bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of Stability of a Sucrose-Formulated, Full-Length rFVIII for Use in Continuous Infusion Therapy.
Authors: Martinowitz Uri , Lubetsky Aaron , Tamarin Ilia , Luboshitz Jacob 
Published: October 2019

Abstract
Introduction: Stability of a FVIII preparation in mini-pumps is an important consideration for its use in a continuous infusion manner. Our goal was to evaluate the stability of a specific recombinant FVIII (Kogenate®FS; KOGENATE®Bayer) in two commonly used infusion mini-pumps, with and without anticoagulant additives that may be used in clinical practice to prevent local thrombophlebitis.
Methods: Kogenate® FS (250 IU/vial and 1000 IU/vial) was reconstituted using aseptic technique according to the manufacturer’s instructions with normal water for injection (100 IU/ml and 400 IU/ml, respectively); an additional dilution of 100 IU/ml was made from the 1000 IU/vial size. Reconstituted material was spiked with unfractionated heparin (UFH) or low molecular weight heparin (LMWH; Enoxaparin/Clexane) to a final concentration ~5 U/ml, or left untreated and then transferred to reservoirs of 2 mini-pumps (Walkmed, Medfusion Inc., Duluth, GA and CADD, Deltec, Inc., St. Paul, MN). All bags were stored in a light-protected environment at room temperature (22°C) for 7 days. Samples were drawn at baseline (immediately post reconstitution), 3h, 6h, 12h, 24h, and days 2–7, frozen at −30°C, and assayed for FVIII activity by one-stage assay. On Day 7, the residual volume of the mini-pump containers was cultured for bacteria.
Results: All samples except one* had FVIII activity >90% of baseline.
Conclusions: Kogenate® FS appears to retain excellent stability at room temperature during 7 days in both CADD and Walkmed infusion pumps with and without addition of UFH or LMWFH. No bacterial growth was observed. These results indicate that Kogenate® FS may be useful in continuous infusion therapy.
Figure Figure
KLF4 as a Mediator of Quiescence in Hematopoietic Stem/Progenitor Cells.
Authors: Alder Jonathan K. , Georgantas Robert W. , Yu Xiaobing , Civin Curt I. 
Published: October 2019

Abstract
At any given time, only a very small fraction of the hematopoietic stem cells (HSCs) in an organism are actively dividing; the vast majority of these cells remain in a quiescent state. HSCs can escape this quiescent state to self-renew and give rise to progenitor cells (HPCs), which expand rapidly to form all of the mature progeny cells of the lympho-hematopoietic system. Despite the centrality of quiescence in hematopoiesis, the mechanisms by which quiescence is regulated are largely unknown. Several studies have shown that p21CIP/WAF1 is required for cell cycle arrest of HSCs, and deletion of p21 results in an increase in HSC pool size. However, little is known about the pathways upstream of p21. Recently, we identified 81 genes that were highly over-expressed by normal HSC-enriched CD34+/CD38− cells compared to HPC-enriched CD34+/CD38+ cells. One of the HSC-over-expressed genes was Krupple-Like Factor 4 (KLF4), a member of the Kruppel-like transcription factor family. KLF4 has been associated with a quiescent phenotype in T and B lymphocytes, and exogenous expression of KLF4 induces growth arrest in a colon cancer cell line. It has been shown that KLF4 is an important mediator of p21 in the G1/S cell cycle arrest following DNA damage. Thus, we hypothesized that KLF4 is an important upstream regulator of HSPC quiescence via p21. To begin to test this, we designed a dual promoter self-inactivating (SIN) lentiviral vector that expressed KLF4 under the control of the EF1α promoter and GFP under the CMV promoter. We transduced primary mouse bone marrow (BM) Lin− or human cord blood (CB) CD34+ HSPCs, and purified the resulting successfully transduced GFP+ cells by fluorescence-activated cell sorting. KLF4-transduced human or mouse HPCs displayed a major reduction in colony forming capacity, as compared to either GFP− HPCs transduced with the vector containing KLF4 and GFP or HPCs transduced with the parental control vector expressing only GFP (Fig 1 shows representative experiments). In suspension cultures, KLF4-transduced cell populations generated fewer total cells, accompanied by an increase in apoptic/necrotic cells. In the KLF4-transduced cells, a higher percent were CD34+. These results are consistent with KLF4 inhibiting HPC differentiation and inducing a p21-like pathway with cell cycle arrest and increased apoptosis. Further investigation of KLF4 cellular and molecular actions may not only lead to enhanced understanding of the molecular basis of HSPC quiescence, but also to novel methods for expansion of normal HSPCs.
Figure Figure
Translational Control of the Hematopoiesis-Specific RhoH Gene Is Mediated by Upstream Open Reading Frames.
Authors: Lahousse Sébastien , Denis Claude , Kerckaert Jean-Pierre , Galiègue-Zouitina Sylvie 
Published: October 2019

AbstractThe hematopoiesis-specific RhoH gene was identified in our laboratory, as a BCL6 partner gene (1), in follicular B-non Hodgkin’s lymphoma associated with the recurring t(3;4) chromosomal translocation (2). The RhoH gene encodes a Rho small G protein supposed to be a Rac antagonist (3). The RhoH protein is always in a GTP-bound state (i.e. constitutively active), which suggests a strongly regulated synthesis. This work is aimed at elucidating molecular mechanisms that may regulate this RhoH protein synthesis.The RhoH gene contains one coding exon only (exon 2) preceded by six uncoding exons, and the RhoH transcripts exhibit an important 5′ UTR heterogeneity, especially in the B-lymphoid lineage. This heterogeneity both reflects (i) an use of multiple transcription start sites and (ii) alternative splicing events of some 5′ uncoding exons (4). The present study is devoted to assess the functional relevance of these 5′ uncoding exons in the RhoH protein synthesis. Some of them (exons 1a, 1b and X4) contain an upstream ORF (uORF) sequence. Ten percent of eukaryotic mRNAs, 2/3 of which are involved in the control of cellular growth and differentiation, contain such uORFs within their 5′-UTR sequence. These uORFs are involved in the translational control of these genes (5). RhoH thus provides an additional opportunity to assess the role of uORFs in the translation of a cellular mRNA. Moreover, uORFs have never been implicated in the regulation of any small G family member gene.The data show that the uORFs sequences from the RhoH gene repress the translation of a downstream reporter gene, in transfected hematopoietic cell lines (erythroid, B- and T- lymphoid). The initiation codon from 1a-, 1b-, and X4- uORFs is strongly involved in this phenomenon, as the inhibitory effect was suppressed by an uAUG invalidation. Targeted mutations (premature Stop, silent or mis-sens) within these uORFs sequences were functionally analyzed in the same reporter LUC system. The results led us to distinguish two uORF-mediated translational regulatory mechanisms: (i) an “uAUG dependent / uORF-peptide dependent” mechanism for the 1a- and 1b- uORFs sequences, and (ii) an “uAUG dependent / uORF-peptide independent” mechanism for the X4- uORF.The RhoH transcripts exhibit a 5′ UTR heterogeneity, which might modulate the protein synthesis. Three main RhoH transcripts are expressed in the different hematopoietic lineages: “1a-X4-2”, “1a-1b-X4-2” and “1b-X4-2” (4), therefore the effects of the 1a-, 1b-, and X4- uORFs were investigated in combination (1a+X4; 1a+1b+X4; 1b+X4), as in the RhoH transcripts. The results convinced us to propose an uORF-mediated translational regulatory mechanism, which is based on a combinatory effect (synergy or antagonism) of three uORFs. This mechanism is called: “translational lock-unlock” model of the RhoH gene. In vitro translation experiments aimed at validating this model will be presented.
Effects of Constitutive Production of Colony-Stimulating Factors by Human Lung Cancer Cells on Invasive Capacity.
Authors: Uemura Yoshiki , Kobayashi Makoto , Nakata Hideshi , Kubota Tetsuya , Taguchi Hirokuni 
Published: October 2019

Abstract
In advanced clinical stage, many cases of lung cancer produce colony-stimulating factors (CSFs), including granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), and macrophage-colony stimulating factor (M-CSF). The biological properties of the overproduction of CSFs by tumor cells are not well understood. We previously found that CSFs produced by two lung cancer cell lines, OKa-C-1 and MI-4, which constitutively produce abundant amounts of G-CSF, GM-CSF, and M-CSF, stimulate the tumor’s own growth through an autoctine mechanism. In this study, we examined whether CSFs produced by tumor cells contribute to the invasion of the tumor itself.
Invasive behaviors of the cancer cells were stimulated by exogenous CSFs but were suppressed by neutralizing antibodies against the CSFs. Matrix-degrading proteinases also are essential for successful tumor cell metastasis. Gelatin zymographs of conditioned media revealed two major bands of gelatinase activity at 68 and 92 kDa. The enzyme activities at 68 and 92 kDa were significantly enhanced in the presence of each CSF in the cell lines but were suppressed by the antibodies against the CSFs. The two gelatinase bands were characterized as matrix metalloproteinase (MMP)-2 and MMP-9, respectively. Our findings suggest that CSFs produced by tumor cells might be closely associated with the tumor invasion through gelatinase activation in CSF-producing lung cancer cells. In addition, an increase of invasion capacity and gelatinase activity by the CSFs might be mediated through the p44/42 mitogen-activated protein kinase (MAPK) signaling pathway.
Figure Figure
Expression of BCR-ABL Does Not Inhibit Apoptosis In Vitro, on a B Lymphoblastoid Cell Line.
Authors: Castro Fabiola A. , Brumatti Gabriela , Amarante-Mendes Gustavo P. 
Published: October 2019

Abstract
The bcr-abl oncogene is generated by the Philadelphia chromosome (Ph) translocation, fusing the BCR gene to the ABL gene and occurs mainly in two different forms. In chronic myelogenous leukemia (CML) a 210kDa Bcr-Abl protein is associated with proliferation and accumulation of myeloid cells and their precursors, whereas a 185kDa form is responsible for the pathogenesis of acute lymphocytic leukemia (ALL). Bcr-Abl not only induces cellular transformation but also regulates cell proliferation and confers resistance to a variety of apoptosis-inducing agents. Much attention has been focused on the development of novel therapies based on mechanistic understanding of Bcr-abl signaling in CML cells. However, we still do not fully understand how this molecule interferes with apoptotic machinery. The aim of this study was to investigate which step of the apoptotic machinery was most influenced by ectopic expression of Bcr-Abl myeloblastic (HL-60) and B lymphoblastic (SKW6.4) cell lines.
Ectopic expression of Bcr-Abl conferred resistance to apoptosis induced by staurosporine and anti-Fas in HL-60 cells, but not in SKW6.4 cells, although increased phosphotyrosine containing proteins could be easily observed in SKW6.4.Bcr-Abl cells. The protein levels of Bcl-xL, Mcl-1 and Flip detected by western-blot were greater in HL-60.Bcr.Abl, when compared to wild type HL-60 cells. In contrast, the proapoptotic protein Bid was considerably reduced in HL-60. Bcr-abl cells. Two independent lines of SKW6.4.Bcr-Abl cells had a small reduction of in Bid levels, but no difference was observed in the expression of the anti-apoptotic proteins Bcl-xL, Mcl-1 and Flip. At the mRNA level SKW6.4 and SKW.Bcr-Abl presented similar expression of Bcl-xL, Mcl-1 and Flip. In addition a greater expression of anti-apoptotitc proteins A1 (IDV A1/actin=0.6) and Bcl-w (IDV Bcl-w/actin=1.6) was found in SKW6.4.Bcr-Abl compare to SKW (0.2 and 0.01, respectively) wild type. Interestingly, the upregulated levels of these anti-apoptotic genes were not sufficient to prevent apoptosis in these cells. Taken together these results suggest that Bcr-abl is not capable of protecting SKW6.4 cells from apoptosis induced by staurosporine and anti-Fas, perhaps because it interacts differently with the apoptotic machinery in B lymphoblasts compared to myeloblast cell lines. Further stydies are underway in our laboratory in order to better understand the Bcr-abl-mediated anti-apoptotic signaling in B lymphoblast cells.
Differential Regulation of Pro- and Anti-Apoptotic Genes by Bcr-Abl in an In Vitro Experimental Model of Chronic Myelogenous Leukemia.
Authors: Ulbrich Axel Gustavo , Bueno-da-Silva Ana Elisa B. , Amarante-Mendes Gustavo P. 
Published: October 2019

Abstract
Leukemic cells from patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) are very resistant to apoptosis induced by DNA-damaging agents and other chemotherapeutic drugs, due to the presence of Bcr-Abl, a chimeric cytoplasmic tyrosine-kinase that confers both malignancy and resistance to apoptosis. Efficient treatment of CML can be achieved with a normal bone marrow transplant, which induces a graft-versus-leukemia response, and more recently by the use of the specific inhibitor imatinib mesylate (glivec. Novartis). Glivec blocks Bcr-Abl kinase activity and, as a consequence, the malignant cell dies by apoptosis. However most glivec-treated patients, mainly in the acute and blast phases, develop resistant forms of the disease. Since resistance to apoptosis in Bcr-Abl+ cells is probably related to the inhibition of mitochondrial release of cytochrome c, an obligatory step in most apoptotic pathways, we sought to investigate expression of Bcl-2 family genes in Bcr-Abl+, glivec-treated cells. By semi-quantitative RT-PCR we analyzed the gene expression of several pro- and anti-apoptotic molecules in the transduced cell line HL-60.Bcr-Abl and the wild-type HL-60, after a 1, 4 and 8h treatment with 10μM glivec. Bcr-Abl′s kinase activity is promptly inhibited by glivec (within 5 to 15min) and HL-60.Bcr-Abl cells begin to show mitochondrial depolarization 24h after treatment with the drug, dying 48h later, whereas no effects are observed in HL-60. Soon after glivec addition some genes are transcriptionally regulated in HL-60.Bcr-Abl cells. The major differences were observed for bcl-xL (2-fold reduction), c-flip (2-fold increase), bcl-w (30% increase) and mcl-1 (20% reduction). Some pro-apoptotic molecules such as noxa also displayed differential regulation in HL-60.Bcr-Abl cells. No differences were observed in HL-60 cells. In conclusion we describe a complex transcriptional regulation mechanism dependent on Bcr-Abl tyrosine-kinase activity, which has not been previously described by the use of microarrays, and could contribute to the understanding of the mechanisms involved in protection of apoptosis and drug resistance of Bcr-Abl+ cells.
Human Fetal Liver Derived Stem Cells Can Be Support the Maintenance of Human Embryonic Stem Cells.
Authors: Baek Jin-Young , Rhee Yun-Hee , Cha Kwang-Yul , Chung Hyung-Min 
Published: October 2019

Abstract
Prolonged propagation of human embryonic stem (ES) cells is currently achieved by co-culture with primary or immortalized mouse embryonic fibroblast (MEF) cells. In order to replace the heterologous with homologous co-culture systems, an attempt was made using mononuclear cells derived from human fetal liver. Human fetal liver-derived mesenchymal-like stem cells (FL-MLSC) can be maintained for the prolonged period of time. They showed the characteristics of mesenchymal stem cells in various aspects. They retained a normal diploid karyotype and growth characteristics over the successive culture. Human ES cells cultured on human FL-MLSC cells up to 8 passages displayed the unique morphology and molecular markers characteristic for undifferentiated human ES cells as cultured on MEF cells. Alkaline phosphatase activity was detected in human ES cells co-cultured on human FL-MLSC. Immunocytochemical analyses showed that expressions of stage-specific embryonic antigen-3, -4 and Oct-4 were not altered on human ES cells cultured on human FLDSC. Reverse-transcriptase PCR analyses showed that similar expressions of Oct-4 and Nanog genes, markers for undifferentiated ES cells, were also observed in human ES cells cultured on both human FL-MLSC and MEF cells. Furthermore, human ES cells cultured on human FL-MLSC retained unique differentiation potentials in culture when allowed to form embryoid body. Results of this study suggest that human FL-MLSC can support the maintenance of human ES cell in vitro.
HTLV-1 Enhances Epstein-Barr Virus (EBV) Infectivity and EBV Infection Attributes to an Aggressive Organ Involvement in Adult T-Cell Leukemia.
Authors: Ueda Satomi , Maeda Yasuhiro , Yamaguchi Terufumi , Hijikata Yasuki , Kanamaru Akihisa 
Published: October 2019

Abstract
The frequent involvement of adult T-cell leukemia (ATL) cells in organs such as the skin and lymph nodes is observed in ATL. Epstein-Barr virus (EBV) infection has been observed in the clinical course of ATL, and ATL cells in all skin cases were strongly positive for EBV nuclear antigen (EBNA)-1 and latent membrane protein (LMP)-1 by immunohistochemical staining. Interestingly, ATL cells in all skin cases were positive for CD45R0 (UCHL1) and CD20 (L26), indicating that they have both T and B cells characterization. We suggested that HTLV-1 enhances the entry of EBV into endothelial cells, epithelial cells and EBV presents for an active role of HTLV-1 in the development of ATL. Here, we established two B-cell lines from peripheral blood of patients with ATL. EBV DNA, proviral DNA for HTLV-1 and Tax mRNA were detected in both B cell lines. On characterization of the cell lines, enhanced expression of intercellular adhesion molecule-1 or-3 (CD54, CD50), lymphocyte function-associated antigen-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) was observed compared to an EBV-infected B cell line, Raji. Moreover, EBV oncogenes such as EBV-encoded small RNA (EBER)-1, EBNA-1,-2 and LMP-2A were also detected. To investigate the role of the interaction of these viruses, we transfected EBV and/or HTLV-1 into a healthy donor’s lymphocytes, Raji cells and a HTLV-1 negative T-cell line, Jurkat. Enhanced expression of adhesion molecules was observed in double transfectants (EBV and HTLV-1). Furthermore, we detected high production levels of interleukin-4 (IL-4) in those B-cell lines and transfectants. These results indicated co-expression of HTLV-1 and EBV may induce aggressive organ involvement by enhanced expression of adhesion molecules via IL-4 signaling. Furthermore, HTLV-1 could greatly enhance EBV infectivity and attribute to increased replication of EBV. EBV also could be a necessary cofactor for ATL development in immunodeficiency states. ATL patients who have been coinfected with EBV and HTLV-1 have a high risk of worse progress of disease. ATL patients have a poor treatment outcome, even with strong chemotherapy, especially who have severe skin involvements have a resistance for chemotherapy. Viewed from a different angle, our observations suggest that EBV infection may be used as one of bad prognostic factors and an identification of bad-risk patients who need to benefit from early high dose chemotherapy. Collectively, a reactivation of EBV infection and EBV oncogenes can provide an acceleration of organ involvement in ATL.These results can explain why EBV-associated ATL is more frequent and clinically more aggressive.
Berbamine Induces Apoptosis and Lowers Expression Levels of Bcr/Abl and Survivin Gene and Bcr/Abl-Related P210 in Different Types of Leukemic Cell Lines Cells.
Authors: Lin Maofang , Wu Dong 
Published: October 2019

Abstract
Berbamine is a kind of bis-benzylisoquinoline extracted from Chinese herb. Berbamine and its derivatives have been proven to be a kind of calmodulin antagonist which could obviously inhibit the proliferation of different kinds of cell lines, such as hepatoma, Hela cells etc. Berbamine has been shown to induce apoptosis of HL-60 cells in vitro in a cell type-specific manner. However, main components of Berbamine induced apoptosis pathway remain to be identified. We studied Berbamine induced apoptosis of different types of leukemic cell lines cells and its possible molecular mechanisms in vitro. The results revealed that Berbamine could significantly induce apoptosis in K562, NB4 and Jurkat leukemia cell lines cells in a time- and dosage-dependent manner. With electron microscope and DNA electrophoresis, the typical apoptosis morphologic changes and DNA ladder were clearly observed. As detected by flow cytometry, the percentage of the apoptotic cells in K562, Jurkat and NB4 cells treated with 8.0μg.L−1 Berbamine increased to 49.07±4.73%, 35.57±5.49%, 54.40±4.56% (P value<0.01), respectively. Using semi-quantity RT-PCR assay, the expression level of survivin gene was lower. in K562 cells treated with 8.0μg.L−1 Berbamine than that in untreated cells (0.69±0.02 vs 1.09±0.01, P<0.01),in a time-dependent manner; as well as the expression level of survivin gene was lower. in Jurkat cells treated with 16.0μg.L−1 Berbamine than that in untreated cells (1.34±0.02 vs 2.18±0.03, P<0.01). Similarly, bcr/abl expression was weaker in K562 cells treated with 8.0μg.L−1 Berbamine than that in untreated cells (0.91±0.02 vs 1.19±0.02, P<0.01), while no difference was found between Ara-C treated K562 cells and untreated treated cells. In the other hand, Using Western Blot assay, the level of bcr/abl-related P210 was lower in K562 cells treated with 8μg.L−1 Berbamine than that in untreated cells (0.63±0.01 vs 1.04±0.02, P<0.01). AS a comparison, the level of P210 was also lower in same cells treated with 1.0μg.L−1 STI571 than that in untreated cells (0.58±0.02 vs 0.93±0.03, P value P<0.01).
In summary, Berbamine extracted from Chinese herb can obviously induce apoptosis in different kinds of leukemic cell lines including K562, Jurkat, NB4 in a time-and dosage-dependent manner in vitro. The down-regulation expressions of survivin gene, bcr/abl gene and bcr/abl -related P210 may play an important role in the apoptotic effect of Berbamine in leukemic cells.