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  • 1
    Electronic Resource
    Electronic Resource
    Focal adhesion formation is associated with secretion of allergic mediators (1995)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 31 (1995), S. 215-224 
    Details
    ISSN: 0886-1544
    Keywords: RBL-2H3 cells ; vinculin ; mast cells ; talin ; cytoskeleton ; permeabilized ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Adherence of cells to the extracellular matrix via focal adhesions is known to modulate many cellular functions. However, the role of focal adhesions in the regulation of secretion is unclear. To examine this we have used the RBL-2H3 rat mast cell line, in which we and others have observed cytoskeletal rearrangements and increased cell spreading during secretion. All activators of secretion examined, whether acting specifically through or bypassing the IgE-receptor, induced the assembly of focal adhesions, as defined by the localization of vinculin and talin. The extent of focal adhesion formation correlated with the extent of secretion and the time course of secretion also correlated with that of the assembly of focal adhesions. To examine the mechanism by which focal adhesion formation occurred, the protein kinase C inhibitor bisindolylmaleimide was used. Bisin-dolylmaleimide caused complete inhibition of both secretion and focal adhesion formation induced by antigen or the calcium ionophore A23187. Although PMA did not induce secretion, it induced focal adhesion assembly which was inhibited by bisindolylmaleimide. The inhibitor had no effect on secretion or focal adhesion formation induced by the ATP analogue, ATPγS in permeabilized cells, indicating ATPγS acts after the activation of protein kinase C in the secretory pathway. These data provide novel evidence that the formation of focal adhesions may have a role in the process of secretion from mast cells.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970310305
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  • 2
    Electronic Resource
    Electronic Resource
    PMA and calcium ionophore induce myosin and F-actin rearrangement during histamine secretion from RBL-2H3 cells (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 354-365 
    Details
    ISSN: 0886-1544
    Keywords: exocytosis ; rat tumor mast cells ; cytoskeleton ; A23187 ; stress fibres ; tubulin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Rat basophilic leukemia (RBL-2H3) cells undergo morphological and cytoskeletal changes during antigen-induced secretion of allergic mediators. The exact role these changes play in the process of secretion is unclear. Using confocal microscopy we now show that PMA + A23187 causes extensive F-actin rearrangements during secretion of [3H] 5-HT. We also describe for the first time the association of myosin with F-actin during this secretory process. In unstimulated cells, myosin and F-actin are concentrated at the plasma membrane with no evidence of stress fibres. Upon addition of PMA or A23187, both F-actin and myosin are rearranged into membrane ruffles and discrete aggregations (foci), followed by the formation of parallel stress fibres located on the ventral membrane. This is in contrast to reports in other cell types in which PMA has been described as causing the disruption of F-actin stress fibres. The time course of secretion coincides with the formation of the foci and ruffles whilst the stress fibres form after the majority of secretion has occurred. These changes are accompanied by a 40% decrease in cell height and a two-fold increase in cell spreading and they occur in the absence of extracellular calcium but are inhibited by the protein kinase C inhibitor, Bisindolylmaleimide, which also inhibits secretion. The formation of myosin-decorated stress fibres, foci, and ruffles is not sufficient to cause secretion, as PMA alone induces these changes without any secretion. The relevance of actin and myosin rearrangements for the regulation of secretion is discussed. © 1994 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970290408
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  • 3
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    Harvest of Autologous Peripheral Blood Stem Cells Using Ifosfamide/Etoposide Regimen Combined with Granulocyte Colony-Stimulating Factor. (2004)
    American Society of Hematology
    Details
    Publication Date: 2004-11-16
    Description: Aim: To make clear the feasibility of Ifosfamide/Etoposide (IE) regimen combined with granulocyte colony-stimulating factor (G-CSF) for the collection of autologous peripheral blood stem cells (APBSC), we evaluated the number of the collected CD34 positive cells, and the regimen-related toxicities. Patients’ characteristics and Methods: 19 Patients (male 12, female 7) were received APBSC harvest in our hospitals during Aug. 2000 to Dec. 2003 with age of 54.3 years on average (30–66). Diagnoses were included HD (n=2), NHL (n=13), MM (n=3), and malignant synovioma (n=1). IE regimen was included with Ifomide 2000 mg/m2 (day 1), and Etoposide 200 mg/m2 (day 1–3) (4cases) or 500 mg/m2 (day 1,2) (15 cases). For the prevention of hemorrhagic cystitis patients were administered with Mesna, and NaHCO3 during and after 1 day of the administration of Ifosfamide. G-CSF was administered at the dose of 10 ug/kg (lenograstim) or 300 ug/m2 (filgrastim) when blood absolute neutrophil count came down to 1000/mm3. When the suppression of bone marrow was recovered and blood WBC count came up to 5000/m3, CD34-positive cells were counted in blood, and APBSC was harvested when blood CD34-positive cells were determined above 0.1 %. APBSC was harvested with CS-3000 plus (Fenwall). Result: The median duration from the start of the administration of G-CSF to the finish of APBSC harvest was 6.5 days (4–11). The median number of CD34-positive cells of the harvested was 4.32 x 106/kg (1.10–13.50). All cases were harvested. The toxicities during from the conditioning to the harvest were included with grade 1 headache (2 cases), grade 2 nausea (6 cases), grade 1 bleeding (1 case), grade 1 constipation (1 case), grade 1 fever (1 case), and grade 1 thrombocytopenia (1 case). Conclusion: IE regimen combined with G-CSF was feasible to harvest APBSC on the yield of the collection of the transplantable stem cells, and on the regimen-related toxicities.
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    Electronic ISSN: 1528-0020
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  • 4
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    Comparison of the Feasibility of Adult Allogeneic Umbilical Cord Blood Transplantation between the Myeloablative Conditioning Regimen and the Non Myeloablative One. (2004)
    American Society of Hematology
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    Publication Date: 2004-11-16
    Description: Aim: To make clear the feasibility of non-myeloablative conditioning on the adult allogeneic umbilical cord blood transplantation, we compared the result between patients receiving the myeloablative (conventional) transplantation (CT) and the reduced intensity stem cell transplantation (RIST) in point of the engraftment, and the incidence of GVHD. Patients’ characteristics and Methods: Patients were admitted to our hospitals to receive umbilical cord blood transplantation during May 1999 to May 2004. CT group (n=23): Median age was 33 years. Diagnoses were included AML (n=9), ALL(n=7), NHL(n=4), MDS(n=2), CML(n=1). Preparative conditioning regimens were; TBI/Ara-C/CY (n=16), TBI/VP-16/CY (n=4), TBI/CY (n=3). GVHD prophylaxis was; Cyclosporine A (CsA) (n=11), CsA with short term Methotrexate (MTX) (n=10), Tacrolimus with short term MTX (n=2). The transplanted median cell number was 2.40 x 10^7/kg. HLA disparities were; 1AMM (n=5), 2AMM (n=18), 3AMM (n=3). RIST group (n=13): Median age was 54 years. Diagnoses were; AML (n=3), NHL (n=5), HD (n=2), ATL (n=1), CLL (n=1), CMMoL(n=1). Preparative conditioning regimens were; Flu/ L-PAM (n=4), TBI/ Flu/ L-PAM (n=1), TBI/ Flu/ CY (n=3), TBI/ Flu (n=1), TBI/ Flu/ BU (n=4). GVHD prophylaxis was; CsA (n=5), CsA with MMF (n=7), Tacrolimus (n=1). The transplanted median cell number was 2.49 x 10^7/kg. HLA disparities were; identical (n=3), 1AMM (n=2), 2AMM (n=6), 3AMM (n=2). Result: The rate of engraftment failure including conditioning failure, and autologous recovery was 14.8 %in CT and 38.1%in RIST, respectively. The median duration of the engraftment of neutrophils in CT and RIST were 24 days and 19 days, respectively. Patients with Grade II-IV acute GVHD were 15 (65.2%) in CT and 8 (61.5%) in RIST, respectively. The incidence of Grade III-IVacute GvHD was 7 ( 30.4%) and 5(38.4%), respectively. Conclusion: Engraftment in RIST was achieved earlier than CT, however the incidence of engraftment failure was increased in RIST. There were no significant differences on the incidence of acute GVHD between two groups. Further observations, especially long-term disease-free survival are seemed to be very interesting to ascertain RIST on adult umbilical cord blood transplantation.
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  • 5
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    Chronic Myelogenous Leukemia Cells Contribute to a Bone Marrow-Stroma in In Vivo NOD/SCID Mouse System. (2009)
    American Society of Hematology
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    Publication Date: 2009-11-20
    Description: Abstract 2191 Poster Board II-168 Aims: The stroma-forming cells in a bone marrow are derived from hematopoietic stem cells. We reported previously that non-adherent leukemia blast cells converted into myofibroblasts to create a microenvironment for proliferation of leukemia blasts in vitro. In this report we demonstrate that with severe combined immunodeficiency (SCID) mouse system chronic myelogenous leukemia (CML) cells are also differentiated into myofibroblasts to contribute to a bone marrow-stroma in vivo. Materials and Methods: Bone marrow cells were collected from informed CML patients, from which mononuclear cells were separated with density-gradient sedimentation method. After discarded an adherent cell-fraction, non-adherent mononuclear cells were injected to the priory 2.5 Gray-irradiated non-obese diabetes (NOD)/SCID mice intravenously. For the inactivation of NK cells, anti-Asialo GM1 antibody was injected intra-peritoneally prior to the transplantation, and on each 11th day thereafter. Blood was collected to monitor Bcr-Abl transcript, and mice were sacrificed after chimeric mRNA was demonstrated. Bone marrow cells were obtained, and sorted with anti-human CD133 antibody and -CD106 to select CML-derived human stromal myofibroblasts referred to the in vitro data. The isolated positive fraction was further cultured, and the biological and the molecular characteristics were analyzed. Results and Discussion: When non-adherent CML cells were transplanted to NOD/SCID mice, CML cells were engrafted after 2 months. In the murine bone marrow human stromal cells were identified, in which BCR and ABL gene was fused with FISH analysis. When the parental CML cells were cultured on the CML-derived myofibroblasts, CML cells grew extensively in a vascular endothelial growth factor-A-dependent fashion. These results indicate that CML cells can create their own microenvironment for proliferation in vivo. Disclosures: No relevant conflicts of interest to declare.
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  • 6
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    Increased Levels of Hepatocyte Growth Factor in Patients with DIC. (2004)
    American Society of Hematology
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    Publication Date: 2004-11-16
    Description: Injury of endothelial cells has been postulated as an initial trigger of the progression of DIC. Although hepatocyte growth factor (HGF) is a member of endothelium-specific growth factor, the relationship between HGF and DIC has not been described. To investigate the role of HGF, we measured plasma levels of HGF in patients with sepsis-associated (n=20) and acute promyelocytic leukemia (APL)-induced DIC (n=6). Plasma samples from those patients groups were assayed for HGF levels by enzyme-linked immunosorbent assay (ELIZA, TECHNE Corporation, USA). The VEGF levels were determined by one-step sandwich enzyme immunoassay (EIA, Chemicon International, USA). The thrombin antithrombin complexes (TAT) levels were higher in both DIC patients as reported by others. In the septic patients with DIC, we found significant elevations in HGF levels compared to normal controls. Also, the HGF levels were elevated in the APL patients with DIC. However, we did not find any difference in plasma levels of VEGF in the APL and septic patients with DIC. There was a slight correlation between the TAT and HGF levels in both (septic and APL) patients groups with DIC. These results suggest that plasma levels of HGF may be candidates for a marker of DIC. It appears that HGF may contribute to the severity of DIC. Sepsis associated DIC APL with DIC Controls (n=10) *(P
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  • 7
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    Interleukin-1β Promotes the Expression of CD34 and Granulocyte Colony-Stimulating Factor-Receptor in Adult Dermal Fibroblasts (2011)
    American Society of Hematology
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    Publication Date: 2011-11-18
    Description: Abstract 4815 Background and Aims: We reported that acute myelogenous leukemia blasts and chronic myelogenous leukemia cells converted to stromal myofibroblasts to create an environment for the proliferation of leukemic cells in vitro and also in a non-obese diabetes/ severe combined immunodeficiency (NOD/SCID) murine bone-marrow in vivo. In normal hematopoiesis, hematopoietic stem cell (HSC) and stromal immature mesenchymal stem cell (MSC) are speculated to have a cross-talk, and some reports indicate that the HSC generates MSC, and also a specific fraction of MSC shares similar molecular expressions to that of HSC. We made a hypothesis that HSC might be generated from MSC. To make clear this issue, expression cloning was performed to isolate a molecule that stimulated bone-marrow stromal myofibroblasts to express hematopoietic stem cell marker, CD34. And, we also observed the effect of the isolated molecule to an adult human dermal fibroblast (HDF). Materials and Methods: cDNA-expression library was constructed using PHA-P-stimulated normal human blood lymphocytes, and the prepared plasmids were transfected to COS7 cells. After 3 days of culture, supernatants were added to the normal human bone-marrow-derived myofibroblasts (final 10%), and cells were further cultured for one week. RNA was extracted from the cultured myofibroblasts, and cDNA was synthesized. Positive clones were selected on CD34-expression with reverse transcription-polymerase chain reaction, and a single clone was isolated. The purified protein from the isolated single clone was added to HDF-culture, and the morphological changes and the expression of specific hematopoiesis-related proteins were analyzed. Results and Discussion: Isolated single clone was human interleukin 1β (IL-1β). When the purified IL-1β protein was added to the bone-marrow-derived myofibroblast cultures, cell growth was increased, and up-regulation of the expression of several hematopoietic specific proteins, including cytokine receptors and transcription factor SCL, was observed. Based on these observations, we determined the effect of IL-1β to HDF. When HDFs were cultured with human IL-1β for 3 weeks, the expression of granulocyte colony-stimulating factor (G-CSF)-receptor, and SCL was increased. When these IL-1β-stimulated cells were cultured in a non-coated dish, cells were floating, and budding of the cells was also observed. When HDF were cultured with IL-1β for 3 weeks, and then G-CSF and erythropoietin were added to the cultures, expression of transcription factor GATA-1 and CEBPA was significantly increased after one week. These observations indicate that IL-1β can stimulate to induce HDF toward hematopoietic cells. Now we determine the precise actions of human IL-1β to HDF using NOD/SCID transplantation model in vivo. Disclosures: No relevant conflicts of interest to declare.
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  • 8
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    Evaluation of Non Overt DIC Diagnostic Criteria. (2009)
    American Society of Hematology
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    Publication Date: 2009-11-20
    Description: Abstract 1297 Poster Board I-319 Introduction Several diagnostic criteria for disseminated intravascular coagulation (DIC) have been proposed by Colman RW et al, Japanese Ministry Health and Welfare (JMHW) and International Society of Thrombosis and Haemostasis (ISTH) etc. The efficacy of treatment in relation to the DIC score when the treatment was begun showed that greater efficacy was achieved in pre-DIC than in DIC patients, suggesting that early diagnosis and early treatment are important. Then, the diagnostic criteria for non-overt DIC was proposed by ISTH/SSC subcommittee in order to diagnose of early phase DIC but it was still not established. In this study, modified non-overt DIC diagnostic criteria using molecular hemostatic markers and antithrombin (AT) has been prospectively evaluated. Total 613 patients suspected to be associated with DIC in nine institutes were registered in this prospective study for DIC diagnostic criteria from January 1, 2005 to December 31, 2008. There were 219 patients with infectious disease, 142 with solid cancer, 115 with hematopoietic tumor, 29 with aneurysm, 10 with obstetrics disease, 23 with trauma, 5 with liver disease, 70 with other disease. Materials and Methods Overt-DIC or non-overt DIC was diagnosed by modified overt-DIC diagnostic criteria or modified non-overt DIC diagnostic criteria using abnormalities or changes of platelet count, prothrombin time (PT), fibrinogen, fibrin and fibrinogen degradation products (FDP), AT, soluble fibrin monomer complex (SFMC), D-dimer and thrombin-AT complex (TAT). Pre-DIC was considered the state within a week before onset of DIC. Measurement of PT, fibrinogen, platelet count, FDP were carried out in each institutes based on numerous previous reports. TAT, SFMC, D-dimer and AT activity were measured in SRL Inc. TAT was measured by enzyme immunoassay (EIA) using. SFMC and D-dimer were measured by latex immune agglutination test using T-test, respectively. AT activity was measured by heparin cofactor activity. Results Frequency of overt-DIC was 29.5 % in 613 patients suspected to be associated with DIC, and the highest frequency of overt-DIC was observed in the patients with obstetrics disease or liver disease. Pre-DIC state was detected in 44 of 613 patients (7.2 %) and the highest frequency of pre-DIC was observed in the patients with hematopoetic tumor (12.2 %). Frequency of non-overt DIC in the patients with overt-DIC, with pre-DIC or without overt-DIC was 97.8 %, 97.7 % or 17.0%, respectively. The mortality of 28 days was the highest in the patients with overt DIC (37.6%), and it was also high in those with non-overt DIC (32.9%) and with pre-DIC (27.3%) in comparison to the patients without overt DIC (15.2%) and those without non-overt DIC (13.8%). Discussion Modified non-overt DIC diagnostic criteria is more sensitive for DIC than overt DIC diagnostic criteria and is related to the outcome. Non overt DIC diagnostic criteria might be able not only to diagnose DIC but also to predict early phase of DIC. Disclosures No relevant conflicts of interest to declare.
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  • 9
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    Increased Levels of Histone In Human Plasma In Septic Patients with DIC. (2010)
    American Society of Hematology
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    Publication Date: 2010-11-19
    Description: Abstract 1422 Sepsis is a life-threatening condition that is characterized by a whole-body inflammatory state (called a systemic inflammatory response syndrome, SIRS). Histones are essential for packing DNA into the cell nucleus and also play role in regulating gene expression but the new study (Jun Xu et al, Nature Medicine 15. 1318, 2009) have shown histones released into blood stream at the onset of sepsis can have devastating effects (endothelial damage and organ failure etc). However, about extracellular histones in the plasma from human, they have shown only one patient's data. Therefore, we measured extracellular hisotones in the plasma from septic patients with DIC (n=20). Also, we investigated extracellular hisotones in the plasma from septic patients without DIC (n=5). Extracellular histones in the plasma from human were examined by western blot analysis for histone H3 (H3). The thrombin antithrombin complexes (TAT) levels were higher in septic patients with DIC as reported by others. We detected high levels of extracellular hisotones (H3) in the plasma of all septic patients with DIC by western blotting. However, we did not find any extracellular histones (H3) in the plasma of fiver septic patients without DIC by western blotting. Moreover, high-mobility group box 1 (HMGB1) concentrations in the human plasma were determined using ELISA kit (Shino-Test, Japan). Plasma levels of HMBG1 were negative for four cases in 20 septic patients with DIC. Also, Plasma levels of HMBG1 were negative in septic patients without DIC. These results suggest that assess of extracellular hisotones in the plasma may be helpful in the making the diagnosis in septic patients with DIC. It appears that extracellular hisotones in the plasma may contribute to develop DIC in patients with sepsis. Disclosures: No relevant conflicts of interest to declare.
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  • 10
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    The Production of Vascular Endothelial Growth Factor Is Decreased in Acute Myelogenous Leukemia Cases That Are Treated with Chemotherapeutic Agents and Show the Prolonged Bone Marrow Suppression. (2007)
    American Society of Hematology
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    Publication Date: 2007-11-16
    Description: Objective: There have been reported that the levels of serum vascular endothelial growth factor (VEGF) were decreased in aplastic anemia cases. We investigated VEGF system after chemotherapy to acute myelogenous leukemia (AML) cases, and determined whether VEGF system influenced the prolonged bone marrow suppression in these cases. Materials and Methods: Sera and bone marrow cells were prepared from 30 AML cases including 10 cases of AML (M3) at the onset of the disease, after chemotherapy, and the recovery periods, and the concentration of VEGF in sera of the patients and in the conditioned media obtained from bone marrow-cell cultures was measured with ELISA kit (Quantikine; R&D Systems). The expression of VEGF, VEGF receptor type-1 and VEGF receptor type-2 was analyzed with RT-PCR. The biological effect of VEGF on the bone marrow cells which showed the prolonged suppression after chemotherapy was assayed with colony-formation with or without any cytokines. Result and Discussion: As was reported previously, VEGF levels were significantly increased in M3 cases. In other types of AML cases the levels of VEGF production varied. When patients were given chemotherapy and the bone marrow suppression was prolonged, the production levels of VEGF were significantly diminished less than that observed in AML cases with normal bone marrow recovery. In M3 cases that were treated with all-trans retinoic acid and the prolonged bone marrow-suppression was observed, VEGF production was also suppressed. The expression of VEGFR-1 and -2 was observed in bone marrow cells from prolonged bone marrow suppression cases. In these cases, when bone marrow cells were cultured with VEGF, synergistic effects with G-CSF and EPO were observed with colony-formation assay. These observations indicate that VEGF works on the important role for the hematopoietic recovery after chemotherapy in AML cases.
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