bluuebunny/crossref_metadata_2025_split huggingface dataset:
Age-Related Homeostasis: Molecular Mechanism, Disease and Predictions from Global Analyses of Mouse Liver Proteins.
Authors: Kurachi Sumiko , Tanaka Taku , Kanai Muneyoshi , Suenaga Emi , Solovieva Elena , Kurachi Kotoku 
Published: October 2019

Abstract
We previously reported the first molecular mechanism underlying the age-related homeostasis, ASE/AIE-mediated genetic mechanism for age-related regulation of gene expression, which turned out to be a mechanism of puberty-onset gene switching, specifically controlling a group of genes for their expression from puberty into old age (Kurachi & Kurachi J Thromb Haemost 2005; Zahng et al J Biol Chem277:4532, 2002; Kurachi et al Science285:739, 1999). This led us to successful construction of a transgenic mouse model of hemophilia B Leyden, a unique subset of hemophilia B with its mechanism being remained mysterious, robustly mimicking its unusual pattern of puberty-onset spontaneous amelioration. With this background, we hypothesized that besides the ASE/AIE-mediated mechanism, there exist more unidentified fundamental regulatory mechanisms for age-related homeostasis, which individually or in various combinations generate age-related complex and dynamic regulatory patterns of liver proteins. We launched a series of global and quantitative analysis of age-related changes in expression of liver nuclear proteins of mice (C57BL/6xSJL, [male], 1 through-24 month of age) by taking a procedure composed of two-dimensional gel electrophoresis (2DE) for separation and quantification of liver nuclear protein spots and of MALDI-TOF/MS PMF analyses to identify proteins in the spots. Out of over 6000 spots recognized and quantified in 2DE, 4547 protein spots were subjected to MALDI-TOF/MS analysis for protein identification. Finally, 2765 protein spots including many isomers were found unique. Systematic analyses of their age-related expression identified several major phases in protein expression throughout the lifespan. These findings supported our hypothesis that there exist multiple novel molecular mechanisms responsible for maintaining age-related homeostasis. The comprehensive liver nuclear protein data set was then used to construct a comprehensive database, which allows rapid and reliable searches for expression of specific proteins, their age-related dynamic profiles, isomers, protein identification from 2DE image, and other related information. This will serve as a valuable platform resource for studying epigenetic challenges, evaluation of drugs as well as gaining further insights into the molecular mechanisms underlying age-related homeostasis.
Characterization of an Ideal Amphipathic Peptide as a Potential Hemostatic Agent.
Authors: Ganopolsky Jorge G. , Charbonneau Sophie , Peng Henry , Blostein Mark 
Published: October 2019

Abstract
We have previously demonstrated that amphipathic helical peptides can accelerate the turnover of the substrate factor X by the enzyme, factor IXa in a gamma-carboxyglutamic acid (Gla) domain dependent manner (Biochemistry,39:12000Biochemistry,39:12000). Such improvement was due to a remarkable decrease in KM and a mild increase in kcat, mimicking the presence of phospholipid membranes. It is hypothesized that amphipathic helical peptides could exert similar activities in other reactions of the blood coagulation cascade, such as thrombin generation and in whole blood clotting. In the present work, we analyze the activity of a 22-amino acid ideal amphipathic helical peptide (IAP) of K7L15 composition, in factor X activation and thrombin generation. The addition of IAP accelerates factor X activation by factor IXa in a concentration dependent manner. IAP also accelerates thrombin generation by factor Xa with a comparable peptide and substrate concentration dependence. Further, the Gla domain is also required for peptide activity confirming the hypothesis this peptide behaves as a membrane mimetic. Using fluorescence spectroscopy and an ELISA-based binding assay, we demonstrate direct binding between IAP and the Gla domain of factor X. Additionally, when IAP is immobilized to a reaction surface, factor X activation and thrombin generation are dramatically enhanced, as compared to the corresponding reactions on untreated surfaces. Finally, activated partial thromboplastin times (APTTs) of pooled plasma are significantly shortened on surfaces treated with IAP, in comparison with clotting times measured on untreated surfaces. The above results suggest that immobilized IAP may serve as a potential hemostatic agent, as demonstrated in isolated critical reactions of the coagulation cascade and in whole plasma.
The Specific Aurora Kinase Inhibitor AZD1152 Significantly Affects the Growth of Human Leukaemic Cells in an In Vivo AML Model.
Authors: Pearce Daniel , Odedra Rajesh , Wilkinson Robert , Bonnet Dominique 
Published: October 2019

Abstract
AZD1152 is a specific Aurora kinase inhibitor with selectivity for Aurora B kinase, designed to target cell division in proliferating tumour cells. It has been shown in model systems that inhibition of Aurora B kinase with AZD1152 reduces histone H3 phosphorylation (phH3) and inhibits cytokinesis, thus inducing cellular multi-nucleation and polyploidy, leading to apoptosis and cell death.
Here, we present results of preliminary studies on the in vivo action of AZD1152 on the growth and development of both a human acute myeloid leukaemia (AML) cell line (HL-60) and primary AML samples in the NOD/SCID xeno-transplantation model. Two weeks of AZD1152 at 25 mg/kg/day is well tolerated in NOD/SCID mice. HL-60 cells were injected iv into sub-lethally irradiated NOD/SCID mice (n=24) and allowed to engraft in the bone marrow. Three weeks later, when malignant cell growth was well established in the bone marrow, mice were split into treatment groups. Either vehicle (n=12) or AZD1152 (25 mg/kg/day via minipump, n=12) was administered for 7 days. One AZD1152-treated group was sacrificed immediately post-treatment (n=9) and another was left for a further 2 weeks before bone marrow analysis for HL-60 cell content (defined by human CD45+/CD33+/CD19− expression). The mean percentage HL-60 cell content in the marrow of untreated mice was 64.5 ± 19.0 (range 14.0–86.6, n=11), whereas in mice that had received AZD1152 and were sacrificed at the end of dosing period, the mean percentage was 0.29 ± 0.74 (range 0–2.26, n=9; p<0.001). In AZD1152-treated mice that were left for 2 weeks post-treatment, the mean percentage of HL-60 cell content in the bone marrow was 0.008 ± 0.74 (n=3).
Similar experiments were performed with two primary AML samples. AML-1 is from a patient with French-American-British (FAB)-type M1 AML and AML-2 is from a FAB-type M2 AML patient. Ten million cells from both AML samples were injected into NOD/SCID mice (n=12 for AML-1; n=6 for AML-2). Ten weeks later, mice were split into treatment groups and dosed with either control vehicle or 25 mg/kg/day of AZD1152 via minipumps for 7 days. All mice were sacrificed after 1 week of treatment in these initial experiments. The mean percentage engraftment in untreated mice was 59.7 ± 36.0 (range 6.2–84.0, n=4) for AML-1 and 21.8 ± 22.5 (range 5.9–37.7, n=2) for AML-2. Whereas, in mice that had received 25 mg/kg/day of AZD1152 for 7 days, the percentage of engraftment was 1.8 ± 1.7 (range 0.38–4.53, n=5; p<0.01) and 0.07 ± 0.07 (range 0.12–0.014, n=2) for AML-1 and AML-2, respectively.
These data indicate that AZD1152 has a profound effect on the engraftment of human AML cell lines and primary AML samples in an in vivo model. Furthermore, initial results may indicate a long-term effect of AZD1152 treatment in vivo. AZD1152 is currently undergoing Phase I clinical trials in AML patients.
Implications of Heparinoid-Induced Inactivation of Coagulation Cascade Factors and In Vivo Proenzyme Concentration.
Authors: Patel Sanjay , Berry Leslie R. , Hatton Mark W.C. , Chan Anthony 
Published: October 2019

Abstract
Heparin is a commonly used anticoagulant in the treatment of thrombosis. We have compared heparinoid-stimulated inhibition rates with the in vivo plasma concentration of antithrombin (AT)-inhibitable coagulation factors. Second order rate constants (k2) for inhibition of activated factors by either AT + unfractionated heparin (AT+UFH) mixtures or a novel covalent complex of AT and heparin (ATH) (Chan et al, J Biol Chem, 272:22111, 1997) were determined by discontinuous assay. A plot of k2 values (mean ± SEM; n ≥5) versus the respective human plasma concentration of coagulation factors revealed a linear correlation (with R2 values of 0.93 for AT+UFH and 0.90 for ATH, excluding factor XII), in which neutralization efficiency was proportional to in vivo factor level (see Figure). Anticoagulant actions of AT+UFH and ATH were more oriented towards treatment than prophylaxis since inhibition of cascade end point enzymes (thrombin and factor Xa) was more rapid than factors involved in coagulation initiation (factors VIIa and XIa). However, ATH exhibited more enhanced inhibition rates against factors VIIa, IXa and XIa than against factor Xa and thrombin, suggestive of an improved prophylactic profile compared to AT+UFH. Intriguingly, factor XII did not follow this trend, further challenging its role in the coagulation cascade. The impact of these assertions requires confirmation by in vivo inhibition studies.
Figure Figure
Interaction of Plasminogen and Angiostatin with Heat Shock Proteins.
Authors: Dudani Anil K. , Mehic Jelica , Martyres Anthony 
Published: October 2019

Abstract
Previous studies from this laboratory have demonstrated that plasminogen and angiostatin bind to endothelial cell (EC) surface-associated actin via their kringles in a specific manner. Heat shock proteins (hsps) like hsp 27 are constitutively expressed by vascular ECs and regulate actin polymerization, cell growth and migration. Since many hsps have also been found to be highly abundant on cell surfaces and there is evidence that bacterial surface hsps may interact with human plasminogen, the purpose of this study was to determine whether human plasminogen and angiostatin would interact with human hsps. ELISAs were developed in our laboratory to assess these interactions. It was observed that plasminogen bound to hsps 27, 60 and 70. In all cases, binding was inhibited (85–90%) by excess (50 mM) lysine indicating kringle involvement. Angiostatin predominantly bound to hsp 27 and to hsp 70 in a concentration- and kringle-dependent manner. As observed previously for actin, there was dose-dependent inhibition of angiostatin’s interaction with hsp 27 by plasminogen. In addition, thirty-fold molar excess actin inhibited (up to 50%), the interaction of plasminogen with all hsps. However, thirty-fold molar excess actin could only inhibit the interaction of angiostatin with hsp 27 by 15–20%. FACS analyses indicated the presence of hsps 27, 60 and 70 on the surface of MCF-7 breast cancer cells but not on human umbilical vein ECs. Polyclonal antibodies to hsp 27 significantly inhibited the interaction of plasminogen and angiostatin with MCF-7 surface-associated hsp27 in a dose-dependent manner. Collectively, these data indicate that
while plasminogen interacts specifically with hsp 27, 60 and 70, angiostatin interacts predominantly with hsp 27 and to some extent with hsp 70; plasminogen only partially displaces angiostatins binding to hsp 27; actin only partially displaces plasminogen/angiostatin binding to hsps and surface-associated hsp 27 can mediate the binding of both plasminogen and angiostatin to MCF-7 cells.
Critical Role of Hem1 in Hematopoietic Cell Chemotaxis and Innate Immunity to S. Pneumoniae
Authors: Lorsbach Robert B. , Kim Yoon-Sang , Moore Jennifer , Smith-Sielicki Hope , Jones Tiffany M. , Sun Weili , Tuomanen Elaine , Downing James R. 
Published: October 2019

Abstract
Hem1 is a hematopoietic specific member of the HEM family proteins, which have been identified as components of the WAVE regulatory complex. To further characterize the role of Hem1 in hematopoietic cell function, we have generated mice deficient in Hem1 using gene targeting methodology. Hem1-deficient mice manifest several phenotypic abnormalities, including peripheral blood lymphopenia and neutrophilia, splenomegaly, premature mortality, and with variable penetrance thymic hypoplasia and alopecia. Flow cytometric analysis of peripheral blood and spleen demonstrated that Hem1−/ − mice have a marked reduction in peripheral B cells, changes that were evident in both adult and neonatal mice; the splenomegaly in Hem1−/ − mice was attributable to increased extramedullary hematopoiesis. The frequency of bone marrow B cell progenitors was also markedly reduced in Hem1−/ − mice. To assess the role of Hem1 in hematopoietic stem cell (HSC) function, competitive bone marrow transplantation assays were performed. In contrast to wild-type HSCs, Hem1-deficient HSCs had poor competitive repopulating activity in irradiated recipient mice. KSL cell analysis demonstrated no significant difference in the frequency of lin-c-kit+Sca1+ HSCs between wild-type and Hem1−/ − bone marrow, suggesting that the defective competitive repopulating activity of Hem1−/ − HSCs is attributable to defective bone marrow homing or stem cell niche interaction. Given the biochemical evidence implicating the HEM proteins as a component of the WAVE regulatory complex, we also assessed the ability of Hem1-deficient leukocytes to undergo cytoskeletal remodeling in vitro. Using a transwell assay, Hem1−/ − bone marrow storage pool neutrophils demonstrated markedly blunted chemotactic responses to formylated peptide which was attributable to defective f-actin formation. Hem1−/ − peripheral CD4+ T cells similarly manifested chemotactic defects in response to SDF-1, and showed blunted proliferation when stimulated with antibodies against CD3 and CD28. Finally, a model of Streptococcus pneumoniae infection was employed to test the role of Hem1 in the in vivo function of neutrophils. Hem1−/ − mice were dramatically more sensitive to S. pneumoniae than wild-type littermates, as manifested by the inability to eliminate S. pneumoniae organisms in vivo and higher mortality. In summary, Hem1 deficiency results in deficiencies and functional defects in multiple hematopoietic lineages due to defective signaling to the actin cytoskeleton, and importantly, Hem1 plays a critical role in innate immunity to S. pneumoniae in vivo.
Do Lipocalins Play a Role in Mast Cell Physiology?.
Authors: Logdberg Lennart E. , Akerstrom Bo , Hair Gregory A. , Allhorn Maria , Vikulina Tatyana , Kirshenbaum Arnold S. , Sundstrom J. Bruce 
Published: October 2019

Abstract
Introduction. Aiming to identify molecular properties of allergens responsible for their “intrinsic allergenicity”, we focus on a subset of xenogeneic lipocalins (LCs) that comprise the major mammalian respiratory allergens. These structurally and functionally homologous molecules likely possess conserved molecular motifs promoting IgE-dependent allergy. We hypothesize that such LC “allergenicity” depends on non-IgE interactions of LCs with components of the innate immune system. Herein we describe a possible basis for such interactions between LCs and mast cells.
Materials and Methods. Two sources of human mast cells were used; primary cultures derived from peripheral blood CD34+ progenitor cells; or the LAD-2 cell line. Cells were cultured in serum-free medium with recombinant human stem cell factor (SCF; 100 ng/ml). Monoclonal antibodies to human gp330/megalin (MAb E11) were a kind gift of Prof. Lars Rask (Uppsala University, Sweden). Cell surface protein expression was assessed by flow cytometry and gene transcription was measured by real-time PCR.
Results. Monoclonal antibodies to an endocytic cell surface receptor (megalin, also known as low-density lipoprotein receptor-related protein (LRP)-2) known to bind multiple LCs stained the mast cell lines. This putative expression of megalin by the mast cells corresponded to their transcription of megalin mRNA as shown as by PCR. Moreover, mast cell megalin gene transcription could be induced (≥ 1000-fold) by overnight culture with monomeric IgE myeloma protein (100 ng/ml), and such induction of the megalin message correlated with both an increase in cell surface expression of the molecule and the specific binding of a purified human LC (a-1 microglobulin).
Conclusion. Megalin, an LC-binding cell surface receptor, appears to be constitutively expressed by both progenitor and mature mast cells, and its expression seems to be strongly upregulated by culture with monomeric IgE. This is consistent with a role for direct mast cell-LC interactions in the development of IgE-dependent allergy. In addition, and also of potential clinical relevance, endogenous LCs may play a functional role in normal mast cell physiology.
Hematopoietic Stem Cells Contribute to Lymphatic Endothelium.
Authors: Jiang Shuguang , Bailey Alexis S. , Swain John R. , Hewett Erin M. , Wong Melissa H. , Streeter Philip R. , Fleming William H. 
Published: October 2019

Abstract
The lymphatic system plays an important physiological role in vascular and immune homeostasis. Lymphatic vessel function is implicated in a number of pathological conditions including tumor metastasis and impaired wound healing. The identity and origin of lymphatic endothelial precursors is poorly understood. Previously we have shown that adult bone marrow-derived, hematopoietic stem cells (HSCs, c-kit+, Sca-1+, lineage−) can differentiate into functional blood vascular endothelial cells. Given the close relationship between the blood and lymphatic vascular systems, we have investigated whether HSCs also give rise to lymphatic endothelial cells (LEC). GFP+ HSCs were transplanted into lethally irradiated (1200 cGy) recipient mice. Donor-derived LEC expressing lymphatic endothelial markers including LYVE-1 and VEGFR3 were clearly distinguished from hematopoietic cells by the absence of CD45 and F4/80 expression. Deconvolution microscopy confirmed the co-localization of donor and LEC marker expression in individual cells. Transplanted HSCs gave rise to LEC in the liver, gut, gastric and kidney. Donor-derived LEC were detected in 2.4% of liver lymphatic vessels at 4 weeks and persisted for at least 12 months (mean of 3.4%). The self-renewal capacity of HSC-derived lymphatic progenitor cells was demonstrated by serial transplantation. The contribution of these progenitors to tumor lymphatics was evaluated. Transplantation of HSCs into young Min−/− mice resulted in the incorporation of donor-derived LEC into the lymphatics of intestinal adenomas that spontaneously develop in these mice. In addition, CD45+F4/80+ leukocytes were detected in the vessel lumens indicating that these are functional tumor lymphatic vessels. Finally, to determine if LEC progenitors contribute to lymphatic vessels in the absence of radiation injury or tumorigenesis, a parabiosis model was evaluated. Donor-derived LEC were detected in parabiotic mice at a frequency similar to that observed for donor-derived blood vascular endothelial cells. This finding suggests that circulating progenitor cells contribute to lymphangiogenesis during steady-state conditions. Our results indicate that hematopoietic stem cells have the potential to contribute to lymphatic endothelium and therefore HSC-derived progenitors may be potential therapeutic targets for hematopoietic and lymphatic disease.
Characterization of Post Natal Human Lymphoid Progenitors.
Authors: Six Emmanuelle , Bonhomme Delphine , Beldjord Kheira , Jurkowska Monika , Cortivo Liliane Dal , Monteiro Marta , Garrigue Alexandrine , Fischer Alain , Cavazzana-Calvo Marina , Andre-Schmutz Isabelle 
Published: October 2019

Abstract
In humans, little is known about post-natal lymphoid progenitors, especially those able to circulate, colonize the thymus and generate T lymphocytes. On the basis of a previous work published by Anne Galy in 1995, we have detected in the human post-natal bone marrow up to 60 yrs of age a population of progenitors characterized by their CD34+Lin-CD10+ phenotype. Their differentiation potential analysed by culture in methylcellulose medium indicated that in contrast with their CD10− counterparts, CD10+ progenitors have lost erythroid and myeloid potential. On the other hand, CD10+ progenitors cultured on MS5 or OP9/hDL1 stroma demonstrated an enriched capacity to generate B, T and NK lymphocytes as compared to CD10− precursors. In limiting dilution assays, the high lymhoid potential of CD10+ population was confirmed, since 1 out 15 of them gave rise to T cells, 1 out 23 to B cells and 1 out 90 to NK cells. Gene expression profile shows that CD10+ cells express both B and T restricted factors, such as RAG, Gata3, Pax 5 and TdT. In addition, recombination at the IgH locus is already going on, with multiple DJ, but also VDJ recombination products detected. More importantly, CD10+ precursors circulate in the peripheral blood and are detected in the thymus where they are part of the most immature thymocytes CD34+CD1a-CD38-. Altogether, our results demonstrate for the first time the existence of a post-natal lymphoid progenitor population with a broad lymphoid potential and the ability to reach the thymus and generate efficiently T cells. On the long term, their full characterization will pave the way for their enrichment and usage in therapy of primary lymphoid immunodeficiencies.