβ activated the RGS4 promoter, while dominant negative NF‐YA and C/EBPβ significantly reduced RGS4 promoter activity. Furthermore, both cyclin D1 and phosphorylated Rb were decreased in confluent cells, indicating cell growth arrest. However, growth arrest induced by serum withdrawal alone did not alter RGS4 expression. Consistent with the fact that RGS4 is a GAP for Gαi and Gαq, LPA‐induced activation of MAPK was attenuated in confluent cells. We conclude that transcriptional activation of RGS4 by NF‐YA leads to an attenuation of G‐protein signaling. Supported by grants from the NIH (GM075033) and AHA (0750057Z).
Exclusive use of ring 2‐[13C]L‐histidine for carbon 2 of the purine ring in purine nucleotide biosynthesis (PNB) de novo in an adult human
Authors: Baggott Joseph E , Gorman Gregory S , Tamura Tsunenobu
Published: June 2021

The ring 2‐carbon of histidine is a source of one‐carbon units (1‐C) carried by folate coenzymes. In theory, this would be metabolically available for carbons 2 (C2) and 8 (C8) of the purine ring in PNB. An adult male ingested 0.71 g of ring 2‐[13C]L‐histidine ?HCl ?H2O (98%, Cambridge Isotope), and the 13C enrichment of urinary uric acid at C2 and C8 of each void was measured by an LC/MS/MS method. The % 13C enrichment at C2 in 33 voids (3 days) ranged from 0.05 to 0.58 with rhythmicity. Means (±SD, number of voids) were 0.15 (±0.09, 12), 0.24 (±0.19, 10) and 0.28 (±0.19, 11) for days 1, 2, and 3, respectively and were greater than zero (P < 0.01). In contrast, the % 13C enrichment at C8 varied from ‐0.12 to 0.09 with the means of 0.00 (±0.06), ‐0.01 (±0.02) and 0.00 (±0.02), respectively and these were not different from zero (P > 0.05). Our data indicate that histidine, like formate, is a source of 1‐C utilized by aminoimidazolecarboxamide ribotide transformylase; thus, C2 is enriched. We reported that the 2‐carbon of glycine is utilized by glycinamide ribotide transformylase for C8 in humans. It is likely in humans that completion of PNB requires different carbon sources for C2 and C8 and that different organs play concerted roles. The seemingly wasteful catabolism of histidine, an essential amino acid, to glutamate, a non‐essential amino acid, may make metabolic sense, when 1‐C produced is used for an essential purpose such as PNB.
Momordica charantia reduces high‐fat‐diet‐associated oxidative stress in mouse brain
Authors: Kipyakwai Gideon Kipkoech , Shah Pranjal , Nerurkar Vivek R. , Nerurkar Pratibha V.
Published: June 2021

Obesity‐associated increase in oxidative stress (OS) has been implicated in reduced cognition and brain function in rodents and humans. Ayurvedic medicine, Momordica charantia (bitter melon, BM) is traditionally used in the management of diabetes and its complications including neuropathies. We recently demonstrated that BM reduces weight gain, improves glucose and insulin tolerance and lowers plasma lipids in high‐fat‐diet (HFD) fed mice. The aim of this study was to investigate the effects of BM on brain antioxidant enzymes in HFD fed mice. Female C57BL/6 mice were randomized into three groups: 1) control, 2) HFD (58% kcal fat), and 3) HFD + 1.5% lyophilized BMJ (W/V). After 16 weeks, brains were excised and analyzed for catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx), mitochondrial manganese superoxide dismutase (Mn‐SOD) and cytosolic copper/zinc‐superoxide dismutase (Cu/Zn‐SOD) activities using commercial kits. HFD‐fed mice demonstrated an initial stress response with increased brain cytosolic Cu/Zn‐SOD activity with a concomitant reduction in GSH, CAT and GPx activities. Feeding of BMJ normalized Cu/Zn‐SOD, CAT and GPx activities in brains of HFD‐fed mice. These data indicate a role for dietary therapies in reducing HFD‐associated OS in brain. [Grants: NCCAM (R21AT003719), and RCMI, NCRR (G12RR/AI03061), NIH].
Inflammatory Cytokine IL‐6 and Cachectic Muscle Oxidative Capacity
Authors: White James P , Sato Shuichi , Baltgalvis Kristen A , Baynes John W , Carson James A
Published: June 2021

Cancer cachexia is a condition of severe wasting of both skeletal muscle and adipose tissue. Circulating inflammatory cytokine IL‐6 can regulate the development of cachexia in the ApcMin/+ mouse, a model of colorectal cancer. The purpose of this study was to determine the effects of cancer cachexia and IL‐6 over‐expression on wasting skeletal muscle oxidative capacity. Gastrocnemius (GAST) muscle mitochondrial number, mitochondrial proteins and PGC‐1α mRNA expression were quantified. ApcMin/+ mice were stratified by cachexia severity. The GAST muscle was sectioned into red (oxidative) and white (glycolytic) portions to determine phenotype specific wasting. IL‐6 was over‐expressed in a subset of ApcMin/+ mice by in vivo electroporation to accelerate wasting. Severely cachectic mice had an 81% reduction in mitochondrial content when compared to mildly cachectic mice. Both oxidative and glycolytic portions of the GAST reduced PGC‐1α mRNA expression approximately 40% in severely wasting muscle. IL‐6 over‐expression resulted in a 63% decrease in PGC‐1α protein expression. Cox IV and Cytochrome C protein expression were decreased 53% and 39% respectively after IL‐6 over‐expression. These results show oxidative capacity in wasting skeletal muscle is reduced in both oxidative and glycolytic muscle and IL‐6 over‐expression can reduce muscle oxidative capacity. Funded by R01CA121249‐01A2
Determining the Binding Affinity Between Calcium and the Phospholipid Analog, Cyclohexyl Phospho‐L‐Serine
Authors: Buytendyk Allyson M. , Amburgey‐Peters Judith C.
Published: June 2021

During apoptosis a ternary complex is formed between the phospholipid membrane surface of a cell, calcium metal ions, and a repair protein. The binding affinity and structural arrangement of this complex is not well characterized. Small molecule analogs of the phospholipid, phosphatidylserine (PS), are proposed to investigate the binding in the ternary complex. A phospolipid analog (PLA) of PS, cyclohexyl phospho‐L‐serine (cycPS), was previously synthesized. Isothermal titration calorimetry (ITC) was used to determine how the anionic phospho‐L‐serine head group of the proposed analog compares to PS in affinity to and selectivity for Ca2+ over Mg2+. The ITC method was first developed to measure Ca2+ binding by a similar commercially available compound, O‐phospho‐L‐serine, and then applied to the proposed analog, cycPS. Comparable Ca2+ binding affinity of cycPS relative to PS allows for further experiments with Mg2+ to evaluate if cycPS is a sufficient structural and biological model of PS. This research was made possible through funding from The College of Wooster.
Lysophosphatidic acid regulates human gingival and periodontal ligament fibroblast expression of PDGF receptors
Authors: Cerutis D. Roselyn , Headen Karmel V. , McVaney Timothy P. , Ogunleye Afolabi , Mattson John S. , Parrish Lawrence C.
Published: June 2021

LPA, a G protein‐coupled lipid mediator involved in wound healing, binds to LPA1‐5 receptors (LPARs). It is co‐released by activated platelets together with platelet‐derived growth factor (PDGF). We have published that human gingival (GF) and periodontal ligament (PDLF) fibroblasts express LPA1‐3, and that LPA positively regulates their responses to PDGF. In this study, we tested the hypotheses that LPA exerts these effects in part by 1) transactivating the PDGFR‐ β, and 2) regulating plasma membrane expression of PDGF receptors. Using immunoprecipitation and Western blotting (IP/WB), antibodies specific for the activated form of the PDGFR‐ β were used to detect if stimulation with LPA transactivates this receptor. Determination of cell‐surface expression of PDGFRs was done by flow cytometry with antibodies specific for PDGFR‐α and ‐ β. Serum‐starved cells in 100‐mm dishes were stimulated with 1 x 10−7 ‐ 1 x 10−5M LPA for 0, 2, 4, and 6h, and the levels of PDGFR ‐α and ‐ β were measured. We were not able to detect transactivation of the PDGFR‐ β in GF or PDLF by IP/WB. However, LPA modulates the cell‐surface expression of both PDGFR‐α and ‐ β, consistently increasing the number of PDGFR‐α and ‐ β‐positive cells between 2‐12‐fold (in a donor‐dependent fashion, compared to untreated controls), between 2 and 4h post‐LPA treatment. From these results, we conclude that the PDGFR‐ β is not transactivated by LPA in GF and PDLF, but that LPA does exert complex regulation of both PDGFR‐α and ‐βin these cells. Support: NIH/NIDCR 1 R15 DE016855‐03 (D.R.C).
Translocator protein 2 (TSPO2), a new family of proteins involved in cholesterol redistribution in erythropoiesis
Authors: Fan Jinjiang , Papadopoulos Vassilios
Published: June 2021

Translocator protein (18‐kDa; TSPO) is a highly conserved cholesterol‐ and drug‐binding protein. Analysis of Tspo‐like sequences from different organisms revealed the presence of evolutionary conserved genes which arose by gene duplications before the divergence of avians and mammals. This new family of genes was named Tspo2 and found to be under strong purifying selection, suggesting that it serves a conserved biological function. The predicted cholesterol‐binding motif at the C‐terminus of TSPO was present in TSPO2, suggesting a role in cholesterol trafficking and/or targeting. In contrast to the mitochondrial TSPO, TSPO2 was found in nuclear and ER membranes. Heterologous expression of TSPO2 in Saccharomyces cerevisiae demonstrated that TSPO2 indeed binds cholesterol with high affinity. However, TSPO2 lost the TSPO drug binding ability. Spatial and temporal distribution of Tspo2 mRNA in the mouse demonstrated its presence in hematopoetic tissues and expression in an erythroid cell type‐specific manner. Overexpression of Tspo2 in erythroid cells resulted in the redistribution of the intracellular free cholesterol which has been linked to the expulsion of the nucleus from erythroblasts leading to erythrocyte maturation. These data suggest that the identified gene expansion in Tspo family supports a specialized function of the TSPO2 in erythropoiesis of endothermic animals, including humans.
ADAM9 Isoforms in Breast Cancer Cell Migration
Authors: Fry Jessica Lynn , Toker Alex
Published: June 2021

Tumor cell migration is a complex event mediated by the genetic characteristics of the tumor cells themselves in cooperation with changes in the tissue microenvironment. ADAM9, a member of a family of transmembrane matrix‐metalloproteases named for their integrin‐binding Disintegrin And Metalloprotease domains, is implicated in cell‐cell and cell‐matrix interactions as well as in shedding of receptors and ligands. ADAM9 is present in breast cancer cell lines as two alternatively spliced isoforms ‐‐ ADAM9‐L and the truncated, secreted, ADAM9‐S. Exogenous and endogenous overexpression of ADAM9‐S increases breast cancer cell migration, leading to our hypothesis that ADAM9 present in the breast tumor environment mediates tumor cell migration via an unknown mechanism. Using lentiviral‐shRNA constructs, we have silenced expression of endogenous human ADAM9 protein in the BT549 and CAMA‐1 breast cancer cell lines, resulting in an increase in cell migration measured by transwell assay. This enhanced migration is reversed by reintroduction of ADAM9‐L protein into these cells, while reintroduction of ADAM9‐S enhances this migration phenotype in a metalloprotease‐dependent manner. Further analysis using functional mutants of the metalloprotease, disintegrin, and cytoplasmic signaling domains of ADAM9‐L will lend insight into the mechanisms by which ADAM9 mediates breast cancer cell migration.
Molecular mechanism and specificity of substrate binding by the E3 Ubiquitin Ligase Pellino
Authors: Huoh Yu‐San , Lin Chun‐Chi , Ferguson Kathryn M.
Published: June 2021

Upon stimulation of Toll‐like receptors or IL‐1 receptors, IL‐1 receptor associated kinases (IRAKs) are activated to initiate cascades of events that are critical in innate immune signaling pathways. The Pellino family of RING E3 ubiquitin ligases is an important regulator of IRAKs. All 3 human Pellino isoforms bind to and polyubiquitinate activated, phosphorylated IRAK1. We have determined the X‐ray crystal structure of the Pellino2 N‐terminal substrate‐binding region, which contains a previously unidentified non‐canonical example of a forkhead‐associated (FHA) domain. FHA domains are phosphoThr (pT)‐binding modules that mediate protein‐protein interactions in a variety of cell‐signaling pathways. We have shown that the canonical pT‐binding region of Pellino mediates interaction with pTs on IRAK1. We expect specificity is defined by non‐canonical features of the Pellino FHA domain and explore this with mutational analysis. We also use IRAK1 truncation variants to define a minimal region of phosphorylated IRAK1 that is sufficient for Pellino binding. This research is supported by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund and by NIH Training Program (T32‐GM08275).
Control of breast cancer by dietary alpha‐lactalbumin is mediated by regulation of retinoid metabolism and signaling
Authors: Sinha Roy Somdutta , Schäffer Michael W , Mukherjee Shyamali , Das Salil K
Published: June 2021

Dietary protein is a significant factor in breast cancer control. However, no information is available concerning the effects of á‐lactalbumin on the expression of certain genes involved in breast cancer control. Breast cancer was developed by gavage administration of DMBA in female rats, maintained on AIN‐76A diet with either casein or á‐lactalbumin as protein source for 120 days. The whey group had less cancer than the casein group. Casein group had a mixture of grade I, grade II and grade III tumors whereas the whey group had mostly grade I tumor. The levels of retinoic acid (RA), retinol (ROH) and retinyl palmitate (RP) were 44, 430, 606 µg/g, respectively in control tissue, and their levels were significantly reduced in tumors. However, the decrease of RA, ROH and RP was significantly less in the whey group (77, 48, 45%, respectively) than that in the casein group (84.5%, 69.5% and 54.6%, respectively). The expression of various binding proteins and mediators of retinoid action like, CRBP I, CRABP II, LRAT and nuclear RAR and RXR receptors also show similar variation among these groups. (Supported by FAMRI Clinical Innovator Award # 62415)
CD137 ligand‐mediated reverse signals increase cell numbers and cytokine expression in murine myeloid RAW264.7 cells
Authors: Lee Hyeon‐Woo , Kim Dong‐Ku
Published: June 2021

Cross‐linking of CD137 ligand (CD137L), a member of the TNF family, with recombinant CD137‐Fc protein (rCD137‐Fc) enhanced adherence of bone marrow‐derived macrophages, and increased the expression of ICAM‐1. In RAW 264.7 cells, a murine myeloid cell line, rCD137‐Fc not only increased adherence but also cell multiplication, in a manner comparable to LPS or M‐CSF. In addition, it up‐regulated expression of IL‐1β, IL‐1 receptor antagonist, IL‐6, COX2, Tenascin C, Neuropeptide Y and M‐CSF mRNA. Neutralization of M‐CSF by incubating the RAW264.7 cells with M‐CSF mAb did not prevent the CD137L signal‐induced viability. Viability was blocked by PP2, a Src tyrosine kinase inhibitor, rapamycin, an mTOR inhibitor and LY294002, a PI3K inhibitor, but not by Wortmannin, another PI3K inhibitor. Cross‐linking of CD137L increased phosphorylation of Akt and p70S6 kinase. The latter was blocked by PP2, rapamycin or LY294002, but not by Wortmannin, whereas phosphorylation of Akt was blocked by LY294002 or Wortmannin. These findings demonstrate that reverse signals evoked by CD137L regulate immune functions in macrophages.
Endothelins derived from vascular endothelial cells promote cortical neurite elongation
Authors: MURAMATSU RIEKO , UESUGI NORIKO , YAMASHITA TOSHIHIDE
Published: June 2021

Branched structure of blood vessels and nerves are similar in adult tissue, suggesting that one may regulate the other patterning during ontogenesis. Given that this regulation continues to work during adulthood, enhancement of this mechanism may contribute to regeneration of injured neural network. To elucidate the crosstalk of vascular system and neurons, we employed co‐culture of cortical neurons and cortical capillary endothelial cells, and found that diffusible factors derived from capillary endothelial cells elongated cortical neurites in vitro. As endothelin receptors were expressed in cortical neurons, we next investigated the effects of endothelins, as factors secreted from capillary, on the nerite length. Treatment with each endothelin isoform promoted cortical neurite extension. These results suggest that endothelins derived from vascular endothelial cells could promote neurite elongation and may contribute to regeneration of the injured CNS.
WT1 regulates expression of pro and anti angiogenic isoform of VEGF
Authors: Amin Elianna Mohamed , Saleem Moin , Bates Dave , Ladomery Michael Robert
Published: June 2021

The Wilms tumour suppressor gene WT1 is mutated in Denys Drash Syndrome (DDS). DDS is characterised by childhood nephrotic syndrome, glomerulosclerosis, end‐stage renal failure and Wilms tumours. VEGF splicing is altered in DDS patients' kidneys (Schumacher et al 2007). The splice factor ASF/SF2 a known proto‐oncogene (Krainer et al), shuttles between the nucleus and cytoplasm promotes the splicing to the pro angiogenic isoform of VEGF (Nowak et al 2008). In DDS podocytes there is a 8.7+0.003 fold reduction in VEGFxxxb compared to wildtype. DDS podocytes stably transfected with a plasmid expressing wildtype WT1 restored the expression of VEGFxxxb to 0.73+0.08. In the DDS podocytes, ASF/SF2 was predominantly localized in the nucleus while the DDS podocytes transfected with wildtype WT1 had ASF/SF2 equally localized between the cytoplasm and nucleus similar to the wildtype podocytes. The localization of ASF/SF2 dependent on phosphorylation by kinases such as SR‐protein‐specific kinase 1 (SRPK1). Western blot and qPCR analysis of SRPK1 levels reveals a higher expression protein and mRNA transcripts in the DDS podocytes. The ''rescued' DDS podocytes had a reduction in both protein and mRNA level of SRPK1 similar to the wildtype podocytes. These findings suggest that the tumour suppressor WT1 regulates splicing through a novel mechanism by transcriptionally regulating a key kinase involved in the RNA machinery.
Isoform‐specific knockdown of monoamine oxidase expression retards murine embryogenesis via inhibition of developmental apoptosis
Authors: Kuehn Hartmut , Borchert Astrid , Wang Chi Chui , Ufer Christoph , Billett Ellen
Published: June 2021

Monoamine oxidases are key enzymes in metabolism of biogenic amines. Their role in the nervous systems is well established but their function in cerebral embryogenesis has not been well characterized. We explored expression of MAO during murine embryogenesis and found that MAO‐A and MAO‐B are expressed as early as E6.5. Expression levels remain constant until E13.5 but then dropped down to almost undetectable levels at birth. In brain similar expression kinetics were monitored. Pharmacological inhibition and siRNA‐mediated knockdown of MAO‐A expression induced severe developmental retardations and significant impairment of developmental brain scores. Such alterations were not observed when expression of MAO‐B was silenced. To explore the molecular basis for these effects we quantified the degree of apoptosis in siRNA treated embryos and found reduced numbers of apoptotic cells in epithelium of the neural tube. These alterations were paralleled by impaired activation of caspase 3 and augmented cyclin D1 expression. These data indicate that knockdown of MAO‐A impairs developmental apoptosis preventing activation of caspase‐3 leading to uncoordinated cell proliferation.
Protein glutathionylation regulates FasL‐induced apoptosis.
Authors: Franco Rodrigo , Cidlowski John A
Published: June 2021

Apoptosis is a basic homeostatic mechanism involved in a variety physiological processes and whose deregulation leads to the progression of several human diseases. It is now clear that changes in the cellular redox homeostasis regulate apoptosis. However, the molecular mechanisms involved have remained largely elusive. Recently, we demonstrated that reduced glutathione (GSH) depletion regulates apoptosis independent of the excessive accumulation of ROS and widespread oxidative stress (J Biol Chem. 2006, 281:29542; J Biol Chem. 2007, 282:30452; J Biol Chem. 2008, Oct 21, Epub) suggesting a direct role of GSH homeostasis in the redox‐dependent regulation of apoptosis. To evaluate potential mechanisms in this process, we studied the role of protein glutathionylation (a reversible oxidative posttranslational modification mediated by changes in GSH homeostasis) on the progression of apoptosis by FasL. FasL induced a dose‐dependent increase in protein glutathionylation in Jurkat cells measured using an anti‐glutathionylated (P‐SSG) residue antibody by western‐immunoblots. FasL‐induced protein glutathionylation was shown to be prevented by inhibition of GSH depletion with high extracellular GSH medium, which suggests that GSH depletion promotes protein glutathionylation during apoptosis. Immunoprecipitation assays demonstrate that FasL induced a dose‐dependent glutathionylation of execution caspases suggesting a regulatory role for protein glutathionylation on caspase activation. GSH depletion is paralleled by a switch in the GSH/glutathione disulfide (GSSG) ratio in favor to an increase in GSSG. Thus, we decided to study if GSSG mediates protein glutathionylation of execution caspases. Using mass spectrometry analysis we demonstrated that in in vitro cell‐free assays, GSSG was able to induce execution caspase glutathionylation at specific cysteinyl moieties with low pKa values distinct from the active site of caspases. In vitro gluathionylation of caspases by GSSG was reversed under reducing conditions (DTT) and in the presence of high GSH concentrations. These results demonstrate that FasL‐induced GSH depletion mediates an increase in glutathionylation of selective proteins. They also suggest that the reduction in GSH/GSSG ratio mediates execution caspase glutathionylation during apoptosis. We propose that GSH homeostasis regulates apoptosis via protein glutathionylation.
EFFECT OF PATHWAY‐INTERCONNECTORS IN SEB INDUCED APOPTOSIS RELATED EVENTS IN HUMAN PBMCS
Authors: Lillge Cayla , Zindel Kristin , Mendis Chanaka
Published: June 2021

Staphylococcal entrotoxin B (SEB) is one of the endotoxins that has been thoroughly investigated; however, little is known about the cascades of signaling events that explain its patho‐mechanism. This study follows a molecular route to better understand the apoptosis related events known to be induced by SEB in human peripheral blood mononuclear cells (PBMC). As SEB is known to induce multiple signal transduction pathways, we have identified a crucial pathway inter‐connector (JNK) that may have an inhibitory effect in SEB induced unwanted cellular activities. Our study was centered on inhibiting pathway inter‐connector JNK by SP600125, followed by an evaluation of the efficacy of the target through alterations to a set of known apoptosis related genes including Caspase 1, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, SOD, and Heparanse precursor (HEP) expression patterns. We further confirmed this effect by carrying out a set of protein expression analysis. As our experimental design specifically targeted pathway inter‐connector JNK, we believe that the target is unique and has the ability to sustain longer lasting inhibitory effects. We are also confident that inhibiting such a component will have a maximum effect on a cell module, terminating the "leaking effect", which may benefit a number of experimental modules focused on disease prevention and rapid diagnostics.
Raising intracellular free magnesium ions antagonizes caspase activation and apoptosis in mammalian cells
Authors: Garcia Patrick L. , Smith Jeffrey B.
Published: June 2021

We have found that free magnesium ions block Apaf‐1 apoptosome activation of caspase‐9 in vitro by competition with cytochrome c (Abstract 2214). Here we show that raising intracellular free Mg2+ inhibits the induction of apoptosis and activation of caspases ‐9 and ‐3. HeLa cells were treated with PS‐341, a proteasome inhibitor, for 28 h to induce apoptosis. Alternatively, apoptosis was induced by a 5 h treatment with TNFα plus cycloheximide. In both cases, apoptosis was observed by characteristic changes in cell morphology, caspase activation, detachment from the substratum, and cell death. Induction of apoptosis increased caspase‐3 like activity, which was assayed in cell lysates with a fluorogenic substrate, by 13‐fold following proteasome inhibition or 43‐fold following the TNFα?treatment??? Intracellular free Mg2+ was raised by the addition of 20 to 60 mM MgCl2 to the normal culture medium, which contained 0.8 mM Mg2+. Increasing external Mg2+ had no effect on cell viability. Intracellular free Mg2+, which was measured with Mag‐Fluo‐4AM, was 0.6 mM in the normal culture medium and increased to 1.6, 2.2, and 2.4 mM after a 5 or 24 h incubation at 21, 41, or 61 mM external Mg2+, respectively. Raising intracellular free Mg2+ essentially abolished caspase‐3 activation following proteasome inhibition and decreased caspase‐3 activation by the TNFα?treatment by ~60%. Western analysis confirmed the activation of caspase‐3 and indicated that procaspase‐9 was processed to subunits following the treatments. Importantly raising free Mg2+ decreased the processing of both procaspases to subunits. Raising external Mg2+ also protected 911 retinoblasts from apoptosis. These findings support the idea that raising intracellular free Mg2+ antagonizes apoptosis, apparently by decreasing caspase‐9 activation via the apoptosome. Supported by grant GM60383 from NIH.
Toward Understanding the Reaction Kinetics of a Coumarin‐Fluorescein FRET based Dithio Probe for Quantitation of Cellular Thiols
Authors: Jonhera Timothy , Sem Daniel
Published: June 2021

Thio containing compounds like glutathione and protein cysteines are essential for the proper function of a cell. The levels of thiols and their oxidation state determine the cellular redox‐potential, which is associated with oxidative stress, and disease state of a cell. We recently reported synthesis and preliminary characterization of a coumarin‐fluorescein‐ reagent (Donor‐S‐S‐Acceptor, DSSA), a FRET‐based fluorescent dithio probe with unusually low reduction potential (‐ 0.6 V) that can detect changes in intracellular thiols and cross membranes of bacterial and mammalian cells. Here we report in vitro reaction kinetics of this probe with glutathione, a major cellular redox potential regulator. The dithio probe was reacted with reduced and oxidized glutathione and their ratiometric mixtures under physiologically relevant conditions. We have demonstrated that this probe is reduced by reduced glutathione (GSH) in a two step process via an intermediate. The first step involves an initial burst, with an intermediate which is more fluorescent than the initial probe, followed by stead state increase in fluorescence in the second step. Reactions with oxidized dithios showed a rapid one step process due to disulfide exchange,while ratiometric mixtures of oxidized and reduced thiols show predictable trends relating to their concentration ratios. Understanding of these mechanisms is critical in utilizing this probe to quantify cellular thiols in real time. This research was supported by a Biomedical Technology Alliance grant (01‐KMS LEG FY06‐12368) to D.S.S.
Assessment of the Effectiveness of Structure Exploration Tools in Biochemistry Education
Authors: Bateman Robert
Published: June 2021

Most biochemistry instructors strive to illustrate complex biomacromolecular structures by both static images and interactive computer graphics. Most textbooks come with elaborate tutorials on CD or websites. Other biochemistry educators have written tutorials for stand‐alone graphics viewers such as Rasmol, Deep View, or kinemages, or for browser‐based viewers such as Jmol. Recent attempts to incorporate virtual reality games into structure exploration also seem promising. A clear measure of the educational effectiveness of these computer‐based renderings, however, will require new assessment tools that will have to account for a variety of factors including learning of content, development of spatial abilities, and effect on student attitudes and motivation levels. This presentation will consider possible approaches to developing such assessment instruments.
Next‐generation Immunoprecipitation Kits Validated for Functional Applications
Authors: Desai Surbhi , Ganapathy Ramesh , Kilmer Greg , Rosenblatt Michael , Deshpande Atul , Wolf Brian D.
Published: June 2021

The study of protein function and interaction is essential for understanding many biological processes. We have developed three new immunoprecipitation (IP) kits with improved protocols and buffers that provide excellent antigen recovery with the least amount of nonspecific binding using 2 to 10 µg of antibody. The Pierce IP Direct kit contains our AminoLink Plus Coupling resin for directly crosslinking purified antibodies to the matrix for specific elution of the antigen. The Pierce IP Classic kit contains Protein A/G Plus Agarose that binds immunoglobulins from a variety of species. The Protein A/G Plus resin is also used in the Pierce IP Crosslinked kit which is designed to purify and crosslink the antibody to the resin before performing the immunoprecipitation procedure. With the new kits, we have demonstrated immunoprecipitations of a diverse selection of proteins including membrane proteins, protein phosphatases and kinases, transcription factors, and cell cycle regulation proteins. We show that the new Thermo Scientific Pierce IP Kits immunoprecipitate protein complexes and are compatible with mass spectrometry and enzymatic assays.
Enhanced Detection of Low Abundant Molecules using Polymerized HRP Conjugates
Authors: Ganapathy Ramesh , Wolf Brian , Nlend Marie , Stanaitis Mike , Schofield Mark , Kilmer Greg , Desai Surbhi
Published: June 2021

Enzyme conjugated secondary detection reagents are routinely used for amplified detection of specific antigens in immunoassays. Horseradish peroxidase (HRP) is the most widely used enzyme in ELISA, Western blotting, immunohistochemistry, Northern and Southern blotting applications and hybridization assays, because it is economical, robust and compatible with both insoluble and soluble substrates.

We have developed a series of Streptavidin, Goat anti‐mouse (GAM) and Goat anti‐rabbit (GAR) Poly HRP for the detection of low‐abundant molecules. The new polymerized HRP conjugates are purified to remove unconjugated HRP, secondary detection reagents and monomer HRP conjugates.

We have demonstrated increased sensitivity and signal intensity with the Thermo Scientific Pierce Poly HRP conjugates in ELISA, Western blotting and immunohistochemistry (IHC) applications. Human IFNγ and Bovine IL4 ELISAs using the Pierce Poly HRP conjugates displayed significant signal amplification over non‐polymerized and polymerized HRP conjugates. Western blotting and IHC detection of low abundant proteins displayed similar signal enhancement with Thermo Scientific Pierce Poly HRP conjugates.
The USDA Nutrition Evidence Library: An Evidence‐Based Resource for Development of National Nutrition Policy
Authors: Fungwe Thomas V , Altman Jean , Blum‐Kemelor Donna , Hopwood Heather , MacNeil Patricia C , McCormick Maura , Wong Yatping , Lyon Joan
Published: June 2021

The USDA Nutrition Evidence Library (NEL) plays an integral role in the development of national nutrition policy. The public health community uses a systematic, evidence‐based approach to review scientific literature for developing health promotion and prevention guidelines. The Dietary Guidelines for Americans provide science‐based advice to promote health and reduce chronic disease risk. They also serve as the foundation of national nutrition policy, programs, and educational efforts. USDA developed the NEL as a web‐based system with a set of tools to support the 2010 Dietary Guidelines Advisory Committee and other efforts to improve the nutritional well‐being of Americans. The NEL provides portfolios of evidence worksheets and management tools to support each step in the evidence‐based process, including research question formulation, literature search and sort plan development, evidence analysis and quality assessment, and rating the strength of the conclusion and recommendations. Completed reports and supporting documents will be archived in the NEL and made accessible to stakeholders and the public. This library will ensure that the relevant, timely, high‐quality, and understandable presentation of evidence is available to support translation of nutrition research into policies that promote the health of the US population.
Snack selection influences nutrient intake, metabolism and bowel habits in adult women
Authors: Howarth Leslie S. , Petrisko Yumi , Furchner‐Evanson Allison , Nemoseck Tricia , Kern Mark
Published: June 2021

Since appropriate snacking may promote a healthy body weight and serve as an important contributor to a healthful diet for women, identification of suitable foods for inclusion between meals is essential. We investigated the influence of the incorporation of 100 kcal servings of snacks of dried plums versus commercial low‐fat cookies twice daily for two weeks with a two‐week washout period between trials on total energy and nutrient intake, biochemical parameters, and bowel habits. Twenty‐nine women aged 25‐54 years with a BMI between 24‐35 kg/m2 participated in a randomized, cross‐over fashion. Incorporation of dried plums or cookies into the diet did not alter energy intake or body weight; however, dried plums promoted greater (p=0.05) intake of fiber, potassium, riboflavin, niacin, and calcium compared to cookies. Total fat intake tended (p=0.094) to drop with dried plum consumption as did cholesterol intake (p=0.098). Plasma TG concentration increased (p=0.05) after consumption of cookies and was lower (p=0.05) for dried plums versus cookies. Dried plums promoted a softer (p=0.05) stool consistency compared to baseline and cookies. Subjects reported more complete evacuation of bowels (p=0.05) with dried plums versus cookies. These results suggest that dried plums improve blood lipids and diet quality and soften stools in comparison to a commercially processed snack.

Supported by the CA Dried Plum Board.