bluuebunny/crossref_metadata_2025 huggingface dataset:
NHLBI‐AbDesigner, artificial intelligence‐based software for design of peptide‐directed antibodies
Authors: Pisitkun Trairak , Knepper M A
Published: June 2021

Although protein mass spectrometry is useful for discovery science, follow up from such studies often requires development of high quality antibodies. Here we describe a new software tool called NHLBI‐AbDesigner written in Java that predicts optimal sequences to be used for production of synthetic peptides for immunization. The choice of the immunizing peptides is based on a need to optimize immunogenicity, antibody specificity, multi‐species conservation, and robustness in the face of post‐translational modifications (PTMs). Current protocols for doing this require multiple manual steps using different computational tools and elements of human judgment that require experience. The new software predicts optimal sites along the primary sequence of proteins by calculating and extracting information as follows: 1) immunogenicity score based on secondary structure prediction and hydropathy; 2) uniqueness score [predicting specificity of an antibody]; 3) conservation score [predicting likelihood of multi‐species recognition of an antibody]; and 4) protein features that describe regions or sites of interest such as PTMs. It displays the prediction in an easy‐to‐view graphic user interface including heat maps for the scored predictors. NHLBI AbDesigner is available in the form of stand‐alone and web applications. It can also perform batch processing for a large‐scale production of antibodies which can be exploited for antibody‐based proteomics tools.
AN INTEGRATED EXPERIMENTAL WORKFLOW TO INCREASE THROUGHPUT AND DATA ROBUSTNESS FOR ANALYSIS OF MAMMALIAN PROTEIN INTERACTION NETWORKS
Authors: Auerbach Daniel , Baumann Lukas , Uhlmann Thomas , Bruderer Roland , Gstaiger Matthias , Aebersold Ruedi , Rezwan Mandana
Published: June 2021

The analysis of protein complexes and how biological information is propagated via protein‐protein interactions is becoming increasingly important to obtain new insights into cellular signaling processes. Affinity purification coupled with mass spectrometry (AP‐MS) has been successfully used in the past to characterize protein complexes. However, mainly due to experimental limitations, the success of the AP‐MS strategy has been somewhat limited to date. Here, we describe an integrated workflow for characterizing mammalian protein complexes. The experimental pipeline comprises rapid generation of isogenic mammalian cell lines expressing a bait protein of interest, protein complex analysis using an efficient double affinity purification strategy and state‐of‐the‐art nano‐LC‐Orbitrap‐MS analysis. We have used this workflow to study interactions associated with various signaling pathways, including the NF‐kB and BMPR pathways. We demonstrate that the improved AP‐MS workflow identifies hitherto unknown protein interactions and can be used to dissect interactions involving membrane bound receptors and associated signaling proteins. The high performance of our strategy is well suited to identifying protein interaction partners that can subsequently be used as entry points for focused research projects and to globally map whole signaling systems linked to important biological processes.
Genomic Analysis of Etoposide Resistance Reveals Multiple Alterations of Gene and microRNA Expression
Authors: Moitra Karobi , McGee Kate , Sawitzke Julie , Yuhki Naoya , Robey Rob , Bates Susan , Dean Michael
Published: June 2021

In order to develop targeted therapeutic strategies to combat drug resistance it is essential to understand the basic molecular mechanisms through which cancer cells control sensitivity to chemotherapeutics. To identify new candidate genes and facilitate the discovery of novel drug resistance pathways we generated differential gene expression profiles of etoposide resistant and sensitive MCF7 breast cancer cells using Affymetrix U133 plus 2 high‐density microarrays. Differential expression of over 4000 genes indicated that several drug resistance mechanisms are operating in etoposide resistant breast cancer cells including upregulation of ABC transporter genes (ABCC1 & ABCC6), upregulation of DNA repair pathway genes (p21, GADD45 & STAT1) and downregulation of apoptotic pathway genes (CASP7, DIABLO etc). Transcription factors SOX9 & RUNX2 were upregulated in the drug resistant cells, suggesting their role in etoposide induced drug resistance. RUNX2 knockdown in the resistant cells increased sensitivity to etoposide and upregulated expression of pro‐apoptotic genes. MicroRNA arrays revealed differential expression of miRNA's in the cells lines. We found that miR‐218 mimic could downregulate the expression of ABCC6 in drug resistant cells indicating it's potential as a therapeutic agent to circumvent drug resistance. This research was supported by the Intramural Research Program of the NIH, NCI.
Preliminary X‐ray Crystallographic and Structural Analyses of a Bacteriophytochrome from Stigmatella aurantiaca
Authors: Woitowich Nicole Catherine , Garrido Carlos , Ozarowski Wesley , Stojkovic Emina A
Published: June 2021

Phytochromes are red‐light photoreceptors found in plants, bacteria, and fungi. Specifically, phytochromes reversibly photoconvert between spectrally distinct red‐light (Pr) and far‐red (Pfr) light absorbing states. Biliverdin (BV), a linear tetrapyrrole is the light‐sensing molecule that initiates the photoconversion in bacteriophytochromes (Bphs). Here we report on the initial structural studies of a Bph from Stigmatella aurantiaca (SaBphP). Sequence alignment of related Bphs revealed that SaBphP is the only Bph that contains a threonine (T) instead of a conserved histidine (H) in the BV‐binding pocket of the protein. This conserved H forms a hydrogen bond with the carbonyl group of the BV D‐ring in the structures of related Bphs. SaBphP crystallized bound to BV in the Pr state in 6 different conditions. In one condition, crystals yielded diffraction to a maximum resolution of 2.65 Å. Data were collected at the BioCARS 14BM‐C beam station at the Advanced Photon Source, Argonne National Laboratory. All images were indexed, integrated, and scaled by using HKL2000. SaBphP crystals are in space group P32 (a = b = 131.1 Å, c = 96.9 Å). Initial SaBphP structure has been determined using molecular replacement method implemented in Phaser by related structure of a homologous Bph from R. palustris (RpBphP3) as a search model. Supported by NIH EARDA pilot grant 5G11HD049644 – 04 to E.A.S.
Immunological and catalytic features of InfA‐15 mAb and the light chain raised against hemagglutinin molecule for Influenza virus A type
Authors: Hifumi Emi , Fujimoto Naoko , Yahiro Taka‐aki , Uda Taizo
Published: June 2021

Through we have been exploring the catalytic antibody (antigenase) in this decade, we have produced a unique monoclonal antibody (InfA‐15) raised against a conserved region of HA2 subunit of the hemagglutinin molecule of influenza virus A type (1). The InfA‐15 monoclonal antibody could recognize all important hemagglutinin molecules such as H1, H3 and H5‐subtype of the virus. Based on the structural analysis, the InfA‐15 monoclonal antibody possesses a catalytic triad‐like structure which is considered to work as the active center of the enzymatic function of the antibody. Thus, we investigated the some features such as amidase activity for synthetic peptide and cleavage activity for the antigen. Moreover the light chain of InfA‐15 monoclonal antibody was genetically synthesized to investigate the detail enzymatic function. It was interesting that the light chain prepared from mouse antibody and the recombinant light chain expressed in E.coli showed the different characteristics for the cleavage reaction of the substrate (antigen). The catalytic function of the antibody or its subunit will be discussed on the molecular basis.
Structural analysis of P6 from nontypable Haemophilus influenzae suggests that it is not a transmembrane protein
Authors: Kalmeta Breanna , Snyder Joy , McCreary Mark , Huddleston Nathaniel , Pichichero Michael , Michel Lea Vacca
Published: June 2021

The objective of our research was to identify an epitope in the outer membrane protein P6 of nontypable Haemophilus influenzae (NTHi). The long term goal of this research is to develop a protein/peptide vaccine against NTHi, as it is one of the major causes of pneumonia, bronchitis, ear infections, and other illnesses. Recombinant DNA technologies, nuclear magnetic resonance (NMR) spectroscopy and ELISA experiments were utilized to determine that residue 40 is part of the epitope to monoclonal antibody 7F3. This result, in light of structural analysis of the protein, suggests that P6 is not a transmembrane protein. From these findings, we were able to develop a novel hypothesis which describes P6 as an intracellular lipoprotein and raises doubt as to whether P6 can still be considered a viable vaccine candidate. This study was funded by NIH NIDCD RO1 08671 and the Rochester Institute of Technology.
MLCK Deletion Enhances Intestinal Smooth Muscle Cells Migration by Reducing Cell Membrane Tension
Authors: Chen Chen , Yu Yang , Wen Cheng , Peng Yajing , He Weiqi , Ye Anpei , Zhu Minsheng
Published: June 2021

Myosin light chain kinase (MLCK), a dedicated Ca2+/calmodulin‐dependent protein kinase that phosphorylates the regulatory light chain of myosin II, has been proposed to play critical roles in many biological processes, including smooth muscle contraction, endothelial cells permeability, stress fiber formation, cell spreading and migration, fibroblasts contractile activity and proliferation, cytokinesis, secretion, neuronal growth cone extension. But the roles of MLCK in regulating the contractility of cortical actomyosin and tension of plasma membrane are still unclear. In this report, we established primary cultured MLCK‐deficient mouse intestinal smooth muscle cells (ISMCs), then investigated the alterations in cell morphology and migrating characteristics. MLCK‐deficient ISMCs showed more lamellipodias and significant increased spreading and random migrating speed according to control cells. Furthermore, tether force measurement by laser tweezers suggested lower cell membrane tension after MLCK deletion. And transmission electron microscopy displayed a possible looser structure of thick filaments nearby MLCK‐deficient SMCs membrane. Thus, we have a conclusion that MLCK contributes to regulating cortical actomyosin based cell membrane tension which affects cell behavior in spreading and migration.
Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors
Authors: Lee Chan‐Soo , Shin Eun‐Young , Hong Jong‐Myeon , Schwartz Martin A , Kim Eung‐Gook
Published: June 2021

Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology‐pleckstrin homology module, the catalytic site of these GEFs, with dissociation constants of ~0.3 microM. Binding to the GEFs required assembly of the MII into filaments and actin‐stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly, inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of bPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells.
Denervation increase Akt phosphorylation and reduce glyceroneogenesis in white adipose tissue from diabetic rats
Authors: Frasson Danubia , Chaves Valéria Ernestânia , Garófalo Maria Antonieta Rissato , Navegantes Luiz Carlos Carvalho , Migliorini Renato Hélios , Kettelhut Isis do Carmo
Published: June 2021

It has been known that adipose tissue (WAT) has three sources of glycerol‐3‐phosphate (G3P) generation and these pathways seem to be reciprocally controlled to maintain the stores of TAG. The aim of this work was to understand the role of SNS and insulin in these processes. WAT from diabetic and controls rats was denervated and in vitro glucose uptake, P‐enol‐pyruvate carboxykinase (PEPCK) activity, a key enzyme of glyceroneogenesis and Akt phosphorylation were evaluated. Chemical denervation (D) of epididymal WAT was performed 7 days before the experiments, using contralateral WAT as control. Diabetes was induced by i.v. STZ injection (40mg/Kg) 72h before the experiments. D caused 88% reduction in WAT NOR content. Diabetes reduced the in vitro glucose uptake (nmol. 106cells−1.min−1) (1.1±0.1 vs control 1.5±0.03) and D caused a further reduction in adipocytes of diabetic rats (0.5±0.1 vs innervated(IN) 1.1±0.1). Diabetes increased PEPCK activity (nmol.mg protein−1.min−1) (12.9±1.1 vs control 2.6±0.1) and PEPCK content (70%), D reduced PEPCK activity (4.0±0.4 vs IN 12.9±1.1) and also PEPCK content (30%). Diabetes reduced pSer473‐Akt (38%) and D increased Akt phosphorylation by 69%. The data suggest that SNS can regulate glyceroneogenesis and denervation seems to increase the insulin response in diabetic animals. Financial support: CNPq and FAPESP.
A modified lipid composition in Fabry's disease leads to an intracellular block of the lipid rafts‐associated dipeptidylpeptidase IV
Authors: Maalouf Katia Elias , Rizk Sandra , Das Anibh , Naim Hassan Y.
Published: June 2021

Fabry's disease (Anderson‐Fabry's disease) is an X‐linked lysosomal storage disorder caused by deficiency of á‐galactosidase A (AGAL), and results in an abnormal accumulation of glycosphingolipids. Here, we show by confocal laser microscopy of Fabry fibroblasts that the colocalization of AGAL with the lysosomal marker LAMP2 is decreased concomitant with a reduced transport of AGAL to lysosomes. Furthermore, analysis of overall composition of membrane lipids in the patients' fibroblasts reveals altered concentrations of glycolipids and phosphatidylethanolamine (PE). The consequences are (i) a decrease in the concentration of lipid rafts as assessed by the reduced levels of the rafts marker flotillin2 in the floating fractions of sucrose gradients of Triton X‐100 lysates of Fabry fibroblasts and (ii) intracellular block and impaired trafficking of lipid rafts‐associated glycoproteins, such as dipeptidyl peptidase IV. The intracellular transport of proteins that are not associated with lipid rafts, such as aminopeptidase N, is not affected. Therefore, the impaired trafficking of AGAL is not only associated with altered glycolipid structure, but elicits also causal effects related to protein trafficking and sorting.
Role of protein kinase D (PKD) phosphorylation of Oxysterol binding protein (OSBP) in sphingomyelin biosynthesis
Authors: Goto Asako , Ridgway Neale
Published: June 2021

Oxysterol binding protein (OSBP) is a high affinity receptor for oxysterols that interacts with the endoplasmic reticulum (ER) and trans‐Golgi network via FFAT and pleckstrin homology (PH) domains, respectively. In vitro, OSBP displays cholesterol extraction and transfer activity and regulates the cholesterol content of Golgi and post‐Golgi membranes. Through this activity, OSBP controls CERT‐mediated ER‐to‐Golgi ceramide transfer, which is the rate‐determining step in sphingomyelin (SM) biosynthesis. CERT and OSBP are phosphorylated by protein kinase D (PKD), thus we investigated whether CERT activity and SM synthesis is regulated by OSBP phosphorylation at serine 242 by PKD. OSBP and PKD are physically associated, however OSBP is not required for Golgi association and activation of PKD. While transfection of wild‐type OSBP restored sterol activation of SM biosynthesis in OSBP‐depleted CHO cells, OSBP S242A and S242D were ineffective. Another OSBP‐related protein ORP9, which transfers cholesterol from ER to Golgi but is not involved in SM biosynthesis, is not a substrate for or associated with PKD. We conclude that an OSBP phosphorylation/dephosphorylation cycle mediated by PKD is necessary and specific for OSBP function in the context of regulating SM biosynthesis. Supported by the Canadian Institutes of Health Research.
Tyrosine Phosphorylation of the hepatic Tocopherol Transfer Protein (TTP)
Authors: Thakur varsha , Ulatowaski Lynn , Manor Danny
Published: June 2021

Alpha‐tocopherol (Vitamin E) is an essential dietary lipid that functions as the principal lipid‐soluble antioxidant in most animal and plant species. The hepatic tocopherol transfer protein (TTP) controls body‐wide levels of vitamin E by regulating the secretion of ‐tocopherol from hepatocytes to circulating lipoproteins which then deliver the vitamin to tissues in the periphery. Mutations in the TTPA gene cause hereditary vitamin E deficiency, characterized by low circulating ‐tocopherol and progressive neurodegeneration cerebellar ataxia. Presently, very little is known regarding the regulation of TTP activity. We found that TTP is subject to phosphorylation on tyrosine residue(s). Tyrosin phosphorylation is regulated by the presence of serum‐derived growth factors, and is inhibited by pharmacological agents that inhibit the tyrosine kinases Src and epidermal growth factor receptor (EGFR). Treatment with these inhibitors caused a marked accumulation of NBD‐tocopherol, a fluorescent analog of vitamin E. These data indicate that tyrosine phosphorylation of TTP is a regulated event, which is required for the protein's activity in stimulating the secretion of vitamin E from hepatocytes.
Purification and characterization of the lipid A 4’‐kinase
Authors: Emptage Ryan , Raetz Christian R. H.
Published: June 2021

The chief component of the outer membrane in Gram‐negative bacteria is lipopolysaccharide (LPS). LPS is anchored to the outer leaflet of the outer membrane by the highly immunogenic acylated disaccharide of glucosamine known as lipid A. Since most Gram‐negative bacteria require lipid A for viability, any enzyme responsible for the biosynthesis of this molecule presents itself as an attractive target for the development of novel antimicrobials. In E. coli, the sixth enzyme in the constitutive pathway of lipid A biosynthesis is an integral membrane protein designated LpxK, which phosphorylates the 4′‐position on the distal glucosamine of disaccharide‐1‐phosphate (DSMP) to form lipid IVA. Previous studies of E. coli LpxK have been hampered due to its instability in the presence of detergents, but the ortholog from the hyperthermophile Aquifex aeolicus (AaLpxK) is stable when separated from bacterial membranes and can readily act upon the E. coli‐derived substrate. These properties have allowed for the purification of AaLpxK, the development of a sensitive radioassay for kinase activity, and the screening of crystallization conditions. Activity of purified AaLpxK in vitro has been shown to be dependent upon maintenance of high pH, select divalent cations, and the presence of detergent in the assay system. This research was funded by N.I.H. grant GM51310 to C.R.H. Raetz.
Enzymatic synthesis of 8‐amino‐3,8‐dideoxy‐D‐manno‐oct‐2‐ulsonic acid (Kdo8N)‐lipid A in Shewanella
Authors: Gattis Samuel Guy , Raetz Christian R.H.
Published: June 2021

The outer membrane of gram‐negative bacteria is primarily composed of lipopolysaccharide (LPS), the hydrophobic anchor of which is lipid A. Lipid A, also known as endotoxin, serves as the permeability barrier for gram‐negative bacteria as well as activating the innate immune system in infection. Modifications to the conserved substructure Kdo2‐lipid A in diverse bacteria have been shown to influence virulence, antibiotic resistance, and immune response. Here we report the discovery of the Shewanella oneidensis genes required for 8‐amino‐3,8‐dideoxy‐D‐manno‐oct‐2‐ulsonic acid (Kdo8N) biosynthesis. Overexpression of a gene cluster of unknown function from S. oneidensis in E. coli resulted in production of (Kdo8N)2‐lipid A, as judged by mass spectrometry and thin‐layer chromatography. The relevant genes were previously annotated as an Fe‐dependent alcohol dehydrogenase and an aspartate aminotransferase, suggesting a two‐step mechanism in which the 8‐OH of Kdo is first oxidized to an aldehyde, followed by transamination to form a primary amine at C8. In vitro enzymology and homologues from the pathogen Leptospira interrogans are being investigated. This work was supported by NIH GM51796 to C.R.H.R. and Lipid Maps GM069338.
Interaction Between Putative Epulopiscium sp. Type B Chemotaxis Proteins
Authors: Meiser Kelly L. , Piefer Andrew J.
Published: June 2021

The main objective of this project is to determine binding between Epulopiscium sp. Type B methyl accepting chemotaxis proteins (MCP) and homologs of E. coli Che adapter proteins. Epulo is a large (up to 250 μm length) bacterium which moves by the use of flagella and lives symbiotically in the brown surgeon fish. Many bacteria that travel by the use of flagella use what is known as chemotaxis, a controlled movement in response to a ligand concentration gradient. E.coli has cell surface receptors that project into the periplasm which interact with Che proteins inside the cytosol when exposed to a specific ligand. The Che proteins then interact with the flagellar motors and cause them to turn in a clockwise or counterclockwise fashion based on the favorability of the ligand. These protein‐protein interactions can be explored by using a yeast two‐hybrid system. PCR was performed on genomic DNA from Epulo and two candidate MCP regions were selected for two‐hybrid analysis. Several putative CheA and W homologs were also cloned to test possible protein‐protein interactions. These studies will further the “interactome” map of Epulo and help elucidate the symbiotic mechanism. This research was funded by the Harwick College Chemistry Department.
Activation of STIM1‐Orai1 involves an intramolecular switching mechanism
Authors: Korzeniowski Marek Krzysztof , Manjarrés Isabel Martín , Varnai Peter , Balla Tamas
Published: June 2021

The cytosolic level of Ca2+ has to be under tight control since Ca2+ is a potent regulator of many pivotal cellular functions. Store operated calcium entry (SOCE) is a major calcium entry mechanism present in virtually all cell types. Its activity is controlled by the luminal Ca2+ concentration of the ER. The stromal interaction molecule (STIM1) located in the ER, which is the Ca2+ sensor, and the highly Ca2+ selective Orai1 channels in the plasma membrane are the two main component of this machinery. Decreased ER luminal Ca2+ concentrations trigger oligomerization and clustering of STIM1, which, in turn, becomes capable of activating Orai1 channels. The Orai1 activating region within the STIM1 cytoplasmic part has been recently identified (CAD/SOAR) Here, we identified an acidic motif in STIM1 that keeps CAD/SOAR domain in an inactive state. Intriguingly, the sequence of the STIM1 acidic motif shows substantial similarity to that of the C‐terminal coiled‐coil segment of Orai1, which is the postulated site of interaction with STIM1. Mutations within this acidic region make STIM1 constitutively active presumably by preventing the intramolecular interaction with a short basic segment within the CAD/SOAR domain that is also essential for Orai1 activation. We propose that the CAD/SOAR domain is released from an intramolecular clamp during STIM1 activation, allowing activation of Orai1 channels.
Improving iron bioavailability with ferritin overexpression in low phytate maize
Authors: Reddy Manju B. , Aluru Maneesha , Weiler Joel
Published: June 2021

Iron deficiency anemia (IDA) is a major global nutritional problem may be due to lack of diversity in the diet, with maize as a staple crop. Iron bioavailability from maize is low due to its high phytate content. Iron content can be increased by expressing ferritin but this in itself may not overcome the inhibitory effect of phytate on bioavailability. We hypothesized that iron from low phytate maize (lpa‐1‐1 with 50–60% lower phytate than wild type) is more bioavailable than from wild type maize (A188), and increasing the iron content of lpa‐1‐1 will further increase iron bioavailability. We generated transgenic maize lines with enhanced iron content by over expressing soybean ferritin, and used the Caco‐2 cell culture model to determine iron bioavailability from these transgenic lines. Total iron content in transgenic maize was 2.5‐fold and 1.8‐fold higher in A188 and lpa‐1‐1 maize, respectively. Iron bioavailability from non‐transgenic lpa1‐1 was 50% (p<0.01) higher than A188 maize. Ferritin over expression improved iron bioavailability by 60% in A188 maize. Overall, transgenic lpa1‐1 showed higher bioavailability (16–38%; p<0.02) when compared to A188 transgenic maize. Our results suggest that over expression of ferritin in low phytate maize significantly improves iron bioavailability from maize, and is a potential approach to alleviate IDA. Supported by NWRC, Iowa State University
14‐3‐3γ Binds to CaM KKα and Blocks Estrogen Signaling in MCF‐7 Cells
Authors: Ankeny Amanda P. , Schmitt John M.
Published: June 2021

Extracellular Signal‐Regulated Kinase (ERK) is activated by estrogen (E2) downstream of CaM KK leading to cell growth in MCF‐7 breast cancer cells. Previous studies have shown that ERK activation may be inhibited by cAMP and PKA. PKA has numerous cellular targets including CREB, Src, Raf‐1, arrestins, and CaM KK. CaM KK is inhibited by direct PKA phosphorylation and the subsequent interaction with 14‐3‐3γ. Agonists that activate cAMP and PKA may block CaM KK activation of ERK and cell proliferation. Our goal was to evaluate the ability of cAMP and PKA to antagonize the effects of E2 on MCF‐7 breast cancer cell signaling specifically, we examined CaM KK phosphorylation and inhibition. Our results suggest that activation of cAMP and PKA with Forskolin, inhibits E2 stimulation of ERK activation. E2 treatment of MCF‐7 cells did not trigger PKA phosphorylation of CaM KK. Forskolin treatment of cells increased CaM KK phosphorylation and its association with endogenous 14‐3‐3. Interestingly, Vitamin D also enhanced 14‐3‐3 binding to CaM KK. Our results suggest that PKA activation, and subsequent phosphorylation of CaM KK, inhibits its activity, and associates with 14‐3‐3, which in turn may remove CaM KK from the ERK signaling cascade in MCF‐7 cells.
TRP Channel Regulation of Estrogen Signaling
Authors: Milligan Jessica , Schmitt John M.
Published: June 2021

Calcium regulates numerous cell functions including growth and development. Calcium can enter cells through transient receptor potential channels (TRPCs). Previous studies in MCF‐7 cells have suggested that the expression of one particular TRPC, TRPC6, correlates with cell transformation and disease progression. Calcium has several cellular targets including the Calcium/Calmodulin‐dependent protein kinases (CaM Ks) and ERK. Previous work has shown that estrogen (E2) may utilize CaM Ks and ERK to promote breast cancer cell proliferation, however the possible involvement of TRPCs in this pathway is currently unknown. Our objective was to understand which E2 receptor is used in our system and if TRPCs participate in the control of CaM Kinase activation of ERK in MCF‐7 cells. Specifically, we wanted to explore if E2 may utilize TRPCs particularly, TRPC6, upstream of the ERK pathway in MCF‐7 cells. E2 stimulation of MCF‐7 cells and the estrogen receptor alpha (α) inhibitor, MPP, completely blocked ERK activity. In contrast, MPP did not block EGF stimulation of ERK. MCF‐7 cells express endogenous TRPC6 protein and TRPC inhibitors, APB and SK&F, both blocked ERK activation downstream of E2. In addition, neither APB or SK&F inhibited EGF activation of ERK. Results from these studies suggest that E2 is capable of activating ERK through the specifically through the alpha form of the estrogen receptor and TRPCs.
Overcoming technical challenges of automating small volume assays in open well format for high throughput drug discovery
Authors: Call Jon , Goloveva Olga , Johnson Edgar
Published: June 2021

The nature of drug discovery creates a demand for instrumentation and experimental protocols for assays with reaction volume of 10 μl or less in high throughput format. These assays require liquid handling equipment that is capable of unattended processing of large batches of plates without a compromise in terms of dispensed volume, flow rate and positioning. In addition, the small assay reaction volumes lead to a significant problem with the reagents evaporation that is magnified by the need for multiple plate processing. To address the evaporation problem, a protective oil layer can be used. This technique allows an open well format and a possibility for adding reagents when performing multi‐step assay protocols. Performance of such assays depends greatly on correct dispensing profiles and rapid penetration of the added reagents through this protective oil layer. In this report we present the study of our MAP‐D liquid handling workstation for performing low‐volume end‐point and kinetics assays in high throughput format. The use of this workstation increases reliability and improved Z‐factor for assays utilizing a protective oil layer.
A novel approach to high‐throughput discovery of anti‐aging drugs identifies lithocholic acid as a longevity‐extending compound
Authors: Titorenko Vladimir , Goldberg Alexander , Kyryakov Pavlo , Bourque Simon , Beach Adam , Burstein Michelle , Richard Vincent
Published: June 2021

Caloric restriction (CR) and dietary restriction (DR) extend life span across species and improve health by delaying the onset of age‐related diseases. All currently known anti‐aging drugs: 1) mimic numerous life‐extending and health‐improving effects of CR and DR without restricting caloric and nutrient intake; and 2) target two signaling pathways that are under the stringent control of calorie and/or nutrient availability. It was believed therefore that all longevity pathways are “adaptable” by nature because they modulate longevity only in response to certain changes in the extracellular and intracellular nutrient and energy status of an organism. However, it is possible that that some longevity pathways could be “constitutive” or “housekeeping” because they control longevity irrespective of calorie and/or nutrient availability. We designed a novel high‐throughput chemical genetic screen for drugs that increase the life span of yeast under CR by modulating such housekeeping longevity pathways and targeting lipid metabolism. Our screen identified lithocholic acid as one of such molecules. We revealed that this bile acid delays aging by suppressing lipid‐induced necrosis, attenuating mitochondrial fragmentation, altering oxidation‐reduction processes in mitochondria, enhancing resistance to oxidative and thermal stresses, suppressing mitochondria‐controlled apoptosis, and enhancing stability of nuclear and mitochondrial DNA. Supported by CIHR and NSERC of Canada.
Analysis of intracellular amyloid‐β as a consistent feature of hippocampal neurons
Authors: Blair Jeffrey Adam , Siedlak Sandra L , Zhu Xiongwei , Lee Hyoung‐gon , Salomon Robert G , Nunomura Akihiko , Castellani Rudy J , Smith Mark A
Published: June 2021

Amyloid‐β, a 39–43 amino acid long peptide, accumulates both intraneuronally (iAβ) and in extracellular senile plaques, the major pathological hallmark in Alzheimer disease (AD). While hypotheses on the role of iAβ are most often derived from etiological approaches regarding AD, iAβ is also commonly reported in the brains of non‐diseased individuals. In this investigation of iAβ, we designed experiments to consider a variety of biological circumstances and relate iAβ to parameters including circumstances of death, use of opiates, gender, postmortem interval, ApoE genotype, disease state, tissue handling and staining methodologies. Ongoing analyses on this large population (n=67) will determine whether iAβ shows a pattern of natural occurrence and whether iAβ plays a role in the risk of or development of AD.
Effect of yhiM expression on antibiotic resistance of E. coli
Authors: Evans Edward J , Niblick Charles J , Taylor Ann T.S.
Published: June 2021

yhiM is an inner membrane protein from E. coli associated with reduced susceptibility to bacteriophage infection. It has also been suggested that it is involved in antibiotic resistance. This hypothesis was tested by examining the zones of clearing obtained by various antibiotics with E. coli that either contained transposon mutants of yhiM or overexpressed yhiM. No significant difference in antibiotic resistance was observed. This work was supported by the Haines Fund for the Study of Biochemistry at Wabash College.
Extracytoplasmic cellular stress responses induced by cationic polyethylenimines and known pore‐forming compounds
Authors: Isaac Daniel , Lander Blaine , Checchi Kyle , Domanski Tammy , Lin Shirley
Published: June 2021

The mechanisms of antimicrobial activity of a cationic polyethylenimine (PEI+) and a small molecule pore‐forming compound, vanillin, are thought to involve disruption of bacterial cell membranes. These proposed mechanisms were studied by measuring the induction of the three known extracytoplasmic stress responses (Cpx, σE and Bae) of the Gram‐negative bacterium Escherichia coli. PEI+ specifically induced the Cpx stress response approximately 2‐fold in a dose‐dependent manner at concentrations ranging from 80–320 μg/mL. Vanillin induced the σE stress response 4–12‐fold at concentrations ranging from 320–960 μg/mL. The implications of the observed differences in the induction of extracytoplasmic stress responses between PEI+ and vanillin on the antimicrobial mechanisms of these compounds will be discussed.
An unassigned DAHP synthase activity found only in pathogenic Escherichia coli strains
Authors: Yep Alejandra , Grmai Lydia , Emanuele Anthony A. , Woodard Ronald W.
Published: June 2021

The enzyme 3‐deoxy‐D‐arabino‐heptulosonate 7‐phosphate synthase (DAHPS) catalyzes the first step of the shikimate biosynthetic pathway, which leads to the synthesis of aromatic amino acids. This pathway is essential in bacteria but absent from mammals, making it a suitable target for antibiotic therapy. Escherichia coli has three DAHPSs, encoded by the genes aroF, aroG, and aroH. AroF, AroG, and AroH are feedback inhibited by Tyr, Phe, and Trp, respectively. BLAST searches identified a previously uncharacterized DAHPS copy present only in extraintestinal pathogenic E. coli strains. The corresponding gene (c1220) was cloned from E. coli CFT073, a strain responsible for most uncomplicated urinary tract infections (UTI). The ORF c1220 is highly upregulated during UTI (Snyder et al. (2004), Infect Immun 72:6373). Purified C1220 displays in vitro DAHPS activity uninhibited by Phe, Tyr, Trp, prephenate, or chorismate. Consistently, a model of C1220 shows conservation of active site topology with AroF, AroG and AroH but no similarities at the inhibitor binding site. Complementation of a triple E. coli knockout strain (ΔaroF, ΔaroG, ΔaroH) with c1220 restores its ability to grow in minimal media. On the other hand, the E. coli CFT073 knock out strain Δc1220 remains capable of growth in minimal media. Our results confirm that C1220 is indeed a DAHPS, but suggest it is not part of the shikimate pathway. NIH AI‐061531 funded.
Blueberries inhibit proinflammatory cytokine TNF‐α and IL‐6 production in macrophages
Authors: Wu Xianli , Xie Chenghui , Kang Jie
Published: June 2021

Blueberries (BB) have been reported to attenuate atherosclerosis in apoE deficient (ApoE−/−) mice. However, the underlying mechanisms are not fully understood. In this study, the effect of BB on proinflammatory cytokine production in macrophages was investigated. ApoE−/− mice were fed AIN‐93G diet (CD) or CD formulated to contain 1% freeze‐dried whole wild BB (BB) for 5 weeks. Peritoneal macrophages were collected and cultured. Protein level and mRNA expression of TNF‐α and IL‐6 were significantly lower in macrophages from mice fed BB. When stimulated by LPS, TNF‐α and IL‐6 mRNA expression increased by ~120 fold and ~550 fold in macrophages from CD fed mice, but only by ~16 fold and ~6 fold in macrophages from BB mice. Similarly, the oxLDL also induced marked increases in TNF‐α and IL‐6 mRNA expressions by ~12 fold and ~70 fold in macrophages from CD mice. While in macrophages from BB mice, TNF‐α expression only went up ~3 fold and IL‐6 was unchanged. Since TNF‐α and IL‐6 are key players in vascular inflammation underlying atherosclerosis, inhibition of TNF‐α and IL‐6 production and enhancement of resistance of macrophages to inflammatory stimuli could be an important mechanism of athero‐protective effects of BB. USDA‐CRIS‐6251‐5100002‐06S.
The effects of manganese (Mn++) supplementation on blood lipids and vascular inflammation in Zucker Diabetic Fatty (ZDF) rats
Authors: Burlet Elodie , Jain Sushil K
Published: June 2021

Vascular inflammation is known to contribute to the development of diabetes complications. Diabetes is associated with low levels of Mn++, an important component of many enzyme systems. Previous studies have shown that diabetics with higher blood levels of Mn++ were protected from LDL‐oxidation compared to those with lower Mn++ levels, and that Mn++ deficiency in animals caused impaired glucose tolerance, which can be reversed with Mn++ supplementation. We examined the hypothesis that Mn++ supplementation lowers blood lipids and vascular inflammation markers in ZDF rats, a model of type 2 diabetes. Starting at six weeks age, rats were daily gavaged with water (D, n=6) or MnCl2 (16 mg/kg BW (D+ Mn++, n=5) for seven weeks. Blood was collected by heart puncture using anesthesia. D+ Mn++ rats showed lower cholesterol (159±6 vs 212±21mg%, p<0.05), triglyceride (651±124 vs 1281±564 mg%, p<0.05), MCP‐1 (4689±753 vs 6033±1235 pg/ml, p<0.05) and ICAM‐1 (22651±717 vs 26969±1827 pg/ml, p<0.05) compared to D rats. Results show that Mn++ supplementation lowers blood levels of lipids and vascular inflammation in ZDF rats. Mn++ supplementation needs to be further explored as an adjuvant therapy to lower vascular inflammation and complications in diabetic patients.
Cardiovascular health promotion in a Mexican population
Authors: Pérez‐Lizaur Ana B , Fernandez Ana Cecilia , Balcazar Hector
Published: June 2021

Health Cardiovascular promotion allows the formation of a community's own resources. The aim of the study was to adapt, validate and develop “Salud para su Corazón” program. The program took place in Alvaro Obregon district in Mexico City. 22 promoters were trained for providing cardiovascular health education and conduct follow‐up sessions of 2 hours per week over a period of 6 months to 225 people of low socioeconomic status. Knowledge and behavior (validated instrument α> 0.6) of the participants were evaluated pre and post, also anthropometric measurements, blood pressure and blood glucose levels.

• Results

Obesity decreases at the end of the workshops (of 48.9% to 43.4%, respectively), increases the overweight (34.0% to 38.6%). Change is observed (p <0.01) in the habits and food consumption and increased physical activity time and frequency. Participants felt that “to be taking action” with regard to their health doubled after the workshops (31.3% to 63.0%) and participants who felt that “fail to take the first step” decreased to a third party after workshops (25% to 6.8%).

Conclusions

Promontion on cardiovascular health is an effective strategy to increase self‐care in this mexican study population
Potential Biological Activities of the Xanthophyll Excentric Cleavage Product 3‐OH‐β‐apo‐10’‐carotenal in vitro
Authors: Mein Jonathan Ryan , Hu Kang‐Quan , Wang Xiang‐Dong
Published: June 2021

Xanthophyll carotenoids, such as lutein and zeaxanthin, may provide potential health benefits in humans against chronic and degenerative diseases. We recently demonstrated cleavage of the xanthophylls lutein and zeaxanthin by carotene‐9’,10’‐monooxygenase (CMO2) at the 9’,10’ and 9,10 double bonds, producing 3‐OH‐β‐apo‐carotenoids. In the present study, we investigated the potential biological activity of the xanthophyll metabolite 3‐OH‐β‐apo‐10’‐carotenal (3‐OH‐ACL) in vitro. We found that incubation of 3‐OH‐ACL with BEAS‐2B, A549 and HepG2 cell lines dose‐ and time‐dependently decreased cell viability. Utilizing High Content Screening (HCS) analysis, we observed that 3‐OH‐ACL treatment significantly increased cytochrome C release and nuclear swelling and decreased mitochondrial potential in HepG2 cells. We further explored the effect of 3‐OH‐ACL on the LPS‐stimulated TNFα, MCP1 and IL‐6 expression in THP‐1 human monocyte/macrophage cells. 3‐OH‐ACL significantly inhibited LPS‐stimulated MCP1 and IL‐6 gene expression but at higher concentrations increased TNFα gene expression in THP‐1 cells. These data indicate that 3‐OH‐β‐apo‐10’‐carotenal may induce apoptotic pathways of cellular death.

Grant Funding Source: R01CA104932
Time‐course of doxorubicin‐induced cardiotoxicity in rats. Morphological study
Authors: Ferreira Ana Lucia Anjos , Reis Raissa Amélia Silva , Rocha Noeme Souza , Pereira Elenize Jamas , Fontes Fernanda , Correa Corina , Correa Camila Renata , Matsubara Luiz Shiguero , Matsubara Beatriz Bojikian , Yeum Kyung‐Jin , Dal‐Pai‐Silva Maeli
Published: June 2021

In order to study doxorubicin‐induced cardiotoxicity Wistar rats were distributed in Control (C, n=80) and Doxorubicin (D, n=70) groups. D received 4 mg doxorubicin/Kg body wt/d intraperitoneally at 3rd, 4th, 5th, and 6th week. Animals were killed 24h before and 24h after 3rd, week and at 8th week for analyses. Myocardium fixed in Karnovsky's solution processed for ultra structural examination. Body‐weight/necropsy‐findings were recorded. C group showed no alterations. However, D exhibited progressive body‐weight loss/nasal bleeding from 4th week. D showed cardiac softness, accumulation of serous fluid in pericardial, pleural peritoneal cavities at necropsy. Mortality was high (39%) in D group. Myocardium from C showed normal ultrastructural aspects; D group showed cardiomyocyte damage. Initially, cardiomyocytes presented focal alterations. At end, alterations were present in the majority of the cardiomyocytes: disorganization or absence of myofilaments, hyper‐contracted myofibrils, polymorphic/swelling mitochondria with disorganized cristae, scarce polymorphonuclear cells in capillaries and in extracellular matrix between cardiomyocytes. Changes in capillaries endothelial cells were frequent, as, loss or dilatation of cytoplasmic material, increase in small vesicles and nucleus heterochromatic with irregular aspect. Supported by:FAPESP#2007/07455‐2;PIBIC/CNPq#11149
Consumption of a tomato carotenoid containing diet reduces UV‐induced inflammation and DNA damage in a Skh‐1 hairless mouse model
Authors: Kopec Rachel E , Schick Jonathan , Tober Kathleen L , Riedl Ken M , Francis David , Oberyszyn Tatiana M , Schwartz Steven J , Oberyszyn Tatiana M
Published: June 2021

Ultraviolet (UV) light exposure is a primary risk factor in the development of skin cancer. Human studies have shown that the consumption of a tomato containing diet for ≥10 weeks reduces the intensity of a UV‐induced sunburn. We hypothesize that deposition of tomato carotenoids in skin might be responsible for this effect. Tangerine tomatoes, containing higher levels of UV‐absorbing carotenoids, were used for this study. Four week old hairless mice (40 females, 40 males) were divided into two feeding groups (0 % or 10 % tangerine tomato powder) and two UV exposure groups (w/ or w/o UV exposure), and maintained on their respective diets for 10 weeks. At week 11, the UV group was exposed to a single dose of UVB radiation. Animals in all groups were sacrificed 48 hrs. post‐UV exposure. Carotenoid levels in backskin samples were analyzed by HPLC‐MS/MS. Amounts were significantly greater in females as compared to males. In addition, carotenoid levels were significantly greater in UV‐exposed animals as compared to no UV exposure. Cyclopyrimidine dimer levels (a marker of direct DNA damage) were lower in both males and females in the 10% tomato group as compared to the 0 % tomato group, and consumption of the 10% tomato diet reduced myeloperoxidase activity (an inflammatory measure) in males. These results support the hypothesis that skin carotenoids may reduce UV‐induced skin damage and ultimately reduce risk of skin cancer.

Grant Funding Source: Center of Advanced Functional Foods Research and Entreprenuership (CAFFRE)
A chemoprotective fish oil/pectin diet regulates the expression of the bcl‐2 oncogene by altering CpG island methylator phenotype (CIMP) in colon cancer
Authors: Cho Youngmi , Turner Nancy D. , Davidson Laurie A. , Chapkin Robert S. , Lupton Joanne R.
Published: June 2021

We have demonstrated that diets containing fish oil and pectin (FO/P) reduce colon tumor incidence relative to those containing corn oil and cellulose (CO/C) in part by inducing apoptosis. CO/C, as compared to FO/P, promotes colonocyte expression of the anti‐apoptotic protein, bcl‐2, and bcl‐2 promoter methylation is altered in colon cancer. To determine if CO/C promotes bcl‐2 expression by reducing promoter methylation in colon cancer, we examined bcl‐2 promoter methylation and mRNA levels, and colonocyte apoptosis (TUNEL assay). Rats were provided diets containing FO/P or CO/C, and were terminated 36 wk after azoxymethane injection (2×, 15 mg/kg BW, sc). DNA isolated from 10 μm sections of PFA‐fixed colon carcinomas was bisulfite modified and amplified by real time qPCR to assess regions of the bcl‐2 CpG islands. Scraped mucosa from the same rats was also used to quantify bcl‐2 mRNA levels. FO/P treatment increased bcl‐2 DNA methylation (p = 0.055) and apoptosis (p = 0.022) as compared to CO/C. Data analysis revealed a negative relationship between bcl‐2 DNA methylation and bcl‐2 mRNA levels. We conclude that dietary FO/P promotes apoptosis by enhancing bcl‐2 promoter methylation relative to a CO/C diet. This study is the first to detect a diet effect on promoter methylation of the bcl‐2 oncogene in the colon. Funded by NSBRI NASA NCC 9‐58 and NIH CA59034.
Validation of a physical activity questionnaire against the doubly‐labeled water standard among a multiethnic population
Authors: Wilkens Lynne R. , Murphy Suzanne P. , Jardack Patricia , White Kami K , Kolonel Laurence N. , Arab Lenore
Published: June 2021

The objective measure of total energy expenditure (TEE) based on doubly labeled water was compared to self‐report variables from physical activity questionnaires in the Energetics Study. An adult population of African Americans and Caucasians, aged 21–70 and living in Los Angeles, were recruited. A subset of 230 adults were administered the one‐page physical activity questionnaire used in the Multiethnic Cohort, from which daily metabolic equivalents (METS) were computed. The correlations (r) between the objective and self‐reported values were reasonable, with r=0.29 for METs. While the differences were not significant, the r's for African Americans were lower than for Caucasians, with r= 0.24 for Caucasian females, 0.27 for Caucasian males, 0.09 for African American females, and 0.13 for African American males. Caucasians tended to be younger, less obese and have more years of education than African Americans in this study, which likely contributed to the ethnic differences, as concordance between the measures varied by these factors: age (0.36 for <50 years of age versus 0.32 for 50+), BMI (0.48 for <25 and 0.21 for 25+), and education (0.32 for college graduates versus 0.25 for those with less education). Doubly labeled water provides an opportunity to validate physical activity instruments and can provide information for improving them. Supported by NIH R01CA105948 and R37CA54281.