bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evolutionarily Conserved Coupling of Transcription and Alternative Splicing in the Protein 4.1R and 4.1B Genes Regulates N-Terminal Protein Structure.
Authors: Tan Jeff , Parra Marilyn K. , Mohandas Narla , Conboy John G. 
Published: April 2020

Abstract
The protein 4.1R gene is regulated by complex pre-mRNA processing events that facilitate the synthesis of protein isoforms with different structure, function, and subcellular localization in red cells and various nucleated cell types. One of these events involves the stage-specific activation of exon 16 inclusion in erythroblasts, which mechanically stabilizes the membrane skeleton by increasing the protein’s affinity for spectrin and actin. Some of the splicing factor proteins and RNA regulatory elements responsible for this tissue-specific alternative splicing event have been defined. Here we focus on another RNA processing event, in the 5′ end of the transcript that can affect the structure and function of the membrane binding domain of protein 4.1R. We have shown that 4.1R transcripts originating at three far upstream alternative promoters/first exons splice differentially to alternative acceptor sites in exon 2′/2 in a manner that suggests strict coupling between transcription and alternative splicing events. A precisely analogous gene organization and RNA processing pattern has also been shown to occur in the paralogous 4.1B gene. Now we demonstrate that this coupling is evolutionarily conserved among several vertebrate classes from fish to mammals. The 4.1R and 4.1B genes from fish, bird, amphibian, and mammal genomes exhibit shared features including alternative first exons and differential splice acceptors in exon 2. In all cases, the 5′-most exon (exon 1A) splices exclusively to a weaker internal acceptor site in exon 2, skipping a short sequence designated as exon 2′ (17-33nt). Conversely, alternative first exons 1B and/or 1C always splice to the stronger first acceptor site, retaining exon 2′. These correlations are independent of tissue type or species of origin. Since exon 2′ contains a translation initiation site, this regulated splicing event generate protein isoforms with distinct N-termini. We propose that these 4.1 genes represent a physiologically relevant model system for mechanistic analysis of transcription-coupled alternative splicing. We have recently constructed a 9kb “minigene” that successfully reproduces the differential splicing patterns of exons 1A and 1B to exon 2′/2 in transfected cells. This minigene will facilitate identification of the determinants that guide coupling. Current experiments are testing the importance for proper splicing of the transcriptional promoter, first exon sequences, length and sequence of the intron, and sequence of a conserved element within exon 2′. Ultimately these studies should provide new insights into the mechanisms of coupling between far upstream, transcription-related processes and downstream alternative splicing.
Differentiation of Systematically Injected Mesenchymal Stem Cells into Retina Cells in the Rat Eye.
Authors: Park Sung-Kyu , Won Jong-Ho , Bae Sang-Byung , Kim Chan-Kyu , Lee Nam-Su , Lee Kyu-Taeg , Hong Dae-Sik , Park Hee-Sook 
Published: April 2020

Abstract
Presently, no effective treatment are available for retinal degeneration but there are a number of lines of investigation which suggest potential approaches. A number of investigations have shown the multilineage potential of mesenchymal stem cells (MSCs) into neural lineages, which prompted us to attempt their induction into photoreceptors. We evaluated whether systematically injected bone marrow-derived MSCs can be incorporated into the neuroretinal tissues and play an important role in healing retinal wound in the laser-induced retinal trauma model. After Nd;YAG laser-induced retinotomies, MSCs prelabeled with GFP were injected via tail vein. The fundus photographs were taken serially, and eyeballs were enucleated for histological studies. In ophthalmic examination, MSCs injected retinas were showed no hemorrhage, retinal detachment, and vitreous opacity at 4 weeks after MSCs injection. At same time, GFP(+) pigmented cells were detected at the damaged sites and observed more plentiful at the base of the laser-induced break sites. In time-dependent manner, more GFP(+) cells were detected around the damaged retina and incorporated into the neuroretinal tissues through the systemic route. We performed immunohistochemical analyses to evaluate the differential potential of MSCs into neuron. Although retinas were rarely stained within 4 weeks, MSCs injected retinas were consistently and strongly stained after 4 weeks in proportion to the amount of MSCs to engraft into retinas. To test whether MSCs treatment could also induced functional rescue of vision in injured rats, we performed electroretinograms on rat 4 weeks after MSCs injection. Detectable, albeit subnormal, electroretinogram recordings are observed in MSCs injected eyes. However, in all cases electroretinogram from the control eyes was non-detectable. Overall, these results are suggestive of some degree of functional rescue in MSCs injected eyes. Conclusively, we demonstrated the successful transplantation of ex vivo expanded MSCs with minimal side effects when injected intravenously into injured rat retina; we also showed the in vivo survival of these cells, their partial integration, and differentiation into the host retina.
Exploring the Molecular Mechanisms for Enhancing MSC Homing and Lodgement within Sites of Liver Damage/Fibrosis.
Authors: Herrbrich Phillip E. , Meyerrose Todd E. , Nolta Jan A. 
Published: April 2020

Abstract
Exogenous stem cells such as human mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC) have been reported to be applicable for tissue repair, as in the case of chronic liver injury. However, one of the limitations in achieving high enough level of stem cell engraftment at these damaged sites is the low level of recruitment of exogenous stem cells. Our goals in the current studies are to enhance recruitment of human MSC to sites of tissue damage and to equip the MSC with high enzymatic activity to digest through areas of fibrosis surrounding of the damaged tissue, in this case, an immune deficient mouse model of CCl4-mediated liver damage as we have previously reported (Wang et al, 2003). Pretreatment of HepG2 cells with hypoxia significantly increased the levels of the hepatocyte growth factor (HGF) receptor, c-met. Since HGF is both a growth factor and a chemotactic agent found at high levels within sites of hypoxic liver injury, we hypothesized that pre-treatment of MSC with hypoxia would increase the expression of c-met and thus, would enhance the sensitivity of MSC to the chemotactic effect of HGF. First, we demonstrated that in the absence of serum and any exogenous growth factor, a 16-hr hypoxic treatment of MSC increased the total cellular protein level of c-met, as well as the total cellular phosphotyrosine activity following HGF stimulation. 3T3 fibroblasts engineered to secrete human HGF were plated in the bottom well of the transmigration assay and human PKH26-labeled MSC pre-incubated in hypoxic vs. normoxic conditions were added to the upper transwell. Transmigrated cells were enumerated after 3 hours to measure the functional role of c-met induction. Preliminary results show enhanced directional transmigration of hypoxia pre- treated MSC toward an HGF gradient, when compared to normoxic treated MSC. Next, we addressed the issue of liver fibrosis as a possible physical barrier that could block the entry of exogenously recruited MSC into the sites of liver damage. We demonstrated that the combination of hypoxia and HGF modulated the degree of matrix metalloproteinase (mmp2 and mmp9) activity in MSC. In addition, recent studies identified the presence of urokinase plasminogen activator receptor (uPAR) on hematopoietic stem cells. We show here for the first time, that uPAR can also be found on MSC. Ongoing studies are assessing the changes in uPAR and uPA activity following hypoxic culture. We hypothesize that changes in MMP and uPA activity can affect the ability of MSC to engraft/lodge within the fibrotic tissue. In conclusion, we propose that the pre-treatment of MSC with hypoxia and HGF might not only enhance the migration of exogenous stem cells to the site of liver damage but also enhance their ability to degrade the accumulated fibrosis at sites of injury for entry into the regions of damaged tissue.
Use of the NOD/SCID/γcnull Mouse Model To Assess the Hepatocyte-Producing Ability of Human Hematopoietic Cells.
Authors: Fujino Hisanori , Hiramatsu Hidefumi , Tsuchiya Atsunori , Noma Haruyoshi , Shiota Mitsutaka , Umeda Katsutsugu , Yoshimoto Momoko , Heike Toshio , Ito Mamoru , Nakahata Tatsutoshi 
Published: April 2020

Abstract
Hematopoietic cells have been shown to generate nonhematopoietic cells, although the true plasticity of stem cells has been questioned. Here we used the NOD/SCID/γcnull mouse model, which permits efficient engraftment of human hematopoietic stem cells and their multi-lineage differentiation including T cells, to investigate whether human hematopoietic stem cells can differentiate into human hepatocytes. Freshly collected cord blood was depleted of phagocytes with Silica® followed by CD34 positive selection using auto MACS®. These cells were intravenously transplanted into irradiated mice, after which the liver was either undamaged or damaged by chemicals. The livers of these mice contained hepatocyte-specific (albumin, CYP family, TAT, alpha1AT, CPSI, prealbumin, transferrin and RBP4), cholangiocyte-specific (CK19) and vascular endothelial cell-specific (eNOS) human mRNAs. Immunohistochemistry detected the human hepatocyte specific antigens, albumin and alpha-1-antitrypsin-positive hepatocytes, cholangiocytes and CD68+ Kupffer cells. We also found human albumin in the murine bloodstream. Human albumin levels in the peripheral blood of transplanted mice correlate with the degree of PB chimerism and increase with time after transplantation. Furthermore, after obtaining liver cells by collagenase perfusion, flow cytometry revealed the presence of human albumin-positive cells that bear both human and murine MHC molecules, suggesting cell fusion occurs. All of the above phenomena were found in both liver-damaged and undamaged mice. In addition, we found human CD34+ cells are recruited from the murine bone marrow to the liver only in the case of acute liver injury but do not acquire hepatic stem/progenitor characteristics. Our observation suggests there are two pathways that yield hepatic cells from hematopoietic stem cells. The first requires liver damage that recruits CD34+ cells from the bone marrow via the circulation while the second pathway does not involve liver damage and appears to represent a constitutive default pathway of hematopoietic to nonhematopoietic transition. Our model is thus a versatile tool for investigating the development of functional human hepatic cells from hematopoietic cells and the feasibility of using hematopoietic cells in clinical situations.
Hepatogenic Potential of Stem Cells and Lymphoid Precursors.
Authors: Yamada Yasuhiro , Miyake Hirosada , Nishimoto Eishi , Mitsuya Hiroaki , Yonemura Yuji 
Published: April 2020

Abstract
Several research groups have recently reported that certain bone marrow cells (BMCs) differentiate into hepatocytes in vitro as well as in vivo in rodents through both transdifferentiation and cell fusion. Hematopoietic myelomonocytic cells are thought to be the major source of hepatocyte fusion partners in the tyrosinaemia type I mouce transplantation model. Transdifferentiation of murine BMCs can be induced with various cytokines and extracellular matrix. We previously reported that when Sca-1+ BMCs were co-cultured with fetal liver cells (FLCs) on laminin-coated dishes, alpha-fetoprotein (AFP)-expressing BMCs became completely adherent by day 4 and expressed albumin as assessed with immunochemistry and RNA-PCR (Yamada et al., Exp Hematol. in press). In the present study, we attempted to further delineate the characteristics of BMCs that differentiate into hepatic-like cells. It was found that AFP-expressing cells were in CD5+ or B220+ lymphoid lineage, mostly Sca-1+CD5+ lineage and that CD5+CD4−CD8− and CD5+CD4+CD8+ thymocytes expressed AFP. When cKit+Sca-1+ lineage BMCs (KSLs) which did not express AFP, CD5+ BMCs, and CD5+ thymocytes, all from green fluorescence protein (GFP)-expressing transgenic mice, were co-cultured with FLCs from ROSA26 mice (X-gal+ FLCs), fractionated cells gave rise to adherent hepatic-like cells, which expressed albumin and cytokeratin 8 (CK 8) as assessed with immunochemistry and AFP, albumin, transthyretin and dipeptidylpeptidase IV as examined with RNA-PCR. The hepatic-like cells from KSLs, CD5+ BMCs and CD5+ thymocytes emerged at the frequency of 1 in 50, 1x103 and 3.5x105 by titration assay. These data suggest that AFP-expressing cells in BMCs were derived from hematopoietic stem cells and that lymphoid precursors differentiated into hepatic-like cells and their hepatogenic ability could diminish over lymphoid maturation.
The Effect of GSK3β Inhibitor on Murine Multipotent Adult Progenitor Cells (mMAPCs).
Authors: Oki Masayuki , Jiang Yuehua , Verfaillie Catherine M. 
Published: April 2020

Abstract
The Wnt/β-catenin pathway is important for maintenance of the undifferentiated state of human embryonic stem cells (ESCs), hematopoietic stem cells (HSCs) and other stem cells. We here tested if β-catenin activation, by inhibiting GSK3 β with BIO (6-Bromoindirubin-3′-oxime), affects the differentiation status of murine multipotent adult progenitor cells (mMAPCs). mMAPCs were cultured with or without 0.1–2 μ M BIO, LIF, EGF and PDGF-BB, at low density (plating density of 100/cm2 and cells passed every 2 days) or at high confluency (like ESCs). Quantitative RT-PCR (Q-RT-PCR) was done weekly for the ESC-specific pluripotency transcripts, Oct4, Rex1, Nanog, UTF1 and E-Ras, and lineage commitment genes, vWF, VE-cadherin, Sox1, Nestin, CK19, Albumin, HNF3β and AFP. After 2–4 weeks, cells were analyzed by FACS for ESC/MAPC specific antigens, and by immunohistochemsitry for Oct4, β-catenin and E-cadherin protein. Furthermore, cells were differentiated into endothelial cells and hepatocytes by using VEGF-A and HGF/FGF4, respectively for 2 weeks. Q-RT-PCR was used to demonstrate lineage differentiation.
BIO caused a dose-dependent clustering of mMAPCs from 4 days after treatment at both cell densities. FACS phenotype of low- and high-density mMAPCs was not affected by BIO. Nanog mRNA was not detected in any of the cell populations. However, Oct4, Rex1, vWF, and CK19 mRNA levels decreased in a BIO dose-dependent manner in mMAPCs maintained at low density, while they acquired higher levels of Sox1 mRNA (P=NS). At high-density, Oct4 and Rex1 mRNA level decreased in a BIO dose-dependent manner for the first week. On the other hand, 0.1 μ M BIO-treatment induced higher levels of Oct4 and Rex1 in cells maintained at high density. β-catenin and E-cadherin protein was highly expressed in clusters of low-density mMAPCs induced by BIO as well as BIO-treated mMAPCs maintained at ESC densities. In both cell densities, only mMAPCs treated with 1 or 2 μM BIO contained large dense mMAPCs clusters that expressed very high levels of β-catenin and E-cadherin. There was no statistical difference of differentiation potential between BIO conditions and cell densities.
BIO may not allow culture of mMAPCs without loss of Oct4 as it did for ESCs, but induces clusters like ESCs in a BIO-dose dependent manner. High concentrations of BIO enhances β-catenin and E-cadherin protein expression. BIO may induce lineage commitment of mMAPCs. Despite loss of Oct4 mRNA and protein, MAPCs treated with BIO continue to have the ability to differentiate. Ongoing studies are testing whether BIO treated cells maintained at high density also maintain in vivo engraftment and differentiation potential.
Identification of Novel Regulators of Hematopoietic Stem Cell Mobilization.
Authors: Voermans Carlijn , Lento William E. , Uqoezwa Mweia , DiMascio Leah N. , Chhotani Ali , Rattis Frederique M. , Reya Tannishtha 
Published: April 2020

Abstract
In an effort to identify the molecular changes during hematopoietic stem cell (HSC) mobilization we have analyzed genome wide changes in gene expression in HSCs in response to the chemotherapeutic agent cyclophosphamide (Cy) and the growth factor granulocyte colony stimulating factor (G). Cy/G treatment leads to an initial loss of proliferating precursors, followed by a rapid expansion of stem and progenitor cells and their migration to the peripheral blood. While this approach has been capitalized on for harvesting hematopoietic stem cells in clinical therapy, the molecular regulation of this process remains less well understood. To analyze changes in gene expression that occur in vivo as HSCs proliferate and prepare to migrate out of the bone marrow, we enriched for HSC fractions by isolating cells that express c-kit (K), Sca-1(S) and low levels of Thy1.1(T) but do not express lineage (L) markers (KTLS) from untreated mice and mice treated with Cy, G or Cy/G. The gene expression profile of these cells was examined by microarray analysis, relative to HSCs isolated from untreated mice. Interestingly, among the genes upregulated by Cy/G, a large majority (&gt;60%) were a consequence of synergistic activity between Cy and G, while the rest of the upregulated genes could be attributed to activation by Cy or G alone. To test whether this screen allowed identification of novel candidates that regulate HSC function we analyzed the role of transforming growth factor beta induced gene (Big-h3), a gene highly upregulated after Cy/G treatment. Big-h3 is an extracellular matrix protein that mediates the adhesion and migration of various cell types, however its role in HSCs is unknown. Our experiments indicate that overexpression of Big-h3 in HSCs leads to enhanced adhesion to fibronectin as well as upregulation of specific integrins. These experiments suggest that changes in adhesion are an integral component of HSC mobilization, and that Big-h3 is one regulator of such processes. Further functional analysis of other candidates will likely lead to identification of other novel regulators of HSC development and mobilization.
Utility of Quantum Dots for Labeling and Tracking Leukemic Cell Lines, Human Bone Marrow and CD 34+ Umbilical Cord Blood.
Authors: Garon Edward B. , Crooks Gay M. , Tcherniantchouk Oxana , Luong Quang T. , Papaioannou Thanassis , Marcu Laura , Koeffler H. Phillip 
Published: April 2020

Abstract
Quantum dots (QDs) are nanometer scale fluorescent semiconductors that have recently garnered much interest as agents for imaging. QDs have several advantages over conventional fluorescent imaging probes, including high quantum yield, broad absorption spectra and minimal photobleaching. QDs have been used little in hematology.
We studied hematologic cells to determine whether they could be labeled with QDs, whether QDs could be seen after multiple cell divisions, whether QDs remained visible as cells differentiate and whether QDs could target specific antigens on the surface of cells. Using QDs attached to an endocytosed protein, we could intracellularly label all hematologic cells tested, including multiple leukemic cell lines (HL-60 and KG-1), normal human and murine bone marrow, leukemic human bone marrow (AML and CML) and normal human CD34+ cells. The QDs are easily detected by FACS and microscopy.
By counting cell divisions using the cell membrane dye PKH26, we show that QDs can be seen in leukemic cell lines and primary cells through more than four cell divisions. Some cells from leukemic cell lines retain QDs for up to two weeks. We are also able to see QDs in cells through differentiation. We labeled HL-60 cells and cultured them with either 1,25 dihydroxy vitamin D or DMSO. QDs were seen in monocytic-like and neutrophil-like progeny of the labeled HL-60 cells. After labeling CD34+ cells and culturing them in appropriate cytokines, QDs were observed in the resultant monocytes.
We were also able to target QDs specifically to cells expressing certain cell surface antigens. QDs complexed to streptavidin and a biotinylated anti-CD33 antibody homed to myelogenous leukemic cell lines but not lymphomatous cell lines. In summary, we show for the first time that QDs effectively label and track hematolgic cells, adding a new option when addressing cell fate questions in hematology.
GABP (GA Binding Protein) Is Required for Formation of a Retinoic Acid-Induced Myeloid Enhanceosome on the CD18 Promoter.
Authors: Resendes Karen K. , Rosmarin Alan G. 
Published: April 2020

Abstract
CD18, the β chain of the leukocyte integrins, mediates cell-cell and cell-matrix interactions of white blood cells and is required for normal innate immunity. Retinoic acid (RA) induces maturation of myeloid cells; mutations in retinoic acid receptors (RARs) account for acute promyelocytic leukemia. CD18 is transcriptionally regulated by RA in myeloid cells. The proximal promoter of CD18 is transcriptionally regulated by RA but, strikingly, this region is not bound or activated by retinoic acid receptors. Rather, the RA-responsiveness of the CD18 proximal promoter is dependent on ets sites that are bound by the ets transcription factor, GA-binding protein (GABP). The transcriptional co-activator, p300, further increases the RA-responsiveness of CD18. We show that p300 and GABPα, the ets DNA binding subunit of GABP, physically interact in myeloid cells. This interaction uses the cysteine/histidine regions of p300, and the pointed domain (PNT) of GABPα. This demonstrates p300 as the first known interaction partner of the GABPα PNT domain. We prepared a GABPα PNT domain construct for expression in mammalian cells and showed that this PNT construct inhibited the GABPα:p300 interaction and dramatically decreased the RA-responsiveness of the CD18 proximal promoter. We used Chromatin immunoprecipitation (ChIP) to demonstrate that RA induces formation of an enhanceosome on the CD18 promoter; thus, GABPα and p300 on the CD18 proximal promoter recruit RAR/RXR bound to a distal enhancer in the presence of RA. We prepared a dominant negative p300 construct and showed that its expression blocks the formation of the CD18 enhanceosome. These results demonstrate that GABP is required for formation of an enhanceosome that mediates responsiveness of CD18 to RA in myeloid cells. Dominant negative forms of GABPα and p300 interfere with RA responsiveness in myeloid cells and disrupt this enhanceosome. The CD18 enhanceosome is the first known RA-induced enhanceosome and demonstrates its role in mediating retinoid responsiveness in myeloid cells.