bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the high affinity choline transporter (CHT1) by ubiquitin ligase Nedd4‐2
Authors: Yamada Haruhiko , Imajoh‐Ohmi Shinobu , Haga Tatsuya
Published: June 2021

The high affinity choline transporter (CHT1) is specifically expressed in cholinergic neurons, and plays a critical role in the regulation of acetylcholine synthesis. Little is known about the mechanisms that regulate the cell surface expression and trafficking of CHT1. Recently membrane proteins such as receptors, channels and transporters have been shown to be ubiquitinated resulting in the regulation of their endocytosis and trafficking. We have examined the role of ubiquitination in the function and trafficking of CHT1, focusing on E3 ubiquitin ligase Nedd4‐2 which is reported to ubiquitinate dopamine transporter and some neuronal membrane proteins. Coexpression of Nedd4‐2 with CHT1 in HEK293 cells was found to decrease the amount of cell surface CHT1 by 40%, which was assessed as the binding activity with hemicholinium‐3 (a membrane‐impermeable, specific inhibitor of CHT1). Choline uptake activity was also decreased by 40% in the same cells, indicating that coexpression of Nedd4‐2 did not affect the specific activity of the cell surface CHT1. Coexpression of catalytically inactive Nedd4‐2 mutant did not affect either hemicholinium‐3 binding or choline uptake activity. These results indicate that the trafficking, but not the specific activity, of CHT1 is regulated by Nedd4‐2.
Intersectin (ITSN) regulation of Rab family GTPases
Authors: Wong Katy Ann , Chen Yun‐Ju , O’Bryan John P.
Published: June 2021

ITSN is a multi‐domain scaffolding protein that regulates endocytosis and signal transduction. In addition, ITSN enhances receptor tyrosine kinase (RTK) trafficking in part through regulation of RTK ubiquitylation. We now report that ITSN regulates Rab‐mediated trafficking of RTKs. ITSN and Rab5 co‐localize on early endosomes. To examine the relationship of ITSN with various Rab family GTPases, we used bimolecular fluorescence complementation (BiFC) in which a YFP variant, Venus, is split into amino (VN)‐ and carboxyl (VC)‐terminal fragments, neither of which is fluorescent. When these fragments are fused to proteins that interact, fluorescence is reconstituted thus allowing for temporal and spatial monitoring of protein:protein interactions in vivo. Using BiFC, we shown that ITSN complexes with Rab5 and Rab7, but not with the dominant negative forms of these GTPases indicating that ITSN preferentially associates with active Rab5 and Rab7. In addition, we have identified a new component of the Rab pathway that forms a complex with ITSN and Rab5/Rab7 suggesting a role for this protein in Rab‐mediated trafficking. The localization of ITSN to vesicles as well as ITSN's interaction with the Rab pathway supports the model that ITSN regulates intracellular trafficking.

K.A.W. supported in part by NIH PSTP Training Grant (5T32GM070388)
Muscle cells have a biphasic response to the LG 4–5 region of laminin (E3)
Authors: Zhou YanWen , Jiang Daifemg , Jarrett Harry W.
Published: June 2021

Studies have shown that laminin or laminin α1‐chain LG4–5 domain (E3) bind to the dystrophin glycoprotein complex (DGC) on the exterior of muscle cells. This binding causes signaling through dystroglycan‐syntrophin‐grb2‐SOS1‐Rac1‐Pak1‐JNK that ultimately results in the phosphorylation of c‐Jun (Oak, Zhou, and Jarrett, J.Biol. Chem. 278:39287). Recently, we reported that laminin or E3 up‐regulates the proliferation of C2C12 myoblasts when a low concentration of E3 (<1 μg/ml) was added to cultured medium (Zhou, et al., Biochemistry, in press). Here, we show that higher concentrations of E3 (>1μg/ml) decrease cell number and cause apoptosis in C2C12 myoblasts. The increase of apoptotic cells was determined using FACS with FITC‐annexin V when 3μg/ml E3 was added to the culture medium while growing C2C12 myoblasts for 24hr. We also found that grb2 was recruited to the DGC at low E3 concentrations but not when toxic amounts of E3, which cause apoptosis, were used. These results show that muscle cells have a biphasic response to E3 and that this biphasic response is also seen in DGC signaling. Another signaling pathway originating at the DGC activates Akt and prevents apoptosis but does not involve grb2. These results suggest crosstalk between the grb2‐Rac1‐JNK pathway and the Akt one. (This work was supported by NIH AR051440 and by the Muscular Dystrophy Association, grant #3789).
KiSS‐1/GPR54 sensitizes TNFα‐induced apoptotic cell death of invasive tumor cells
Authors: Cho Sung‐Gook , Lewis Stafford Joe , Li Dali , Luo Jian , Liu Mingyao
Published: June 2021

TNFα can induce NF‐κB transcriptional activity which positively regulates metastatic processes. KiSS1/GPR54 signaling pathways have been suggested to play a role in suppressing cancer metastases. However, the molecular mechanism remains unknown. To understand whether KiSS1/GPR54 signaling pathways regulate tumor metastasis through the inhibition of NF‐κB, we first examined the known KiSS1/GPR54 function such as the inhibition of cell migration and NF‐κB‐dependent gene expression. We demonstrated that KiSS1 via endogenous GPR54 suppressed TNFα‐induced chemotactic migration of MDA‐MB‐231 and reduced the expression levels of NF‐κB‐mediated genes in cell proliferation and migration. Furthermore, we found that KiSS1/GPR54 could inhibit TNFα‐dependent NF‐κB transcriptional activity by blocking p65 nuclear translocation in both MCF‐7 and MDA‐MB‐231 cells. However, KiSS1/GPR54 sensitized TNFα‐mediated cell apoptosis only in the metastatic MDA‐MB‐231 cells, but not in non‐metastatic MCF‐7 cells. Taken together, our data suggest that KiSS1/GPR54 suppresses the metastatic nature of TNFα‐stimulated invasive tumor cells through the inhibition of NF‐κB pathway and the induction of apoptotic cell death.
Histone Deacetylase Inhibition Augments the Inhibitory Effect of Leptomycin B on Proinflammatory Cytokine Release from Stimulated Human Monocytes.
Authors: Ramaswami Sitharam , Ghosh Chandra C , Vu Hai‐Yen , Juvekar Ashish P , Vancurova Ivana
Published: June 2021

Release of proinflammatory cytokines tumor necrosis factor‐alpha (TNFα), interleukin (IL)‐8 and IL‐6 from stimulated monocytes plays a major role in inflammatory disorders such as sepsis, arthritis, or chronic lung disease. Synthesis of these proinflammatory cytokines is regulated at the transcriptional level by the transcription factor NFκB. There is a compelling evidence that transcriptional regulation is controlled by histone acetylation, and histone deacetylase (HDAC) inhibitors are emerging as a new class of promising agents for the treatment of certain forms of cancers as well as inflammatory disorders. We have recently shown that the release of TNFα, IL‐8 and IL‐6 from stimulated monocytic U‐937 cells is inhibited by the inhibitor of CRM1‐dependent nuclear export, leptomycin B (LMB). In this study, we investigated whether the HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaB) may potentiate the inhibitory effect of LMB on proinflammatory cytokine release from lipopolysaccharide (LPS)‐stimulated U‐937 monocytic cells. Our results show that TSA and NaB significantly augment the LMB inhibitory effect on TNFα, IL‐8 and IL‐6 release from LPS stimulated U‐937 cells, and suggest that the underlying mechanisms involve the transcription factor NFκB. Understanding the precise mechanisms by which LMB and HDAC inhibitors down‐regulate the proinflammatory cytokine release may lead to the design of novel combination strategies for inflammatory disorders.
The Role of Neuregulin/erbB Signaling in Endothelial Progenitor Cell Biology
Authors: Safa Radwan Nabeel , Lamparter Mathias , Pentasugglia Laura , Peng Xu Yang , Hatzopoulos Antonis , Sawyer Doug
Published: June 2021

Neuregulin (NRG) is a ligand for the erbB3 and erbB4 members of the EGF family receptors, and is known to activate signaling pathways that mediate proliferation, survival, migration and differentiation in various cell types. Stimuli such as exercise and ischemia‐reperfusion, which induce NRG shedding also induce endothelial progenitor cell (EPC) mobilization and recruitment. We hypothesized that NRG signaling may a play role in EPC mobilization and homing.

Using western blot and immunostaining, we found that a murine embryonic endothelial progenitor cell line expresses NRG ligand and the ErbB‐2, 3 and 4 receptors. Treatment with recombinant NRG led to ErbB2/B3 phosphorylation as well as downstream Akt and endothelial nitric oxide synthase phosphorylation. NRG activated signaling was blocked by the ErbB receptor inhibitors AG1478 and Lapatanib. To examine the role of NRG/ErbB signaling in EPC biology we performed a matrigel angiogenesis assay. In contrast to mature rat cardiac microvascular endothelial cells (CMEC), EPC's in 3 dimensional monoculture do not form microvessel networks. However when EPC's are co‐cultured with CMEC's in matrigel, EPC's incorporate into the developing microvessel network within 24 hrs. This effect was completely inhibited by the erbB receptor inhibitor AG1478.

These results support a role for Neuregulin/ErbB signaling in EPC recruitment. A clearer understanding of what role NRG signaling may play in EPC biology has important implications for cardiovascular biology, as well as the pathophysiology of cardiac dysfunction seen in the setting of erbB2 targeted cancer therapeutics.
Progesterone and subsequent polar metabolites are essential for completion of the first meiotic division in amphibian oocytes
Authors: Kostellow Adele B , Morrill Gene A
Published: June 2021

The amphibian oocyte remains arrested in prophase of the first meiotic division until released by the gonadotropin surge which initiates ovulation. Prior studies indicate that the gonadotropins act on the follicle cells surrounding the oocyte to release progesterone, which targets receptors on the oocyte surface. We present evidence that completion of the first meiotic division in Rana pipiens oocytes requires the sequential action of at least two steroids: progesterone and one or more subsequent polar metabolites of progesterone. Progesterone binding to oocyte surface receptors increases during the first 4–5 h, followed by internalization of the plasma membrane together with membrane‐bound progesterone over the next hour. Internalized progesterone is metabolized to highly polar polyhydroxylated steroid(s) prior to nuclear membrane disappearance which occurs at 8–9 h. Polar steroids alone cannot induce meiosis, but can do so in oocytes pretreated with physiological progesterone levels for 1 h. Similarly, the non‐metabolizable progestin R5020 will not induce meiosis but does if oocytes are subsequently exposed to polar steroids. An inhibitor of steroid ‐reductase (4‐methyl‐4‐aza‐steroid) inhibits both progesterone metabolism and progesterone‐induced meiosis. Thus, progesterone binding at the oocyte surface initiates a process which requires polar progesterone metabolites to advance to metaphase of the second meiotic division. Since polar steroids are essential for meiosis and are known to stimulate the Src pathway in other cells, the newly formed polar steroids may initiate the Src signaling pathways associated with cell cycle control in oocytes.
Cofilin translocation to the nucleus is a key feature of decidualization
Authors: Ihnatovych Ivanna , Livak Mark , Strakova Zuzana
Published: June 2021

Decidualization (D), the differentiation of stromal fibroblasts (SF) into decidual cells, is critical for pregnancy establishment. D requires the reorganization of the actin cytoskeleton that is governed by phosphorylation (P) of myosin light chain and actin dynamics. Manipulation of actin dynamics by jasplakinolide prevents D of human SF. Actin dynamics are regulated by actin‐binding proteins, one of which is cofilin. The activity of cofilin depends on its P, which is regulated by LIMK1 and LIMK2. D of human SF is associated with changes in the distribution and activity of the proteins of the LIMK‐cofilin pathway. D induced by a stimulus of embryonic origin, interleukin‐1β (IL‐1β) and steroid hormones (H), is accompanied by an upregulation of LIMK1, a downregulation of LIMK2 and the translocation of inactivated (phosphorylated) cofilin from the nucleus into the cytosol. On the other hand, D induced by an artificial stimulus cAMP (with H) is characterized by a significant decrease in phosphorylated cofilin and unchanged amounts of LIMK1 and LIMK2. Despite the differences in molecular mechanisms underlying the D induced by H alone, IL‐1β+H or cAMP+H, the most striking observation during D is the translocation of cofilin from the cytosol to the nucleus. Taken together, the stabilization of F‐actin significantly inhibits D; and the translocation of cofilin to the nucleus is a key feature of D. Supported by HD044713
The role of phosphorylation in the regulatory mechanism of the Diaphanous‐related formins
Authors: Rawson Jonathan M. , Veltman Katherine J. , Wallar Bradley J.
Published: June 2021

Diaphanous‐related formins (DRFs) are a conserved family of Rho GTPase effector proteins that coordinate the actin and microtubule cytoskeletal networks. This situates the DRFs in a key position to influence cellular shape, motility, and cytokinesis; therefore, it is critical that these proteins are tightly regulated. Normally, DRFs are maintained in an inactive state via intramolecular interactions and can be activated by the binding of Rho GTPases. However, recent studies of mammalian DRFs have demonstrated that Rho GTPase binding is necessary, but not sufficient for DRF activation. Based upon the hypothesis that a second activation ‘signal’ might be phosphorylation, we show here that the mouse DRF, mDia2, is phosphorylated by p21‐activated kinase 1 (PAK1). In vitro kinase assays using purified PAK1 and mDia2 mutants have identified a serine residue in mDia2 as the site of phosphorylation. The main activity of PAK1 is to coordinate extracellular signals with the reorganization of the actin cytoskeleton, and mDia2 phosphorylation may be one way this is accomplished. Currently, we are probing the role of PAK1 phosphorylation in mDia2 regulation by using fluorescence anisotropy to monitor mDia2 intramolecular binding. The cytoskeletal effects of PAK1‐mDia2 regulation, as well as the effects of PAK1 and Rho GTPases on mDia2‐mediated actin nucleation will provide insight into the mechanism of mDia2 regulation.
Molecular basis of transcriptional coactivator recruitment by ARNT PAS domains
Authors: Partch Carrie Lee , Card Paul B. , Gardner Kevin H.
Published: June 2021

PAS (Per/ARNT/Sim) domains are used throughout all kingdoms of life to monitor diverse environmental stimuli, including light, oxygen, and small molecule ligands. ARNT (Aryl Hydrocarbon Nuclear Translocator) forms a heterodimeric complex with the sensor bHLH‐PAS protein HIF‐α; to mediate hypoxia adaptation ineukaryotes. Aberrant activation of the hypoxia response in solid tumors is a key predictor of poor clinical outcome in cancer patients. We are studying the molecularbasis of transcriptional coactivator recruitment by ARNT to understand how PAS domains coordinate responses to environmental stimuli. Using NMR spectroscopy, we have characterized the binding sites of both ARNT PAS domains on a minimal fragment of one coactivator, TRIP230. The interaction is mediated in part by ahighly conserved LXXLL‐like motif on TRIP230 that targets the a‐helical interface of ARNT PAS‐B. Two other coactivators target a similar interface on the PAS‐Bdomain, suggesting that this interface is critical for coactivator recruitment. Further studies suggest that the minimal TRIP230 peptide makes contacts with both PASdomains that stabilize formation of a proteolytically protected core complex. Using an NMR‐based screen, we have identified exogenous small molecule ligands thatbind internally to ARNT PAS‐B and disrupt the ARNT:TRIP230 interaction in vitro in an effort to develop novel chemotherapeutic agents.
Evaluation of gender differences in methylation‐sensitive immune gene expression in T cells by cDNA microarray
Authors: Hewagama Anura , Patel Dipak , Yarlagadda Sushma , Strickland Faith M. , Richardson Bruce C.
Published: June 2021

Previous studies in our laboratory have demonstrated that DNA methylation inhibitor 5‐aza‐2′‐deoxycytidine (5‐aza) demethylates DNA. T cell DNA hypomethylation has been implicated in the pathogenesis of idiopathic and drug‐induced human lupus which is 9 time more common in women than men. The genes affected by DNA hypomethylation are largely unknown. We report here a cDNA microarray analysis of T cell gene expression from men and women after treatment with the 5‐aza. PBMC isolated from 3 men and women were stimulated with PHA for 24hrs. T cells were isolated and cultured with or without 5‐aza for 3 days, then restimulated or not for 6 hr with PMA + ionomycin. We identified at least 268 up‐regulated T cell genes in women upon 5‐Aza treatment. 167 of these upregulated genes were uniquely expressed in women compared to men. CTLA4, IL6R and TNFSF14 were among the immune genes uniquely upregulated in women but not in men by 5‐Aza that were chosen for further study. These studies may provide insight into the molecular basis of autoimmune susceptibility in women. Supported by the National Institutes of Health Grant AR 42525 and the US Department of Veterans Affairs.
Determining Role of O‐GlcNAcylation During T‐cell Activation
Authors: Shimoji Shino , Hart Gerald W
Published: June 2021

O‐GlcNAcylation is a dynamic post‐translational modification of serine/threonine with β‐linked N‐acetylglucosamine. This cyto‐nuclear modification is found in all multi‐cellular organisms. Regulatory factors NFκB, NFATc1, and Elf‐1 have been identified to be O‐GlcNAc modified upon activation. O‐GlcNAcylation of Elf‐1 is necessary for translocation into nucleus. OGT expression has been positively linked to IL‐2 production. These data identify O‐GlcNAcylation as an integral part of signal transduction during T‐cell activation. We examined O‐GlcNAcylation changes upon PMA/ionomycin activation. Preliminary study showed cyto‐nuclear O‐GlcNAcylation levels increased through the first 2 hrs of the activation. The changes appeared to be protein specific. SILAC was used to identify the proteins with altered modifications. 30 proteins with increased modifications and 18 proteins with decreased modifications were identified.

(Supported by NIH grants CA42486 and HD13563. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody. Terms of this arrangement are managed by JHU.)
FAK Interacts With MEF2 and Drives the Stretch‐Induced Activation of an Intronic Enhancer of Phospholamban Gene in Cardiomyocytes
Authors: Cardoso Alisson C , Judice Carla C , Deckmann Ana C , Clemente Carolina F.M.Z. , Pereira Ana HM , Franchini Kleber G
Published: June 2021

Aim: FAK re‐locates at the nucleus and mediates the hypertrophy of cardiomyocytes in response to mechanical stress. Herein, we investigated possible regulatory role of FAK in gene expression in cardiomyocytes.

Methods: We identified an intronic sequence of phospholamban gene (Pln‐i1), containing a MEF2 consensus site, by a chromatin immunoprecipitation (ChIP) approach with anti‐FAK antibody. We then used a combination of electrophoretic mobility shift (EMSA), pulldown and reporter genes assays to examine possible FAK/MEF2 interaction and FAK/MEF2 regulation of Pln‐i1 expression.

Results: Fractional centrifugation and confocal microscopy confirmed the presence of FAK in the nuclei of stretched cardiomyocytes. EMSA indicated an interaction between MEF2 and Pln‐i1, but not with the fragments or the full‐length FAK. Pulldown assays performed with recombinant fragments of FAK (GST‐FERM and GST‐C‐TERM domains), nuclear extracts from nonstretched and stretched cardiomyocytes and anti‐MEF2 antibody indicated an association between FAK and MEF2. Further assays performed with recombinant GST‐MEF2 and full‐length FAK confirmed the interaction of MEF2 with phosphoFAK but not with non‐phosphorylated FAK. Transfection studies with Pln‐i1 and mut‐Pln‐i1 reporter constructs indicated that the MEF2 site is sufficient to direct the baseline and the stretch induced expression of luciferase. FAK silencing was demonstrated to markedly reduce the stretch‐induced expression of Pln‐i1 reporter construct.

Conclusion: These data identify the interaction of C‐terminal FAK with MEF2 and indicate that active FAK may modulate the MEF2‐dependent transcriptional regulation of phospholamban gene.
Bradykinin B2 Receptor Interacts with Integrin α5β1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells
Authors: Kramarenko Inga I , Bunni Marlene , Raymond John R , Garnovskaya Maria N
Published: June 2021

The vasoactive peptide bradykinin (BK) stimulates proliferation of the inner medullary collecting duct (mIMCD‐3) cells via transactivation of epidermal growth factor receptor (EGFR) by an unusual mechanism that involves collagenases‐2 and ‐3. Since collagenases lack an integral membrane domain, we hypothesized that integrins may target collagenases to the membrane to form a functional signaling complex.

We supported a role of integrins in BK‐induced signaling by showing that RGD peptides and neutralizing antibody against α5β1 integrin partially (∼50%) decreased BK‐induced phosphorylation of extracellular signal‐regulated protein kinase (ERK) in mIMCD‐3 cells. Silencing of α5 and β1 expression by transfecting cells with siRNA also caused a significant (∼40%) decrease in BK‐induced ERK activation and EGFR phosphorylation. The effect was more pronounced (∼70% inhibition) in cells that were co‐transfected with siRNAs directed against collagenases. Using immunoprecipitation/Western blotting, we demonstrated association of BK B2 receptor with α5β1 integrin upon BK treatment. Furthermore, we showed that BK induced complex formation between α5β1, collagenases and EGFR.

These data provide the first evidence that integrins are involved in BK‐induced proliferation of kidney cells, and might ultimately lead to development of new strategies for treatment of renal tubulointerstitial fibrosis.
5‐HT induces threonine (T375) phosphorylation of ADAM17/TACE cytoplasmic tail
Authors: Gooz Monika , Rocha Claudia , Bell Hannah , Gooz Pal , Luttrell Louis M , Raymond John R
Published: June 2021

We have shown recently that TACE transactivates epidermal growth factor receptor (EGFR), and induces ERK phosphorylation (ERK‐P) and cellular proliferation upon 5‐HT2A receptor stimulation in renal mesangial cells.

In this study our aim was to identify kinases responsible for TACE phosphorylation and potential activation during inter‐receptor cross‐talk. We detected robust threonine phosphorylation of TACE as early as 6 minutes following 5‐HT stimulation using a phospho‐threonine/proline antibody. This suggested phosphorylation of threonine 375, as this is the only threonine next to a proline present in the cytoplasmic tail of TACE. Using chemical inhibitors (naltrindole and PDK1/Akt/Flt dual pathway inhibitor) we showed that ERK phosphorylation is PDK1 dependent. Additionally, a MAPK cascade seems to have a role in TACE phosphorylation in that a MEK inhibitor (10 μM of PD989059) significantly attenuated 1 μM 5‐HT‐induced TACE phosphorylation. To study kinase‐induced activation of the enzyme we used a quenched fluorogenic TACE substrate. We found attenuation of 5‐HT‐induced TACE activity in PD989059‐pretreated cells. No serine phosphorylation of TACE upon 5‐HT stimulation was detected. Our data suggest that (1) either MEK itself or members of the MAPK family can be (directly or indirectly) responsible for TACE phosphorylation and activation during inter‐receptor crosstalk, and (2) multiple kinases contribute to TACE phosphorylation.

This work was supported by NIH DK070054‐03 to M.G., T32 DK07752‐08 to C.L.R., NIH NCCR P20 RR016434 to P.G., NIH R01 DK55524 to L.M.L. and REAP from the VA Research Service.
Nitrosative Stress Triggers Inhibition of DNA Synthesis and Translational Suppression of Positive Cell Cycle Regulators
Authors: Tomko Robert J. , Lazo John S.
Published: June 2021

Nitrosative stress or aberrant accumulation of intracellular nitroso‐species has recently emerged as a candidate regulator of cellular processes such as transcription, cell signaling, and apoptosis. We report herein that nitrosative stress, generated by expression of human inducible nitric oxide synthase (hiNOS) or by treatment with the cell‐permeable S‐nitrosating agent S‐nitrosocysteine ethyl ester (SNCEE), induced hyperphosphorylation of the translational regulator eIF‐2α at the inhibitory residue Ser51, repression of protein translation as measured by 35S‐incorporation, and inhibition of DNA synthesis as measured by bromodeoxyuridine incorporation. As inhibition of protein translation would initially alter the levels of rapidly turned‐over proteins, we hypothesized that loss of one or more short half‐life cell cycle proteins was responsible for the observed halt of DNA synthesis under nitrosative stress. Indeed, we observed decreased expression of the Cdc25A protein, which drives progression through S‐phase by activating cyclin‐dependent kinase 2 complexes, as well as decreased protein levels of two other cell cycle proteins, c‐Myc and Cyclin D1, with kinetics that precede DNA synthesis inhibition. Together, these results suggest that nitrosative stress may initiate a program to stop progression through S‐phase until perturbation of the intracellular redox status has been resolved.
Differential Sensitivities of Cytotoxicity Assays on C6 Glioma Cells Exposed to Dinitrobenzenes
Authors: Rivera Berenise , Miller Richard Timothy
Published: June 2021

Dinitrobenzene (DNB) refers to a mixture of three isomers: 1,2‐, 1,3‐ and 1,4‐DNB. These DNBs are used as common intermediates in the dye and plastic industries. 1,2‐ and 1,4‐DNB cause methemoglobinemia, in contrast 1,3‐DNB is toxic to cerebellum and certain brainstem nuclei in rats. To our knowledge, no studies have been conducted to determine if the cytotoxic effects of 1,2‐ and 1,4‐DNB parallel those of 1,3‐DNB in neuronal or glial cell models. The purpose of this in vitro study was to compare the relative ability of different cytotoxicity assays to detect early changes in cell viability. C6 Glioma cells were exposed to 10–25 μM DNBs for up to 6 hours. Cytotoxicity was then determined using MTT reduction for mitochondrial integrity, and neutral red (NR) uptake for lysosomal integrity. C6 Glioma cells exhibited a concentration‐ and time‐dependent decrease in NR uptake following a four hour exposure to 1,2‐ and 1,4‐DNB. Effects were most evident between 50–250 μM (80% and 8%, respectively). Under the same conditions, no evidence for NR uptake was observed upon exposure of C6 glioma cells to 1,3‐DNB. In contrast to results obtained with NR, no reduction of MTT by C6 glioma cells was observed at any time or concentration used in these studies. These preliminary data indicate that under the conditions used in our system, NR is a more sensitive indicator of changes in cell viability. This project was supported in‐part by 5G12‐RR008124 from NCRR at UTEP and by NIEHS/NIH grant ES011982 to RTM.
Characterization of Proteins in Commerical Preparations of Allergens by SDS‐Polyacrylamide Gel Electrophoresis
Authors: Martinez Adela M. , Gomez John Eric , Foglesong Paul David , Gonzalez Edward E.
Published: June 2021

Extracts of plant pollens and spores of a fungus that are used for immunotherapy of allergies were obtained commercially from Greer and analyzed for protein composition by SDS‐polyacrylamide gel electrophoresis. The extracts analyzed were pecan (Carya illinoensis), mesquite (Prosopis glandulosa), short ragweed (Ambrosia artemisiifolia), white oak (Quercus alba), mountain cedar (Juniperus ashei), and the fungus Aspergillis fumigatus. The extracts were resuspended in deionized water at a concentration of protein = 1.0 mg/ml. Samples containing 1–5 μg of protein were incubated for 2 min. at 100°C with SDS buffer and loaded on 4–20% gradient polyacrylamide gels. Electrophoresis was performed at 75 V for 2 hrs. Broad range markers (Bio‐Rad) were electrophoresed in two lanes on each gel in parallel with the samples. The gels were stained with Coomassie Brilliant Blue. The values of Mr (kDa) for the markers were 7.1, 21, 35, 49, 80, 124, and 209. The Mr (kDa) of the observed proteins for each of the allergens was determined by comparison to the migration of the markers: Carya illinoensis (10, 13, 17, 20, 27, 39, 41, 50, 51, 56, 71, 105, 119); Prosopis glandulosa (20, 27, 44, 50, 57, 63, 78, 115, 139, 146); Ambrosia artemisiifolia (16, 20, 25, 29, 42, 55, 62, 69, 84, 111); Quercus alba (12, 23, 40, 44, 63, 115, 135, 141) Juniperus ashei (14, 25, 37, 51, 66); Aspergillis fumigatus (10, 24, 27, 51, 59, 79, 85, 91, 100, 108, 124, 129, 140). The extracts will be fractionated in order to isolate and identify the proteins responsible for allergic reactions. This work was supported by a grant from the Welch Foundation to the Department of Chemistry at University of the Incarnate Word and by CA74388 to PDF.
Role of BAFF/APRIL in GALT Development
Authors: Yeramilli Venkata A. , Knight Katherine L.
Published: June 2021

In rabbits, B cells develop in the bone marrow and migrate to GALT where they proliferate, form organized follicles, and develop the primary antibody repertoire by Ig gene diversification. While the molecular events leading to Ig gene rearrangements in the bone marrow are well understood, little is known about the mechanisms regulating B cell proliferation and homeostasis in GALT. We investigated the role(s) played by BAFF and APRIL in stimulating GALT B cells. By in situ hybridization, we found BAFF transcripts in the appendix to be highly expressed in B cell follicles. In striking contrast to human B cells, we detected BAFF expression on both naïve peripheral and GALT B cells. In vitro, purified rabbit BAFF induced splenocytes to undergo proliferation, but not B cells isolated from GALT. However, addition of BAFF to cultures of GALT B cells transiently prolonged their survival. Following in vivo injection of neonatal rabbits with a soluble decoy receptor (TACI‐Ig), we found a marked reduction in both the size and number of B cell follicles and also in Ki‐67 expression, demonstrating that BAFF/APRIL is required for B cell proliferation in GALT. We are currently investigating whether B cells residing in GALT are immature or mature B cells. Since rabbit B cells express BAFF, we propose that B cells in GALT are stimulated in an autocrine fashion.

Supported by NIH AI50260
The Appendix as a Site of Immune Development in Response to Select Bacterial Species
Authors: Severson Kari M. , Mallozzi Michael , Driks Adam , Knight Katherine L.
Published: June 2021

The human appendix was recently proposed to function as a reservoir for beneficial, commensal bacteria (Randal Bollinger, R. et al. J. Theor. Biol. (2007), doi: 101016/j/ktbo/2007.08.032). The only other mammalian species with an appendix is rabbit, which may provide a model system to study the function of the human appendix. In rabbits, the appendix is a site where B cells proliferate and diversify Ig genes in response to the intestinal microbiota.

Our goal is to determine the mechanism by which the intestinal microbiota stimulates B cell proliferation in the appendix. We injected individual bacterial species into germ‐free appendices, and found that the spore‐former, Bacillus anthracis, stimulates B cell proliferation. We hypothesize that B. anthracis stimulates B cells in the appendix via a B cell superantigen. We probed western blots of bacterial lysates with rabbit IgM and identified a 30kD protein in B. anthracis spores that bound IgM. A purified, recombinant form of this protein stimulated calcium flux in appendix B cells, indicating that it can activate B cells. These data suggest that the appendix develops in part, through superantigen‐like stimulation of B cells by the intestinal microbiota. We propose that, in addition to serving as a reservoir for beneficial microbes, the human appendix, like that of rabbits, is a site of immune development in response to select bacterial species.

Supported by NIH AI50260
In vivo positive selection determines the peptide and MHC specificity of the CD8 T cell repertoire
Authors: Wang Baomei , Lybarger Lonnie , Primeau Tina , Hansen Ted H. , Connolly Janet M.
Published: June 2021

The nature of the peptides that drive positive selection of T cells in the thymus remains poorly defined. To examine the role of peptides during positive selection in vivo, we generated single‐chain‐trimer (SCT) transgenic mouse strains that express only the OVAp/Kb/β2m complex or the VSVp/Kb/β2m complex. Although the SCT transgene is expressed at levels that are comparable to Kb expression in B6 mice, very few CD8 T cells are selected by the SCT compared to B6 mice, a finding consistent with positive selection being peptide specific. Furthermore, the SCT selected CD8 T cells are highly reactive with Kb loaded with endogenous peptides, but are not reactive with Db. This demonstrates that endogenous Kb self peptides are effectively excluded by the covalently attached peptide, and that CD8 T cells preferentially react with the selecting class I molecule. A T cell clonal analysis revealed that SCT selected T cells were highly specific for Kb and were not more MHC cross‐reactive than T cells from B6 mice. These results suggest that CD8 T cells selected on a single peptide/MHC ligand are MHC specific and possess at least some peptide cross‐reactivity. The extent of this peptide cross reactivity is under investigation to define the relationship between the selecting and agonist peptides. In sum, these data strongly suggest that positive selection determines the peptide and MHC specificity of the CD8 T cell repertoire.
Melanocortin signaling via the MC4 receptor regulates T cell function
Authors: Youm Yun‐Hee , Yang Hyunwon , Butler Andrew , Dixit Vishwa Deep
Published: June 2021

The central melanocortin pathway is critical for regulating food intake and energy expenditure. However, precise role of melanocortins and MC4 receptor in regulation of thymic function remains to be elucidated. Here we demonstrate that MC4R are highly expressed in thymus and lymphoid organs. Compared to wild type control mice, genetic deletion of MC4R led to reduced cellularity and loss of cortico‐medullary junctions suggesting increased thymic involution. Interestingly, the absence of MC4R mediated signals caused a marked increase in lipid bearing thymic fibroblasts and reduction in medullary thymic epithelial cells with reduced thymic output. Analysis of lymphoid progenitors revealed significant reduction in LSK cells in bone marrow and ETP cells in thymus. Using complementarity determining region 3 (CDR3) length polymorphism analysis we next demonstrated that absence of MC4R causes a severe contraction of peripheral T cell receptor diversity suggesting reduced immune‐surveillance. Collectively, these data demonstrates a novel role of melanocortin ‐MC4R pathway in coupling metabolic and immune system. In addition, our findings suggest that specific Mc4R agonists may serve as an important target for rejuvenating thymic function.
Microbial Induction of T Cell Areas in the Rabbit Appendix
Authors: Lanning Dennis Keith , Hanson Nicholas Buck
Published: June 2021

The rabbit appendix contains multiple B cell follicles separated at their sub‐apical regions by well‐defined T cell areas. We found, by RNA in situ hybridization, that the T cell chemoattractant SLC (secondary lymphoid tissue chemokine, or CCL21) is highly expressed in the T cell areas of adult rabbit appendix. We unexpectedly also found, however, that the localized expression of SLC differs dramatically in early neonatal appendix. SLC was first expressed about two days after birth in a continuous layer beneath the B cell clusters. As B cells accumulated and began proliferating over subsequent days, developing follicles grew down through the layer of SLC expression, ultimately generating the characteristic organization of B and T cell areas seen in adult appendix. Furthermore, by introducing bacterial isolates into appendices rendered germfree through ligation at birth, we found that T cell area development is dependent on intestinal commensals, and that individual commensals differed in their ability to induce T cell areas. Microbial induction of T cell areas in the rabbit appendix might be important for supporting B cell proliferation and V(D)J gene diversification in appendix follicles, which generate the primary antibody repertoire. This work was supported by NIH grant 1 RO1 AI49458‐01A1.
The kinase activity of ITK is not required for iNKT cell development and function
Authors: Qi Qian , August Avery
Published: June 2021

Invariant natural killer T (iNKT) cells are a unique subset of innate T lymphocytes that are selected by CD1d. They have diverse immune regulatory functions via the rapid production of interferon‐gamma (IFN‐gamma) and interleukin‐4 (IL‐4). In the absence of ITK, a Tec family non‐receptor tyrosine kinase, mice exhibit a significant defect in iNKT cell development, and residual iNKT cells have defective cytokine secretion. Although the kinase activity is important for ITK, several studies have shown that ITK can activate pathways that are independent of its kinase activity. Whether the requirement for ITK in iNKT cells development and function is dependent on its kinase activity is not clear. We report here that the kinase activity of ITK is not fully required for the mature and activation of iNKT cells. In mice which carry a kinase‐domain deleted ITK in the ITK null background (ItkΔKinTg/Itk−/ −), we observe significant rescue of mature iNKT cells in the thymus and spleen compared with ITK null mice. Furthermore, unlike ITK−/ − iNKT cells, iNKT cells from ItkΔKinTg/Itk−/ − mice were able to secrete IFN‐gamma and IL‐4 upon exposure to α‐GalCer. These data indicate that ITK uses a scaffolding function in the signaling pathway leading to the maturation and activation of iNKT cells.
Signal via IL‐12Rβ2 Negatively Controls Intrathymic Maturation
Authors: Zhang Guang‐Xian , Yu Shuo , Gran Bruno , Rostami Abdolmohamad
Published: June 2021

Mice lacking IL‐12 and its responsiveness exhibited more severe inflammatory responses and were more susceptible to autoimmune diseases than wild type mice, yet the mechanism underlying this phenomenon is not clear. An increased number of CD4+ T cells has been observed in the periphery of IL‐12Rβ2−/− mice compared to wild type mice, indicating that IL‐12 responsiveness is involved in either decreased T cell death and/or increased T cell generation. We report here that thymocytes in mice deficient in IL‐12Rβ2, thus lacking IL‐12 responsiveness, undergo dramatically accelerated maturation. This mainly occurs at the stage from double positive (DP) to single positive (SP), resulting in higher numbers of CD4 and CD8 SP cells in these mice. No difference was observed in percentage and absolute number of apoptotic cells between the two mouse trains. Accelerated maturation also occurs at the stage from double negative (DN) to DP, given the significantly lower CD24 and higher TCRαβ and IL‐7Rα expression in these subsets of IL‐12Rβ2‐deficient mice. This occurs not only in mice injected with anti‐CD3 to mimic pre‐T cell receptor stimulation, but also in mice immunized with autoantigen to induce EAE. Thus, signals through IL‐12Rβ2 negatively regulate thymocyte maturation and play a crucial immunoregulatory role in controlling autoimmunity.
An assay for biotinylated‐erythropoietin (bio‐EPO) suitable for application in tracer pharmacokinetic studies of erythropoietin (EPO)
Authors: Forestiere Matthew J , Bogusiewicz Anna , Mock Nell I , Veng‐Pedersen Peter , Widness John A , Mock Donald M
Published: June 2021

Although low birth weight infants often receive EPO therapy for anemia, optimum pharmacokinetic and pharmacodynamic properties of EPO under various physiological conditions have not been determined for this group. Here we describe the development and validation of an assay capable of detecting tracer concentrations of bio‐EPO. This sequential, solid‐phase assay used streptavidin‐coated 96‐well plates to capture bio‐EPO. Captured bio‐EPO was specifically detected using europium‐filled nanoparticles coated with covalently bound monoclonal antibody to EPO. Total nanoparticle binding to the well was quantitated by time‐resolved fluorescence (TRF). The TRF signal was linear with concentrations of bio‐EPO from 0.5 to 5 fmol per well; correlation coefficient = 0.93 and the sensitivity was 1.5 fmol per well (defined by signal/noise = 2). These observations provide evidence that this assay for bio‐EPO is accurate and sufficiently sensitive to allow tracer studies with normal endogenous concentrations of EPO, which are ≅ 2 fmol/mL. If this assay can be validated in serum, in vivo pharmacokinetic and pharmacodynamic studies using bio‐EPO as a tracer should be possible in populations in which radiolabeled tracer EPO is ethically precluded. Funded by NIH Program Project Grant HD 046925.
Regulation of B cell fate specification and VH gene rearrangements by Ikaros
Authors: REYNAUD Damien , REDDY Karen , SCHJERVEN Hilde , SPOONER Chauncey , BERTOLINO Eric , SMALE Stephen T , WINANDY Susan , SINGH Harinder
Published: June 2021

Genetic analyses have established that the transcription factor Ikaros plays a critical role at the earliest step in B lymphopoiesis. We show that EBF expression in Ik−/− hematopoietic progenitors can rescue B cell development in vitro. The Ik−/− EBF‐rescued B cells are IL7Rα+, CD43+ and CD19+ and have turned on the expression of various early B cell genes including mb1, B29, λ5, VpreB, Pax5 and Aiolos. However, despite normal expression of Pax5, these cells fail to properly repress the expression of Csf1r gene and as a result can differentiate into macrophages in the presence of M‐CSF. Moreover, Ik−/− pro‐B cells, in contrast to their wild type counterparts, express very low levels of the Rag and TdT genes and fail to undergo V‐to‐DJ rearrangements in the immunoglobulin heavy‐chain loci. We establish that Ikaros is a direct activator of the Rag locus in B‐lineage cells. Finally, we show that Ikaros is required for the proper rearrangement of the distal VH genes and controls both the accessibility of these genes and the compaction of the IgH locus. Collectively, our data reveals two unexpected functions of Ikaros proteins during early B cell differentiation: (i) as repressors of the myeloid program independently of Pax5 and (ii) as activators of Rag gene expression and V‐to‐DJ rearrangements.
Human red blood cells (RBCs) labeled at discrete 5 densities can be used to independently determine blood volume in vitro
Authors: Matthews‐Mock Nell I. , Widness John A. , Schmidt Robert L. , Mock Donald M.
Published: June 2021

We have previously shown that blood volume can be independently and simultaneously measured in vivo using 2 discrete densities of biotinylated RBCs (2004, Mock, et al. Transfusion 44: 431). We further have shown that we can label RBCs at up to 5 discrete densities (2005, Mock and Mock, FASEB J 19:A260). Here we sought to determine whether human RBCs biotinylated at 5 densities can be used to independently determine blood volume in vitro. We used the biotinylation reagent s‐NHS‐biotin in increasing amounts from 5.8 to 634 μg/mL of RBC to biotinylate RBC surface proteins; each population of RBCs was enumerated by flow cytometry after complexing with fluorescein‐labeled avidin. Mean error in blood volume was 4%, SD = ±7%. These data provide evidence that blood volume can be accurately measured simultaneously and independently using up to 5 biotin densities. We speculate that different densities of biotinylated RBCs can be used to determine human blood volume in vivo at intervals over time. Further, we speculate that discrete populations of labeled RBCs can be used to determine survival of multiple populations of transfused RBCs. This work was supported by NIH Program Project Grant HD 046925.
Regulation of myeloproliferation and M2 macrophage programming by Lyn/Hck, SHIP and Stat5
Authors: Xiao Wenbin , Hong Hong , Kawakami Yuko , Lowell Clifford A. , Kawakami Toshiaki
Published: June 2021

Homeostasis of hematopoietic stem cells (HSCs) is finely regulated by extrinsic and intrinsic factors via various signaling pathways that control their proliferation, survival, and differentiation. Here we showed that loss of two Src family kinases, Lyn and Hck, or SH2‐containing inositol 5′‐phosphatase (SHIP) resulted in accelerated cell cycling, increased proliferation, reduced apoptosis, growth factor‐independent colony formation, and myeloid and M2 macrophage‐skewed differentiation of HSCs, eventually leading to unlimited myeloproliferation by activating Stat5 at least in part through secreted IL‐3 and GM‐CSF. The clinical features of these knockout mice are a myeloproferative disease and M2 macrophages‐associated lung inflammation, both originating from long‐term HSCs. Expression of a membrane‐bound form of SHIP in Lyn/Hck deficient HSCs restored the homeostasis of HSCs and prevented myeloproliferation and lung inflammation. Thus this study has defined a myeloproliferative transformation‐sensitive signaling pathway, composed of Lyn/Hck, SHIP, autocrine/paracrine cytokines, and Stat5, that regulates the homeostasis of HSCs and M2 macrophage programming.
Study of the Role of the HSC Niche in HSC Migration from Fetal Liver to Fetal Bone Marrow
Authors: Ciriza Jesús , Hall Dominique , Wang Shelley , Carroll Tania , García‐Ojeda Marcos
Published: June 2021

Hematopoietic stem cells (HSC) play an important role in the production of blood cells. During embryogenesis, HSC migrates through a well‐established pathway along different organs: from the AGM to the Fetal Liver, then to the Fetal Spleen, to finalize their migration at the Fetal Bone Marrow. HSC from the AGM region colonize the Fetal Liver (FL) by day 11.5 of gestation. By day 17.5, these cells have colonized the Fetal Bone Marrow (FBM) where blood cells will be generated throughout the life of the organism. In order for this migration to occur, signals from chemokines, the calcium sensing receptor (CaR) and adhesion molecules guide the HSC to fetal BM niches. We have tested the expression of several genes involved in migration and adhesion of HSC to elucidate the molecular control of migration from the fetal liver to the fetal bone marrow. This expression has been studied by real time PCR analysis from single cells isolated by FACS from fetal organs at different stages of gestation. Furthermore, we will use confocal microscopy to study the architecture of the fetal HSC niche as well as determine the expression of molecules involved in adhesion and migration. These results will help to further understand the mechanism regulating the emigration of HSC from the FL and colonization of the FBM. This research has been funded by UC Merced startup funds.