bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phosphorylation of the calpains
Authors: Vazquez Randy , Wendt Amanda S , Thompson Valery F , Novak Stefanie M , Ruse Cristian , Yates John R , Goll Darrel E 
Published: June 2021

The calpains are phosphorylated and phosphorylation affects their activities, but no attempts have been made to determine which calpain resides are phosphorylated. Ca2+ requirements of the calpains are much higher in in vitro assays than would ever be encountered in living cells, and cells must contain a mechanism to reduce this requirement; the crystallographic structure of m‐calpain suggests that phosphorylation at selected residues in either domain IIa or IIb of the calpains may lower their Ca2+ requirement. We have used anti‐phospho‐Tyr, anti‐phospho‐Ser, and antiphospho‐Thr antibodies and mass spectrometry to characterize phosphorylation of calpains purified from bovine skeletal muscle or human placenta. Anti‐phospho antibodies show that both μ‐ and m‐calpain have at least 6 phosphorylated sites; 2 each phospho‐Tyr, phospho‐Ser, and phospho‐Thr. The N‐terminal part of the 80‐kDa subunit from residues 6‐266 (μ‐calpain) or residues 9‐265 (m‐calpain) have at least 1 each phospho‐Tyr, phospho‐Ser, and phospho‐Thr. The C‐terminal part of the 80‐kDa subunit from residues 245 to 714 (μ‐calpain) or from residues 265‐700 (m‐calpain) also have at least 1 phospho‐Tyr, 1 phospho‐Ser, and 1 phospho‐Thr. Mass spectrometry has thus far identified 4 phosphorylated sites in the 80‐kDa subunit of μ‐calpain (only 1 in the N‐terminal 266 residues), 8 phosphorylated sites in the 80kDa subunit of m‐calpain (5 in the N‐terminal 265 residues), 1 phosphorylated site in the small subunit of μ‐calpain, and 3 phosphorylated sites in the small subunit of m‐calpain. Four of the sites identified are in domains IIa or IIb.Supported by NIH and MDA
TTC4 provides a functional link between RASSF1C and DNA primase
Authors: Dmitriev Ruslan , Shakhparonov Mikhail I , Pestov Nikolay B 
Published: June 2021

TTC4 protein was identified as a putative tumor suppressor since its gene is often mutated in some cancers. Also, its drosophila homologue was shown to interact with DNA primase (p180). Recently, we observed that murine TTC4 interacts with hampin, a component of MYST1 histone acetyltransferase complex. This interaction may provide a link between DNA primase (p180) and other tumor supressor proteins, such as RASSF1C, in the complex interactome network. We observed that in cultured 3T3 fibroblasts and other mammalian cell lines TTC4 localizes during cell cycle progression similarly to DNA primase (p180). In addition, the nuclear pool of endogenous TTC4 has a punctate pattern partly co‐localizing with endogenous RASSF1C and PML nuclear bodies.Upon overexpression, TTC4 has almost exclusive cytoplasmic localization and its transport to nucleus occurs only in the presence of overexpressed RASSF1C. Interestingly, overexpressed TTC4 with RASSF1C also co‐localize on microtubules. Therefore, RASSF1C appears to be required for nuclear sorting of TTC4. We propose that this regulation of TTC4 nuclear sorting by RASSF1C may be important for its association with DNA primase (p180) and, therefore, may constitute a part of unknown signaling pathway leading to regulation of DNA synthesis.
Molecular Approaches to Promote Wound Healing in Sulfur Mustard Exposed Human Keratinocytes In Vitro: Role of Nitric Oxide in Wound Healing
Authors: Ishida Hiroshi , Ray Radharaman , Ray Prabhati 
Published: June 2021

Sulfur mustard (SM), a chemical warfare agent, produces burns with sever skin blistering. Promoting re‐epithelialization is a challenging issue in the SM skin injury treatment. We reported that human inducible nitric oxide (NO) synthase (iNOS) was a factor for skin wound healing after SM exposure in the normal human epidermal keratinocytes (NHEKs) scratching‐wound model (Ishida et al., J Dermatol Sci, 2007). In this study, we carried out experiments to further elucidate the roles of iNOS/NO by manipulating iNOS gene expression with siRNA and adenovirus constructs for gene knockdown and overexpression, respectively. NOC‐12, a NO donor drug and L‐NIL, an iNOS inhibitor, were also tested to study the roles of iNOS. The siRNA transfection significantly reduced both the gene and protein expression levels followed by a complete inhibition of wound healing. Infection of NHEKs with the adenovirus constructs resulted in a marked increase of iNOS expression in a time‐ and dose‐dependent manner. NOC‐12 reversed wound healing suppressed by the siRNA or L‐NIL; however, neither overexpression nor NO donor application caused significant improvement of wound healing inhibited by SM.These statistics suggest that SM causes not only the pathway involved NO production, but also another pathway independent from NO production.This work was supported by JSTO‐CBD/DTRA award # 2.T0002_05_WR_C to Prabhati Ray.
Phosphorylation of the autoinhibitory insert in endothelial nitric oxide synthase promotes calmodulin binding at low free Ca2+ concentrations
Authors: Tran Quang‐Kim , Leonard Jared , Black DJ , Persechini Anthony 
Published: June 2021

The 45‐residue autoinhibitory insert in the endothelial nitric oxide synthase (eNOS) reductase domain inhibits electron transfer, and this effect is counteracted when CaM is bound to the enzyme. We have investigated the effects of phosphorylation at Ser‐617 and Ser‐635 in this region on the Ca2+ sensitivities of CaM binding and CaM‐dependent enzyme activation and on reductase and synthase activities. The mean EC50(Ca2+) values (± SD) for CaM binding to bovine eNOS and mutants with S617D, S635D, S617D/S635D substitutions or lacking the entire autoinhibitory insert were determined to be 220 ± 25, 109 ± 7, 202 ± 21, 117 ± 14, or 47 ± 9 nM, respectively. Corresponding mean EC50(Ca2+) values for eNOS activation were found to be 440 ± 55, 240 ± 28, 461 ± 56, 263 ± 28, or 124 ± 16 nM. The S617D substitution and deletion of the insert were found to increase CaM‐dependent synthase and cytochrome c reductase activities 2‐fold, and to respectively increase CaM‐independent reductase activity 3‐ and 6‐fold. The S635D substitution was found to have no significant effect on any property examined. These results indicate that phosphorylation at Ser‐617 acts synergistically with CaM to counteract the effects of the autoinhibitory insert. Given a resting free Ca2+ concentration in endothelial cells of 50 – 150 nM, phosphorylation at this site also appears to promote binding of CaM to the synthase under basal conditions.Source of research support: NIH Grant GM074887.
IL‐6 reveals an intrinsic polyspecific phenotype of the HEL T cell repertoire in BALB/c mice
Authors: Gabaglia Claudia Raja , Menezes Juscilene , Braciak Todd , Sercarz Eli 
Published: June 2021

The T cell repertoire raised by immunization with hen egg lysozyme (HEL) in H‐2d mice has been well characterized by proliferation studies, as presenting a single dominant determinant (peptide108–116) and 2 subdominant determinants (p11–25 and p20–35). T cells directed to other areas on the molecule do not arise after HEL‐CFA immunization as detected in a pepscan by proliferation and, in our hands, by ELISAspot assays.In previous experiments, IL‐6 had been shown to alter the hierarchy of peptide responses to HEL (Drakesmith et al, PNAS, 2000). We therefore immunized BALB/c mice with a CFA emulsion containing HEL with recombinant IL‐6 (or HEL‐CFA co‐immunized with an adenovirus vector containing IL‐6) to ask which T cells would be primed that could be recalled in vitro by a panel of overlapping 15 aa peptides all across the HEL molecule. IL‐6 plus HEL induced co‐dominant responses recalled by pepscan peptides in the regions 103–121, 67–85 as well as 87–103, as measured by proliferation and ELISAspot. Other, weakly subdominant determinants were also detected by proliferation and cytokine analysis in distinct areas: 9–25, 27–45, 37–51 and 47–61. Surprisingly, immunization with p61–85 primed for a response to p106–120 as well as to HEL. T cell hybrids generated after immunization with p106–120 and selected on the basis of reactivity to HEL, confirmed these results in that crossreactive responses to 106–120, 85–101 and 67–85 regions were displayed by public T cell clones. This phenomenon may be more frequent than previously imagined, and in this case extended to three determinants on the same molecule. Such polyspecific clones may achieved dominance owing to their polyreactive phenotypes. In conclusion, the inherent degenerate recognition of particular T cells can be revealed during inflammatory processes upregulated by agents such as IL‐6.Grant support NIH R0142396
Costimulatory Requirements for the Expansion of Human T Regulatory Cells
Authors: Golovina Tatiana Nikolaevna , Mikheeva Tatiana , Shan Xiaochuan , Suhoski Megan , Liu Ronghua , Case Chanelle , June Carl , Riley James 
Published: June 2021

We used cell‐based artificial APCs (aAPC) to explore the costimulatory requirements to expand functional T regulatory cells (Treg) enriched from human peripheral blood. Only CD28 costimulation maintained high levels of FoxP3 expression and suppressive function in enriched T regulatory cultures. Interestingly, CD4 T cells grown in the presence of CD28 costimulation and rapamycin displayed significant in vitro suppressive activity. However, when these cells were evaluated for suppressive activity in vivo, only the cultures that were initially enriched for Tregs suppressed. We investigated whether additional costimulatory pathways could augment CD28's ability to promote further Treg expansion in rapamycin. In a minority of donors OX40 costimulation was able to augment Treg expansion and maintain function; however, in the majority of donors these cultures often lost suppressive function. In contrast, we found that re‐stimulation of the Treg cultures after 8–10 days of culture with CD28 costimulation alone resulted in 1000 fold expansion of Treg in less than 3 weeks. These highly expanded Tregs retained potent in vitro and in vivo suppressive function. These studies indicate the optimal costimulatory requirements to expand Tregs differ from T effector cells and establish the feasibility of using expanded Tregs for adoptive T cell therapy.The project is supported by JDRF for Cell Therapy grant.
Formation of in vivo immunological synapses (IS) between CTLs and virally infected astrocytes leads astrocyte to polarization mediated by the activation of a Rho‐GTPase signaling pathway.
Authors: Lowenstein Pedro R , Puntel Mariana , Barrett Robert , Yang Jieping , Sanderson Nicholas , Bondale Niyati , Assi Hikmat , Kroeger Kurt M , Castro Maria G 
Published: June 2021

Immunological synapses (IS) mediate intercellular communication between T cells and APCs. T cell polarization at the Kupfer‐type IS consists of a peripheral supramolecular activation cluster (pSMAC), which is high in LFA‐1, and a central SMAC, high in TCR. Target virally infected astrocytes also polarize towards the immunological synapse in vivo. Astrocyte polarization in response to physical lesions in vitro is mediated through the activation of a signaling pathway mediated by a family of Rho‐GTPases, and coordinated by Cdc42. Here we tested the hypothesis that that stimulation of a Rho‐GTPase cascade underlies astrocyte polarization in response to an immune attack in vitro and in vivo. To do so, we infected brain astrocytes with a novel adenoviral (Ad) vector expressing a dominant negative variant of Cdc42 (Cdc42DN) and Thymidine Kinase (TK), a marker of infected cells, and analyzed their responses to a T cell attack. We show that during an anti‐viral immune response, infected astrocytes contacted by CTLs become polarized through the activation of a Rho‐GTPase signaling pathway.This work was funded by grants from NINDS/NIH.
Vα14i NK T Cells Participate to the Early Response to MCMV Infection.
Authors: Brossay Laurent , Wesley Johnna D. , Tessmer Marlowe S. , Chaukos Deanna 
Published: June 2021

Immunity to the well‐characterized virus MCMV is critically dependent on the innate response for initial control of viral replication. The contribution of innate immune cells, other than natural killer (NK) cells, to the propagation of an optimal anti‐viral immune response has not been fully examined. Therefore, we sought to determine the role and function of a unique, innate subset of T cells, Vα14 invariant natural killer T (Vα14i NK T) cells, in the anti‐viral response to MCMV infection in vivo. Vα14i NK T cells become activated and produce significant levels of IFN‐γ but not IL‐4 following MCMV infection, particularly in the liver. Adoptive transfers of Vα14i NK T cells in MCMV infected CD1d17−/− mice demonstrate that CD1d1 is not critical for their activation. Interestingly, both IFN‐α/β and IL‐12 are partially required for their activation status. Vα14i NK T cell derived IFN‐γ is highly dependent on IL‐12 but surprisingly partially dependent of IFN‐α. Collectively, these findings highlight the unique activation requirements of these innate T cells in the induction of an anti‐viral immunity.Supported by NIH grant AI46709.
CD70 antibody therapy for the prevention and treatment of experimental Inflammatory Bowel Disease (IBD).
Authors: Manocha Monika , Rietdijk Svend , Laouar Amale , Bhan Atul , Terhost Cox , Swamy Manjunath N 
Published: June 2021

BackgroundInflammatory bowel disease is a chronic disorder of the gastrointestinal tract, characterized by an aberrant mucosal T cell‐mediated inflammation. The costimulatory molecule CD70 is constitutively expressed by a unique antigen presenting cell in the lamina propria and its interaction with T cells is critical for the gut mucosal T cell activation.ObjectiveTo test if blockade of CD70 reduces intestinal inflammation in an experimental model of colitisMethodsColitis was induced by adoptively transferring CD45RBhiCD4+ T cells into Rag 1−/− mice, the mice were treated with control Ig or a blocking anti‐CD70 antibody twice weekly for 6‐8 weeks and the animals were monitored for disease development.ResultsTreatment with CD70 blocking antibody dramatically reduced the colitis‐associated weight loss, clinical symptoms and intestinal hisopathology. Importantly, anti‐CD70 antibody treatment initiated after onset of the disease was also able to partially reverse the features of colitis. The Th‐1 related cytokine IFNγ as well as key proinflammatory cytokines of the Th17 pathway, including IL‐23, IL‐17, IL‐6, and TNFα were significantly reduced after CD70 antibody treatment as compared to control group (p<0.001). Conclusion: This study shows that both Th1 and Th17 related cytokines play a dominant role in the pathogenesis of IBD and that CD70 blockade can effectively suppress these cytokines leading to reduction of inflammation.Source of Research supportCrohn's and Colitis foundation of America (CCFA)
Melan‐A‐specific T cells derived from A2+ or A2‐umbilical cord blood (UCB) units proliferate and exhibit specific cytolytic function following in vitro stimulation
Authors: Merindol Natacha , Gimmig Sylvain , Duval Arnaud , Duval Michel , Champagne Martin A , Soudeyns Hugo 
Published: June 2021

UCB is increasingly used as a source of haematopoietic progenitors to treat a variety of blood disorders requiring bone marrow transplantation. Advantages of UCB include availability and reduced HLA matching requirements as compared with bone marrow. To characterize functional properties of UCB T cells in the context of allogeneic UCB transplant, naïve CD8+ T cells specific for the HLA‐A2‐restricted Melan‐A‐derived peptide ELAGIGILTV were sorted from A2+ and A2− UCB units using A2/Melan‐A tetramers and were cultured in the presence of IL‐2, IL‐7, PHA, and peptide. Microcultures were phenotyped and tested for Melan‐A‐specific cytotoxicity by 51Cr‐release assay. Clonal diversity was estimated by T cell receptor (TCR) β chain sequencing. 83.3% (15/18) of clinical units tested were positively stained by tetramers (median frequency of 0.11% regardless of HLA type). Following in vitro stimulation, the median frequency of tetramer+ T cells was increased (p<0.0001), T cell microcultures exhibited Melan‐A‐specific cytolytic activity (median = 11.98% of positive cultures at an input of 1000 cells/well), and the TCR β chain repertoire showed evidence of oligoclonality consistent with antigen‐driven expansion. This study will bring new light on the ability of the preimmune T cell repertoire to participate in antitumoral and antiviral activity following UCB transplantation.Supported by FRSQ/Héma‐Québec.
Sulfur Amino Acids Are Necessary for Normal Intestinal Mucosal Growth in Neonatal Piglets
Authors: Bauchart‐Thevret Caroline , Stoll Barbara , Chang Xiaoyan , Cui Liwei , Burrin Douglas 
Published: June 2021

Sulfur amino acids (SAAs), methionine and cysteine, play important metabolic and functional role in human health and disease. Gastrointestinal tract is an important site of transmethylation and transsulfuration of methionine and metabolizes ∼20% of the dietary methionine intake (Riedijk et al. PNAS, 2007, 104: 3408). Our aim was to determine intestinal mucosal growth in neonatal piglets (3‐d‐old) enterally‐fed a control diet (n=9) or SAA‐free, elemental, isonitrogenous diet (n=7) for 7 days. After 7 d, both small intestine (jejunum and ileum) mass (−33%; g/kg body weight) and weight gain (−70%; g/kg/d) were decreased in SAA‐free vs. control pigs. Also, protein and DNA mass in jejunum and ileum was also lower in SAA‐free pigs vs. control pigs (−50 to −60%). Histological analysis of jejunum, ileum and colon showed lower (−20 to −40%) cross‐section area, number of villi, villus height and crypt depth in SAA‐free pigs vs. control pigs. Moreover, absolute number of goblet cells and Ki‐67 positive proliferative cells in jejunum and colon were lower in SAA‐free vs. control pigs (−40 to −60%). These results show that SAA deficiency significantly alters intestinal epithelial cell proliferation and survival. We conclude that SAAs are necessary for normal intestinal mucosal growth in piglets.(Supported by ADREN/SFNEP and Nutricia Research Foundation).
Culturing conditions conducive to the differentiation of hESCs along a hepatic lineage
Authors: Zamule Stephanie Mary , Weyant Denise M , Omiecinski Curtis J 
Published: June 2021

The limited availability of hepatic tissue suitable for the treatment of liver disease and for research makes the generation of hepatic‐like cells from alternative sources an attractive proposition. Although a number of studies have demonstrated that human embryonic stem cells (hESCs) are capable of differentiating into hepatic precursors, the methodology has yet to be fully elucidated. To this end, we cultured hESCs for 10 days on collagen substrata in our primary hepatocyte maintenance media comprised of William's E media supplemented with 25 nM dexamethasone, 10 nM insulin, 5 ng/ml selenium, 5 μg/ml transferrin, and 1% linoleic acid. Using quantitative RT‐PCR assays, we found that hESCs cultured under these conditions exhibited a 2‐fold decrease in mRNA levels of the stem cell pluripotency marker, Oct4, a ~30,000‐fold increase in levels of the endodermal marker, α‐fetoprotein, and a ~475‐fold increase in the hepatic marker, albumin. Further, levels of the constitutive androstane receptor, CAR, were increased ~530‐fold, to levels comparable to those in primary cultures of human hepatocytes. These results define a novel, straightforward methodology that may be useful in facilitating hESC differentiation along the hepatic lineage within a relatively short period of time and provide a framework for subsequent studies seeking to develop a renewable supply of human hepatocytes for research applications.Supported by NIH grant, GM66411.
Shear stress‐dependent suppression of endothelial angiopoietin‐2 is mediated via transcription factor FOXO1
Authors: Pries Axel Radlach , Chlench Sven , DaSilva‐Azevedo Luis , Hoffmann Christian , Hohberg Margret , Baum Oliver , Bongrazio Mauro , Zakrzewicz Andreas 
Published: June 2021

While vascular endothelial cells are normally exposed to shear stress, there is no flow and consequently reduced shear stress in capillary sprouts. To explore, if this lack in mechano‐stimulation alters the endothelial phenotype, we analysed the influence of shear stress on the activity of transcription factor FOXO1 and the expression of its target gene angiopoietin‐2 (ang2), an antagonist of the endothelial tyrosine kinase receptor tie2.Endothelial cells were exposed to shear stress using a cone‐and‐plate system with or without inhibition of PI3K (using LY294,002) or FOXO1 (using siRNA) and analysed by real time RT‐PCR, Western blots, immunoprecipitation, and fluorescence microscopy.Shear stress (6°dyn/cm2) increased phosphorylation of Akt and FOXO1 in a PI3K‐dependent manner and FOXO1 was translocated out of the nucleus. In addition, expression of FOXO1 on the mRNA‐ and protein level was strongly reduced by shear stress. Suppression of FOXO1 by shear stress was accompanied by a decrease of ang2‐mRNA and ‐protein, which was similarly be achieved by siRNA against FOXO1. Following flow stop, ang2‐mRNA expression increased within two hours to steady state values under static conditions.So, in capillary sprouts, lacking shear stress activates endothelial FOXO1 by which ang2 is rapidly induced. Thus, the tie2‐system will be inactivated during capillary sprouting, which may turn the vessel wall more susceptible to additional angiogenic factors. By contrast, the onset of flow will activate the tie2‐system and thereby contribute to stabilize the vessel wall.
Uptake of Zn2+ across the basolateral membrane of the gastric parietal cell is regulated by [Ca2+]i
Authors: Liu JingJing , Mathew Jeff , Kohler Jonathan , Blass Amy , Soybel David 
Published: June 2021

Aims: Our recent work has suggested that basolateral to apical movement of Zn2+ across the gastric parietal cell (PC) is matched to its secretory state. In this study, we tested the hypothesis that changes in [Ca2+]i may regulate uptake of Zn2+ across the basolateral membrane.Methods: Isolated rabbit gastric glands were loaded with a Zn2+‐sensitive fluorescent dye, fluozin‐3. Cytoplasm loading of Zn2+ in PCs was monitored during a 15 min exposure to solutions containing a “free” [Zn2+] of 10μM. To lower [Ca2+]i, glands were pre‐treated with Ca2+‐free Ringer's (containing 0.3mM EGTA) and thapsigaragin (TG, 2μM), which irreversibly depletes intracellular stores of Ca2+ and opens store‐operated channels (SOCs) that permit Ca2+ to escape from the cytoplasm. To raise [Ca2+]i, glands were pre‐treated with Ca2+‐Ringer's and TG, which permits Ca2+ loading of the cytoplasm through the SOCs.Results and Conclusions: When exposed briefly to Ringer's without TG (n= 21), [Zn2+]i increased 157% ± 2.4 above baseline, indicating PCs have a capacity for Zn2+ uptake. Exposure to Ca2+‐free Ringer's containing TG abolished this response (3.75%± 0.2, n= 42). When exposed to Ca2+‐containing Ringer's and TG, [Zn2+]i increased 60% ± 0.6 (n=25), indicating that extracellular Ca2+ partially restores Zn2+ loading and that uptake of Zn2+ across the basolateral membrane of the PC is regulated by [Ca2+]i.