bluuebunny/crossref_metadata_2025_split huggingface dataset:
The effects of taurine depletion on bile acid composition and its amino acid‐conjugation in the bile of cats
Authors: Miyazaki Teruo , Sasaki Sei‐ich , Toyoda Atsushi , Shirai Mutsumi , Ikegami Tadashi , Matsuzaki Yasushi , Honda Akira 
Published: June 2021

AimThe most established effect of functional amino acid‐like compound taurine is the conjugation with bile acids. In addition, taurine ameliorates hypercholesterolemia through promotion of hepatic bile acid synthesis from cholesterol by enhancing of CYP7A1. On the other hand, the conjugations of bile acids with taurine as well as glycine and the biosynthesis of taurine are also promoted by bile acids through the nucleus farnesoid X receptor. These findings imply that there is an interactive relationship on the metabolism of taurine and bile acids. Contrary to rodents, the ability of taurine biosynthesis is very poor in humans, and therefore, the body pool of taurine in humans depends on the oral ingestion. Furthermore, the ratio of conjugation of taurine, but not glycine, with bile acids also depends on taurine ingestion. This study evaluated the influence of depletion by taurine non‐ingestion on the metabolism of bile acid in bile in cats with poor taurine‐synthesizing ability as in humans.MethodControl (N=4) and Depletion(N=6) groups of adult cats were fed the soy protein‐based diet containing 0.15% and 0% taurine, respectively, for 30 weeks. Thereafter, taurine in the serum and tissues and bile acids in the bile were measured by LC‐MS/MS system.ResultSerum taurine concentration in the Depletion group significantly decreased to one tenth compared to that in the Control group after the 2nd week, and was undetectable since the 4th week. After 30 weeks, taurine in the various tissues was depleted in the Depletion group. Over 99.5% of bile acids in the bile were conjugated with taurine in the Control group. In the Depletion group, taurine‐conjugated bile acids significantly decreased to 35.2%, and instead, unconjugated bile acids significantly increased to 62.9%. Bile acid concentration in the bile significantly decreased to approximately 40% compared to that in the Control group. Bile acid types in cats were same to those in humans. In the composition of bile acids, cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acids (CDCA) were 83%, 12%, and 5%, respectively, in the Control group. On the other hands, CA significantly increased to 98%, and DCA and CDCA significantly decreased to 0.6% and 1.2%, respectively, in the Depletion group.ConclusionTaurine depletion caused the decrease of taurine conjugated bile acids in the bile, and instead, the increase of unconjugated bile acids. Consequently, the level of bile acids significantly decreased and the composition has changed. These results suggested that the synthesis of bile acids in humans might be regulated by taurine ingestion through the direct effects of taurine or the state of taurine conjugation with bile acids.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Novel Calreticulin Reporter for Determination of Immunogenic Cell Death
Authors: Bocian Rebecca , Franco April , Gurzell Eric , Soendergaard Mette 
Published: June 2021

Immunogenic cell death (ICD) initiates a series of damage associated molecular patterns (DAMPs) that are able to activate an immune response. Of these, translocation of calreticulin (CRT) from the endoplasmic reticulum (ER) to the outer leaflet of the cell membrane is a hallmark event that leads to activation of antigen presenting cells (APC), thus priming an immune response. Induction of ICD in cancer cells may provide a manner of activating an immunological anti‐tumor response, which may be utilized in immunotherapy. Currently, a few chemotherapeutic drugs, such as doxorubicin, are known to induce ICD and have been proposed to be repurposed for this use. Thus, it is likely that other known drugs may exhibit ICD induction and may be utilized in immunotherapy. However, to evaluate such drugs, there is a need to develop a high throughput method of detecting ICD.Here, a novel ICD reporter based on translocation of CRT and the HiBit‐tag expression system is described. This system utilizes a recombinant HiBit‐tag that associates with cell impermeable LgBit, thereby forming a bioluminescent protein complex. HiBit‐tagged CRT will complex with LgBit when translocated to the outer leaflet of the cell membrane, which may be detected bioluminescently by addition of D‐luciferin. Thus, the intensity of bioluminescence may be correlated to translocation of CRT, a hallmark of ICD.To create the novel ICD reporter, the human CRT gene was amplified using polymerase chain reaction (PCR) from plasmid HsCD00322958 (Harvard Medical School, MA), and inserted onto the 3′ end of the HiBit tag (pBiT3.1‐N, Promega, WI) using restriction enzyme digestion (PspXI and SbfI) followed by ligation. The recombinant HiBiT‐CRT plasmid was transformed into competent DH5α E. coli cells using the heat‐shock method. The HiBit‐CRT cassette was validated by DNA Sanger sequencing (Genscript, Piscataway, NJ). In order to transfect human ovarian adenocarcinoma cells (SKOV‐3) with the ICD reporter, the optical concentration of geneticin was determined by performing a kill curve. In brief, SKOV‐3 cells were maintained in McCoy's 5A cell medium supplemented with 10 % fetal bovine serum (FBS) at 37°C and 5% CO2, and then treated with varying concentrations of geneticin (0.05 mg/mL, 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL, 1.25 mg/mL, 1.5 mg/mL, 1.75 mg/mL, 2 mg/mL). The cell viability was evaluated daily for 7 days using bright field microscopy. The optimal concentration was determined to be 1.25 mg/mL. Next, SKOV‐3 cells will be transfected with the ICD reporter, and used to screen the Approved Oncology Drug Set VI (kind gift from the National Cancer Institute) for compounds that induce ICD.Support or Funding InformationWestern Illinois University Research Council GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Biochemical Program of Extreme Terminal Differentiation
Authors: Finley Daniel 
Published: June 2021

As cells undergo extreme forms of terminal differentiation, they are able to accumulate specific proteins to exceptionally high levels–hundreds of milligrams per ml in the case of globins and crystallins. During the time in which these proteins are being generated in high amounts, almost all other components of the cell, numbering many thousands, are eliminated. The mature red blood cell has one of the simplest cellular proteomes known, with hemoglobin concentrated to a remarkable ~98% of soluble protein by some estimates. How can cells carry out such vast programs of biosynthesis and degradation simultaneously? We proposed in 1995 that the ubiquitin‐proteasome system (UPS) may play a central role in this process. We have addressed this problem through quantitative global proteomics in collaboration with the laboratories of Drs. Mark Fleming, Mitch Weiss, and Peng Ji. We found that ubiquitinating factor UBE2O, which is strongly induced in late erythroid differentiation, functions as an E2–E3 hybrid enzyme to mediate the elimination of ribosomes and myriad other proteins in maturing red cells. UBE2O recognizes substrates directly, and its unexpectedly broad specificity appears to reflect the presence of multiple substrate recognition domains within the enzyme. Additional ubiquitinating factors–TRIM10, TRIM58, and UBE2H–are, like UBE2O, specifically induced in late erythroid differentiation (during this period, most UPS components disappear). Applying quantitative global proteomics to mouse mutants in these enzymes, we find that the ubiquitin ligase TRIM10 eliminates dynactin, many myosins, actin crosslinking proteins, and CopI vesicles, while in contrast TRIM58 eliminates dynein and centrosome components, and UBE2H eliminates ER components. In general we find that erythroid lineage inducible ubiquitinating factors have quite distinct specificities, as if each were tasked to eliminate its own part of the cell. Together the eliminate many of the basic functionalities of cell biology. The UPS has been conceived of as having two major types of function: the selective degradation of regulatory proteins, and protein quality control. Our work indicates a new capacity of the UPS, to effect global and developmentally controlled proteomic remodeling. We propose that ubiquitin‐dependent proteome remodeling is likely to be an important new aspect of the biology on eukaryotic cells, critical for the generation of highly differentiated cell types.Support or Funding InformationR01 HL125710This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screening Tools for Efficient Delivery of siRNA and Plasmid into Mammalian Cells
Authors: Collins Michelle , Jordan Elizabeth , Terefe Joseph , Pineda Maxinne , Ugozzoli Luis , Rubio Teresa 
Published: June 2021

Modulating gene expression is essential to achieve a better understanding of gene function. The transfer of exogenous nucleic acids such as plasmids or small interfering (si)RNAs into mammalian cells is an important technique for the study and analyses of gene function, expression, regulation, and mutation, and it has advanced basic cellular research, enabled drug target identification, and validation. Electroporation is a well‐established gene transfer method, and an effective means of transferring nucleic acids into cells. The shift of today's research to more scientifically relevant cell lines has created a void for efficient delivery tools. In addition, the wide adoption of RNAi technology, has led to a demand of screening tools to identify the most effective siRNA. In order to obtain consistent downstream results, it is critical that delivery conditions be highly efficient.Here, we show data that demonstrates the benefits of using a high throughput electroporation system to develop optimal conditions for delivery of plasmids and (si)RNA into mammalian cells. We demonstrate that the use of this instrument allows for rapid screening as well as the ability to identify the most efficient delivery conditions into a difficult‐to‐transfect cell line. Finally, we present quantitative PCR data that validates the benefits of this technique.
Rap1 promotes Prostate Cancer metastasis
Authors: Bailey Candice L , Casey Patrick J 
Published: June 2021

Elucidating the mechanisms of prostate cancer (CaP) survival and metastasis are critical to the discovery of novel therapeutic targets. Increasing evidence demonstrates significant roles for integrins and matrix metalloproteinases (MMP) in CaP metastasis, making them attractive targets for therapy. The monomeric G protein, Rap1 activates integrins and has been implicated in cancer tumorgenesis. Rap1 signals to pathways involved in cell adhesion, migration, and survival, suggesting Rap1 may promote several processes associated with CaP metastasis. Thus, we sought to explore the functional significance of Rap1 activation in CaP by examining its impact on invasion and migration of CaP cells during metastasis.Results from in vitro assays demonstrate activation of Rap1 increases CaP cell migration toward bone‐secreted factors. Similarly, results show activation of Rap1 enhances CaP cell invasion, and may involve modulation of MMPs. Inhibition studies indicate that integrin activation is a mechanism of Rap1‐mediated CaP migration and invasion. Additional studies will explore whether Rap1 influences MMPs directly or through its subsequent activation of integrins. The in vivo impact of Rap1 in CaP is being evaluated in the mouse and preliminary results demonstrate activated Rap1 drives the rate and incidence of CaP metastasis.Support for CLB: Department of Defense PCRP #DAMD17‐0510560
Expression of NUANCE, a potential novel oncogene, is inhibited by nonsteroidal anti‐inflammatory drugs (NSAIDs) in human colorectal cancer cells
Authors: Liggett Jason Lee , Choi Chang Kyoung , Donnell Robert L , English Anthony E , Kihm Kenneth David , Baek Seung Joon 
Published: June 2021

NSAIDs are well known for treating inflammatory disease and have been reported to have an anti‐tumorigenic affect. Our lab has previously shown that NSAIDs also induce other anti‐tumorigenic genes including NSAID activated gene (NAG‐1). This makes NSAIDs a prime candidate for further study. HCT‐116 cells treated with sulindac sulfide (SS) showed dramatic morphological changes under differential interference contrast microscopy as well as weakened cellular adhesion as measured by micro‐impedance. Microarray analysis was performed using HCT‐116 cells treated with SS, and NUANCE (NUA), a novel connection between the actin skeleton and the nuclear envelope, was selected based on its novelty in relation to cell adhesion and biogenesis. SS diminished NUA mRNA expression by one third and various other NSAIDs also showed inhibition of NUA. Additionally, immunohistochemistry showed higher levels of NUA in tumors of many tissues. Further micro‐impedance experiments on cells with shRNA NUA showed a reduction in cellular adhesion. Thus, our data suggest that NUA may be a potential novel oncogene in human colorectal cancer cells and NSAIDs could decrease its expression.ACS RSG‐06‐124‐01
Autophosphorylation of Protein Kinase C Facilitates its Ubiquitination and Down‐regulation
Authors: Gould Christine , Newton Alexandra 
Published: June 2021

Protein kinase C (PKC) is a Ser/Thr kinase that tranduces signals generated from the second messengers, diacylglycerol and calcium, the products of receptor‐mediated lipid hydrolysis. PKC has three modes of regulation: phosphorylation, cofactor binding, and localization. Phosphorylation of PKC regulates its maturation, activation, and signaling properties. Autophosphorylation at two conserved sites in the carboxyl‐terminus of PKC is a major step in the generation of catalytically competent enzyme. PKC has been show to readily autophosphorylate at additional sites in vitro, and their physiological role is unknown. Chronic activation of PKC that occurs with phorbol esters, functional analogues of diacylglycerol, results in the degradation and “downregulation” of the enzyme. Here, we examine the role of autophosphorylation at two sites preceding the autoinhibitory pseudosubstrate sequence in the phorbol ester‐mediated downregulation of PKC βII. We find that mutating the in vitro‐identified autophosphorylation residues, Ser16 and Thr17, to generate a phospho‐mimic (S16E/T17D) increases the rate of PKC degradation following phorbol ester stimulation. This mutant was more rapidly dephosphorylated and ubiquitinated in comparison to wildtype PKC. In contrast to its altered degradation, the phospho‐mimic was processed by phosphorylation similarly to wildtype and had similar activity. Generation of a phospho‐specific antibody to Thr17 indicated that PKC autophosphorylates in vitro as well as in vivo in response to phorbol esters in both an overexpression system as well as with endogenous PKC in the K562 lymphona cell line. Based on these data, we propose that PKC is additionally autophosphorylated following activation by phorbol esters, an event that promotes the downregulation of the enzyme.This work was supported by NIHGM43154.
Antibacterial Activity of New Coumarins
Authors: Govori Sevdije R. , Spahiu Sabrije , Haziri Arben 
Published: June 2021

4‐(4‐Bromo‐thiazol‐2‐ylamino)‐3‐coumarincarbaldehyde (1), 4‐(4‐Methoxy‐benzothiazol‐2‐ylamino)‐3‐coumarincarbaldehyde (2) and 4‐(2‐Chloro‐6,7‐dimethoxy‐quinazolin‐4‐ylamino)‐3‐coumarincarbaldehyde (3) are synthesized in very good yields, by nucleophilic substitution with aminoheteroaryl derivatives on 4‐chloro‐3‐carbaldehyde. The antibacterial activity of coumarin was tested against: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853.The compounds showed strong to moderate activity in reducing the microbial growth. The inhibitory effects of compounds were affected by their substitution patterns. All compounds showed the most effective antibacterial activity against Escherichia coli ATCC 25922.
Itk regulates T cell signaling through localization of active Cdc42
Authors: Singleton Kentner L. , Fowell Deborah J. , Wuelfing Christoph 
Published: June 2021

The tyrosine kinase Itk can regulate actin accumulation at the T cell/APC interface, elevation of the T cell intracellular calcium concentration, and IL‐4 secretion. The cellular mechanism Itk uses to control these diverse functions is unresolved. Here we have identified Itk as a general regulator of the spatiotemporal organization of T cell activation: Itk controls the localization of the TCR, PLC&#947;, Cdc42, and Actin. Cdc42 localization is of particular interest, as not the cellular activity of Cdc42 but only its localization to the center of the T cell/APC interface was dependent on Itk. This control of the localization of active Cdc42 is a central part of the cellular mechanism of Itk function: Providing active Cdc42 selectively at the center of the T cell/APC interface in Itk‐deficient T cells could restore Itk‐dependent aspects of T cell activation. Providing Cdc42 without such targeting as a means to raise Cdc42 activity in the entire T cell could not. These data are of wider relevance, as they prove that the control of the localization of a signaling intermediate without affecting its activity can be a central part of the regulation of cellular activation.Supported by the NIH.
Proinflammatory signaling via the neurokinin 1 receptor promotes dendritic cell survival resulting in robust cellular immune responses
Authors: Janelsins Brian Mark , Mathers Alicia R , Tkacheva Olga A , Erdos Geza , Larregina Adriana T 
Published: June 2021

Inflammatory and immune responses are regulated by the secretion of cytokines and neuropeptides released in immune‐competent organs. Substance P, the prototype proinflammatory neuropeptide secreted by nerve fibers, promotes T cell proliferation by binding the neurokinin 1 receptor (NK1R). The initiation of T cell responses requires the activation and survival of dendritic cells (DC). Since DC home strategically to richly innervated organs, we hypothesized that signaling via the NK1R will promote DC survival resulting in robust DC‐T cell stimulatory functions. Using a model of murine DC generated from bone marrow precursors we investigated the: i) expression of the NK1R, ii) ability of the NK1R agonist [Sar9Met(02)11]SP to activate intracellular pathways involved in DC survival and iii) in vivo DC stimulatory function and survival of NK1R‐signaled DC homing to local draining lymph nodes. Our data show that DC expressed functional NK1R and agonistic signaling prevented DC apoptosis via PI3K and ERK signaling pathways and mobilization of intracellular Ca2+. Adoptive transfer of NK1R‐signaled DC favored lymph node homing and survival of DC which resulted in enhanced and prolonged Ag specific DTH responses. We conclude that signaling DC with a NK1R agonist enhances DC survival and their ability to stimulate cellular immunity.This work was supported by NIH grant R01CA100893 to ATL
Adjuvant abolishes immune regulatory effect mediated by type II NK T cells
Authors: Maricic Igor , Halder Ramesh C. , Kumar Vipin 
Published: June 2021

Myelin‐derived sulfatide (3′‐sulfagalactosyl ceramide) is a self‐glycolipid ligand recognized by a subset of CD1d‐restricted type II NKT cells. Here we show that treatment of mice with an immunodominant isoform of sulfatide inhibits the chronic and relapsing course of experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. However the regulatory effect is lost upon administration of sulfatide lipid with complete Freund's adjuvant.Using PLP139–151/As‐tetramer and intracytoplasmic cytokine staining techniques we have examined the frequency of and cytokine secretion by pathogenic T cells in lymph nodes as well as in CNS. Our data indicate an increased number of PLP‐reactive Th1‐like cells and enhanced microglial activation in mice co‐challenged with lipid and adjuvant. These results indicate that activation of type II NK T cells in the presence of strong adjuvant is ineffective in the treatment of T cell‐mediated autoimmune diseases. (Supported by grants from the NIH, MSNRC to VK).
A Rapid New Procedure for Basophil Isolation from Human Peripheral Blood
Authors: Fairhurst Maureen , Nauer Melany , Eaves Allen , Thomas Terry 
Published: June 2021

Basophil research has been limited by the difficulty in isolating pure basophils in large numbers. Typically, basophils comprise < 1% of peripheral blood leucocytes in non‐allergic, healthy humans. Current protocols for their isolation are time consuming involving multiple steps and often special equipment. Prolonged manipulation of basophils can lead to undesirable activation and spontaneous histamine release.We describe a rapid (within 1.5 hours) and simple method for the enrichment of basophils from normal blood that does not require Ficoll or lysis steps and yields good purity and recovery. Blood was collected with heparin and red blood cells were removed by HetaSep sedimention. The basophils were then enriched using immunomagnetic, column‐free negative selection (EasySep®). Briefly, unwanted cells were specifically labeled with dextran‐coated magnetic particles using a cocktail of bispecific tetrameric antibody complexes. The labeled cells were then removed using a magnet leaving unlabeled basophils. The entire separation procedure can be automated with a pipetting robot (RoboSep®).Final basophil purity range as assessed by flow cytometry was 92–99% CD45+IgE+ CD123+. May‐Grunwald stained cytospins showed typical morphology and characteristic blue‐black granules. Expression of cell‐surface CD203c was similar between whole blood and enriched basophils. Recovery averaged 49 ± 17 % (n=7).
Cross‐Talk Between NK Cells and Adenovirally‐Engineered Dendritic Cell Vaccines
Authors: Vujanovic Lazar , Newton Sarah , Landsberg Jennifer C. , Vujanovic Nikola L. , Butterfield Lisa H. 
Published: June 2021

Recombinant adenoviral vectors (AdV) are an effective modality for antigen engineering of dendritic cells (DC) in order to harness DC immunostimulatory ability and activate tumor antigen‐specific T cells. Little is known about the capacity of AdV‐transduced DC (AdV/DC) to activate natural killer (NK) cells, and stimulate their cytotoxic and regulatory abilities. Here, we have investigated the effects of AdV transduction on the ability of DC to activate, and become activated by NK cells. We showed that AdV transduction does not enhance DC sensitivity to Granzyme B‐mediated lysis by NK cells when compared to immature DC (iDC). AdV/DC and matured DC both enhance activation of CD56hiCD16− and CD56dimCD16+ NK cell subsets, based on the expression of Granzyme B, CD25, and CD69 activation markers. We detected this effect in short and long‐term cultures, as well as in clinical trial patient PBMC samples post‐AdV/DC vaccination. Reciprocally, NK cells enhance the state of AdV/DC maturation in short‐term co‐cultures according to surface marker expression and cytokine profile evaluation. Moreover, NK cells pre‐activated with AdV/DC kill K562 tumor cells more efficiently than those from iDC co‐culture by Granzyme B ELISPOT. Together, this data demonstrates that there is a positive cross‐talk between AdV/DC and NK cells, which may be an additional pathway by which this vaccine platform may induce anti‐tumor activity.This work was supported by the University of Pittsburgh Cancer Institute and the Henry L. Hillman Foundation (to L.H.B.)