bluuebunny/crossref_metadata_2025_split huggingface dataset:
PTH Receptor Responds To Shear Stress Perturbation of Plasma Membrane
Authors: Chachisvilis Mirianas , Frangos John A , Zhang Yan‐Liang 
Published: June 2021

The precise molecular mechanisms by which bone cells transduce mechanical stimulus into intracellular biochemical response have not been established yet. Here we show that mechanical perturbation of the plasma membrane leads to ligand‐independent conformational transitions in G protein coupled receptor (GPCR) such as parathyroid hormone type 1 receptor (PTH1r). By using picosecond time‐resolved fluorescence microscopy and GPCR conformation‐sensitive fluorescence energy transfer (FRET) we found that stimulation of MC3T3 cells with fluid shear stress or membrane fluidizing agent leads to a significant changes in conformational equilibrium of PTH1r in MC3T3 cells. The PTH1r conformational dynamics was detected by monitoring redistribution of GPCRs between inactive and active conformations in a single cell under fluid shear stress in real time using genetically engineered PTH1r containing intramolecular FRET pair. Our data demonstrate that changes in cell membrane tension and membrane fluidity affect conformational dynamics of PTH1r. Therefore we suggest that PTH1r is involved in mediating primary mechanochemical signal transduction in MC3T3 cells. Furthermore we show that conformational transitions induced by mechanical stress are different from those induced by its ligand PTH(1‐34).This research was supported by the NIH grant HL086943.
Maintenance and dynamic of the chromatin structure in Trypanosoma cruzi
Authors: Flawiá Mirtha María , Alonso Guillermo D. , Meyer Cristian G. , Vilchez Larrea Salomé C. , Fassolari Matías , Torres Héctor N. , Fernández Villamil Silvia H. 
Published: June 2021

Dynamic structural changes on the chromatin occur throughout the cell cycle. These changes range from local ones, necessary for transcriptional regulation and DNA damage repair machinery accessibility, to global changes necessary for chromosome segregation. We demonstrated that the poly(ADP‐ribose)polymerase from T. cruzi (TcPARP) is strongly activated by nicked DNA and attaches polymers of ADP‐ribose (PAR) to itself and to T. cruzi histones. We assayed several DNA damaging agents that trigger different DNA repair mechanisms and confirmed an increase in PAR synthesis in every case. However, when the induced damage led to cell death, the amount of PAR detected decreased. To study in depth the chromatin remodeling we cloned two T. cruzi HATs (TcHAT1 and TcElp3). TcElp3 failed to rescue a thermosensitive mutant yeast strain. We also analyzed the expression and localization of TcHAT1 and TcElp3 by using specific antibodies. We also analyzed the effect of HDACs inhibitors Trichostatin A (TSA) and sodium butyrate on epimastigotes. When parasites were treated with 50 nM TSA strong proliferation inhibition was observed and a sub G1 peak appeared on DNA histogram when analyzed by flow cytometry. This evidence not only supports a physiological relevance of chromatin remodeling in Trypanosoma cruzi but also opens new insights in drug therapy against Chagas′ disease.Supported by CONICET, FONCYT and UBA‐Argentina.
Interactions of HP1 with other chromosomal proteins
Authors: Mendez Deanna Lynn , Stephens Gena , Volpi Gina , Kim Daesung , Khorasanizadeh Sepideh , Elgin Sarah CR 
Published: June 2021

To investigate the molecular basis of HP1a binding with a number of non‐histone chromosomal proteins in D. melanogaster, we are focusing on three examples: HP2, PIWI, and dAF10. Using co‐immunoprecipitation assays we demonstrate that an HP2 region containing an LCVKI sequence is responsible for binding to the chromo shadow domain (CSD) of HP1a. HP2 co‐precipitates with native HP1, but not with mutatants in HP1 that disrupt dimerization (I191E) or disrupt the PXVXL platform (W200A); mutation of the HP2 peptide at V2468E also abrogates binding. Using fluorescence polarization binding assays, we measure the observed dissociation constant of a partner peptide interacting with the HP1a‐CSD. We find that all three peptides bind with micromolar affinity and that the HP2 peptide binds the tightest followed by PIWI and the dAF10 peptides. Using 2D NMR spectroscopy we show that binding of any of the three peptides causes similar chemical shift perturbations in the portion of HP1a encompassing the CSD and CTE (C‐terminal extension) regions. Together our studies show that different short linear hydrophobic peptides can bind to the HP1a CSD‐CTE region, and potentially influence its localization in heterochromatin.Supported by NIH GM068388‐18 to SCRE and GM064786 to SK.
Characterization of the specific role and activation of the High Mobility Group A1 (HMGA1) in colon cancer
Authors: Bush Bethany Marie , McKinney Kimberly Q , Sumter Takita Felder 
Published: June 2021

The High Mobility Group A1 (HMGA1) proteins are a class of oncogenes whose specific role in colon and other cancers is currently emerging. Our analysis of the HMGA1 promoter region revealed several putative sites for beta‐catenin/tcf binding suggesting that HMGA1 may be activated by the adenomatous polyposis coli (Apc)/beta‐catenin pathway in colon cancer. To better understand the specific role of HMGA1 in colon carcinoma formation, we evaluated the expression of HMGA1 in the Apc/min colon cancer mouse model. Results of quantitative real‐time RT‐PCR (qRT‐PCR) show a 9‐fold increase over normal intestine in HMGA1 mRNA where Apc is truncated and in tumors. Consistent with qRT‐PCR data, HMGA1 protein levels were also significantly higher in Apc‐min intestinal tumors than those from normal mouse tissues. To determine if Apc/beta catenin signaling directly impacts HMGA1 expression, we tested the effect of Apc recovery on HMGA1 expression. Preliminary results indicate that zinc‐induced full‐length Apc expression suppresses HMGA1 levels and that truncations in Apc may activate HMGA1 to promote cancer initiation and progression. Collectively, these results provide the first line of evidence supporting the cooperation of truncated Apc and HMGA1 in colon cancer.This project was supported by NIH grant P20 RR‐16461 in support of SC INBRE.
Amyloid‐β degradation by insulin‐degrading enzyme is inhibited by nitric oxide
Authors: Cordes Crystal M , Hamel Frederick G 
Published: June 2021

Amyloid beta (Aβ) accumulation is a crucial step in Alzheimer.s disease (AD) development. Insulin‐degrading enzyme (IDE) degrades Aβ. We examined the effect of nitric oxide on IDE‐mediated degradation of Aβ. Partially purified rat liver IDE was treated with NO donors S‐Nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) and sodium nitroprusside (SNP), or control compounds N‐Acetyl‐D‐penicillamine (NAP) and potassium ferricyanide (KFC). IDE activity was measured by trichloroacetic acid precipitation of 125I‐amyloid β. IDE cysteine nitrosylation was measured using the biotin switch assay. Degradation of Aβ was specifically mediated by IDE, as unlabeled insulin inhibited Aβ degradation with an approximate Km 10−7 M. SNP and SNAP inhibited IDE degradation of Aβ by 50% and 25% respectively. KFC and NAP had no effects. As a possible mechanism for the inhibitory effect on Aβ degradation, we found IDE to be nitrosylated by SNAP, indicating a new posttranslational modification of the enzyme. Aβ accumulation in the AD brain contributes to the disease progression and pathology, and IDE dysfunction has been implicated as a possible reason for this accumulation of Aβ. We have shown that increased NO levels, which have been observed in patients with AD, can decrease IDE enzymatic function, potentially resulting in increases in Aβ deposition in the brain and development of AD.Funding: Veterans Affairs Merit Review
Phospholipase D and Phosphatidylinositol 3‐kinase are Activated by Oxidative Stress in Central Nervous System.
Authors: Salvador Gabriela Alejandra , Uranga Romina María , Mateos Melina Valeria , Giusto Norma María 
Published: June 2021

The purpose of the present work was to study the involvement of phospholipase D (PLD) and phosphoinositide‐3‐kinase (PI3K) signaling in synaptic endings incubated under oxidative stress injury. For this purpose, synaptosomes purified from rat cerebral cortex were exposed to the oxidative insult (FeSO4, 50 μM) or vehicle and subsequently phosphatidylethanol (Peth) and phosphatinolsitol‐3‐phosphate generation were evaluated. Peth formation was increased after 5, 30, and 60 min of Fe2+‐exposure with respect to the controls, whereas PI3P levels were only increased after 5 min of Fe2+ exposure. PI3K activity was also measured by using anti‐p85 immunoprecipitates. Peth generation was not altered by the presence of the tyrosine kinase inhibitor, genistein, whereas PI3K activation was dependent on tyrosine phosphorylation. Synaptosomal viability was evaluated by using MTT reduction and LDH leakage measurements under all experimental conditions. These viability parameters were significantly affected in the presence of FeSO4, with respect to control conditions. Neither ethanol nor LY294002 were able to prevent the deleterious effect of free iron. Our results demonstrate that oxidative stress activates PLD and PI3K pathways in cerebral cortex synaptic endings.Supported by Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional del Sur and Agencia Nacional de Promoción Científica y Tecnológica.
Roles of two GDP‐mannose transporters in cell wall stability in the filamentous fungus Aspergillus nidulans
Authors: Gordon Barbara , Fay Lauren , Martin Stuart , Nkashama Sonia , Patel Ravi , Hill Terry W. , Loprete Darlene M. , Jackson‐Hayes Loretta 
Published: June 2021

In order to identify genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans. We have identified two distinct genes (GMT1, GMT2) in one such mutant (R205) that are homologous to the yeast VRG4 GDP‐mannose transporter genes. GDP‐mannose transporters carry nucleotide sugars from the cytosol across the Golgi apparatus membrane in various eukaryotic organisms including plants and a variety of fungi. We have observed punctate patterns of fluorescence of GMT1 and GMT2 GFP chimeras, indicative of Golgi localization in A. nidulans. Cloned GMT1 and GMT2 complement hypersensitivity to the chitin binding agent calcofluor white in strain R205, and ameliorate hyper‐branching and septation abnormalities R205 displays under normal growth conditions. GMT1 in R205 contains a genetic mutation that causes an A315P amino acid substitution, and the amino acid sequence of GMT2 in R205 is identical to wild type. However, both GMT1 and GMT2 appear to be essential. These findings suggest that GMT1 and GMT2 are involved in cell wall maintenance in A. nidulans.Funded by Research Corporation and the Merck/AAAS Undergraduate Science Research Program
Neuregulin 1β/erbB signalling in cardiac myocytes enhances integrin based myocyte‐matrix interactions via src
Authors: Pentassuglia Laura , Arteaga Carlos , Sawyer Douglas 
Published: June 2021

The Neuregulin‐1β (Nrg)‐erbB2/ErbB4 (E2/E4) system is critical for the heart and it is needed for myocyte survival in stress conditions. Nrg activates Src and FAK, both part of integrin signaling. Hyp.: Nrg signaling regulates myocyte matrix interactions, critical for coupling force generation to the extracellular matrix.Immunostaining of mouse hearts showed E4 and E2 are at intercalated discs and have a t‐tubule like striated pattern. Isolated myocytes were incubated 30 min or 24h with an E2 monoclonal antibody (Ab) or an E2 tyrosine kinase inhibitor (TKI). Western‐blot showed E2 TKI blocks activation of FAK, Erk1/2, and Akt by Nrg; Ab is a weak inhibitor. ErbB activation in myocyte‐matrix coupling was tested by plating myocytes onto untreated culture dishes spot‐coated with laminin, with or without Nrg (10ng/ml). After 30 minutes unattached cells were stained and counted. Attachment was enhanced 4‐fold by Nrg vs. laminin alone. Cells did not attach to Nrg coated plates in absence of laminin. Pre‐treatment of myocytes with E2 inhibitors did not prevent Nrg‐enhanced attachment; pre‐treated with Src inhibitor PP2 (10 μM) Nrg effect was inhibited.Nrg‐1β treatment of cardiac myocytes enhances integrin mediated myocyte‐matrix coupling via Src dependent signalling. This appears to be E2 independent pathway, involving E4 signaling, supported by e4 localization to the intercalated disks in vivo.Support by SNSF, NIH.
Insights into the structure and function of the extracellular domain of alpha‐neurexin by single particle EM and surface plasmon resonance
Authors: Miller Meghan Thorne , Comoletti Davide , Wilson Jennifer , Nakagawa Teru , Taylor Palmer 
Published: June 2021

Neurexins are a family of presynaptic cell‐adhesion membrane proteins that are important for synapse maintenance through contact with the postsynaptic neuroligin molecules. Several studies have indicated that mutations in these genes may play an important role in autism and mental retardation. Each neurexin gene contains two promoters corresponding to the α‐and β‐neurexins. While the extracellular domain of β ‐neurexin contains only a single LNS/LG domain, α ‐neurexin consists of six LNS/LG domains, including the β ‐neurexin domain, separated by three EGF domains. To investigate the conformation of the extracellular domain of α ‐neurexin we imaged purified protein using negative‐stain EM. Raw particle EM images suggest that the LNS/LG domains of α ‐neurexin exist in a mixture of different conformations. Additional experiments to obtain class averages and to investigate the effect of calcium and ligands on conformation are ongoing. Using surface plasmon resonance (SPR) we report a rank order of specificity for α ‐neurexin binding to the different neuroligins. Furthermore, we are using mutagenesis and SPR to help us understand the contribution of the different LNS/LG domains of α‐neurexin to neuroligin binding.This work was supported by: USPHS Grant R37 GM‐18360 and NIEHS to PT, and the Pharmacology Training Grant
The cell cycle inhibitor p21(WAF1/Cip1) as a transcriptional regulator of pigment cell‐specific gene expression
Authors: Vachtenheim Jiri , Sestakova Blanka 
Published: June 2021

The cyclin dependent kinase inhibitor p21(WAF1/Cip1) has been shown previously to regulate expression of a number of genes related to cell cycle regulation, DNA repair, senescence, differentiation and apoptosis. Recently, transcription of p21 was found to be activated by MITF (microphthalmia‐associated transcription factor), a factor which plays a central role in melanocyte‐specific transcription. Here we investigated the expression and transcriptional activity of p21 in human melanoma cells. We found no correlation between proliferation and p21 protein level in unsynchronized melanoma cells. p21 protein levels were surprisingly higher in low‐pigmented, faster proliferating normal melanocytes than in dark‐pigmented slowly proliferating melanocytes. Furthermore, clones of melanoma cells with p21 level stably knocked‐down by siRNA had even decreased growth rates when compared to controls. We further found that promoters of several genes related to differentiation were stimulated by p21 overexpression, including the MITF promoter. Mutational analysis of p21 suggested that mechanisms other than cell cycle inhibition, PCNA binding, and derepression of p300/CBP repression domain CRD1 underlie the transcriptional effects. These results suggest that p21 may have a role in transcription rather than in cell cycle regulation in melanoma cells.Grant support: MZO00064211, NR/9319‐3, MH, Czech Rep.
Cilostazol reduces prolifferation in MDCK cells through down‐regulation of c‐Myc
Authors: Muñoz Rodrigo , López‐Bayghen Esther 
Published: June 2021

Polycystic kidneys are inherited diseases characterized by excessive proliferation and apoptosis of renal tubular epithelia, cysts development and progressive renal insufficiency. Emerging therapies are c‐myc antisense oligonucleotides or phosphodiesterases (PDEs) inhibitors. Cilostazol is a specific inhibitor of PDE3, used as antithrombotic and antiplatelet agent because inhibits vascular cell proliferation increasing cAMP levels, downregulating E2F activity and inhibiting cell cycle associated kinases.Treatment of MDCK cells (cystogenesis model) with Cilostazol results in decrease of proliferation rates and downregulation of transcription factor c‐Myc. Reduced expression and DNA/binding were observed. Decreased transcriptional activity of E2F and cyclin D1, promoters with E‐Boxes was recorded. In contrast DNA/binding and activity of the differentiation related factor Sox10 increased. Using cytometry flow analysis we found arrest in G1/S phase and apoptosis; DAPI staining analysis showed that this drug does not cause complete DNA damage. Response to Cilostazol is probably mediated by an increase in cAMP, PKA activation and inhibition of ERK. Results indicate that cilostazol downregulates c‐Myc and other elements controlling cell cycle progression in MDCK, reducing proliferation rates, activity that may contribute to kidney cyst treatment.Support: CONACyT 50414
A Novel Model of IVIG Immunosuppression: Activation of Natural Regulatory T cells by IgG‐Derived Peptides
Authors: Rivera Daniel S , Martin William , Wambre Erik , Van Overvelt Laurence , Scott David W , Knopf Paul M , Moingeon Philippe , De Groot Anne S 
Published: June 2021

Background. Intravenous IgG (IVIG) is widely used to suppress immune responses in autoimmune diseases, allergy and transplantation. However, the exact mechanism responsible for its immunosuppressive/tolerogenic activity remains unknown. We hypothesized that the immunosuppressive effect of IVIG may be due to the presence of T cell epitopes in IgG that are capable of activating natural regulatory T cells (Tregs), leading to the suppression of T effector immune responses.Methods and Results. Using the EpiMatrix algorithm, we identified three highly promiscuous MHC Class II T‐cell epitopes in both the heavy and light chain constant domains of IgG. These epitopes were shown to bind multiple HLA Class II molecules with high affinity and specifically upregulated FoxP3 expression in CD4+CD25high T cells. Co‐administration with various antigens led to suppression of effector T cell responses an increase in IL‐10 and CTLA‐4 expression.Discussion. We believe that these data point to a previously undescribed mechanism for the immunosuppressive activity of IVIG. In this model, regulatory T‐cell epitopes (Tregitopes) in IgG are co‐processed and co‐presented with immunogenic epitopes derived from an antigen. The IgG Tregitopes activate a subset of natural regulatory T cells that can, at low dose of antigen, tip the resulting immune response towards tolerance rather than immunogenicity.All work was funded by EpiVax Inc.
The p85α regulatory subunit of phosphatidylinositol 3‐kinase critically modulates class switch DNA recombination
Authors: Xu Zhenming , Zhang Jinsong , Ho Yin‐Wei , Ho Yin‐Wei , Al‐Qahtani Ahmed , Pone Egest , Zan Hong , Casali Paolo 
Published: June 2021

Somatic hypermutation (SHM) and class switch DNA recombination (CSR) are central to the maturation of the antibody response. The PI 3‐K signaling plays an important role in the B cell development, proliferation and survival. PI 3‐K was also suggested to negatively regulate CSR by inhibiting AID expression and/or promoting plasmacytoid differentiation. Here, we directly addressed the role of PI 3‐K in CSR and, thereby, the maturation of the T‐dependent antibody response, using mice that are deficient in p85α, the predominant Class I PI 3‐K regulatory subunit. We found that the p85α‐deficiency did not significantly alter proliferation of in vitro stimulated B cells, but significantly decreased CSR to IgG1 or other isotypes, mainly due to a decreased proportion of switched B cells that had undergone five or more divisions. While B cell receptor (BCR)‐crosslinking increased the overall degree of CSR inp85α−/− B cells, it did not rescue the defective CSR in p85α−/− B cells at high division numbers, suggesting a BCR signaling‐independent function of PI 3‐K in CSR. Accordingly, CSR in p85α−/− mice immunized with a T‐dependent antigen was impaired, leading to a three‐ to five‐fold decrease of antigen‐specific IgG1, as compared to their wildtype counterparts. Thus, p85α and, therefore, PI 3‐K signaling, plays an important role in CSR and an effective antibody response.Supported by NIH grants AI 45011, AI 60573 and AR 40908.
Kinetic Analysis of a Complete Poxvirus Transcriptome Reveals a Novel Class of Genes
Authors: Assarsson Erika , Greenbaum Jason A. , Sundstrom Magnus , Schaffer Lana , Pasquetto Valerie , Oseroff Carla , Tscharke David , Sidney John , Grey Howard , Head Steven , Peters Bjoern , Sette Alessandro 
Published: June 2021

Vaccinia virus is the prototypic orthopoxvirus and was the vaccine used to eradicate smallpox, yet the expression profiles of many of its genes remain unknown. Using a genome tiling array approach, we simultaneously measured the expression levels of all 223 annotated vaccinia virus genes during infection and determined their kinetics. Almost all of the genes were transcribed, and for 62 of these, this is the first empirical evidence of expression. Most remarkably, classification of the genes by their expression profiles revealed 35 genes exhibiting immediate‐early expression. Although a similar kinetic class has been described for other virus families, this is the first demonstration of its existence in orthopoxviruses. Despite expression levels higher than for genes in the other 3 kinetic classes, the functions of more than half of these remain unknown. Additionally, genes within each kinetic class were spatially grouped together in the genome. This genome‐wide picture of transcription alters our understanding of how orthopoxviruses regulate gene expression.This work was supported by National Institutes of Health grants ROI‐AI‐56268 and HHSN266200400124C.
The missing link in the transfer of antigen between the dendritic cells after HSV infection.
Authors: KUMARAGURU UDAY , Woolard Stacie N , Nandakumar Subhadra 
Published: June 2021

NK cells can be recruited to inflamed tissues and meet with the immature DC within a short period of time after HSV infection. Evidence from a recent study suggests that antigen bearing DC (CD205+CD11c+CD11b‐CD8a‐) that migrate to the lymphoid organs do not prime the T cells. They transfer the antigen to lymphoid resident DCs (CD205+CD11c+CD11b‐CD8a+) which prime a potent T cell response. The mechanism of transfer is still not clear. We believe the missing link in enabling this transfer is the NK cell. We propose that the NK cell uses the TNF receptor apoptosis inducing ligand (TRAIL) to deliver the death signal to the antigen bearing migratory iDC.We used ex vivo and in vivo assays to assess the role of NK‐TRAIL. DC and NK isolated from mice infected on FITC painted skin were used. They were cocultured in the presence of anti‐TRAIL antibody or control IgG. The skin DCs were analyzed for apoptosis and uptake by LN‐ DC by gating on FITC positive cells. NK cells isolated from TRAIL knock out bone marrows were assessed for their ability to lyse HSV infected migratory DCs. The LN‐DCs were then assessed for their ability to stimulate T cells in vitro and in vivo. Results suggest a series of events, which include DC‐induced NK‐cell proliferation, NK‐mediated killing of antigen bearing DCs, NK‐dependent DC maturation and cross‐presentation.We suggest the following sequence of events to take place after HSV infection. HSV prevents maturation of DCs, but upon arrival at the lymphoid organ stimulate the NK cells, they reciprocate by killing them through TRAIL‐induced apoptosis. The resulting inflammatory milieu than aids the lymphoid resident DC to take up the antigen bearing dead DC, processes and cross‐presents the viral epitope resulting in optimized immune activation.