bluuebunny/crossref_metadata_2025 huggingface dataset:
Gga/Clathrin‐Dependent Transport from the TGN to the Late Endosome/Prevacuolar Compartment
Authors: Fuller Robert S. , Abazeed Mohamed E. , Hoppe Adam D. , Zhang Xuefeng , Kumar Anuj
Published: June 2021

Localization of membrane proteins (Kex2p, Vps10p) to the trans Golgi network (TGN) in yeast requires vesicular transport between the TGN and endosomes. While Vps10p follows a direct pathway between the TGN and the late endosome/prevacuolar compartment (PVC), Kex2p partitions between this pathway and an indirect pathway through the early endosome. Using cell‐free reconstitution of TGN‐PVC transport [Abazeed, M.E., et al. (2005) J. Biol. Chem., 280:4442–50], we now find that the direct pathway requires the Gga1/2 clathrin adapators, in addition to clathrin, the dynamin homolog Vps1p, the Vps21p GTPase, the SM protein Vps45p and the PVC syntaxin Pep12p, but is independent of AP‐1 2‐hybrid analysis identifies sites in the Kex2p and Vps10p C‐tails that bind the Gga2p VHS domain. Mutation of the Gga binding site in Kex2p alters Kex2p localization as judged by a sensitive mating assay. To study TGN‐PVC transport in vivo, we are employing quantitative fluorescence resonance energy transfer (FRET) microscopy using donor and acceptor C‐terminal fluorescent protein fusions created in haploids by integrative recombination of Venus YFP and mCherry RFP. Microscopic analysis utilizes “Fast 4D” FRET stoichiometry combined with algorithms for mathematical elimination of autofluorescence. Using this method we have visualized the real‐time interaction between Gga2p and the Arf1 GTPase. Support was from NIH RO1 GM50957.
Regulation of Cytokinesis by GTPase Flux
Authors: Miller Ann Louise , Bement William
Published: June 2021

How do microtubules of the mitotic spindle specify the actomyosin‐based contractile ring during cytokinesis? Prior to contractile ring assembly, spindle microtubules precisely localize proteins that regulate the small GTPase Rho to the cell equator. These Rho regulators then direct formation of a zone of high Rho activity, which promotes actomyosin contractility. We are examining the roles of the Rho regulator MgcRacGAP during cytokinesis in Xenopus embryos. We find that MgcRacGAP is required for cytokinesis, as knock‐down of MgcRacGAP leads to multinucleate cells. This multinucleate phenotype can be rescued by re‐expressing wild type MgcRacGAP but cannot be rescued fully by either of two GAP‐DEAD MgcRacGAP mutants. Expression of the GAP‐DEAD mutants leads to Rho activity zones that are broad and spatially unstable compared to controls, suggesting that GAP activity is necessary to maintain a focused Rho activity zone. Additionally, disruption of MgcRacGAP's GAP activity promotes increased accumulation of downstream targets of Rho at the furrow. We propose that during cytokinesis the Rho activator Ect2 locally activates Rho, while MgcRacGAP locally inactivates Rho, resulting in the constant flux of Rho through the GTPase cycle. By using the GTPase flux mechanism, cells can maintain a tightly focused zone of Rho activity, which is necessary to drive contractile ring formation.

Funding: American Cancer Society and Helen Hay Whitney Postdoctoral Fellowships to ALM. NIH grant to WMB.
Methionine synthase reductase undergoes phosphate‐sensitive loss of flavin mononucleotide upon dilution in vitro
Authors: Elmore Calvin Lee
Published: June 2021

Hyperhomocyst(e)inemia is a metabolic derangement that is linked to the distribution of folate pools, which provide one‐carbon units for biosynthesis of purines and thymidylate and for remethylation of homocysteine to form methionine. In humans, methionine synthase deficiency results in the accumulation of methyltetrahydrofolate at the expense of folate derivatives required for purine and thymidylate biosynthesis. Methionine synthase reductase (MSR) is absolutely required for methionine synthase activity in vivo. MSR is a dual flavoprotein, containing one mol of FAD and one mol of FMN per mol of enzyme. While free FMN is highly fluorescent, enzyme‐bound FMN fluorescence is quenched. Flavin mononucleotide (FMN) binding in MSR is weak compared to other dual flavin‐containing enzymes (Kd = ~85 μM; 0 mM Pi), especially in the presence of phosphate (Kd = ~450 μM; 50 mM Pi) at constant ionic strength. Release of FMN from MSR is associated with loss of enzyme activity, as determined by cytochrome c reductase assay, while FMN‐reconstituted enzyme regains all activity. We found that the presence of 50 mM phosphate increased the overall loss of reductase activity upon dilution by ~50% and decreased the rate of rebinding of FMN ~5‐fold compared to phosphate free buffer. These findings point to a mechanism by which MSR may be inactivated under conditions that limit the availability of FMN in vivo in a phosphate sensitive manner.
Structure Function Studies of “CGxxxM” NADH Binding Motif of Cytochrome b5 Reductase
Authors: Roma Glenn William , Melichar Jamie , Barber Michael
Published: June 2021

Rat hepatic cytochrome b5 reductase (cb5r), a member of the ferredoxin:NADP+ reductase (FNR) family of flavoprotein transhydrogenases, catalyzes the NADH‐dependent reduction of cytochrome b5 (cb5r). Using site directed mutagenesis, the roles of three residues comprising a conserved pyridine nucleotide‐binding motif, “CGxxxM”, present in FNR family members, were probed. Alanine screening suggested residues C273 and G274 play a crucial role in conserving WT functionality. Thus, four more variants, C273S, C273M, G274P, and G274S, were analyzed. Additionally, two insertion mutants, CGPPPaM and CGPPPgM, were generated to further probe the role of M278. All cb5r variants were constructed, produced in E. coli and purified to homogeneity. Compared to the wild‐type (WT) cb5r, all mutants were efficiently expressed, showed the appropriate molecular masses. Absorption and CD spectra were comparable to the wild type rat cb5r diaphorase domain, suggesting similar protein folding and flavin incorporation. In contrast, all mutants exhibited decreased NADH:ferrricyanide reductase (NADH:FR) activities with the greatest impairment shown in the C273 and G274 variants and the two insertion mutants. These results confirm that the conserved C273, G274 and M278 residues play important roles in both modulating the protein's affinity for NADH and regulating its subsequent rate of oxidation.
Characterization of the human Ki67 promoter for high‐throughput functional genomic screens of G1‐S phase cell‐cycle regulation
Authors: Zambon Alexander C.
Published: June 2021

Recent studies have shown how a gene's promoter can be used to drive expression of a selectable marker for functional genomic screens to identify cDNAs that can activate transcription of a target gene [Takahashi et. al, Cell, 2006 126: 1–14]. Our goal was to identify a promoter that was specifically active as cells enter the cell cycle, but that was inactive in arrested or quiescent (G0) cells. We chose the human Ki67 gene because it is a commonly used marker for cellular proliferation and used PCR to clone the Ki67 1.5 kb proximal promoter (−1233–+291). Comparative genomic analysis of this fragment reveals several highly conserved E2F transcription factor binding sites, suggesting that it is under control of E2F transcription factors, which are known to regulate the G1‐S phase of the cell cycle. Expression of GFP driven by the 1.5 kb Ki67 promoter results in cellular colocalization of GFP with endogenous Ki67 protein in HT1080 cells. Furthermore Ki67 promoter activity is attenuated by serum starvation or treatment with mitomycin C, evidence in support of its specificity for proliferating cells. In conclusion, the Ki67 promoter provides a useful molecular tool that could have a variety of applications, including rapid functional genomics screens that seek to identify novel oncogenes and tumor suppressors as well as for in vivo identification of cellular proliferation in transgenic animals (Supported by UCSD IRACDA training fellowship).
Black tea polyphenols suppress hepatic lipid accumulation through activating LKB1‐AMPK pathway in human HepG2 cancer cells
Authors: Lin Jen‐Kun , Lin Chih‐Li , Huang Hsiu‐Chen , Lin‐Shiau Shoei‐Yn
Published: June 2021

Tea is one of the world most popular beverage and approximate 80% of global harvested tea have been consumed as black tea. The anti‐obesity and hypolipidemic effects of black tea have been intensively investigated, but the mechanism underlying these phenomena remain unclear. In the present study,the black tea polyphenols theaflavins were assessed for their hepatic lipid–lowering potential when administered in fatty acid overload conditions both in cell culture and in an animal experimental model. We found that theaflavins significantly reduced lipid accumulation suppressed fatty acid synthase and stimulated fatty acid oxidation. Furthermore, theaflavins also inhibited acetyl‐coenzyme A carboxylase activities by stimulating AMP‐activated protein kinase (AMPK) through the LKB1 and reactive oxygen species pathways. These observations support the idea that AMPK is a critical component of decreased hepatic lipid accumulation by thaflavin treatments. Our results show that theaflavins are bioavailable both in vitro and in vivo and may be active in the prevention of fatty liver and obesity. (This study was supported by the research grants from National Science Council, NSC95‐2320‐B002‐111 and NSC96‐2311‐B‐002‐020).
Role of CCTα in B‐lymphocyte Development from Hematopoietic Stem Cell to Plasma Cell
Authors: Fagone Paolo , Jackowski Suzanne
Published: June 2021

B‐lymphocytes, or B‐cells, originate in the bone marrow and mature in the spleen and lymph nodes. Upon activation with ligand, mature B‐cells proliferate and differentiate either into memory B‐cells or immunoglobulin (Ig)‐secreting plasma cells. Plasma cells are distinct from mature or memory B‐cells and possess a more extensive endoplasmic reticulum (ER) and Golgi compartment to sustain high‐capacity Ig synthesis and secretion. Phosphatidylcholine (PtdCho) is the major lipid component of B‐cell membranes and the CDP‐choline pathway is the major source of PtdCho during B‐cell differentiation after LPS stimulation. Choline cytidylyltransferase (CCT) activity regulates the rate of PtdCho synthesis through the CDP‐choline pathway in B‐cells. Mice were derived which lacked B‐cell expression of CCTα the predominant isoform, to define the role(s) of this protein in B‐cell development and function. Knockout (KO) and wild‐type (WT) mice showed no difference in spleen structure or the total number of splenic B‐cells as determined by immunohistochemistry and flow cytometry. However, KO mice lacked the subpopulation of marginal zone B‐cells and had lower basal serum IgG levels, whereas IgM levels were comparable to genetically matched WT mice. Immunization with a T‐cell dependent antigen in vivo should elicit an IgG response, but KO mice were unable to switch from IgM to IgG production and had elevated levels of IgM. B‐cells lacking CCTα expression had intact signaling responses to stimulation of the B‐cell receptor with anti‐IgM, but were unable to synthesize DNA or proliferate following stimulation with LPS or anti‐IgM in vitro, in contrast to WT cells. Our working model proposes that the requirement for PtdCho synthesis for the combined tasks of proliferation, clonal expansion and ER biogenesis cannot be met by the reduced CCT activity in the KO B‐cells. (Supported by NIH GM45737 and ALSAC.)
Intracellular HIV‐Nef Downregulates LPS‐induced IL‐12p40 Expression by Inhibiting JNK‐activated NFkB in Human Monocytic Cells
Authors: Ma Wei , Mishra Sasmita , Mishra Jyoti P , Angel Jonathan B , Kumar Ashok
Published: June 2021

Impaired cellular immunity caused by decreased production of Th1 type cytokines including IL‐12 is a major feature of HIV‐associated immunodeficiency and AIDS. IL‐12p40, an inducible subunit shared between IL‐12 and IL‐23, plays a critical role in the development of cellular immunity and its production is significantly decreased during HIV infection. The mechanism by which HIV induces loss of IL‐12p40 production remains poorly understood. We have previously shown that LPS‐induced IL‐12p40 production in monocytic cells is regulated by NFκB and AP‐1 through the activation of JNK and CaMK‐II. Herein, we show that intracellular Nef expressed through transduction of monocytic cells with retroviral‐mediated Nef gene inhibited LPS‐induced IL‐12p40 transcription by selectively inhibiting the JNK pathway without affecting the CaMK‐II‐activated pathway. In addition, Nef inhibited JNK‐activated NFκB without affecting the AP‐1 activity. Overall, our results suggest for the first time that intracellular Nef inhibited LPS‐activated JNK which may cause inhibition of IL‐12p40 expression in human monocytic cells by selectively inhibiting NFκB without affecting the CaMK‐II‐activated pathway. Furthermore, the inhibitory effect of Nef on JNK may have broad implications with respect to the inhibition of Th1 responses. The research was supported by NSERC, OGS, CIHR, and OHTN, Canada.
Regulation of Mitogen‐Activated Protein Kinase and Down‐Stream Signaling by Guanylyl Cyclase‐A/Natriuretic Peptide Receptor‐A
Authors: Tripathi Satyabha , Kimonis Katerina O. , Vellaichamy Elangovan , Pandey Kailash N.
Published: June 2021

Atrial natriuretic peptide (ANP) is a member of the natriuretic peptide hormone family that maintains cardiovascular homeostasis and exerts antiproliferative effects in a variety of cell types. ANP mediates its effects by binding guanylyl cyclase‐A/natriuretic peptide receptor‐A (GC‐A/NPRA) and subsequently activating cGMP‐dependent protein kinase (PKG), but the signaling pathways regulated by this system have not yet been elucidated. The objective of this study was to determine the effects of ANP‐NPRA system on mitogen‐activated protein kinases (MAPKs) and proliferative transcription factors AP‐1 and CREB in murine Leydig tumor (MA‐10) cells. Measurement of phorphorylated protein levels by Western blot demonstrated that vascular endothelial growth factor (VEGF) treatment caused a 3‐ to 4‐fold increase in phosphorylation of MAPKs (Erk1, Erk2, JNK, and p38). ANP treatment inhibited VEGF‐stimulated phosphorylation of MAPKs by 50–65%. VEGF treatment caused a 3‐ to 5‐fold increase in c‐jun, c‐fos, and CREB protein levels, an effect which was significantly inhibited by ANP treatment. ANP treatment inhibited VEGF‐stimulated AP‐1 and CREB transcriptional activity by 60–70% in a PKG‐dependent manner. Thus, the present study delineates that ANP/NPRA signaling can exert an inhibitory effect on the proliferative cellular mechanisms that play a critical role in angiogenic response in disease states.
Expression and characterization of human membrane progestin receptors in Saccharomyces cerevisiae
Authors: Smith Jessica L , Garitaonandia Ibon , Kupchak Brian , Lyons Thomas
Published: June 2021

Recent studies indicate that a few members of the Progestin and AdipoQ Receptor (PAQR) family (PAQR5, PAQR7, and PAQR8) act as membrane progestin receptors (mPRs) and signal in a G‐protein dependent manner, but these studies have been disputed. To clarify the role of human mPRs in progestin signaling, we are using the yeast Saccharomyces cerevisiae as a simple model organism. When mPRs are expressed at low levels, we observe repression of the yeast promoter FET3 in a FET3‐lacZ promoter‐reporter assay only when certain progestins are present. To explore the molecular mechanism by which mPRs affect FET3 promoter activity, we used genetic mutational analyses in combination with our FET3 activity assay. We show that the progestin‐mediated effect of mPRs on FET3‐lacZ is not dependent on the presence of human or yeast G‐proteins. Thus, we present evidence that human mPRs are able to signal in a G‐protein independent manner. We also show that all human PAQRs and their yeast homologues share a common intracellular signaling mechanism in yeast involving tpk2, the yeast homologue of human PKA. In addition, we provide evidence that human PAQR6 and PAQR9 act as progestin receptors, which confirms functional predictions based on bioinformatic sequence analyses. Funding for this study was provided by the National Institutes of Health and by the University of Florida Department of Chemistry.
Gαz negatively regulates insulin secretion and glucose clearance
Authors: Kimple Michelle E. , Joseph Jamie W , Bailey Candice L. , Fueger Patrick T. , Hendry Ian A. , Newgard Christopher B. , Casey Patrick J.
Published: June 2021

Relatively little is known about the in vivo functions of the alpha subunit of the heterotrimeric G protein Gz (Gαz). Clues to one potential function recently emerged with the finding that activation of Gαz inhibits glucose‐stimulated insulin secretion (GSIS) in an insulinoma cell line [Kimple et al. (2005) J Biol Chem 280:31708]. To extend this study in vivo, a Gαz knock‐out mouse model was utilized. No differences were discovered in the islet physiology or morphology or the insulin sensitivity of Gαz‐null mice and wild‐type controls. Gαz‐null mice did, however, display increased plasma insulin concentrations and glucose clearance following glucose challenge as compared to wild‐type controls. The increased plasma insulin observed in Gαz‐null mice is likely a direct result of enhanced insulin secretion, as pancreatic islets isolated from Gαz‐null mice exhibited significantly higher GSIS than those of wild‐type mice. Finally, the increased insulin secretion observed in Gαz‐null islets appears to be due to the relief of a tonic inhibition of adenylyl cyclase, as cAMP production was significantly increased in Gαz‐null islets in the absence of exogenous stimulation. These findings indicate that Gαz may be a potential new target for therapeutics aimed at ameliorating β‐cell dysfunction in Type 2 diabetes. This work was supported by NIH grants DK67799 to M.E.K, DK42583 to C.B.N., and GM55717 to P.J.C.
Apoptosis of Breast Cancer Cells by L‐PPMP and cis‐Platin: Translational and Transcriptional Regulation of Glycosyltransferase Genes
Authors: Ma Rui , Hopp Elizabeth E , Decker N. Matthew , Loucks Audrey J. , Thomas Christopher , Banerjee Sipra , Moskal Joseph , Basu Subhash
Published: June 2021

Apoptotic effects of anti‐cancer agents (glycolipid biosynthesis inhibitor L‐PPMP, DNA biosynthesis inhibitor cis‐Platin) in human breast cancer cells (MCF‐7, MDA‐468, and SKBR‐3) were identified with techniques including phase contrast microscopy, DNA laddering, and the flopping of PS with PSS‐380 fluorescent dye studies. Using a new fluorescent Dye (AKS‐0) in the presence of these two apoptotic reagents, we observed a time‐dependent scrambling of membranes of mitochondria, ER, and Golgi bodies. SA‐LeX (neolacto‐series glycolipids) is distributed mostly on the outer lamella of plasma membranes of human breast and colon metastatic cells. Although exact functions of these glycoconjugates are not yet known, direct analyses of these glyco‐molecules suggest they alter their structures during the onset of oncogenic processes. A time‐dependent down‐regulation of glycosyltransferase (GLTs) activities involved in the biosynthesis of SA‐LeX [GalT‐4, GalT‐5, and FucT‐3] and gangliosides [SAT‐4 and SAT‐2] was observed. By using novel DNA‐microarrays specifically designed for screening 359 Glyco‐related genes, transcriptional up‐regulation of several GLTs involved in the biosyntheses of Sialo‐LeX and Sialo‐Lea (cancer cell surface antigens) was observed. Down‐regulation of GLT activities and up‐regulation of some GLT mRNA suggest a tight regulation of these enzymes by signal transduction pathways.
The effects of pro‐apoptotic DNA vaccinations on Dendritic Cells
Authors: Perez Jessica Marie , Li Alice , Escher Alan
Published: June 2021

Pro‐apoptotic DNA vaccines specifically pND2‐SGAD55‐BAX, have been found to suppress diabetes and insulitis in non‐obese diabetic (NOD) mice, ameliorate new‐onset diabetes, and induce foxp3 regulatory T cells in pre‐diabetic mice with early diabetes diagnosis.(1,2)The effects of the apoptotic DNA vaccines are now being studied. This study focuses on the effects of apoptotic DNA vaccines on Dendritic cells (DCs). This study will focus on effects of the vaccines on DCs gene expression level of mRNA. Two methods of transfection were used to introduce three different plasmid constructs, BAX, GAD65‐BAX, and SGAD55‐BAX. These methods were lipofection and electroporation. The results of the Real Time Polymerase Chain Reaction (RT‐PCR) were used to measure the mRNA expression level of seven different genes including: CD40, CD80, CD86, TGFβ1, FCγIIB, IL‐1RA, and GAPDH, as house‐keeping gene control. Results showed that the delivery of SGAD55‐BAX induced significant changes in gene expression in DCs when compared to GAD65‐BAX. DCs transfected via lipofection with the SGAD55‐BAX caused decreased expression of CD80, CD86, TGFβ1, FCγIIB, and IL‐1RA compared to the delivery of BAX alone. However delivery of the same plasmid via electroporation showed increased expression of the same genes compared to BAX alone. These results may be due to different efficiencies of transfection of plasmid DNA or due to the capacity of DCs to respond rapidly to different stimuli.

National Institute of Health‐National Center On Minority Health and Health Disparities through grant 5P20MD001632 (LLU Center for Health Disparities Research)
Alpha‐tocopherol induces procaspase‐3 expression mediated by the transcriptional factor Sp1
Authors: Miyoshi Noriyuki , Nakamura Yoshimasa , Ohshima Hiroshi
Published: June 2021

Alternative roles of alpha‐tocopherol have been proposed that are independent of its antioxidant/radical scavenging ability. In this study, we reveal that alpha‐tocopherol enhances cellular susceptibility to both oxidative and non‐oxidative apoptosis‐inducing stimuli through up‐regulation of procaspase‐3 expression in several human cell lines. Exposure of alpha‐tocopherol pretreated cells to the Fas, hydrogen peroxide, or etoposide, resulted in an increase of apoptotic cells. In addition, alpha‐tocopherol elevated the procaspase‐3 protein level and mRNA expression in a time‐ and dose‐dependent manner, while other tocopherol analogues showed no effect. Experiments using a GC‐specific DNA binding agent, mithramycin A, and an electrophoretic mobility shift assay demonstrated that Sp1 might mediate the enhanced expression of procaspase‐3. However, although we detect the increasing level of intracellular tyrosine phosphorylation after the treatment with alpha‐tocopherol, protein kinase C (PKC) inhibitor did not show any effect on alpha‐tocopherol induced procaspase‐3 expression. Additionally, our results confirmed that alpha‐tocopherol promotes the expression, but not the activation, of caspase‐3 in various human cell lines. These findings provide biological evidence showing that alpha‐tocopherol can amplify the apoptotic response by up‐regulating the expression of pro‐caspase‐3.
Investigation of apoptotic related genes in SEB induced human PBMC module
Authors: Mendis Chanaka , Jett Marti
Published: June 2021

Our study is to characterize a set of apoptotic related genes that are key in the pathogenesis of Staphylococcal Enterotoxin B (SEB) induced lethal shock in human peripheral blood mononuclear cells (PBMC) and then utilize the gene expression pattern to further investigate the effectiveness of potential signal blockers. By using a set of genes previously identified through differential display (DD) and micro‐array techniques we carried out a conformational study of SEB induced apoptotic events in the following manner. First, we carried‐out an extensive literature search to characterize apoptosis related genes identified by DD and micro‐arrays. Second, we designed primers using stringent parameters, third we confirmed the gene expression pattern using RT‐PCR and then validated the gene expression patterns by further investigation of protein expression using ELISA assays and finally utilized the known gene expression patterns to evaluate the efficacy of potential signal blockers. We were able to confirm the previously observed gene expression patterns of heparanase precursor, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Ubiquitin specific protease and SOD genes through RT‐PCR, protein expression pattern of a set of caspases through ELISA assays and correlated the functions of genes to SEB induced apoptosis related events. Our study also confirmed the effectiveness of the inhibitors as potential target to block SEB induced apoptotic events. We believe that our investigation may reveal crucial information to better understand the mechanisms as well as the regulation/inhibition of SEB induced apoptotic events.
Leptomycin B Inhibits NFκB Activity in Stimulated Monocytic Cells by Increasing Nuclear Accumulation of IkBa
Authors: Ghosh Chandra Chur , Ramaswami Sitharam , Vu Hai‐Yen , Juvekar Ashish P , Vancurova Ivana
Published: June 2021

Monocytic cells are the main producers of the pro‐inflammatory cytokine tumor necrosis factor alpha (TNFα). Synthesis of TNFα is regulated by the transcription factor NFκB, which is critically involved in numerous inflammatory disorders. We have previously shown that leptomycin B, an inhibitor of CRM1‐dependent nuclear export, inhibits the release of TNFα from stimulated monocytic U‐937 cells by inhibiting activity of NFκB. In this study, we investigated the mechanism by which LMB inhibits the NFκB activity in U‐937 cells stimulated with lipopolysaccharide (LPS), and peptidoglycan (PGN). We show that while LMB does not inhibit degradation of the NFκB inhibitor, IκBα, it induces IκBα nuclear accumulation in LPS and PGN‐stimulated monocytic cells, thus inhibiting NFκB DNA binding. Our results suggest that the increased nuclear accumulation of IκBα could be used as a therapeutic target for disorders characterized by high levels of NFκB activity and TNFα release.
Determination of the Substrate Envelope for Multidrug‐Resistant HIV‐1 Protease
Authors: Liu Zhigang , Yedidi Ravikiran , Brunzelle Joseph , Kovari Iulia , Kovari Ladislau
Published: June 2021

Objectives: Determine the substrate envelope for MDR HIV‐1 protease to guide antiviral drug design.

Background: HIV‐1 protease, required for HIV replication, is a critical therapeutic target in the treatment of AIDS. Under drug selection pressure, HIV‐1 protease mutations give raise to multiple drug resistance. Crystallographic studies on substrate complexes of wide‐type HIV‐1 protease revealed a consensus substrate‐shape which is defined as the ‘substrate‐envelope’. Many drug resistant mutations occur where inhibitors protrude from this substrate‐envelope and may predict potential resistance.

Methods and Results: We focus on MDR HIV‐1 protease to propose atomic details of drug resistance and the drug design of novel inhibitors. We co‐crystallized HIV‐1 protease with nine natural Gag and Gag‐Pol cleavage site peptides and solved the structures of these nine complexes. Based on these structural studies we determined the substrate envelope.

Conclusions: All the nine natural substrate peptides show induced fit to MDR HIV‐1 protease, although the extent of induced fit varies. Furthermore, the positions of the substrate peptides are different from those in wide‐type HIV‐1 protease, which gives a unique substrate envelope of MDR HIV‐1 protease. This project is funded by NIH.
Immunology Investigators: Why the Lyme Disease Vaccine Didn't Work
Authors: Troy Scott Maresh , Torres Raul
Published: June 2021

The goal of this curriculum is to help students solve a fictional medical case. Using the current immunological understanding of vaccination and information concerning the role of Toll‐like receptors (TLRs) in vaccination, students will work collaboratively in groups to analyze a written case study concerning patients who have received a Lyme disease vaccine. Next, students will be provided with the background information of the fictional patients, including medical records, biographical data, and simulated serum samples. Background information concerning Lyme disease and the Lyme disease vaccine (LYMErix) will also be provided. Following an introductory discussion and comparison of these patients, students will conduct simulated ELISA tests on the serum samples of the patients and discover that one of the patients has contracted Lyme disease despite their vaccine. The collaborative student groups will then attempt to explain the surprising test result by learning about the current immunological understanding of vaccination, including the role that TLRs may play in vaccination. Students will conclude through their own research and provided materials that this particular case of reduced responsiveness to vaccination is attributed to a decreased expression of particular TLRs. In particular, the patient who contracted Lyme disease had a decreased expression of particular TLRs and a corresponding decreased recognition of a cell surface protein on the Lyme disease‐causing Borrelia spirochete. Finally, students will apply their new knowledge to current topics in vaccination, including possible connections with the development of an HIV vaccine. The Biological Sciences Curriculum Study (BSCS) 5E instructional model will be used throughout this instructional unit.
Role of Phosphoinositide 3‐kinase in induction and maintenance of B cell self tolerance
Authors: Kitahara Seiji , Suzuki Harumi , Oda Hiroyo , Sakai Kouhei , Tsuchida Masahiro , Koyasu Shigeo , Naito Katsusuke , Shirai Mutsunori
Published: June 2021

Phosphoinositide 3‐kinase (PI3K) plays important roles in BCR‐dependent signal transduction, therefore it is critical in B cell development and activation. B cell tolerance to self antigen is induced and maintained mainly by clonal deletion and peripheral anergy. To assess the function of PI3K in the regulation of B cell self tolerance, mice lacking the gene for p85α (p85α−/−), the most important regulatory subunit of the class IA PI3Ks, were bred with anti‐hen egg lysozyme (HEL) immunoglobulin (HEL‐Ig) and membrane HEL (mHEL) or soluble HEL (sHEL). We found that development of monoclonal anti‐HEL‐Ig B cells was severely impaired in p85α−/− HEL‐Ig mice. Massive deletion of HEL‐specific B cells were observed in HEL‐Ig/mHEL double‐transgenic mice, even in the absence of p85α. Furthermore, anergy induction in HEL‐Ig/sHEL mice, represented by reduced production of anti‐HEL IgM antibody and impaired proliferative responses of B cells was maintained in p85α−/− double‐transgenic (HEL‐Ig/sHEL) mice. These results suggest that PI3K is essential for B cell maturation but not required for clonal deletion and anergy induction of self‐reactive clones.
In‐vitro Effects of the Anti‐Retroviral Protease Inhibitor Lopinavir on CD4+ Lymphocyte Activation
Authors: Ho David Kien , Scott James D. , O'Barr Stephen A.
Published: June 2021

HIV infection is epidemic with over 39.5 million people infected worldwide. Protease inhibitors (PIs) remain the mainstream therapy for the treatment of HIV infection. At the 2006 International AIDS Society conference it was reported that patients who receive PIs as part of their therapy show a trend toward shorter virologic failure times as well as significant increases in CD4+ cell count. Similar results are reported in a case ‐ control study in which significantly increased CD4+ cell counts are measured in patients receiving PIs as part of their Highly Active Anti Retroviral Therapy (HAART) compared to those receiving Non ‐ Nucleoside Reverse Transcriptase Inhibitor (NNRTI) therapy. As the molecular mechanisms explaining these clinical observations are still unknown, basic scientific studies leading to the understanding of how PIs enhance T cell activation would allow for more effective therapeutic management. During a viral infection, T ‐ cells are first activated through the T ‐ cell receptor complex which induces the expression of the cytokine interleukin 2 (IL ‐ 2) and the IL ‐ 2 high affinity receptor, CD25. IL ‐ 2 induces both autocrine and paracrine activation of T ‐ cells, resulting in clonal expansion and differentiation, and ultimate viral clearance. Apart from CD25, several other cell surface markers are upregulated during T ‐ cell activation including the adhesion molecule CD38; the signal transmitting receptor CD69; and the antigen presenting complex, HLA ‐ DR. While previous attempts to show changes in T ‐ cell surface markers due to PI treatment have been unsuccessful, we show for the first time that the PI, lopinavir (lop) increases CD25+ T ‐ cell levels, independent of viral load.
Endogenous galectin‐1 induced by activated CD8 T cells limits CD8 burst size
Authors: Liu Scot Daren , Tomassian Tamar , Bruhn Kevin W , Poirier Francoise , Miceli M Carrie
Published: June 2021

CD8 burst size is regulated by the balanced control of CD8 activation, expansion, and apoptosis. While it is clear the length of antigen exposure and continued and sustained TCR signaling regulate these processes, molecular regulators controlling this balance are not well elucidated. Here, we report that galectin‐1 (gal‐1) deficient CD8 cells hyper‐proliferated upon TCR engagement. Dividing gal‐1 deficient CD8 cells were less susceptible to apoptosis, suggesting gal‐1 promotes apoptosis to limit burst size. Coinciding with induced expression of gal‐1 in CD8 cells within 48 hours of activation, TCR signaling was sustained in gal‐1 deficient CD8 cells over that period, reflected in more pERK and pS59 lck, and less lck‐associated SHP‐1. Consistently, treatment of gal‐1 deficient CD8 cells with recombinant gal‐1 antagonized sustained pERK. These data suggest gal‐1 may regulate burst size through antagonism of continued TCR signaling by modulating the ERK positive/SHP‐1 negative feedback pathways. Adoptive transfer of gal‐1 deficient TCR transgenic cells into wild type hosts indicates autocrine CD8 gal‐1 production is responsible for limiting CD8 expansion in vivo. These findings identify gal‐1 as a novel negative regulator of CD8 burst size, which functions by opposing continued TCR signaling and promoting apoptosis. Supported by NIH RO1A1056155 to M. C. Miceli, T32 AI07323‐15, AI07126‐30, and Warsaw Fellowship to S. D. Liu.
Aging‐Induced NF‐κB‐Dependent Alterations in T Cell Cytokines
Authors: Huang Mei‐Chuan , Liao Jia‐Jun , Bonasera Stephen , Longo Dan L. , Goetzl Edward J.
Published: June 2021

Immunosenescence (IS) encompasses decreases in protective immune responses and increases in inflammation and autoimmunity. To elucidate cytokine components of IS, splenic CD4 T cells from 22 to 24 month‐old (old, O) mice and 6 to 10 week‐old (young, Y) mice were incubated for up to 4 days on adherent anti‐CD3 plus anti‐CD28 Abs with daily quantification of cytokines and their mRNAs. After 96 hr, T cells of O C57BL/6 and CBA mice generated more IL‐17 (up to 20‐fold) and IL‐6 (up to 3‐fold) than those of Y mice, T cells of Y mice generated up to 5‐fold more IL‐21 than those of O mice, and no difference was found for IFN‐γ. At 24 hr, cytokine mRNA levels paralleled 96 hr cytokine concentrations. Naïve (CD62L‐high) CD4 T cells from O mice on Abs with TGF‐β, IL‐1, IL‐6 and IL‐23 expressed more IL‐17 mRNA and IL‐17 at days 3 and 5 than those of Y mice. The nuclear levels of NF‐κB were lower in CD4 T cells of O than Y mice, but further decreases of their NF‐κB by BAY11‐7082 reduced IL‐17 and IL‐6 levels to those of CD4 T cells from Y mice. High reversibility of enhanced generation of T cell cytokines in IS suggests specific therapy for aging‐induced inflammation. (NIH grant RO‐1 HL31809)
CD27 plus CD28 Cross‐linking Induces Phosphorylation of p38 in CD4+CD45RO+ T Cells
Authors: Tamma Seetha M. , Balan S. P. , Akshintala S. Deepika
Published: June 2021

CD27 antigen is a 120 KDa transmembrane protein and a member of nerve growth receptor family. Its expression is predominantly confined to lymphocytes and equally distributed between CD4 and CD8 lymphocytes. It has been reported that CD27 is required for long term maintenance of T cell immunity and was found to rescue

CD28(−/−) T cells from death at the onset of division. We investigated the role of CD27 on resting and memory CD4+ T cells. We isolated human CD4+CD45RA+ and CD4+CDRO+ subsets using Dynal magnetic beads and cultures were set up by cross‐linking with antibodies as indicated: CD3 + CD28; CD27 alone; CD27 + CD3; CD27 + CD28; CD28 alone and control cells. MAPK ERK2 phosphorylation appears to be similar in both RA and RO populations when stimulated with CD27 plus CD28. We report that CD27 plus CD28XL resulted in enhanced and accelerated phosphorylation of p38 MAPK, activation peaked at 10 minutes and decreased by 2 hours in RO+ cells when compared with that in RA+ cells. CD27 on its own induced phosphorylation of ERK2 slightly higher than that in control, where as no phosphorylation of p38 was observed in both populations following cross‐linking with CD27 alone. This data suggests that RA and RO populations respond differently to signals transmitted through coreceptors CD27 plus CD28.

Grant from C.W. Post Campus Research Committee of Long Island University, New York supported this work.
ENDOTHELIAL CELL PRESENTATION OF ANTIGEN INITIATES THE ADAPTIVE‐INNATE IMMUNE INTERACTION SEQUENCES IN T CELL MEDIATED RESPONSES IN THE SKIN
Authors: Kish Danielle D. , Fairchild Robert L.
Published: June 2021

Contact hypersensitivity (CHS) is a CD8 T cell‐mediated response to skin sensitization and challenge with hapten. Hapten‐primed CD8 T cell recruitment into the challenged skin to mediate CHS requires prior CXCL1/KC and CXCL2/MIP‐2 directed neutrophil infiltration. The factors inducing KC and MIP‐2 to challenge were investigated. KC and MIP‐2 were produced 3–6 h after challenge with dinitrofluorobenzene (DNFB) in sensitized, but not unsensitized, mice and were dependent on hapten‐specific CD8 T cells. KC and MIP‐2 production was accompanied by expression of IFN‐g and IL‐17. DNFB sensitization induced hapten‐specific CD8 T cells producing IFN‐g and IL‐17. CHS responses and KC production were at naïve levels in sensitized and challenged IFN‐γ−/− and IL‐17−/− mice. DNFB‐induced KC in naïve mice was restored by co‐transfer of CD8 T cells from sensitized IFN‐γ−/− and IL‐17−/− mice but not each population alone. In vitro, primed CD8 T cells stimulated hapten‐labeled EC to produce KC which was blocked by either anti‐IFN‐γ or anti‐IL‐17 mAb. Addition of rIFN‐γ and rIL‐17 synergized to stimulate EC to produce KC. These results indicate that hapten sensitization induces populations of CD8 T cells producing IFN‐γ and IL‐17. Activation of both T cell populations is required to induce EC in the challenge site to produce KC and MIP‐2 to initiate elicitation of the CHS response.
Osteoclast differentiation through rheumatoid synovial fibroblasts could be regulated by TLR‐3 pathway
Authors: Lee Sang‐Heon , Kim Hae‐Rim , Kim Kyoung‐Woon , Cho Mi‐La , Kim Ho‐Youn
Published: June 2021

This study was undertaken to determine the effect of toll‐like receptor‐3 (TLR‐3) on the regulation of osteoclastogenic activity in rheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLS).

The expression of receptor activator of nuclear factor kappa B ligane (RANKL) mRNA and protein in RA FLS after TLR3 activation were determined using RT‐PCR, real time PCR, western blot and immunostaining. Human monocytes were co‐cultured with RA FLS which were pre‐stimulated by polyI:C, the TLR3 ligand, and subsequently stained for tartate‐resistant acid phosphatase (TRAP) activity. The expression of TLR3 and RANKL was much higher in RA synovium than in OA synovium. TLR3 activation induced RANKL expression in RA FLS, but not in OA FLS or normal skin fibroblasts. TLR3 activation also induced the production of IL‐1beta, but no effect on TNF‐alpha in RA FLS. Inhibition of IL‐1beta reversed the TLR3‐induced upregulation of RANKL expression. When human monocytes were co‐cultured with TLR3‐activated RA FLS, the expression of TRAP, RANK, cathepsin K, calcitonin receptor, and MMP‐9 was upregulated, reflecting the osteoclast differentiation.

Our results suggest that the RA FLS promotes osteoclastogenesis via TLR3 both directly and indirectly by RANKL induction. Targeting between TLR3 pathways and osteoclastogenesis of RA FLS may be a promising approach to prevent joint destruction in RA.
Site‐specific targeting of autoimmunity in mice induced by the localized production of IL‐6
Authors: Campbell Iain Leslie , Quintana Albert , Müller Marcus , Frausto Ricardo , Getts Daniel , King Nicholas J. , Hofer Markus J. , Hidalgo Juan
Published: June 2021

IL‐6 is required for the induction of experimental autoimmunity. The role of IL‐6 in EAE was examined in transgenic mice (GF‐IL6) with cerebellum specific production of IL‐6. MOG‐immunized (Mi‐) GF‐IL6 mice developed severe ataxia which was in contrast to Mi‐wild type (WT) mice that developed classical EAE with hind limb paralysis. Immune pathology and demyelination were markedly decreased in the spinal cord but significantly increased in the cerebellum of Mi‐GF‐IL6 mice. Tissue damage in the cerebellum at peak disease in the Mi‐GF‐IL6 mice was accompanied by increased total numbers of infiltrating leukocytes and increased proportions of both neutrophils and B‐cells. During peak disease, the expression of various cytokines was found at comparable levels in the cerebellum between Mi‐GF‐IL6 and Mi‐WT, whereas significantly higher levels were found in Mi‐WT spinal cord. In conclusion, site‐specific production of IL‐6 targets and modifies the autoreactive inflammatory response and creates a leukocyte “sink” that retards trafficking to normally dominant antigenic sites. The mechanisms underlying this response to IL‐6 likely include the induction of specific chemokines as well as increased activation and loss of integrity of the BBB in the cerebellum of the GF‐IL6 mice observed prior to the induction of EAE. Support:NSW Spinal Cord Injury and Other Neurological Conditions Project Grant.
Pulmonary arterial muscularization induced by a Th2 immune response
Authors: Grunig Gabriele , de Waal Malefyt Rene , Emson Claire , Louten Jennifer , Voelkel Norbert , Guignabert Christophe , Rabinovitch Marlene , Grunig Ekkehard , Daley Eleen
Published: June 2021

Pulmonary hypertension and pulmonary arterial muscularization (PAmusc) are seen in many parasitic and autoimmune diseases and in chronic inflammatory lung diseases. The goal of this study was to determine the relationships between the immune response, PAmusc, and pulmonary hypertension. Mice were immunized intraperitoneally and exposed to inhaled soluble antigen intermittently for a prolonged period of time or acutely. The prolonged antigen exposure induced dramatic PAmusc that was dependent on the presence of CD4 T cells, IL‐4 and IL‐13. PAmusc was characterized by neo‐intima formation, cell proliferation, and the expression of smooth muscle cell actin. Surprisingly, PAmusc was not associated with increased right ventricular pressures at rest or following challenge with acute hypoxia. The possibility that PAmusc does not necessarily cause pulmonary hypertension has been suggested by clinical observations in children that may quickly regain normal pulmonary arterial pressures after surgical repair of cardiac defects despite the presence of muscularized pulmonary arteries. In conclusion, our data are the first to show experimentally that the adaptive immune response can cause PAmusc and that severe PAmusc and pulmonary hypertension can be dissociated.

Supported by: Flight Attendant Medical Research Inst., Stony Wold Herbert Fund, Am. Lung Association of the City of New York
B‐cell activating factor (BAFF) in a model of systemic lupus erythematosis: expression and rabbit homologue identification
Authors: Yang Jiahui , Pospisil Richard , Ray Satyajit , Mage Rose
Published: June 2021

To gain further insight into the contribution of BAFF to B‐cell hyperactivity in systemic lupus erythematosus (SLE), we studied BAFF expression in our noninbred rabbit lupus model (Rai et al. J. Immunol. 179: 660, 2006). We also cloned cDNA encoding the rabbit homolog (rabBAFF) and identified a predicted rabbit BAFF cDNA and genomic exons in the Ensembl database. Before immunization with a peptide sequence from the rabbit N‐methyl‐d‐aspartate receptor NR2b on multiple Ag‐branched polylysine backbones (GR‐MAP8), rabBAFF was most strongly expressed on IgM+ B cells, and intermediate on CD14+ cells from peripheral blood mononuclear cells (PBMC). In spleen, rabBAFF can be detected on IgM+ B cells, and CD4+ T cells. After 1st and 2nd boosts with GR‐MAP8, BAFF detected on PBMC was significantly decreased; it then started increasing after 3rd through 5th boosts. This was negatively correlated with the levels of autoantibodies in serum. Real‐time PCR assays of mRNA from PBMC and spleen are in progress. This study broadens our knowledge of the expression of this important member of the TNF superfamily in a noninbred pedigreed rabbit model of SLE where it may specifically regulate B lymphocyte proliferation and survival.

This research was supported by the intramural research program of the NIH, NIAID and the Oak Ridge Associated Universities.
Genetic and environmental factors complement EAE susceptibility in tyk2 mutant B10.DI‐H2q/SgJ (DI) mice
Authors: Spach Karen M , Noubade Rajkumar , Blankenhorn Elizabeth P , Teuscher Cory
Published: June 2021

Multiple sclerosis (MS), and its mouse model EAE, are autoimmune (AI) diseases that depend on both genetic and environmental factors. Shared immunopathology genes, e.g., tyrosine kinase‐2 (Tyk2), may also be shared AI disease genes. DI mice have a single 2538 G → A missense mutation in tyk2 making them AI arthritis resistant and T. gondii susceptible, unlike the wild‐type B10.Q/Ai (Ai) substrain. We induced EAE in DI and Ai mice with MOG79‐96‐CFA using a double injection protocol (2×). DI mice were resistant (0/46) to EAE while Ai (32/36) and F1 hybrids (43/50) were susceptible (p < 0.0001). Antigen specific production of RANTES and IFN‐γ were specifically increased in Ai and F1 mice. Thus, tyk2 is a shared AI susceptibility gene, and EAE can be complemented genetically by one wild‐type Tyk2 allele. Next, we tested whether EAE in DI mice could be complemented by environmental factors using MOG79‐96‐CFA + pertussis toxin (1× + PTX). DI mice immunized with 1× + PTX were significantly more susceptible to EAE than 2× immunized mice (33/36 vs. 0/46; p < 0.0001). Only RANTES changed significantly with both genetic and PTX complementation. In summary, tyk2 is both a shared immunopathology and AI disease susceptibility allele that can be complemented both genetically and environmentally. Such SNPs are of considerable significance given the potential role of gene‐by‐environment interaction in MS susceptibility.

Research supported by NIH/NINDS R01 NS36526, R01 AI058052, R01 AI41747, P01AI45666‐08P4, and National Multiple Sclerosis Society RG3575A5
CD40L is crucial for the development of autoimmunity induced by TLR7 overexpression
Authors: Pisitkun Prapaporn , Deane Jonathan A. , Bolland Silvia
Published: June 2021

Antibodies against nuclear antigens secreted by autoreactive B cells are a hallmark of systemic autoimmunity. Failure to eliminate autoreactive B cells in the germinal center reaction has been reported in lupus disease. We have shown that control of TLR7 expression is essential to restrict B cell autoimmunity and DC expansion. However, the underlying mechanisms of TLR7‐induced autoimmunity still need to be scrutinized. We found increased spontaneous germinal center formation in Tg.TLR7 mice. B cells from these mice showed enhanced H2‐Ab1, stat1, T‐bet, AID expression and increased IgG1 and IgG2b immunoglobulin transcript. To test whether these B cell phenotypes required T cell help, we bred Tg.TLR7 with CD40L deficient mice. In the absence of CD40L, Tg.TLR7 mice did not develop spontaneous germinal center or any autoimmune phenotypes. Reconstitution experiments with an equal mix of Tg.TLR7.CD40L−/− and WT bone marrow showed that CD40L provided in trans can induced the disease. All of these results suggest that autoreactive B cell and autoimmune phenotype of Tg.TLR7 mice is CD40L‐dependent. Future experiments will determine whether T cells are needed to provide help through CD40L in this system.