bluuebunny/crossref_metadata_2025_split huggingface dataset:
Absence of intestinal gluconeogenesis in type 1 diabetic rats: Arteriovenous and carbon 13 NMR measurements.
Authors: Baverel Gabriel , Gadet Rudy , Nazaret Rémy , Martin Mireille , Boghossian Michelle , Martin Guy , Ferrier Bernard , Conjard Agnès 
Published: June 2021

Recent reports have indicated that type 1 diabetes induces gluconeogenesis in the small intestine of adult Sprague Dawley rats in vivo. Since this would (i) represent a dramatic revision of the prevailing view that only the liver and the kidneys are gluconeogenic, and (ii) have major consequences in the diabetes fields, we have thoroughly re‐examined this question. For this, metabolically viable small intestinal segments from fed and 36h‐fasted streptozotocin‐diabetic Sprague Dawley rats were incubated with [3–13C]glutamine as substrate. After incubation, substrate utilization and product accumulation were measured by enzymatic and NMR methods. Although the segments utilized 13C‐glutamine and accumulated 13C‐labeled products at high rates for 30 min in vitro, no substantial glucose synthesis could be detected. This was not due to the re‐utilization of 13C‐glucose initially synthesized. Arteriovenous metabolite concentration difference measurements across the portal vein‐drained viscera of 36h‐fasted streptozotocin‐diabetic Sprague‐Dawley rats clearly indicated that glutamine, the main if not the only gluconeogenic precursor taken up, could not give rise to detectable glucose production in vivo. Therefore, we challenge the view that the small intestine of the type 1 diabetic rat is a gluconeogenic organ.Supported by the INSERM.
Preparation of immobilized bovine deoxyhypusine hydroxylase and its applications
Authors: Soustek Meghan S , Murtaugh Megan L , Wang Yinglu , Smith Joshua L , Huang Jenq‐Kuen , Wen Lisa 
Published: June 2021

Deoxyhypusine hydroxylase (DOHH) catalyzes the second and the final step of eukaryotic initiation factor 5A (eIF5A) maturation which is essential for eukaryotic cell proliferation and survival. Modification of eIF5A has been suggested to be a potential target for anticancer and antiviral therapy. A detailed study of the structure and properties of DOHH is essential. We have previously partially purified DOHH from bovine testis. Our results suggested possible presence of multiple forms of DOHH. However, the native DOHH protein has never been fully purified and characterized from any tissues. Antibody probes specifically recognize DOHH would be a value tool for detection and purification of native DOHH protein. We have elicited polyclonal antibodies against recombinant bDOHH. Here, we report the preparation of immobilized recombinant bDOHH by coupling the protein to CNBr‐activated agarose. The bDOHH‐agarose has been used as an affinity column to purify antibodies from the immune serum. The affinity purified antibodies have been used in detecting recombinant bDOHH in a Western blot. These antibodies are being used to study native DOHH proteins in bovine tissues by Western blotting of bovine tissue extracts after separation by 1D or 2D gel electrophoresis.Supported by grants from National Institutes of Health 1R15 GM60266‐01A1 and the University Research Council at WIU.
Leukocyte 12‐lipoxygenase: expression, purification and physical characterization
Authors: Funk Max O. , Rapp Johanna , Sharp Allan M. 
Published: June 2021

The expression and purification procedure [Richards, K.M. and Marnett, L.J. (1997) Biochemistry 36, 6692–6699] for leukocyte 12‐lipoxygenase was updated for the production of the large quantities of protein necessary for the characterization of its physical properties. A two‐step FPLC ion exchange chromatography procedure was adequate for purification as evaluated by SDS PAGE, specific activity measurement, isoelectric focusing, and electrospray ionization mass spectrometry. The solution properties of the enzyme were examined by variable temperature circular dichroism spectroscopy, differential scanning microcalorimetry, and dynamic light scattering measurements. The redox characteristics of the active site iron atom were determined by EPR spectroscopy following treatment with a variety of fatty acid hydroperoxides. Leukocyte 12‐lipoxygenase was found to be more similar to reticulocyte 15‐lipoxygenase than to soybean lipoxygenase‐1 or human 5‐lipoxygenase.(Supported by the National Institutes of Health GM 62140)
Gene Repair in sub‐S phase Cell Populations and Cell Cycle Response to Quadruplex Oligonucleotides
Authors: Warriner Melissa , Schwartz Timothy , Engstrom Julia , Kmiec Eric 
Published: June 2021

Targeted nucleotide exchange (TNE) relies on the use of a short synthetic oligonucleotide (ODN) designed to be complementary to a target sequence in the genome except for a centrally located mismatch, which directs the desired base change in the DNA. The repair reaction is believed to be enhanced by mechanisms that promote a more open conformation of the DNA, thus, enabling the target site to be more accessible for the binding of the oligonucleotide. One such method is through the modulation of cell cycle progression to increase the population of cells in S phase, wherein targeting of a single base pair is enhanced. Studies aimed at demonstrating the variations in correction in relation to cell cycle measured repair frequency late in the reaction cycle. Our work aims to discern variations in repair levels in subphases of S as cells enter and progress through this phase, in the presence of the oligonucleotide. In addition, we evaluate gene expression patterns of several cell cycle genes that may help denote the changes in cell cycle distribution as a function of gene repair events.Additional research has been performed analyzing gene expression patterns in malignant versus non‐malignant esophageal cancer cells in response to treatment with G‐rich oligonucleotides (GRO). It has been shown that these single‐stranded DNA molecules which are comprised primarily of guanosine residues induce retardation in the progression of the cell cycle, and result in a sub‐G1 population of apoptotic cells. Successful inhibition of growth of malignant, but not nonmalignant, cells has been seen. Thus, G‐rich ODNs may be guiding the selective killing of tumor cells. I am currently analyzing the RNA levels of apoptosis genes incubated with GROs to understand how the cells are responding, and which pathway may be responsible for the induction of apoptosis. Funded by Howard Hughes Medical Institute.
VEGF‐B is an apoptosis inhibitor via suppression of BH3‐only protein gene
Authors: Li Xuri 
Published: June 2021

Despite its early discovery and high sequence homology to the other VEGF family members, the function of VEGF‐B thus far elusive.We have recently found a novel function of VEGF‐B as a potent apoptosis inhibitor. VEGF‐B treatment significantly rescues neurons from apoptosis in both the retina and brain. Mechanistically, we reveal that VEGF‐B inhibits apoptosis via suppressing the expression of the BH3‐only protein and other apoptotic/cell death‐related genes. Remarkably, the anti‐apoptotic/survival effect of VEGF‐B is not associated with undesired angiogenesis. VEGF‐B thus appears to be the first member of the VEGF family that has a potent anti‐apoptotic effect, while lacking a general/universal angiogenic activity.
Neutralization of the transcription factor nuclear factor erythroid derived‐2 (NF‐E2) causes robust increases in the anti‐apoptotic protein Mcl‐1 in human neutrophils
Authors: Johnson Paul Richard , Kausar Hina , Wu Rui , Rane Madhavi J 
Published: June 2021

Human neutrophils (PMNs) are short‐lived cells that undergo constitutive apoptosis in vivo. When challenged by pathogens, cytokines, or ROS, PMNs survive longer. One hallmark of prolonged PMN lifespan is up‐regulation of the anti‐apoptotic protein, Mcl‐1.We previously showed that GM‐CSF delays PMN apoptosis via the Akt pathway. Proteomic analysis of neutrophil Akt immunoprecipitates identified NF‐E2 p45 as an Akt binding protein. To explore the possible role of NF‐E2 in regulating PMN apoptosis, neutrophils collected from healthy human donors were subjected to NF‐E2 or isotope control (IC) antibodies or control buffer (Krebs) for 1 hr at 37°C. LPS, TNFα, or control buffer was then added to cells and incubated overnight at 37°C.PMNs pre‐incubated with NF‐E2 antibody showed an increase in NF‐κB activation and Mcl‐1 protein versus IC or control buffer. LPS overnight incubation caused a slight increase in Mcl‐1 protein, whereas TNFα showed no effect. Also, we demonstrated that Akt phosphorylates NF‐E2 on Ser170 and that additional Akt sites exist. Akt‐mediated NF‐E2 phosphorylation mediates its nuclear localization and DNA‐binding activity.Collectively, our results suggest that Akt regulates NF‐E2 activity and that NF‐E2 plays a role in constitutive apoptosis of human neutrophils. Neutralization of NF‐E2 causes a robust increase in PMN survival as determined by levels of the anti‐apoptotic protein, Mcl‐1.This work supported by AHA (G030700) Scientist Development Grant (MJR).
Single Cell q‐PCR Analysis of The Molecular Regulation of T Cell Commitment
Authors: Al‐Kuhlani Mufadhal M , Carroll Tanya , Garcia‐Ojeda Marcos E 
Published: June 2021

Hematopoietic stem cells (HSC) differentiate into all mature blood cells, including common lymphoid progenitors (CLPs) that can give rise to NK, dendritic, B and T cells. The final fate of CLP depends on the signals and growth factors they receive during development. Notch‐1 is a highly conserved transmembrane receptor that instructs CLP to differentiate into T cells while blocking B cell development. However, the molecular mechanism that regulates this process is not completely understood. Notch‐1 induction of T cell development has been shown to be dose‐dependent, suggesting regulation by a threshold mechanism. In order to elucidate the molecular mechanism exerted on Notch‐1 signaling we use single‐cell Real Time PCR to establish the gene expression level of the regulators of Notch‐1 Deltex‐1, Mint, Nrarp and Numb. Lymphocytes development will be monitored when these regulators are expressed at their normal level and then compared to their development when these regulators over‐expressed, through retroviral infection, or down regulated through the use of siRNA. Understanding the threshold level of the expression of these genes that regulate T cell development will allow us to predict the interaction mechanism of those genes on the CLP cells and the decisions made to determine the fate of these cells.Source of Support comes from Start‐up Fund from University of California‐Merced
Modulation effect of in vivo induced type I immune responses on airway inflammation in an asthma model
Authors: Tang Yafang 
Published: June 2021

Asthma is a Th2‐mediated inflammatory disease of the airways characterized by reversible obstruction of respiratory airway flow caused by airway thickening, mucus hyper‐secretion and bronchoconstriction. Production of antigen‐specific IgE, type II cytokines such as IL‐4, IL‐5 and IL‐13 as well as eosinophil infiltration into the lung are important markers of asthma. It is widely accepted that Th1 responses induced by viral or bacterial infections can antagonize allergic diseases. However, there is also evidence showing that virus or bacteria‐induced Th1 immune responses can promote the development or exacerbation of allergic diseases. Genetic background was recognized as a possible explanation on this contradictory issue. We show here that ovalbumin (OVA) induced stronger Th2 immune in Th2‐biased BALB/c mice while Th1 responses induced by influenza A virus were similar in both BALB/c and Th1‐biased C57BL/6 mice. When the asthmatic mice were infected with Influenza A, despite for the severer netrophilia induced in BALB/c mice than in C57BL/6 mice, OVA‐induced eosinophilia, IgE production and Th2 cytokine secretion were not affected by viral infection in either strain of mouse. Apart from Th1 cells, cytotoxic CD8 T cells (CTLs) also play an important role in immune defence against viral infection. To study the role of CTLs in asthma development, OVA‐specific CTLs isolated from OT‐I mice were activated in vitro and then intravenously injected into OVA sensitized C57BL/6 mice before intranasal challenge with OVA. We found that transferred CTLs led to an increase in IFN‐γ level in the bronchoalveolar larvage (BAL) fluid without apparent inhibition effect on IgE production.This work is funded by Immunology Programme (Grant no. C182‐002‐002‐001). Tang YF is supported by NUS Graduate School for Integrative Science and Engineering Scholarship.
Adjuvants enhance the cognate function of aged CD4 T cells via a mechanism involving IL‐17 production
Authors: Eaton Sheri , Lanthier Paula A , Maue Alexander C , Haynes Laura 
Published: June 2021

The humoral response in aged humans and mice is significantly reduced compared to young and is a contributing factor in the reduced efficacy of vaccines seen in aged populations. Using an adoptive transfer model comparing young and aged T cell receptor transgenic (TCR Tg) CD4 T cells in young hosts, we have shown that age‐related declines in CD4 T cell function contributed greatly to the reduced humoral responses observed in the aged. This decline leads to reduced B cell expansion and differentiation as well as reduced IgG production. The helper function of aged CD4 T was enhanced using a combination of inflammatory cytokines (TNF alpha, IL‐1 and IL‐6) or adjuvants that induce these cytokines. In addition, the helper function of aged CD4 T cells was also enhanced when the inflammatory cytokines were added during in vitro CD4 effector generation. In both of these cases, the helper activity of aged CD4 T cells was significantly enhanced, leading to better expansion and differentiation of antigen‐specific B cells. Using real time PCR, we found that these cytokines induced significant production of IL‐17 by the aged CD4 T cells in vitro. We have also gone on to show that Th17 effectors generated in vitro from naive aged CD4 T cells exhibit potent helper activity in our adoptive transfer model. We propose that one of the major mechanisms involved in the ability of adjuvants to enhance T cell responses involves induction of IL‐17 production by the responding CD4 T cells.This work was funded by NIH grant AG02160 to LH.
Bim and Fas Function Concurrently to Control Autoimmunity and CD8+ T Cell Contraction
Authors: Weant Ashley , Michalek Ryan D , Khan Islam U , Holbrook Beth C , Willingham Mark C , Grayson Jason M 
Published: June 2021

Throughout most of adult life lymphocyte number remains constant due to a balance between proliferation and death. To determine whether loss of genes in both the extrinsic and intrinsic cell death pathways would exacerbate defects in immune homeostasis we created Bim−/−Faslpr/lpr mice. Mice were born at the expected Mendelian ratios and appeared to develop normally. However, by 14 weeks of age they developed massive lymphadenopathy compared to either single mutant or wildtype animals. Loss of both Bim and Fas also resulted in dramatic increases in autoantibodies and deposition of immunoglobulin in the kidneys. When antiviral immune responses were quantitated following acute LCMV infection, double mutants contained 100‐fold more antigen‐specific memory CD8+ T cells in their lymph nodes compared to wildtype mice. This increase in cell numbers was not the result of increased proliferation, therefore it must be due to decreased apoptosis. Although contraction was blocked in the lymph nodes of double mutants, it was similar to the Bim parental strain in the spleen and liver. Following chronic LCMV infection, the number of antigen‐specific CD8+ T cells was similar between Bim−/−Faslpr/lpr and the Bim−/− parental genotype. Our results demonstrate that multiple death pathways function concurrently to prevent autoimmunity and downsize T cell responses.Supported by ACS‐ #RSG‐04‐066‐01‐MBC to Jason Grayson.
G‐CSF induces leukocyte recruitment in vivo via the leukocyte G‐CSF receptor
Authors: Hickey Michael , Norman M. Ursula , Croker Ben , Roberts Andrew , Wicks Ian , Eyles Jo 
Published: June 2021

Granulocyte colony stimulating factor (G‐CSF) is in routine clinical use but has been shown to activate neutrophil adhesion in vitro. The aim of this study was to determine if G‐CSF is capable of inducing leukocyte‐endothelial cell interactions in vivo. Mice were treated with G‐CSF either by acute, local administration, or systemically over 4 hrs, and leukocyte‐endothelial cell interactions assessed via intravital microscopy of the cremasteric microcirculation. Local application of G‐CSF caused a reduction in leukocyte rolling but induced a significant increase in leukocyte adhesion within 15 mins, a process dependent on Mac‐1. The adherent cells were exclusively Gr‐1+ neutrophils. Adherent leukocytes also underwent transmigration within 45 mins. To assess the effect of G‐CSF under conditions mimicking its clinical use, G‐CSF was also administered systemically. Systemic G‐CSF induced increased leukocyte rolling, adhesion and transmigration after 4 hrs. Using mice rendered chimeric for the G‐CSF receptor (G‐CSFR) via bone marrow transfer between wild‐type and G‐CSFR−/− mice, this response was found to be dependent on leukocyte‐expressed G‐CSFR. These findings indicate a pro‐inflammatory role for G‐CSF and provide a potential mechanism for the association of G‐CSF administration with exacerbation of diseases such as rheumatoid arthritis.Supported by the NHMRC (Australia)
Gender specific disease propensities of bone marrow cells from New Zealand hybrid mice
Authors: Jorgensen Trine N. , MacLeod Megan K.L. , David Alexandria , Kappler John W. , Marrack Philippa 
Published: June 2021

The (NZBxNZW)F1 (BWF1) hybrid mouse is the best analyzed mouse model of SLE. Most female, but only ∼25% of male, BWF1 mice develop SLE‐related symptoms by 1 year of age.Sex hormones may play a role in this disease. To test this, we transferred BM cells from 9 wk old BWF1 mice into lethally irradiated, age‐matched recipients of the same or opposite gender, thus obtaining four groups of chimeras: F→F, M→F, F→M and M→M. Eighteen weeks later, F→F and F→M chimeras had higher levels of serum IgG (3.7 ± 1.8 and 3.4 ± 2.6 mg/ml respectively) than M→F or M→M chimeras (1.9 ± 1.2 or 1.9 ± 1.1 mg/ml respectively). The same was true for the development of renal disease, as it developed statistically earlier in F→F and F→M mice (27 and 30 wk post transfer, respectively) than in M→M and M→F chimeras (>33 wk, p < 0.02). Thus, BM cells from 9 week old female mice have the capacity to drive autoantibody production and renal disease regardless of their subsequent hormonal environment.This study was funded by The Denver Autoimmune Center of Excellence and HHMI.
Toll‐like receptor control of IL‐6 production by dendritic cells and B cells
Authors: Sisk Bryan , Krogstad Aric , Kannan Krishnaswamy , Ortmann Robert 
Published: June 2021

Collagen‐induced arthritis can be elicited in resistant C57BL/6 mice by increasing the amount of adjuvant (dessicated Mycobacterium tuberculosis) during immunization. This adjuvant contains ligands for toll‐like receptors (TLRs), especially TLR2 and TLR9, which are known to be critical for innate immune responses and are expressed on Antigen Presenting Cells (APCs) such as dendritic cells and B‐cells. Our current studies are aimed at elucidating mechanisms by which TLR signaling may promote arthritis. We investigate how cross‐linking TLR2 and TLR9 receptors might promote pathogenic Th‐17 cells by regulating the production of IL‐6 by these APCs.We isolated bone‐marrow derived dendritic cells (BM‐DCs) and splenic B‐cells from wild type, TLR2 and TLR9 deficient B6 mice. Wild type BM‐DCs produce IL‐6 in response to TLR2, TLR4, and TLR9 cross‐linking. Immune (but not naïve) B‐cells make IL‐6 in response to TLR2, TLR4 and TLR9 ligands. As compared to B6 wt, however, TLR2‐KO derived DCs produced several fold more IL‐6 when stimulated with TLR4 or TLR9 ligands and TLR9‐KO derived DCs produced several fold more IL‐6 when stimulated with TLR4 or TLR2 ligands. These findings suggest that DCs and B‐cells are potent cellular sources of IL‐6. IL‐6 production in response to adjuvants appears to be regulated by the coordinated input of TLR signaling and may be important in the generation of pathogenic Th‐17 cells.
Pristane‐induced alveolar hemorrhage in C57BL/6 mice is not mediated by immune complex, T or B‐cells, or Fas
Authors: Barker Tolga , Lee Pui Y. , Weinstein Jason S. , Nacionales Dina C. , Kelly‐Scumpia Kindra M. , Sobel Eric S. , Reeves Westley H. , Satoh Minoru 
Published: June 2021

Intraperitoneal (ip) injection of pristane in normal mice induces antinuclear antibodies and immune‐complex (IC) glomerulonephritis similar to human systemic lupus erythematosus (SLE). In addition, C57BL/6 (B6) mice frequently develop alveolar hemorrhage (AH), leading to 10–50% early mortality. AH is an uncommon but often fatal complication of human SLE and is thought to be mediated by IC. The role of IC, apoptosis and T‐ and B‐cells in pristane‐induced AH was investigated. B6, B6 RAG1−/−, B6 FcR γ1 −/−, and B6/lpr, received ip pristane and the lungs were examined for pathology, IC deposition, apoptosis and cytokines and chemokines. In some experiments, bone marrow, spleen, or peritoneal cells from pristane‐treated B6 mice were transferred to RAG1−/− mice to examine their ability to induce AH. Two‐weeks after pristane ip, nearly all of B6, B6 RAG1−/− or B6 FcRγ1 −/− mice developed AH, indicating IC, autoantibody‐binding to lung tissue, or T‐ or B‐cells is not necessary for AH. B6/lpr are resistant to pristane‐induced lupus yet developed AH, indicating that Fas‐mediated apoptosis is not essential and that AH and other pathology induced by pristane are regulated differently. Short term transfer of pristane treated B6 cells into RAG1−/− mice did not induce AH while some long term transfers did induce AH. We conclude that the induction of AH by pristane does not require IC or antibodies, B‐ or T cells, or Fas.Supported by NIH.
Characterization of the Specificity of Anti‐Neuronal Antibodies in Sera of Multiple Sclerosis Patients
Authors: Takalo Elisa , Fox Autumn , DeVries George H. , Kennett Roger H. 
Published: June 2021

Screening of 30 sera from multiple sclerosis (MS) patients against enriched axolemma fractions (AEF) indicated that 11 could be classified as having high titers, 14 as intermediate and 5 as low or negative. (J. of Neurochem. 96 (Suppl. 1), 134) Two of these sera were pooled, diluted, bound to microtiter plates and used to screen a phage display library expressing random peptide epitopes 5–7 amino acids in length. Peptides bound corresponded to sequences in several proteins including ankyrin (3 epitopes), spectrin, K+ and Ca++ ion channels and the extra‐cellular matrix protein AGC1 (2 epitopes). We have continued these experiments by binding antibodies in MS sera to human neuroblastoma cells and using the eluted antibodies to screen a phage display library expressing cDNA clones from human brain. This new procedure detects antibody epitopes on cell surface proteins that may represent targets for antibodies found in MS sera. Screening of the phage display targets with additional MS sera should provide an indication of whether or not they may be involved in the pathogenesis of MS.We thank D. L. Feinstein and C. Kaiser, Department of Anesthesiology, Univ. of IL for the MS sera samples used in the study. The work was supported by Wheaton College Ruth Kraft Strohschein research funds and a VA Merit Grant†.