bluuebunny/crossref_metadata_2025_split huggingface dataset:
Runx1 Deficiency Predisposes Mice to T-Cell Lymphoblastic Lymphoma.
Authors: Kundu Mondira , Compton Sheila , Garrett Lisa , Stacy Terryl , Starost Mathew , Eckhaus Michael , Speck Nancy A. , Liu Pu Paul 
Published: October 2019

Abstract
Chromosomal rearrangements affecting RUNX1 and CBFB are common in acute leukemias. These mutations result in the expression of fusion proteins that act in a dominant negative manner to suppress the normal function of the CBF β/RUNX1 complex. In addition, loss-of-function mutations in RUNX1 have been identified in sporadic cases of acute myeloid leukemia (AML) and in association with familial platelet disorder with propensity to develop AML (FPD/AML). In order to examine the hypothesis that decreased gene dosage of RUNX1 may be a critical event in the development of leukemia, we treated chimeric mice generated from Runx1lacZ/lacZ embryonic stem (ES) cells that have homozygous disruption of the RUNX1 gene, as well as Runx1+/lacZ mice with N-ethyl-N-nitrosurea (ENU). The heterozygous Runx1+/lacZ mice did not show increased incidence of any malignancy. On the other hand, we observed an increased incidence of precursor T-lymphoblastic lymphoma in Runx1lacZ/lacZ compared to wild-type chimeras, and confirmed that the tumors were of ES cell origin. It was determined by PCR that Runx1lacZ/lacZ ES cells contributed to the T cell progenitor population in the chimeras prior to leukemia development, which may explain the tissue-specificity of the malignancy we observed. Our results suggest that deficiency of Runx1 can indeed predispose mice to hematopoietic malignancies.
Gender-Dependent Up-Regulation of the VWF-Cleaving metalloprotease ADAMTS-13 in Mice with Obesity and Hypercholesterolemia.
Authors: Liu Anne , Wu Huaizhu , Tao Zhenyin , Choi Hiuwan , Bergeron Angela , Ballantyne Christie M. , Dong Jing-Fei 
Published: October 2019

Abstract
The development of atherothrombosis is associated with hypercholesterolemia and systemic inflammation. Recent studies have shown that adipose tissue may directly promote inflammation. Since one of the hallmark events of atherosclerosis is the deposition of von Willebrand factor (VWF) on vascular endothelial cells, the release and proteolysis of ultra-large (UL) and hyperreactive VWF may play an important role in the development of thrombotic diseases. We therefore investigated how the synthesis of ADAMTS-13, the newly characterized metalloprotease that cleaves the hyperreactive ULVWF to smaller and less active form, may be regulated in mice with obesity and hypercholesterolemia. Wild-type C57BL/6 and Apo E −/− C57Bl/6 mice (18 in each group with equal gender distribution) were fed with regular Chow or western-type high fat diet for 6 months and then analyzed for ADAMTS-13 mRNA in the liver (by quantitative PCR), the primary site of ADAMTS-13 synthesis, and ADAMTS-13 activity under flow condition. We found that ADAMTS-13 mRNA was more than doubled for wild-type mice on high fat diet than those on regular diet (4.14±0.46 vs. 1.72±0.57 units, respectively, p < 0.01). The increase was 47.9% for Apo E−/− mice on high fat diet compared to mice on regular diet (2.61±0.31 vs. 3.86±0.72 units, respectively, p = 0.05). When the data were stratified for gender difference, the increase was primarily observed in male mice. For male mice, ADAMTS-13 mRNA on high fat diet was more than four and two folds of those on regular diet for wild-type and Apo E−/− mice, respectively. , For female mice, however, the increase was less than 40% and only detected in wild-type mice fed with high fat diet. Consistent with this observation, ADAMTS-13 activity in general was lower in female (61±12%) than males (81±10%, p < 0.05). For both wild-type and Apo E−/− mice, we detected no difference in ADAMTS-13 activity in males, but a significantly lower activity in the female Apo E−/− mice on high fat diet (19.6±10.5% vs. 49.1±9.2% for high fat and regular diets, respectively, p < 0.05). These results demonstrate that the synthesis of ADAMTS-13 is up-regulated in obese mice (wild-type on high fat diet) and mice with hypercholesterolemia (Apo E−/− mice on high fat diet). The increase may serve as a protective measure to encounter the likely increase in ULVWF release due to systemic inflammation and endothelial cell stimulation seen in these mice. Interestingly, male mice showed a much stronger up-regulation than females. The low rate of ADAMTS-13 synthesis may contribute to the lower ADAMTS-13 activity found in female mice, suggesting that the machinery of ULVWF proteolysis in female mice may be less responsive to obesity/hypercholesterolemia and systemic inflammation.
Oral Mucositis Incidence and Severity after Methotrexate and Non-Methotrexate Containing GVHD Prophylaxis Regimens.
Authors: Cutler Corey , Li Shuli , Kim Haesook T. , Laglenne Patricia , Ford Colleen , Ho Vincent , Lee Stephanie J. , Alyea Edwin , Soiffer Robert , Antin Joseph H. , Sonis Stephen 
Published: October 2019

Abstract
Oral mucositis (OM) occurs in up to 75% of recipients of high-dose chemoradiotherapy conditioning regimens prior to allogeneic hematopoietic stem cell transplantation (HSCT). OM affects patient quality of life by causing oropharyngeal pain, and by impairing communication and swallowing. As a result, narcotic analgesia and total parenteral nutrition (TPN) are commonly required in the recovery period after HSCT. Methotrexate (MTX), an antiproliferative agent used as GVHD prophylaxis, impairs mucosal regeneration after conditioning-related injury, and worsens and prolongs OM. We assessed the impact of sirolimus, a novel immunosuppressive agent used in lieu of MTX for GVHD prophylaxis, on outcomes associated with OM.
Methods: Two cohorts of patients were prospectively analyzed for OM severity, and chart reviews were performed to assess correlative outcomes. All patients underwent HLA-matched sibling PBSC transplantation after Cy-TBI conditioning. GVHD prophylaxis consisted of sirolimus/tacrolimus (ST) in the study group and tacrolimus/methotrexate (TM) in the control group. OM was assessed 3x/week using a prospectively validated 6-point assessment tool, by members of the Oral Medicine Service. At each assessment, the evaluators systematically evaluated for the presence of erythema and ulceration of the oral mucosa at nine pre-defined locations within the oral cavity. The use of narcotics and TPN was recorded from the time of transplantation to hospital discharge.
Results: 30 patients received ST and 24 patients received TM as GVHD prophylaxis after HLA-matched PBSCT between 10/2000 and 5/2003. The two groups were balanced for demographic variables, including age, sex, and disease status at transplant. OM severity was reduced in the ST group. Mild, moderate and severe OM was noted in 37, 57 and 7% of the ST group and 8, 42 and 50% of the TM group (p=0.0002). As a result, TPN use was reduced in the ST group (17 vs. 43% of hospital days, p=0.02), and a higher proportion of subjects required no TPN in the ST group (47 vs. 21%, p=0.08). The total number of hospital days where narcotics were required for pain control was lower in the ST group in comparison with the TM group (14 vs. 16 days median, p = NS). The time from transplant to first hospital discharge was shorter in the ST group compared with the TM group (18 vs. 22 days median, p= 0.07)
Conclusions: The use of ST for GVHD prophylaxis is associated with less severe oropharyngeal mucositis than TM, despite equivalent conditioning regimen intensity. As a result, TPN use was reduced, narcotic use was reduced and the duration of hospitalization was shortened. The use of less toxic GVHD regimens without MTX may have significant impact on patient quality of life, patient outcomes and economic outcomes associated with allogeneic stem cell transplantation.
An Externally Active Endogenous Platelet Galactosyl Trasferase (Gal-T).
Authors: Hoffmeister Karin M. , Wandall Hans H. , Sankaranarayanan Vasanthi , Clausen Henrik , Stossel Thomas P. , Hartwig John H. 
Published: October 2019

Abstract
Over 30 years ago, Jamison and Barber proposed that externally disposed glycosyl transferase (GT) activity mediates platelet adhesion and other functions. Subsequent work ruled out ecto-GT activity in nucleated cells and established the Golgi as the site of such enzymes, although no further studies examined platelets. We recently reported a platelet-associated galactosyl transferase (β4Gal-T) catalyzing galactose coupling in a β1,4 linkage to exposed N-acetylglucosamine (GlcNAc) residues on N-linked glycans of the GPIbα subunit of the von Willebrand factor receptor complex. This activity, also present in plasma, mediates galactose transfer to GPIbα following addition of UDP-galactose (UDP-Gal) alone. Since galactosylation of GPIbα prevents clearance of chilled platelets by liver macrophages after transfusion, sufficiently active endogenous endogenous β4Gal-T could facilitate platelet cold storage after the mere addition of substrate UDP-Gal (Science301: 1531–1534, 2003). We now give evidence that human platelets contain a highly active (0.05 nmol/min) plasma membrane-associated ecto-β4Gal-T with properties of an isoform designated as type 1 (β4Gal-T1). A specific β4Gal-T1 antibody immunoprecipitates β4Gal-T1 activity from platelet lysates and the inhibition profile activity by α-lactalbumin and benzyl-β-D-GlcNAc are characteristic of β4Gal-T1. β4Gal-T1 has a transmembrane domain, and platelet β4Gal-T, although releasable by non-ionic detergent, remains cell- and membrane associated despite extensive washing. The platelet enzyme incorporates UDP-[C14]Gal into a single polypeptide with the apparent Mr of GPIbα, identifying this protein as its unique endogenous target. Nevertheless, washed platelets are as active as recombinant β4Gal-T1 in galactosylating exogenous substrates, benzyl-β-D-GlcNAc and ovalbumin. In conclusion, we have revived the concept of an ecto-glycosyl transferase on blood platelets and demonstrated that platelet β4Gal-T is probably sufficiently active to accommodate reliable enzymatic conversion of platelets by simple addition of UDP-Gal.
Characterization of Regulatory Elements in the 5′-Upstream Region of the Human Gαq Gene in a Human Megakaryocytic Cell Line.
Authors: Gauthami Jalagadugula S. , Dhanasekharan Danny N. , Rao A. Koneti 
Published: October 2019

Abstract
GTP-binding protein Gαq plays a major role in platelet signal transduction. We studied the transcriptional regulation of the human Gαq gene in a megakaryocytic cell line, human erythroleukemia (HEL). 10 nM phorbal myristate acetate (PMA) was used to induce megakaryocytic lineage in HEL cells. Western blot analysis on untreated vs PMA-treated HEL lysates showed enhanced Gαq expression with PMA. Firefly luciferase reporter gene constructs carrying various lengths of the Gαq gene upstream promoter region −1/−2727 bp from ATG codon, were transiently transfected into PMA-treated HEL cells. Gαq promoter driven luciferase expression was measured by Dual Luciferase Reporter Assay system (Promega). These studies indicated that the Gαq gene is regulated by positive and negative elements. Two positive regulatory sites were identified in the proximal region (−138/−238 bp) and distal region (−731/−1116 bp); a negative regulatory site was observed further upstream (−1117/−2727 bp). The proximal region −138/−238 was resolved into a repressor (−207/−238) and a positive (−138/−207) site. Consensus sequences for two well recognized megakaryocytic transcription factors (TFs) PU.1 (−225/−230) and GATA-1 (−208/−211) in the repressor site were deleted; this had no effect on promoter activity. The positive region (−138/−207) was further resolved into repressor (−162/−186) and positive (−187/−207) regions by functional studies. In the repressor region a CCACC (−175/−179) consensus motif for Sp/XKLF family of TF was found and deleted; the repressor activity was lost indicating a functional effect of the CCACC-motif. The positive region contained a Sp1/AP2 consensus sites between −152 and −203. Gel shift and super shift experiments on double stranded DNA oligo 1 (−175/−203) and oligo 2 (−152/−174) using HEL extracts demonstrated nuclear protein binding to these regions. However, antibodies against Sp1 and AP2 or the consensus oligos did not alter the binding. Gel shift mutant analysis was performed on both oligos to define the functional sequences mediating the nuclear protein binding. Mutations made at −190/−194, which has a consensus sequence for EGR1/EGR2 and, at −180/−184 and at −156/−160, abolished the protein binding suggesting that they are preferred sequences for DNA-protein interaction. The distal upstream positive region −731/−1116 also revealed positive and negative elements. Mutation in the EGR-2 consensus sequence (−909/−919) decreased the promoter activity to 50% and mutation in the AP2/EGR-1/Sp1 consensus sequence (−885/−893) abolished the promoter activity. Our results suggest that the sequences −156/−160, −180/−184, and −190/−194 in the proximal region, and −885/−893 and −909/−919 in the distal region have a critical role in the transcriptional regulation of Gαq in megakaryocytic cells.
Differential Agonist Stimulated Cytokine Release from Platelets: A Proteomics Approach.
Authors: Coppinger Judith A. , O Connor Roisin I. , Cagney Gerard , Cox Dermot , Maguire Patricia 
Published: October 2019

Abstract
Dept of Clinical Pharmacology, Royal College of Surgeons in Ireland, Dublin 2 Coppinger JA, O Connor RI, Cagney G, Cox D & Maguire PB. Recent evidence suggests that certain cytokines secreted by activated platelets can promote the development of atherosclerosis and thrombosis e.g the platelet specific cytokine platelet factor 4 (PF4) facilitates macrophage differentiation during inflammation and has shown to be incorporated into atherosclerotic plaques. In this study, we examined the release of several cytokines and other secretory markers from platelets stimulated by three different agonists in the presence and abscene of the anti-platelet drug aspirin. Using antibody array technology we detected the presence of 22 cytokines in the platelet releasate. High levels of abundant platelet cytokines RANTES, PDGF and EGF were seen throughout the arrays for all releasate fractions. The interleukin family were generally underrepresented with the exception of interleukin 8 and GRO. Several interesting angiogenic factors such oncostatin M (not previously reported in platelets) were present in some of the releasate fractions. Higher levels of cytokines were detected in the Collagen and TRAP stimulated fractions than in the ADP fractions suggesting that platelet secretion is an agonist dependent response. The levels of platelet secretory markers thrombospondin and PF4 identified from one dimensional gels by mass spectrometry were stronger in the Collagen and TRAP releasate fractions in keeping with the microarray results. Interestingly, unique protein profiles were obtained for the three different agonists. As platelet activation is COX-dependent we examined the effects of aspirin on cytokine release. Certain cytokines for example GRO were down regulated in the ADP and Collagen treated fractions but no significant decrease in cytokine levels were detected in the TRAP releasate. Cytokines released during platelet activation play diverse roles in vascular biology such as smooth muscle cell proliferation. These results indicate that that the release of platelet cytokines can be in part COX dependent, which may explain some of the anti inflammatory properties of aspirin.
A Possible Mechanism for Synergy in Transcriptional Activation between AML1 and PU.1: Exclusion of the Co-Repressor CBFA2T3 (ETO-2, MTG16).
Authors: Baraoidan Kristine , Ibanez Vinzon , Mittal Chetna , Yimer Habte , Chakraborty Suman , Sinha Kisle , Ackerman Steven , Nucifora Giuseppina , Saunthararajah Yogenthiran 
Published: October 2019

Abstract
AML1 and PU.1, important regulators of hematopoietic differentiation, interact with each other and are known to synergize in transcriptional activation (Zhang et al, 1996). PU.1 and AML1 also interact with mSin3, a component of a co-repressor complex that can include N-CoR, HDAC and CBFA2T1 (ETO, MTG8) or CBFA2T3 (ETO2, MTG16). CBFA2T3 is highly expressed in hematopoietic cells and is a target of a chromosomal translocation found in acute myeloid leukemia (t(16;21)). In transfected 293T cells, we demonstrate that both AML1 and PU.1 co-immunoprecipitate with the conserved N-terminal TAFH domain of CBFA2T3 but not the C-terminal MYND domain. Although AML1 and PU.1 independently co-immunoprecipitate with CBFA2T3, when all three proteins are over-expressed in 293T cells, AML1 and PU.1 co-immunoprecipitate with each other while excluding CBFA2T3. CBFA2T3 interacts with the non-runt portion of AML1 (AML1 C-terminus) while PU.1 can interact with both the runt domain of AML1 and AML1 C-terminus. Presumably, the interaction between AML1 and PU.1 shields the CBFA2T3 binding sites on both proteins. Since this region includes the binding site for mSin3, other co-repressors may also be excluded from an AML1/PU.1 complex. This may be one basis for the co-operation between AML1 and PU.1 in transcriptional activation.
Molecular Characterization of a PU.1 Transcription Complex Formed on the IL-1β Proximal Promoter.
Authors: Hoang Trang , Grondin Benoit , Lefrancois Martin , St Denis Marianne , Tenen Daniel G. , Waga Kazuo 
Published: October 2019

Abstract
The gene coding for the pro-inflammatory cytokine IL-1β is induced at the transcription level in differentiating macrophages and in stress response. Interestingly, PU.1 and C/EBPβ, two transcription factors implicated in IL-1β gene expression are not induced by stress exposure, while c-Jun is strongly induced. Strikingly, this upregulation of c-Jun is required for IL-1β induction, as cells expressing a c-Jun antisense construct fail to respond to stress exposure. We have mapped the induction of IL-1β gene expression to its proximal promoter and show that it is mediated by the transcriptional synergy between C/EBPβ, c-Jun and PU.1 via specific DNA binding sites for C/EBPβ and PU.1 only. To elucidate how PU.1 and C/EBPβ cooperate with c-Jun at the molecular level, we have optimized a DNA binding assay based on IL-1β promoter fragments immobilized on beads to isolate protein complexes from nuclear extracts, which were subsequently eluted and identified by Western blotting. We show that PU.1 or C/EBPβ alone directly bind this promoter fragment via specific sequences while purified recombinant c-Jun fails to do so. However, the presence of either PU.1 or C/EBPβ on the promoter allows for a recruitment of c-Jun to the DNA template, mediated by direct protein-protein interaction. Interestingly, the leucine zipper domain of c-Jun is essential for its interaction with C/EBPβ while dispensable for PU.1 interaction in vitro whereas its basic domain is required for both interactions. Furthermore, we show that PU.1 and C/EBPβ cooperatively bind the IL-1β promoter, resulting in a synergistic recruitment of c-Jun. Finally, we show that the strength of interaction of c-Jun mutants with PU.1 or C/EBPβ determine the strength of transcription output and c-Jun mutants that fail to associate with either PU.1 or C/EBPβ are transcriptionally inactive. In contrast, c-Jun mutants exhibiting increased homodimerization are more active that the wild type protein. Taken together, our data suggest that c-Jun homodimers can be targeted to the IL-1β promoter in the absence of a specific DNA binding element, and conclude that PU.1 and C/EBPβ are specifically tethered to the IL-1β promoter while c-Jun cooperatively binds these proteins and acts as a transcriptional co-activator. We propose a mechanism based on an initial binding of PU.1 and C/EBPβ to the IL-1β promoter followed by a cooperative recruitment of c-Jun, resulting in transcriptional synergy and IL-1β gene expression in stress response.
Identification of Common Target Genes of the BCR/ABL and FLT3-ITD Tyrosine Kinases by Microarray Analysis.
Authors: Banerji Lolita , Scheijen Blanca , Griffin James D. 
Published: October 2019

Abstract
Human myeloid leukaemias are commonly associated with activating mutations in tyrosine kinases. The BCR-ABL oncogene causes CML and is generated by the Philadelphia (Ph) chromosome translocation t(9;22)(q34;q11). The FLT3 tyrosine kinase receptor is mutated in about 30 % of all cases of AML, most often through a mechanism that involves an internal tandem duplication (ITD) of a small number of amino acid residues in the juxtamembrane domain. Both BCR/ABL and FLT3 (ITD) produce a constitutively active tyrosine kinase that enhances proliferation and viability of myeloid cells, and induce a similar myeloproliferative disorder when transplanted into mice. In an effort to identify any common downstream targets of BCR/ABL and FLT3(ITD) we generated paired Ba/F3 cell lines in which either Bcr/Abl or FLT3-ITD could be induced under the control of a tetracycline-inducible promoter. Treatment of these cells with doxycycline rapidly induced expression of BCR/ABL or FLT3-ITD proteins, respectively proteins. The gene expression profile of these conditionally expressing BCR/ABL and FLT3-ITD BaF3cells was examined using the U430A gene chip (Affymetrix), which contains more than 34000 well substantiated genes. With a 2.5 fold cut-off, Bcr/Abl altered the expression of 336 genes and FLT3-ITD altered expression of 231 genes at 24 hours after induction. Fifty one genes were regulated by both Bcr/Abl and FLT3-ITD, including Cyclin G2, cyclin D2, CXCR4, osteopontin and FKBP12. Following induction of BCR/ABL and FLT3-ITD, cyclin D2, osteopontin and FKBP12 are strongly upregulated, whereas cyclin G2 and CXCR4 are downregulated. We validated the expression pattern of these genes by real-time PCR analysis. To further validate that these genes are directly regulated by the kinase activity of BCR/ABL and FLT3-ITD, cell lines were treated with specific small molecule kinase inhibitors, resulting in both cases in a significant decrease in cyclin D2, osteopontin, and FKBP12 and an increase in cyclin G2 and CXCR4 expression. The functional role of several of these genes is under investigation. For example, we found that the PI3K/AKT pathway is important for regulating osteopontin gene expression, and that osteopontin is involved in mediating the increase in cell growth caused by both BCR/ABL and FLT3-ITD. The identification of downstream targets, such as cyclins, osteopontin, and others, that are shared in common by several oncogenic tyrosine kinase oncogenes suggest that they could have value as therapeutic targets.