bluuebunny/crossref_metadata_2025 huggingface dataset:
Systematic quantitative function annotation of mouse genes
Authors: Roth Frederick
Published: June 2021

Several years after sequencing human and mouse genomes, much remains to be discovered about the functions of most genes. Computational prediction of gene function promises to help focus limited experimental resources on the most likely hypotheses. In this study, a standardized collection of mouse functional genomic data was assembled, and nine bioinformatics teams used this dataset to independently train classifiers and generate predictions of function for 21,603 mouse genes. We compared the performance of function prediction algorithms and identified strengths and weaknesses of current functional genomic datasets. Quantitative function predictions may be useful, e.g., in the study of complex disease in human populations.
Optimization is Essential to Increase Transfection Efficiency and Cell Viability in Primary and Difficult‐to‐Transfect cell lines
Authors: Jordan Elizabeth T. , Collins Michelle , Terefe Joseph , Pineda Maxinne , Ugozzoli Luis , Rubio Teresa
Published: June 2021

Electroporation is a valuable tool for nucleic acid delivery because it can be used for a wide variety of cell types. Many scientists are shifting towards the use of cell types of increasing scientific interest, including lineage‐specific cell types, bone marrow cells and stem cells, but these cells can be difficult to transfect. The ability to transfect these cell types with nucleic acid molecules of interest at a relatively high efficiency while maintaining cell viability would offer a new tool towards the elucidation of the pathway(s) in which a gene product is involved.

In this poster, we present data demonstrating that optimization of electroporation parameters is essential to deliver molecules in a highly efficient manner. We display results of transfection of several difficult to transfect cell types, including Human Primary Fibroblasts, HUVEC, Jurkat and (mouse) Neuro‐2A neural cells with plasmid DNAs and siRNAs. We used multiple detection methods to determine transfection efficiency, including flow cytometry, real‐time PCR and the luciferase assay. Gene Pulser electroporation buffer and square or exponential wave pulses were used to convey molecules of interest into the cells. Optimization of electroporation parameters was carried out using the high throughput Gene Pulser MXcell electroporation system. Just 4 hours post electroporation with a GAPDH siRNA, Jurkat cells expressed 88% less GAPDH mRNA than controls transfected with scramble siRNA. The same degree of silencing persisted at 24 and 48 hours post electroporation. Transfection of cells with fluorescently labeled siRNA resulted in 75% transfection efficiency for Neuro‐2A, 93% for HPF and 98% for HUVEC cells, as analyzed by flow cytometry. We provide optimized electroporation conditions for these and other difficult to transfect cells.
Differential placental gene expression in preeclampsia
Authors: Enquobahrie Daniel A , Meller Margaret , Rice Kenneth , Psaty Bruce M , Siscovick David S , Williams Michelle A
Published: June 2021

Objective: Candidate genes associated with preeclampsia have not been fully described. We conducted a microarray study to investigate global placental gene expression in preeclampsia.

Study design: Cases (n=18) and controls (n=18) were selected among patients receiving care at Swedish Medical Center, Seattle, WA. Oligonucleotide probes representing 22,000 genes were used to measure gene expression. Differentially expressed genes were selected using criteria based on Students T‐test, fold change and Significance Analysis of Microarrays (SAM) analysis. Functions and functional relationships of differentially expressed genes were evaluated using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA) tools.

Results: Genes (n=58) participating in immune system, inflammation, oxidative stress, signaling, growth and development pathways were differentially expressed in preeclampsia. These genes include previously described candidate genes (such as leptin, LEP), potential candidate genes with related functions (such as cytochrome P450 family 11 subfamily A, CYP11A) and novel genes (such as cyclin‐dependent kinase inhibitor 1C, CDKN1C).

Conclusion: Expression of genes (both candidate and novel) with diverse functions is associated with preeclampsia risk, reflecting the complex pathogenesis.
Kinetic characterization of arylamine N‐acetyltransferase 2
Authors: Baker Inesha , Parker Chasta L
Published: June 2021

N‐acetyltransferase 2 (NAT 2) is an enzyme that catalyzes the acetylation of harmful arylamines and has been implicated in various types of cancer. NAT 2 is primarily expressed in the hepatic system and intestinal epithelium and is encoded at two polymorphic loci that give the phenotypic characteristics of slow and fast acetylation. The purpose of this experiment was to study the stability of the NAT 2 over time in order to determine if rate data was affected by a time lapse between purification and rate determination. The NAT 2 gene was amplified from both pENTR 221 and genomic DNA using PCR. NAT 2 was ligated into a pET28b+ vector containing an N‐terminal hexahistidine tag and transformed into E. coli Rosetta 2 cells. Protein expression was carried out under different conditions to determine the correct culture and IPTG concentrations necessary for protein production. The protein was isolated from the cells and purified using FPLC. The enzyme was then kinetically characterized using sulfamethazine and aspartame immediately after purification as well as one week post – purification. Preliminary results indicate that a one week time lapse results in a 2 to 4 fold decrease in the activity. This project was supported by the Winthrop University Research Council and NIH Grant Number P20 RR‐16461 from the NCRR for support of the program entitled “South Carolina IDeA Networks of Biomedical Research Excellence (SC‐INBRE)”.
Substrate Loop in Glutathione Synthetase: Mutation Analysis
Authors: Selman Kerri Diane , Brown Teresa , Anderson Mary
Published: June 2021

Glutathione (GSH), a small tripeptide, is a required cellular antioxidant. Cellular GSH is synthesized by two ATP‐dependent enzymes. The first enzyme, gamma‐glutamylcysteine synthetase, ligates glutamate and cysteine to form gamma‐glutamylcysteine. The second enzyme, glutathione synthetase (GS), ligates glycine to gamma‐glutamylcysteine to form GSH. Human GS, a homodimer, contains a conserved eleven amino acid residue loop region (266‐FRDGYMPRQYS‐276) called the Substrate Loop (S‐loop). In order to study the role of the conserved amino acid residues, several GS mutant enzymes, such as Gly269Val and Tyr270Phe, within this region were prepared using PCR site‐directed mutagenesis. These were expressed, purified and assayed for activity. Our findings show that mutations at Gly269 cause a significant decrease in GS activity (< 5% wild type). (Supported by Welch Foundation Chemistry Department grant; TWU A&S Research Development Fund (MEA).)
The influence of protein nucleocytoplasmic transport on expanded polyglutamine‐induced neurodegeneration
Authors: Chan Wing Man , Shaw Pang Chui , Chan Ho Yin Edwin
Published: June 2021

Polyglutamine (polyQ) disease is a group of inherited neurodegenerative disorders caused by abnormal elongation of an existing polyQ domain in disease proteins. Proteins that harbor the expanded polyQ domain cause progressive dysfunction and death of neurons. The nucleocytoplasmic transport of disease proteins displays a close correlation with polyQ toxicity. Classical nuclear localization signals (NLSs) and nuclear export signals (NESs) have been reported on different polyQ disease proteins and they play roles in regulating the subcellular localization and functions of disease proteins. In addition to classical NLSs and NESs, our preliminary data demonstrated that naked expanded polyQ domain also possesses nuclear transport signal activities. Elucidating the transport pathways that govern the nuclear translocation of polyQ proteins would provide extra insights into the pathogenesis of polyQ diseases.
Characterization of the metabolic remodeling in liver by genetic mutation of ALDH2 in muscles
Authors: Hishiki Takako , Adachi Takeshi , Sano Motoaki , Katayama Takaharu , Endo Jin , Fukuda Keiichi , Suematsu Makoto
Published: June 2021

Objective: Aldehyde dehydrogenases 2 (ALDH2) is the detox enzyme to catalyze aldehydes‐carboxylation, which modulates reactive oxygen species in mitochondria. We created transgenic (TG) mice expressing mutant ALDH2 in muscles, which exhibited the phenocopy of senescence. To test the mechanism of detoxification against the systemic aldehydes toxicity, we characterized the compensational metabolic remodeling in liver from TG mice.

Methods: ALDH2 expression and phosphorylation of acetyl‐CoA calboxylase (P‐ACC) were assessed with immuno‐blotting. Gene chip analysis and metabolome analysis with capillary electrophoresis mass spectrometry were performed.

Results: In TG mice, the mutant protein was highly expressed in heart and skeletal muscles, but not in liver. The upregulation of enzymes for fatty acid oxidation and an increase in the P‐ACC caused the elevation of citrate levels. The inhibition of phosphofructokinase by citrate caused the elevation of glucose 1‐phosphate (G1P) levels and the reduction of metabolites in glycolysis below fructose 1,6‐bisphosphate. The elevated G1P levels could increase the utilization for glucuronide conjugation to detoxify aldehydes.

Conclusion: We concluded that the metabolic remodeling of fatty acid oxidation and glycolysis to augment the flux for glucuronide conjugation contributed to the detoxification in liver against the systemic aldehyde toxicity.
In vitro oxidant stress causes preferential loss of IRS proteins in rat skeletal muscle.
Authors: Archuleta Tara Lakshimi , Lemieux Andrew , Teachey Mary , Kim John , Henriksen Erik
Published: June 2021

Oxidant stress, the imbalance of oxidant production and the antioxidant defenses in the cell, can impair the functionality of elements of the insulin signaling pathway that regulates glucose metabolism in skeletal muscle. In this context context, the primary purpose of the present investigation was to characterize the effects of shorter‐term (2 hr) and longer‐term (4 hr) oxidative stress on protein expression of critical insulin signaling elements in skeletal muscle. Soleus muscles from insulin‐sensitive lean Zucker rats were exposed in vitro to an oxidant stress (60–90 μM H2O2 produced by glucose oxidase) for 2 or 4 hr. The oxidant stress reduced (p<0.05) insulin‐stimulated glucose transport activity after shorter‐term (50%) or longer‐term (53%) exposure. Moreover, the diminution of insulin action in the presence of the oxidant stress after 4 hr was associated with decreased protein levels of the primary insulin receptor substrate (IRS) isoforms expressed in mammalian skeletal muscle, IRS‐1 (59%) and IRS‐2 (33%). In contrast, this longer‐term oxidant stress did not alter the protein expression of IR‐β, PI3‐kinase, Akt, and GSK‐3. In addition, the stress‐activated p38 mitogen‐activated protein kinase (p38 MAPK) was activated after both 2 hr and 4 hr of oxidant exposure. The selective p38 MAPK inhibitor (10 μM A304000) prevented a significant portion (40%) of the oxidant stress‐induced loss of IRS‐1, but not IRS‐2, protein in skeletal muscle.
Toxicogenomic Response of Staphylococcus aureus to Triclosan
Authors: Jang Hyeung‐Jin , Chang Matthew Wook , Toghrol Freshteh , Bentley William E
Published: June 2021

For the first time a genome‐wide transcriptional analysis was performed to elucidate the cellular response of S. aureus to triclosan. Our results indicate that triclosan effects are widespread on the metabolism, affecting many vital cellular processes. Triclosan downregulated the transcription of genes involved in virulence factor and energy metabolism such as amino acid, carbohydrate, lipid transport and metabolism, while multidrug resistance genes, coenzyme transport and metabolism and transcription genes were upregulated. Furthermore, triclosan downregulated transcription of genes encoding major lipid metabolism enzymes such as 3‐hydroxyacyl‐CoA dehydrogenase, acetyl‐CoA acetyltransferase, acetyl‐CoA synthetase, and acetyl‐CoA carboxylase, which all play essential roles in S. aureus lipid metabolism. Interestingly, the expression of enoyl‐ACP reductase gene, fabI, was not changed after exposure of S. aureus with 0.05μM triclosan at 10 and 60min in our study. This work also implies that triclosan may kill S. aureus, by interfering with the ability to form cell wall membrane the same as penicillin. Another important implication of our result is that S. aureus may generate resistance factors under triclosan stress.
Characterization of 4‐Hydroxy‐2‐Nonenal and 4‐Hydroxy‐2‐Hexenal modified proteins by Data Dependent Neutral Loss‐Triggered MSn
Authors: Rauniyar Navin , Stevens Stanley M , Prokai Laszlo
Published: June 2021

End‐products of lipid peroxidation, 4‐hydroxy‐2‐nonenal (HNE) and 4‐hydroxy‐2‐hexenal (HHE), react with proteins to form covalent adducts. Mass spectrometric characterization of these adducts using a “bottom‐up” proteomic approach has been challenging because of the favored cleavage of the HNE/HHE modifications prior to backbone fragmentation of the peptide, preventing identification of the actual site of carbonylation due to the predominant neutral loss signal(s) observed in the MS/MS spectra. Our study involves characterization of HNE/HHE‐modified proteins using data dependent neutral loss‐triggered MSn with either collision induced dissociation (CID) or electron capture dissociation (ECD) via a hybrid linear ion trap FT‐ICR mass spectrometer. The general utility of NL‐driven MS3 acquisition using CID is evident from the enhanced characterization of protein‐HNE/HHE adducts observed by improving search algorithm scores to acceptable probability thresholds for identification. In the ECD method, peptide fragmentation is performed in the FTICR cell based on the neutral loss of HNE/HHE (156/114 Da) after MS/MS in the linear ion trap. The NL‐ECD method allowed for confident validation of protein modification by HNE/HHE and also identified several novel sites which were not definitively localized via NL‐CID. This research was supported by the grant AG025384 from the National Institutes of Health.
Lysine acetylation: a new protein modification in PPAR‐alpha agonist induced peroxisomes
Authors: Contreras Miguel A. , Baatz John E. , Singh Inderjit
Published: June 2021

Peroxisomes are ubiquitous subcellular organelles that play important role in cellular metabolisms and related disease processes. To date, very few of the peroxisomal proteins have been reported to be posttranslational modified. In this study we screen PPARα‐induced peroxisomal proteins for the presence of acetyl‐modifications. Subcellular organelle proteins from a rat liver peroxisomes‐enriched fraction separated by a linear density gradient were analyzed by Western blot using acetylated‐lysine‐specific antibody. A single 80kDa band was identified in an area where the peroxisomal enzymatic marker catalase localized. LC‐MS/MS analysis of the bands excised from SDS‐PAGE and from 2D resolved immunoprecipitated proteins were then used to identify peptides chemically modified. A peptide containing acetylated lysine having the sequence (69avanydsgeagek81) was identified and consistent with the peroxisomal multifunctional enzyme‐2 (MFE‐2) protein sequence. In silico analyses for acetyl‐modification of MFE‐2 sequence supported our finding. Analysis of MFE‐2 tertiary protein structure resulted in mapping the modified residue (81Lys) to the MFE‐2 surface, specifically on the (3R)‐hydroxyacyl‐CoA dehydrogenase domain. In summary, this is the first report of acetyl‐modification of a protein in PPARα‐induced peroxisomes. (Supported by NIH grant NS‐34741 and NCRR grants C06 RR018823 and C06 RR015455).
Separation of aminoacids and selenoaminoacids by capillary electrophoresis
Authors: Bautista María Esther , Revilla Alma Luisa
Published: June 2021

Selenium (Se) is an essential micro‐nutrient. Which is important for the normal functioning of muscle, heart, liver, kidney and other organs. It is incorporated within the molecule of the enzyme glutathione peroxidase and it is vital to protect red blood cells and cell membranes against the adverse reactions of peroxide that accumulates in the tissues. The qualitative and quantitative determination of particular species of this element like selenoaminoacids is important in order to understand its role in metabolism and physiology human. Experiments were performed on a Beckman P/ACE MDQ capillary electrophoresis system. Silica capillaries with different total lengths 77 or 83cm, 50 microm internal diameter. Use of 30 mM borates buffer at several pH values (9.0, 10.0, and 10.5), addition of SDS at different concentrations 10, 20, 30, 50, 60 and 70 mM and two separation temperatures 20 and 25oC, with hydrodynamic injection 0.5 psi for 25 s. and 25 kV of applied voltage and detection at 210 nm were studied. Adding 8.7 microg/ml of platinum to a mixture of aminoacids and selenoaminoacids (20 microg/ml) a partial separation was obtained using borates buffer pH 10.5, SDS 30 mM, capillary 77 cm and temperature 20oC. The detection limit for the analytes under study is 5 microg/ml.
Mammalian Luciferase Detection Vectors and Applications
Authors: Naleway John Joseph , McGibney Mischa Taylor , Coleman Daniel John , Cook Gabriele Maria
Published: June 2021

The light‐emitting reaction of the marine bioluminescent bacterium Vibrio harveyi is catalyzed by the bacterial luciferase enzyme which exists as an alpha‐beta heterodimer encoded by the luxA and luxB genes with subunit molecular weights of 42K and 37K respectively. The enzyme catalyzes a reaction with FMNH2, oxygen and a long‐chain aldehyde as substrates to yield visible light at 490 nm. A new luciferase marker gene detection system has been developed based upon this bacterial luciferase isolated from Vibrio harveyi. Sequences encoding the two luciferase subunits, luxA and luxB have been cloned into two separate vectors. These vectors also include a CMV promoter for expression in mammalian cells as well as an ampicillin resistance gene (amp) for selection and amplification, the SV40 polyadenylation sequence and the SD/SA‐RNA splice donor and acceptor sequence for maximum expression. The vectors were tested for light emission parameters in a live mammalian cell format and with cell lysate samples from NIH3T3 murine fibroblasts in conjunction with transfection using vector GC:11726 Mus musculus biliverdin reductase B (flavin reductase (NADPH)) sequence for production of the reduced flavin mononucleotide (FMNH2) cofactor intracellularly. These systems are being developed to monitor regulation of expression for two independent vector constructs, upon the dual expression.
Dispensable role of focal adhesion kinase in integrin alpha9beta1 mediated cell migration
Authors: Gupta Shiv Kumar , Vlahakis Nicholas E
Published: June 2021

Focal adhesion kinase (FAK) is a key signaling protein for integrin mediated cell migration. Auto phosphorylation of FAK at tyrosine‐397 is induced upon integrin activation, which then serves as a high affinity binding site for src tyrosine kinase forming a FAK‐src complex. Fak‐Src complex is a potent regulator of subsequent downstream signaling for enhanced cell migration. Using stable lines of SW480 colon carcinoma cells expressing integrin subunit α9, we explored the importance of the FAK‐Src complex in α9β1‐dependent cell migration. We found that ligand binding of α9β1 rapidly activated src tyrosine kinase; with concomitant tyrosine phosphorylation of FAK, p130Cas and Erk MAP kinase; inhibition of src tyrosine kinase or ErkMAP kinase activity decreased α9β1‐dependent cell migration. Surprisingly, activated α9β1 induced phosphorylation of iNOS resulting in NO production and associated NO‐mediated cell migration. Src tyrosine kinase appeared to signal upstream of iNOS, Erk, FAK and p130Cas as its inhibition blocked α9β1‐dependent tyrosine phosphorylation of these molecules. FAK binding to src was however not required for integrin α9β1 induced cell migration; and FAK inhibition (by siRNA) did not alter integrin α9β1 induced src activation and phosphorylation of Erk, p130Cas, iNOS. Analysis of Chinese Hamester Overian cells that expressed intact or mutant forms of integrin α9‐subunit showed that the α9‐cytoplasmic domain was critical for α9β1 induced src tyrosine kinase activation, downstream signaling events and ultimately cell migration. Taken together these results suggest that src, in a FAK independent manner, serves as a key early signaling protein for α9β1‐dependent cell migration; it is activated at the α9 ‐cytoplasmic domain and subsequently initiates multiple signaling intermediates, including NO, to induce cell migration.
NHE1 is critical in urokinase plasminogen activator‐mediated invasion and migration processes in lung cells
Authors: Rastedt Danielle E , Wallert Mark A , Provost Joseph J
Published: June 2021

Cancer cell motility is a complex series of events, which includes the coordination of proteases and cytoskeletal proteins at the leading edge of the tumor. Matrix metalloproteinases (MMP) play a critical role in digesting the extracellular matrix (ECM). Degradation of the extracellular matrix by MMP in migrating cells provides a vital function for tumor metastasis. The sodium hydrogen exchanger (NHE), functions as a coordinator of motility by acidification of the ECM and as a docking site for cytoskeletal proteins. CCL39 cells were used as a model to dissect the role on NHE in UPA signaling. In these cells, 10 nM UPA gave rise to a two fold increase in pH, activated MMP9 and doubled the number of cells invading through matrigel. EIPA, a NHE1 inhibitor blocked the UPA‐mediated activation of MMP in CCL39 and H1299 cells. UPA activation of MMP9 and CCL39 cells expressing mutant NHE was also determined. UPA stimulated tumor formation in both H1299 and H358 cancer cells well over controls. EIPA inhibition of cell proliferation and growth in a soft‐agar assay was investigated in both human lung cancer cell lines. We show that urokinase plasminogen activator (UPA) stimulates MMP activity in an NHE dependent fashion and that UPA activation of tumor formation and invasion required NHE. NIH 1 R15 HL074924‐01A1
Inhibition of fatty acid synthase activates the unfolded protein response in tumor cells
Authors: Kridel Steven J.
Published: June 2021

Fatty acid synthase (FAS), the enzyme that catalyzes the de novo synthesis of palmitate, is highly expressed in most tumors, with little or no expression in the corresponding normal tissue. Considering the strong link between FAS expression, tumor progression and disease recurrence, it is critical to develop FAS inhibitors and understand their anti‐tumor mechanisms. Because of previous links between lipid homeostasis and endoplasmic reticulum (ER) function in multiple models systems, we hypothesized that the unfolded protein response (UPR) plays a role in the anti‐tumor mechanism of FAS inhibitors. Indeed, FAS inhibitors induce 1) a robust PERK‐dependent phosphorylation of the translation initiation factor eIF2α and 2) the IRE1‐mediated processing of XBP1 specifically in tumor cells, consistent with UPR signal transduction. Induction of the UPR precedes activation of the canonical markers of apoptosis. Inhibition of FAS also antagonizes turnover of ubiquitin‐modified proteins. Interestingly, the proteasome inhibitor Velcade, an FDA‐approved drug that also induces the UPR, induces fatty acid synthesis by up‐regulation of acetyl‐CoA carboxylase (ACC) expression and synergizes with FAS inhibitors to increase UPR signaling. These data provide a link between the fatty acid synthesis pathway and ER homeostasis in tumor cells and demonstrates crosstalk between the fatty acid synthesis and proteasome pathways. These data also suggest that combining FAS inhibitors with other drugs that affect ER homeostasis may translate into more effective tumor therapies.

Research supported by NIH CA114104 and the Department of Defense Prostate Cancer Research Program W81XWH‐07‐1‐0024.
Glucocorticoids: Lipolytic or Lipogenic?
Authors: Campbell Jonathan Edward , Ceddia Rolando , Hawke Thomas , Riddell Michael
Published: June 2021

The effects of glucocorticoids (GCs) on lipid metabolism are controversial with some studies indicating that GCs stimulate lipolysis, while others suggest lipogenesis. The purpose of this study was to clarify the role of GCs in lipolysis by utilizing a range of GC concentrations and to assess the long‐term effects of GCs on basal lipolytic rates. In study #1, both 3T3‐L1 were treated for 24h with increasing concentrations of GCs (0.01‐100 uM) and subsequently assayed for glycerol concentration as a marker of lipolysis. Lipolysis increased above control cells in a parabolic fashion to a maximum of 1.9±0.2 fold with 0.1 uM GC (P<0.01). In study #2, 3T3‐L1 were treated with varying GC doses for 24 hrs, then washed and resuspended in fresh GC‐free media. Glycerol was measured 1hr post GC removal. Basal lipolytic rates increased in a dose‐dependent and linear manner, with 100uM GC treatment resulting in a 4.6±0.47 fold increase in lipolysis. The responses in both studies were specifically related to GC treatment as co‐treatment with the GC receptor antagonist mifepristone abolished the effects. These results were confirmed in primary adipocytes (subcutaneous and epididymal). Based on these findings, we hypothesize that GCs can cause both in a lipolytic and anti‐lipolytic manner depending on the concentrations present and their duration of exposure.
The Ras‐GTPase activating proteins Ira1p and Ira2p are required for proper control of phosphatidylinositol deacylation and glycerophosphoinositol transport in Saccharomyces cerevisiae.
Authors: Patton‐Vogt Jana , Anand Puneet , Bishop Andrew , Hofer Katherine
Published: June 2021

Phospholipase B‐mediated deacylation of phosphatidylinositol (PI) results in the formation of extracellular glycerophosphoinositol (GroPIns). GroPIns is transported across the Saccharomyces cerevisiae plasma membrane into the cell via the transporter encoded by GIT1. We screened the yeast viable knockout set for mutants unable to grow when GroPIns was added as sole phosphate source. Our selection criteria identified strains bearing deletion mutations in IRA1 and IRA2 as being defective for growth on GroPIns. Ira1p and Ira2p are known to act as Ras‐GTPase activating proteins. Not only do ira1Δ, ira2Δ and ira1Δira2Δ mutants display decreased GroPIns transport activity, but also an increase in extracellular GroPIns production, with the ira1Δira2Δ double mutant displaying the most extreme phenotypes. These results suggest that hyperactivation of the RAS/cAMP signaling pathway upregulates the production of extracellular GroPIns via phospholipase B‐mediated PI turnover and downregulates the transport of GroPIns into the cell via Git1p. This research was supported by NIH grant GM59817 to J.P.‐V.
Deciphering the Role of Disulfide Bonds in RIalpha
Authors: Day Michele Elizabeth , Koller Antonius , Taylor Susan S.
Published: June 2021

The dimeric regulatory subunits (R) of cAMP‐dependent protein kinase (PKA) are multidomain proteins with diverse functions. Other than inhibiting the catalytic subunit of PKA, one isoform (RIalpha) has been implicated in diseases. Though much is known about RIalpha, the role of the disulfide bond at the N‐terminus is poorly understood. Studies have shown that the cysteines involved in the disulfide bond contribute to its binding to A Kinase Anchoring Proteins (AKAPs) and may influence its localization to discrete sub‐cellular locations. One mutant of RIalpha, R74C, has been found in Carney complex patients. To date, the mechanism of action of the R74C mutation remains unknown. We determined if the disulfide bonding is altered in the R74C mutant and found a novel dimer. We are using mass spectrometry to analyze the disulfide bonding profile of this dimer. Additionally, studies are currently underway to see if there are changes in its binding to proteins, such as AKAPs, and whether there are changes in the sub‐cellular localization of this mutant.
Differential Contribution of G‐protein Coupled Receptor Kinase 2 and 3 in Regulating Structurally Related Human Dopamine D1 and D5 Receptors
Authors: Muntasir Habib Abul , Tiberi Mario
Published: June 2021

Dopamine (DA) D1‐like receptors (D1R and D5R) are seven‐transmembrane proteins regulating important brain and peripheral functions. Impairment in D1‐like responsiveness may play a crucial role in the phenotypic expression of several brain disorders such as schizophrenia and Parkinson's disease. Agonist‐induced receptor phosphorylation mediated by G‐protein coupled receptor kinase isoforms (GRK1–GRK6) curtails G‐protein signaling and target receptors for internalization. Here we investigated the role of GRK2 and GRK3 in regulating D1R and D5R desensitization in a subtype‐specific manner. Human embryonic kidney 293 cells expressing the D1R or D5R alone or with either GRK2 or GRK3 were analyzed for DA‐mediated receptor activation using whole cell cAMP assays. We demonstrate that GRK2 shows little effect in promoting desensitization of D1R and D5R, whereas GRK3 promotes a significantly more robust desensitization of D1R than D5R as assessed by the extent of DA‐induced maximal stimulation of adenylyl cyclase. However, expression of GRK2 and GRK3 leads to a more reduced DA potency for D5R than for D1R. These findings point to the existence of marked differences in the ability of GRKs to promote D1R and D5R desensitization. Our studies thus provide insight into potential distinct GRK2 and GRK3 molecular mechanisms through which D1‐like receptor signaling is regulated. Supported by a CIHR grant (MOP‐81341) to MT.
Signaling from β1‐ and β2‐adrenergic receptors is defined by differential interactions with PDE4
Authors: Richter Wito , Day Peter , Agrawal Rani , Bruss Matthew D. , Granier Sébastien , Wang Yvonne L. , Rasmussen Søren G.F. , Horner Kathleen , Wang Ping , Lei Tao , Patterson Andrew J. , Kobilka Brian , Conti Marco
Published: June 2021

β1‐ and β2‐adrenergic receptors (βARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by β1AR but not β2AR signaling and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here we demonstrate that β1AR forms a signaling complex with a cAMP‐specific phosphodiesterase (PDE) in a manner inherently different from a β2AR/β‐arrestin/PDE complex reported previously. The β1AR binds a PDE variant, PDE4D8, in a direct manner and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the β2AR is a prerequisite for the recruitment of a complex consisting of β‐arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of β1‐ and β2‐adrenoceptor signaling.

This research was supported by Fondation Leducq and NIH grants HD20788 to M.C. and HL71078‐01 to B.K.
A novel biological role of tachykinins as an upregulator of oocyte growth: evolutionary origin of tachykinergic functions in the ovary
Authors: Satake Honoo , Aoyama Masato , Kawada Tsuyoshi , Fujie Manabu , Hotta Kohji , Sakai Tsubasa , Sekiguchi Toshio , Oka Kotaro Kotaro , Satoh Nori
Published: June 2021

Tachykinins (TKs) and their receptors have been shown to be expressed in the mammalian ovary. However, the biological roles of ovarian TKs have yet to be verified. Ci‐TK‐I and Ci‐TK‐R, characterized from the protochordate (ascidian), Ciona intestinalis, are prototypes of vertebrate TKs and their receptors. We show a novel biological function of TKs as an inducible factor for oocyte growth using C. intestinalis as a model organism. Immunostaining demonstrated the specific expression of Ci‐TK‐R in test cells residing in oocytes at the vitellogenic stage. DNA microarray and real‐time PCR revealed that Ci‐TK‐I induced gene expression of several proteases including cathepsin D, chymotrypsin, and carboxypeptidase B1 in the ovary. The enzymatic activities of these proteases in the ovary were also shown to be enhanced by Ci‐TK‐I. Of particular significance is that the treatment of Ciona oocytes with Ci‐TK‐I resulted in progression of growth from the vitellogenic stage to the postvitellogenic stage. The Ci‐TK‐I‐induced oocyte growth was blocked by a TK antagonist or by protease inhibitors. These results led to the conclusion that Ci‐TK‐I enhances growth of the vitellogenic oocytes via upregulation of gene expression and enzymatic activities of the proteases. This is the first clarification of the biological roles of TKs in the ovary and the underlying essential molecular mechanism.
Localized PKD activity at PDZ domain‐containing scaffolds
Authors: Kunkel Maya T. , Garcia Erin L. , Hall Randy A. , Newton Alexandra C.
Published: June 2021

The protein kinase D (PKD) family plays a role in many cellular functions including the regulation of Golgi sorting, apoptosis, cell growth and cell survival. The mammalian PKD family consists of three isoforms, PKD1, PKD2 and PKD3, and of these, PKD1 and PKD2 contain PDZ‐binding motifs at their C‐terminus. Using a proteomic array containing 96 purified PDZ domains, we have identified domains that interact with the 25 C‐terminal residues of PKD1 or PKD2. We show that PKD activity localized at the PDZ domain scaffold is significantly greater than bulk cytosolic activity. Specifically, a FRET‐based kinase activity reporter designed to report PKD signaling, DKAR (D Kinase Activity Reporter), when targeted to the scaffold protein, reveals enhanced amplitude and duration of signaling compared to cytosol. Thus, this study unveils novel protein scaffolds that localize PKD activity.

This work was supported by NIH P01 DK54441
Ras/Raf/MEK/ERK signal attenuation by the PHLPP Phosphatases
Authors: Niederst Matt , Enserink Jorrit , Newton Alexandra C
Published: June 2021

The recently discovered PH Domain Leu‐Rich Repeat Protein Phosphatase (PHLPP) family of proteins was found in a search to understand how Akt signaling is terminated. The two PHLPP isoforms control the amplitude of agonist‐induced signaling by directly dephosphorylating and inactivating Akt, thus suppressing cell proliferation and inducing apoptosis. Given the dramatic effect of PHLPP in antagonizing cell proliferation, we asked whether PHLPP plays a role in regulating additional proliferative signaling pathways, specifically the Ras/Raf/MEK/ERK pathway. siRNA experiments revealed that loss of PHLPP1 or PHLPP2 leads to increased amplitude and duration of MEK and ERK phosphorylation following EGF stimulation compared to vector controls. Further investigation of upstream pathway components showed that PHLPP1/2 also suppress Ras activation following agonist stimulation. Thus, PHLPP isoforms also control signal termination of the Ras/Raf/MEK/ERK pathway. These results strengthen the case for PHLPP1/2 as putative tumor suppressor proteins, as the loss of PHLPP1/2 in cells leads to increased activity of the pro‐survival Akt and ERK pathways, which are commonly up‐regulated in many human cancers, and in several instances mutations in these pathways alone are sufficient to drive tumorigenesis. This work was supported by GM NIH 067946.
Identification of novel substrates of S6 kinase 2
Authors: Quintanilla Rene Harold , Peng Yen‐Lin , Lee‐Fruman Kay K
Published: June 2021

Ribosomal S6 kinase 2 (S6K2) has been shown to act downstream of the mammalian target of rapamycin (mTOR). Although S6K2 shows high homology to S6 kinase 1 and shares in its ability to phosphorylate the ribosomal protein S6, evidence suggest that they may have different cellular function, indicating that the two kinases may have different sets of substrates. Although several substrates of S6K1 have been identified, S6K2 substrates other than S6 are not known to date. In order to identify possible substrates of S6K2 we carried out a phosphorylation screening of 1,800 human proteins via protein microarray. We identified three possible substrates of S6K2: protein Kinase C zeta (PKCζ), casein kinase 1 gamma 1 (CK1γ1) and casein kinase 1 gamma 2 (CK1γ2). Biochemical analysis of in vitro phosphorylation of these potential targets by S6K2 will be presented, as well regulation of phosphorylation of these proteins by the mTOR pathway. The in vitro and in vivo analyses of phosphorylation of these proteins by S6K2 will help shed light on the complexity of S6K2's function within the cell. This research was supported by NIH SCORE Grant 2 S06 GM063119.
Metabolic Lysosomal Enzyme Probes
Authors: Naleway John Joseph , Coleman Daniel John , Guzikowski Anthony Peter , Williamson Staci Patricia
Published: June 2021

Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells. Lysosomes have been found to be involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of diseases including Parkinson's, Tay‐Sachs, Sandhoff, Krabbe and Gaucher's syndromes. New lysosomal staining compounds that are useful for labeling lysosomes in a live‐cell format and are capable of monitoring lysosomal metabolic activity have been developed. These new targeted substrates are based upon fluorescent probes that have a low pKa value for optimum fluorescence at the lower physiological pH values found in the lysosomes as well as targeting groups to direct their accumulation to the lysosomes using a live‐cell staining format. Application to the staining of cells derived from patients with Krabbe's, Gaucher's I and II, Tay‐Sach's and Sandhoff Diseases as well as healthy human and murine fibroblast control cells are presented. In addition the ability to monitor the effect of secondary therapeutic agents on staining is presented. This work was supported by NIH Grant 5R43MH079542‐02.
Functional and Molecular Characterization of a Newly Discovered Bacteriocin‐like Substance
Authors: Brock Susan R. , Knadler Judith , Triplet Meredith , Baker Jason C.
Published: June 2021

Bacteriocins are antimicrobial peptides produced by bacteria while antibiotics are non‐proteinacous small organic antimicrobial molecules. They also differ from antibiotics in modes of action. Their wide range of molecular and functional characteristics include: molecular weight, structure, temperature/pH stability, and killing spectrums of susceptible species. Bacteriocins present potential for diverse applications as treatment/prevention of infectious diseases, food preservers, ect. In the spring of 2006, a bacteriocin‐like substance, currently referred to as Bacteriocin X (Bac. X), was discovered from a putative strain of Bacillus amyloliquefaciens. The objective of this study was to discern the functional and molecular characteristics of Bac. X. It demonstrated a wide spectrum of activity against an array of bacterial organisms (both Gram(+) and Gram(−)), including a variety of potential pathogens. It displayed stability under extreme temperature and pH conditions. It was not notably susceptible to Proteinase K or Trypsin; but its activity was destroyed by Pronase E. Bac. X was also able to destroy mammalian cells. This study is still pursuing the purification of Bac. X from cell filtrate or perhaps a plasmid isolation of its gene to facilitate sequencing and homology comparisons, looking for the identification of the bacteriocin or documentation of a new one.

MWSU Summer Research Institute
Spin label studies of interactions between the estrogen receptor and coactivator peptides
Authors: Rupert Samantha , Barhite Kelly , Gulla Stefano , Budil David Edward
Published: June 2021

The estrogen receptor (ER) is a member of the family of nuclear receptors that has become an important therapeutic target for the treatment of osteoporosis and a variety of cancers. The response of ER to estrogenic ligands is mediated by a wide range of coactivator and corepressor proteins with the NR box sequence motif that binds to helix 12 (H12) of ER. We have studied ER‐coactivator interactions by the spin labeling method, in which a nitroxide reporter group is attached either to H12 or to a peptide sequence containing the NR box. Lineshape analysis of the electron spin resonance (ESR) spectra of such labels allows them to be characterized in terms of local mobility and ordering, providing new physical details about the binding of different coactivator sequences. Research supported by Army BCRP award W81XWH‐06‐1‐0551 and a Merck Undergraduate Summer Research Fellowship.
The role of E proteins in the CD8+ T cell immune response
Authors: D’Cruz Louise M , Camfield Kristin A , Wu Bei Bei , Pham Thomas , Goldrath Ananda W
Published: June 2021

CD8+ T cells are essential cells of the immune system because they mediate control of infection by intracellular pathogens such as bacteria, viruses and protozoan parasites. Naïve CD8+ T cells, upon encounter with antigen, differentiate into effector T cells, which rapidly elicit an immune response to eliminate infected cells. Upon removal of the pathogen, a subset of effector cells will further differentiate into memory T cells, remaining to respond to subsequent infection. E proteins and their inhibitors, the Id proteins, have been shown to be crucially important in regulating the development of T cells in the thymus. We have also recently shown that Id2 protein, an inhibitor of E protein DNA binding, is essential for CD8+ T cell responses. We speculate that E proteins, and their transcriptional targets, regulate the activation and formation of CD8+ effector T cells during an immune response. Using E2A‐GFP reporter mice and EMSA studies, we show that E proteins are up‐regulated and bound to DNA during the early stages of the T cell immune response. Using E protein conditional knock‐out mice, we are currently studying the role of E proteins in the in vivo CD8+ T cell response.
Catecholamine activation of p38 MAPK in S49 T lymphosarcoma cells is dependent on Protein Kinase A
Authors: LaJevic Melissa Dawn , Suleiman Samia , Cohen Rhonna L. , Chambers Donald A.
Published: June 2021

The catecholamine norepinephrine (NE) functions through a β2AR/cAMP/PKA pathway in T cells leading to altered immune function. p38 MAPK is an important intracellular signaling mediator for environmental stressors and plays a role in cell survival, apoptosis, and inflammation. To determine a relationship between NE‐mediated immune dysfunction and the p38 MAPK pathway, S49 wild type cells were incubated in the presence and absence of NE or a cAMP analogue, and the levels of activated (dually phosphophorylated, pTpY180/182) p38 MAPK were detected by ELISA. Activated p38 MAPK increased upon NE or cAMP treatment within 5 min and was sustained for 30 min. When the cells were treated with nadolol, a β2AR antagonist, the activation of p38 MAPK by NE was inhibited. Treatment of an S49 PKA mutant cell line (kin‐) with NE or cAMP, unlike wt, showed no change in activated p38 MAPK levels. These results suggest that NE can signal through the β2AR/cAMP/PKA pathway to activate p38 MAPK and reveal a new signaling pathway for engagement of p38 MAPK through a G protein‐coupled receptor/PKA dependent mechanism. Supported by NIH.
γδ T‐cell dysregulation in the cutaneous diabetic environment
Authors: Taylor Kristen R. , Mills Robyn E. , Jameson Julie M.
Published: June 2021

Skin γδ T‐cells are specialized intraepithelial lymphocytes that express a canonical γδ T‐cell receptor (TCR), which recognizes stressed or damaged epithelial cells. Once activated, skin γδ T‐cells release cytokines and growth factors that facilitate cutaneous wound healing. In diabetes, wound healing is delayed and patients often suffer from chronic infections. We hypothesize that skin γδ T‐cells are dysregulated in diabetes, contributing to complications in wound healing. Using the leptin receptor deficient mouse model of Type 2 diabetes, we have determined that skin γδ T‐cell homeostasis is altered in the diabetic environment. Skin γδ T‐cells in diabetic mice display decreased levels of T‐cell activation markers including CD69, CD25 and the γδ TCR, indicating a decreased activation status. Following the onset of diabetes, the number of skin γδ T‐cells decrease two‐fold. In vivo wound healing assays reveal that diabetic γδ T‐cells have delayed rounding following wounding, suggesting an inability to respond to epithelial cell damage. Taken together, our studies suggest that the diabetic environment has negative effects on the activation status of skin γδ T‐cells and ultimately renders them unable to properly respond to damaged epithelial cells. Alterations in skin γδ T‐cell function may influence the production of growth factors and contribute to immune and repair deficiencies in diabetic patients.
Identification of a biological ligand for human plasmacytid dendritic cell receptor ILT7
Authors: Cao Wei , Bover Laura , Wang Yi‐Hong , Rosen David B , Arai Naoko , Yao Zhengbin , Lanier Lewis L , Liu Yong‐Jun
Published: June 2021

Human plasmacytoid dendritic cells (pDCs) are instrumental for systemic type I IFN production during viral infections. How these cells crosstalk with other cell types during steady state and/or after the initial IFN burst is not clear. Using a reporter cell system, we discovered that a list of breast cancer cell lines were potent in triggering human pDC specific receptor ILT7, indicating the presence of a potential biological ligand on these cells. Monoclonal antibodies were generated against ILT7 ligand positive cancer cells and two clones were able to neutralize the abilities of cancer cells to activate ILT7. Using these mAbs, we found that, in addition to transformed cells, normal peripheral hematopoietic and non‐hematopoietic cells up‐regulate the expression of such molecule in response to IFN exposure. We have screened a cDNA library using the mAbs and identified a putative candidate gene. Confirmation is underway to establish the role of this molecule as the ligand for ILT7. As ILT7 activates a potent ITAM‐mediated negative regulatory pathway in pDCs, our findings may suggest a converged molecular mechanism modulating pDC function, which may operate in a tumor microenvironment or in a negative feedback manner post IFN production.
MSC license CD34‐DC into regulatory DC via Notch pathway activation
Authors: Yinping LI , Sophie Paczesny , Evelyne Lauret , Sonia Poirault , Ping OuYang Jing , Jean‐François Stoltz , Assia Eljaafari
Published: June 2021

In this study, we intent to clarify these mechanisms by examining the immunosuppressive effect of human adult MSC on adult DC differentiated from CD34+ hematopoietic progenitor cells (HPC). We showed that MSC halt both interstitial and Langerhans cell differentiation from adult CD34+HPC, as assessed by the decreased expression of CD1a, CD14, CD86, CD80 and CD83 antigens on their cell surface. Accordingly, the allostimulatory capacity of these CD34‐DC was impaired. Furthermore, we showed that (i) MSC inhibited commitment of CD34+HPC into immature DC, but not maturation of CD34‐DC (ii) this inhibitory effect was reversible and (iii) MSC‐DC induced the generation of allo‐Ag specific regulatory T cells following secondary allo‐stimulation. Direct coculture of MSC with CD34+HPC resulted in much stronger immunosuppressive effect and led to an activation of the Notch pathway as assessed by the overexpression of Hes1 in MSC‐DC. Finally, DAPT, a gamma‐secretase inhibitor that inhibits NOTCH signaling was able to overcome MSC‐DC defects. In conclusion, our data suggest that MSC licence adult CD34+HPC to differentiate into regulatory DC, through activation of the NOTCH pathway.