bluuebunny/crossref_metadata_2025_split huggingface dataset:
NF90/NF45 complexes regulate cell division
Authors: Pe'ery Tsafi , Guan Deyu , Altan‐Bonnet Nihal , Parrott Andrew M , Arrigo Cindy J. , Lee Chee‐Gun , Mathews Michael B. 
Published: June 2021

Nuclear factor 90 (NF90) and its C‐terminally extended isoform, NF110, belong to a family of nucleic acid binding proteins. NF90/110 are isolated with the less‐studied protein NF45, as complexes interacting with highly‐structured and double‐stranded RNAs. They have been implicated in the regulation of cellular and viral genes at several levels but it remains unknown whether NF90 and NF110 form functionally distinct complexes. We studied the composition of the complexes in vivo and used siRNA strategies to elucidate their function in cells.NF90 and NF110 form heterodimeric core complexes with NF45, and large in vivo complexes in which multiple NF90/110 isoforms interact with each other. NF45 knock‐down by RNA interference led to decreased expression of NF90 and NF110. This co‐regulation is at the protein level: the stability of NF90 family proteins is dependent on binding to their NF45 partner. Reciprocally, NF90 knock‐down reduced the expression of NF45 as a result of its accelerated degradation. Cell growth is retarded and giant multi‐nucleated cells accumulate when the expression of NF45 or NF90, but not NF110, is reduced in HeLa cells. The defect leaves nuclei attached by constrictions. Thus, NF90 family members form complexes inside cells whose stability is dependent on their binding to NF45. NF90 and NF45 are essential for cell growth and their depletion results in defects in nuclei division and cytokinesis.Support: NIH AI034552
CD27 plus CD28 Cross‐linking Induces Phosphorylation of STAT‐6 and Synthesis of IL‐4 in CD4+CD45RO+ T Cells
Authors: Tamma Seetha M. , Lee H. J. , Dandamudi Durga B. 
Published: June 2021

Expression of CD27, a 120 KDa transmembrane protein, is predominantly confined to lymphocytes and equally distributed between CD4 and CD8 lymphocytes. We investigated the role of CD27 in phosphorylation of STAT‐6, and in induction of IL‐4 synthesis in 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. We report that CD27 plus CD28XL resulted in enhanced and accelerated phosphorylation of STAT‐6 in RO+ cells when compared with that in RA+ cells. CD27 on its own failed to induce STAT‐6 phosphorylation in both populations. We also analyzed the cytokine profiles following stimulation of both populations as indicated and observed that RA+ cells responded by producing TNF‐alpha and RO+ cells responded by producing IL‐4. We conclude that CD27 plus CD28 cross‐linking induces TH2 differentiation in RO+ cells and that augmentation of STAT6 would be one of the underlying mechanisms of induction of IL‐4 synthesis. The data may suggest that CD4+CD45RA+ T cells act as TH1 type and CD4+CD45RO+ cells act as TH2 type.Grant from C.W. Post Campus Research Committee of Long Island University, New York supported this work.
Cyclophilin A and MIP‐2 synergize to promote greater neutrophil migration
Authors: Heine Shannon Jennifer , Olive Denise S. , Constant Stephanie L. 
Published: June 2021

Chemokines induced during inflammatory diseases contribute to the inflammatory response by inducing leukocyte migration and extravasation. Extracellular cyclophilins are an alternative group of proteins with chemokine activity. In previous studies, we have shown that extracellular cyclophilins are highly elevated during inflammatory responses and their inhibition reduces leukocyte infiltration by 40–50%. Such a major contribution by cyclophilins to leukocyte recruitment led us to investigate whether they might function synergistically with other chemokines. Using a mouse model of acute lung inflammation we first established the chemokines present concurrently with cyclophilin A (CypA) in inflamed lung airways and selected two (MIP‐2 and Gro1‐α) for further testing. Using in vitro Boyden chamber chemotaxis assays we determined that MIP‐2 synergizes with CypA to induce augmented neutrophil migration, but that Gro1‐α does not. The observed in vitro synergistic effects were confirmed in vivo by co‐injection of CypA and MIP‐2 into air pouches. These data provide evidence that CypA has the capacity to synergize with other chemokines present during inflammatory responses, potentially explaining the significant reduction in cell recruitment observed when cyclophilin activity is inhibited in vivo.This work was funded by NIH AI067254.
Dendritic cells infected with Listeria monocytogenes undergo a maturation response distinct from neighboring uninfected dendritic cells
Authors: Cook Anne Stanner 
Published: June 2021

Dendritic Cells (DC) are considered the sentinels of the immune system due to their ability to activate naïve T‐cells, which is dependent on their maturation state. Listeria monocytogenes (L.m.) induces a maturation response marked by costimulatory molecules, cytokines, and T cell priming in a population DC if bacteria enter the cytosol. However, microscopic examination of L.m.‐infected DC both in vitro and in vivo revealed that a larger percentage of uninfected DC expressed CD86 than infected DC. Two color flow cytometric analysis of fluorescently labeled L.m. vs. intracellular IL‐12 confirmed that the majority of infected cells failed to express IL‐12 while the majority of uninfected cells produced IL‐12. However, infected DC expressed IL‐6, TNFα, and CD40, while uninfected DC expressed CD86, CD80, and IL‐12. Based on these findings, we hypothesize that Lm infected DC undergo a different maturation response than neighboring uninfected DC. Consistent with this hypothesis, we have shown that uninfected DC can be stimulated to produce IL‐12 and CD86 when treated with supernatant from Lm‐treated DC. Additionally, uninfected DC required less antigen to stimulate naïve CD 8+ T cells than infected DC. These data illustrate the cooperative nature of the immune response to a bacterial infection.Research supported by NIH/NIAD grant 1R01AI057770‐01A1
Highly pure, viable and functional intact human Natural Killer (NK) cells isolated in a single isolation step using Dynabeads® FlowCompTM Technology
Authors: Schjetne Karoline Western , Kullman Anette , Møller Anne Sophie , Kierulf Bente , Lycke kirsten , Karlsson Malin , Neurauter Axl 
Published: June 2021

BACKGROUNDNK cells contribute to a variety of innate immune responses to viruses, tumors and allogeneic cells. In humans, NK cell express CD56. However, this surface marker is not exclusive for NK cells and separation technology focusing on this marker will potentially result in contamination of non‐NK cells or involve several isolation steps. Here we present a single step, positive isolation protocol for human NK cells.METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from healthy individuals. NK‐specific antibody conjugated to a modified biotin was added and incubated at 2–8 °C. In the next step modified streptavidin coated Dynabeads were added and placed in the magnet after incubation at room temperature. After resuspension with FlowComp release buffer the bead‐free cells were analyzed on flow cytometry and tested in cytotoxicity assays.RESULTS: Highly pure human CD56+CD3− NK cells were isolated from healthy blood donors using Dynabeads® FlowComp™ Human NK Cell Kit. Using the single step positive isolation protocol, very few CD3+ NKT/T cells were present after isolation and viability was >95%. Isolated NK cells were used in functional assays where LAMP‐1 expression was assayed by flow cytometry and where cytotoxicity was measured in a 51Cr release assay using K562 as target cells. We demonstrate that isolated NK cells were activated and kill target cells.
Expression and Purification of native TL1A (TNFSF15) to generate TL1A Aptamers.
Authors: Majid Ayaz Mahmood , Deyev Vadim , Podack Eckhard R 
Published: June 2021

The TNF‐like cytokine TL1A (TNFSF15) has gained significant immunological interest since its characterization as a ligand for TNFRSF25. We have shown that TL1A is intimately involved in the pathogenesis of inflammatory lung disease. Moreover, TL1A up‐regulation on macrophages and lymphocytes has been implicated in human inflammatory bowel disease (IBD) and rheumatoid arthritis.The pMAL‐2 protein fusion and purification system (New England Biolabs) was initially invented for obtaining recombinant protein targets in E. coli. We have devised a strategy to utilize that system in eukaryotic cells to express native TL1A. Since active TL1A is proposed to be in a soluble form, an expression cassette containing the IgK secretion signal, maltose binding protein (MBP) and human TL1A (hTL1A) was created in a pBMG‐Neo plasmid. Transfection and selection of NIH 3T3 cells or 293SF suspension cells resulted in active secretion of MBP‐hTL1A as shown by an ‘in‐house’ TL1A ELISA, and MBP‐based Western blots. Secreted MBP‐TL1A proteins were purified on Amylose columns under serum free conditions and analyzed by SDS‐PAGE or Agilent protein electrophoresis. Furthermore, an enterokinase cleavage site allowed cleavage of hTL1A from MBP. Chromatography techniques were applied to further investigate native TL1A monomers and trimeric complexes. These targets will be utilized to generate TL1A aptamers using SELEX based or capillary electrophoresis applications. Selected aptamers may allow further understanding of TL1A immunology and provide useful immunotherapeutic interventions.This work was supported by NIH grants; AI061807 and AI068515.
The plasma concentration‐time profile of beta‐alanine using a controlled‐release formulation (Carnosyn®)
Authors: Harris Roger C , Jones Glenys , Wise John A 
Published: June 2021

Carnosine (β‐Alanylhistidine) synthesis in muscle cells is limited by the availability of β‐Alanine, but can be increased 80% or more when the diet is supplemented with β‐Ala. This is of interest to athletes since increased levels of muscle Carnosine have been shown to improve exercise performance. Above 10mg/kg bwt, however, β‐Ala in solution or rapidly‐dissolving capsules causes symptoms of paraesthesia, despite the amount being less than that available from normal‐size portions of meat.PURPOSETo investigate β‐Ala absorption using a controlled‐release formulation.METHODThree males and 3 females were given 1600mg β‐Ala dissolved in water (C), 2 × 800mg β‐Ala controlled release tablets (T2) and 4 tablets (T4), on separate occasions. Controlled release tablets were Carnosyn™ supplied by Collegiate Sports Nutrition. Plasma from 100μl capillary blood samples was collected over 6h, initially every 0.25h, and analysed for β‐Ala by HPLC.RESULTSSubjects reported significant paraesthesia with C, no symptoms with T2 and zero/trace symptoms with T4. Peak β‐Ala (P) with C was 219±78μM with return to ~10uM at 3h. Increases in P were greatly attenuated with T2 (126±36μM) and T4 (177±123μM) but greatly extended (>50μM at 3h). Areas under the plasma concentration curve were C: 286±48; T2: 309±154; T4: 518±220 μM.h. with C or T2 vs T4: P<0.05; C vs T2: P>0.05.CONCLUSION: T2 was bioequivalent to C but did not cause paraesthesia.
Expression and localization of connexin 43 in glioblastoma
Authors: Gould Peter Vincent , Cottin Sylvine Carrondo , Caruso Manuel , Cantin Léo 
Published: June 2021

The bystander effect is essential to the success of the Herpes simplex virus thymidine kinase (TK)/gancyclovir (GCV) strategy for cancer therapy. It consists of the transfer of phosphorylated GCV from TK+ cells to neighbouring TK− cells via gap junctions made up of connexin molecules. Down‐regulation and/or perinuclear localization of connexins should decrease the efficacy of TK/GCV treatment. The status of Cx43, the connexin expressed in astrocytes, in glioblastoma is unclear. In this study we have evaluated Cx43 expression, localization and functionality in glioblastoma cell lines, biopsies and primary cell cultures.In three glioblastoma cell lines, Cx43 was the major connexin subtype, and accumulated mainly in a perinuclear location. Nevertheless, gap junction permeability was preserved and a strong bystander effect was seen. Cx43 expression was observed in 77% (57/74) of glioblastoma biopsies and seven of eight primary cell cultures derived from surgical resection. Four of seven cultures showed cytoplasmic localization of Cx43 with preservation of gap junction permeability. These results suggest that glioblastoma is a suitable candidate for the TK/GCV approach. Supported by the CTECM/FRSQ/CIHR.
Asymptomatic AD is associated with hypertrophy of CA1‐hippocampus neurons in the NUN study
Authors: Iacono Diego , Pletnikova Olga , Rudow Gay , Patel Ela , Tudor Dianna , Markesbery William , Snowdon David , Troncoso Juan C 
Published: June 2021

Objective: a) to measure differences in the volume of CA1 hippocampal neurons, and their nuclei and nucleoli in four different groups: subjects with Alzheimer's Disease (AD) pathology and no cognitive deficits (ASYMAD), mild cognitive impairment (MCI), definite AD (AD) and age‐matched controls (C).Introduction: the pathology of AD evolves decades before the disease becomes clinically manifest. Consequently, it is common to find substantial amounts of Aβ ‐plaques (NP) and neurofibrillary tangles (NFT) in autopsy brains of older subjects with normal cognition briefly before death (ASYMAD).Methods: we used designed‐based stereology to measure the volume of neuronal cell bodies, nuclei, and nucleoli in CA1. We examined postmortem brains of 13 C, 10 ASYMAD, 5 MCI, 10 AD subjects from the NUN study.Results: We found a significant hypertrophy of the neuronal cell bodies, nuclei, and nucleoli in CA1 of ASYMAD subjects compared to controls (p<0.01) and MCI (p<0.01). The hypertrophy of CA1 neurons in ASYMAD may represent a very early pathologic effect of A‐beta and tau on neurons or a reflection of cellular mechanisms that prevent the progression of the disease into dementia. Studies of the correlation of ApoE genotypes and ASYMAD are in progress.
Reversible posterior leukoencephalopathy syndrome: Report of 3 autopsy cases
Authors: Takei Hidehiro , Bhattacharjee Meenakshi B , Fuller Gregory , Powell Suzanne Z 
Published: June 2021

Background: Reversible posterior leukoencephalopathy syndrome (RPLS) is an increasingly recognized clinicoradiological entity associated with multiple pathologies, the most common being hypertensive crisis. Because of the “reversible” nature of this syndrome, few autopsy/histological findings have been reported to date.(Case 1) A 48 y/o woman with a history of allogeneic renal transplant and hypertension presented with headache, blurred vision, confusion and died of pneumonia. (Case 2) A 48 y/o man who had a history of malignant lymphoma treated developed seizures and died of fungal pneumonia. (Case 3) A 74 y/o woman with a past medical history significant for hypertension, idiopathic thrombocytopenic purpura treated with splenectomy followed by Rituximab and steroid died of multiorgan failure. Radiologically, these 3 patients showed MR image findings characteristic of RPLS. Postmortem examination of these brains revealed leukoencephalopathic changes including diffuse white matter rarefaction with mild gliosis (Case 1) and relatively well‐circumscribed perivascular rarefied foci without gliosis (Cases 2, 3).Discussion: Our cases support the two proposed pathophysiologic mechanisms for PRLS; a breakdown in cerebrovascular autoregulation with ensuing interstitial accumulation of fluid (Cases 1), and cerebral vasospasm with resulting ischemia within the involved territories (Cases 2, 3).
TDP‐43 immunoreactivity in anoxic, ischemic and proliferating lesion of the central nervous system
Authors: Lee Edward B. , Lee Virginia M.‐Y. , Trojanowski John Q. , Neumann Manuela 
Published: June 2021

TDP‐43 proteinopathies are a newly categorized group of neurodegenerative diseases characterized by progressive cognitive and motor impairments associated with the abnormal accumulation and mislocalization of the nuclear TAR‐DNA‐binding protein‐43 (TDP‐43) protein in neurons and glia. Little is known about the expression and distribution of TDP‐43 in normal and pathologic states. A panel of clinical neuropathologic specimens was examined for TDP‐43 expression by immunohistochemistry. TDP‐43 inclusions like those seen in neurodegenerative frontotemporal dementia and motor neuron disease were not seen in a variety of anoxic, ischemic and proliferating lesions. However, TDP‐43 was found in Rosenthal fibers and eosinophilic granular bodies associated with low grade tumors and reactive brain tissue. Furthermore, cytoplasmic TDP‐43 was seen in M‐phase tumor cells, but not in mitotic spindles. These findings expand our knowledge of the distribution and localization of TDP‐43 in pathologic specimens, and indicate that the TDP‐43 inclusions seen in frontotemporal dementias and motor neuron diseases are specific to a neurodegenerative process.Supported by an intradepartmental grant from the Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania (to EBL) and a grant by the Federal Ministry of Education and Research (BMBF: “Degenerative dementias: Target identification, validation and translation into treatment strategies” to MN).
Investigating DAT uptake inhibitors as potential replacement therapies for cocaine
Authors: Batman Angela , Dutta Aloke , Reith Maarten , Beardsley Patrick M. 
Published: June 2021

There are no approved therapies for cocaine dependence. Our objective is to develop potential replacement medications that possess the subjective effects of cocaine but with longer durations of action and the ability to reduce cocaine self administration.Cocaine blocks the dopamine (DAT), serotonin (SERT), and norepinephrine (NET) transporters. We have reported that our lead compound, D84, a 3‐hydroxy substituted analogue of 1‐[2‐(diphenylmethoxy)ethyl]‐4‐(3‐phenylpropyl) piperazine (GBR 12935), is one of the most potent and selective inhibitors of the DAT so far disclosed [Ki's (nM): 4.05 (DAT):1274 (SERT):164 (NET)]. D84 possesses several of our target properties and here we continue its evaluation, as well as that of new analogues. D84 dose‐dependently (9.6–30.4 mg/kg) reduces cocaine self‐administration in rats. Previously undisclosed compounds, D225, D232, and D233, elevated locomotor activity for several hours, and occasioned cocaine's discriminative stimulus effects. Compound D232 was almost 10 fold more potent than D233 in this latter regard, perhaps attributable to its greater SERT potency. The results show these new GBR 12935 analogues possess features desirable for a cocaine replacement therapy.This research was supported by NIDA Grant DA012449, A. Dutta (PI).
The effect of paraquat on the expression of D2 dopamine receptor in CAD cells
Authors: Kim Ok‐Jin J , Megaris Michael Paul , Azadani Zahra Crystal 
Published: June 2021

We identified a protein kinase Czeta interacting protein (ZIP1) as an interacting protein of the D2 dopamine receptor (DAR) through a yeast two hybrid assay. We confirmed that the functional interaction between D2 DAR and ZIP1 in human embryonic kidney 293 (HEK 293) cells by using immunoprecipitation/western blot analysis, cAMP assays and radioligand binding assays. Significantly, we also demonstrated that D2 DAR and ZIP1 were coimmunoprecipitated from mouse brain and colocalized in mouse brain. The role of ZIP1 has not been clearly elucidated although overexpression of ZIP1 resulted in the reduction of membrane associated D2 DAR in HEK 293T cells. Our studies also demonstrated that ZIP1 was induced by an herbicide paraquat in HEK 293T cells and Cath.a.Differentiated (CAD) dopaminergic neuronal cells. The expression of membrane associated D2 DAR was reduced in CAD cells stably transfected with D2 DAR (CAD‐D2 DAR) in the presence of paraquat by radioligand binding assays. Subsequent radioligand binding assay and Western blot analysis studies also appeared to indicate that paraquat reduces the expression of membrane associated D2 DAR in the presence of ZIP1 in CAD‐D2 DAR cells. The further characterization of the effect of paraquat on the expression and trafficking of D2 DAR are still in the progress.This work was supported by the University of Kansas General Research Fund to OJK and Undergraduate Research Assistantship fund to MM.
Selective regulation of chemokine CXCL6 by estrogen receptorβ (ERβ)
Authors: Scotland Ramona Sumintra , Roy Bornali , Duchene Johan , Ahluwalia Amrita 
Published: June 2021

Endothelium‐derived chemokines are integral mediators of inflammatory cell recruitment and have been implicated in the pathogenesis of cardiovascular disease (CVD). Estrogens are thought to underlie the protective effects of female sex against CVD, although the exact mechanisms of this effect are uncertain. We investigated the hypothesis that estrogens inhibit endothelial chemokine production in response to an inflammatory stimulus.qPCR of human umbilical vein endothelial cells (HUVECs) confirmed presence of ERα (46kD variant), ERβ and GPR30 mRNA. IL‐1β (1ng/ml, 8h) treatment elevated CXCL1 (Groα, 370‐fold), CXCL5 (ENA78, 310‐fold), CXCL6 (GCP2, 1030‐fold), CXCL8 (IL8, 150‐fold), and CCL2 (MCP1, 30‐fold) expression in HUVECs. Whilst pre‐treatment of cells with the ERα agonist 4,4′,4″(4‐Propyl‐[1H]‐pyrazole‐1,3,5‐triyl)trisphenol (PPT, 1nM, 16h) had no effect on IL‐1β‐induced chemokine expression, treatment with the ERβ agonist 2,3‐bis(4‐hydroxyphenyl)‐propionitrile (DPN, 1nM, 16h) suppressed CXCL6 expression only (P<0.01, n=7). This effect was abolished by 1h pre‐treatment with ER antagonist ICI 182,780 (100nM, n=5).Thus our results show that activation of ERß but not ERα selectively inhibits endothelial CXCL6 expression; a response that likely contributes to the anti‐inflammatory actions of estrogens in vivo.Supported by Wellcome Trust.
Synthesis and theoretical calculations of 5‐aminosalicylic acid derivatives as potential analgesic agents
Authors: Correa Jose , Ramirez Eduardo , Badillo Abigail , Gomez Carlos , Fabila Humberto L , Trujillo Jose G 
Published: June 2021

5‐Aminosalicylic acid is one of the drugs most commonly used for inflammatory bowel disease treatment, although its use is limited due to side effects. The aim of this work was to synthesize four 5‐ASA derivatives (1–4) and analyze their pharmacological effects. The compound structures were elucidated by spectral (IR and 1H and 13C‐NMR) analysis, and their analgesic effects and lethal doses 50 (LD50) were evaluated in the mouse model. In addition, their Log Ps and affinities for both cyclooxygenase enzymes (COX I and COX II) were evaluated through theoretical calculations.All compounds showed analgesic activities from 0.1 mg/Kg to 16 mg/Kg in the mouse model. The imides showed more affinity by COX enzymes and their Log Ps were the highest. The docking calculations showed that all compounds have good affinities for COX I and COX II ((1 ?M), making ????, van der Waals interactions and hydrogen bonds. The toxicities of all compounds were low, judging by the LD50.Finally, the docking analysis show that the compounds act on COX enzymes and their analgesic effects could be mediated in part by the inhibition of these enzymes.