The BCR/ABL preleukemic fusion gene (PFG) is one of the most frequent fusion genes in acute lymphoblastic leukemia (ALL) and was also detected in hematopoietic cells from umbilical cord blood (UCB) ...of healthy newborns. Since hematopoietic stem/progenitor cells (HSPC) are considered to be a critical cellular target for origination of leukemia, we have studied the presence of BCR/ABL PFG in expanded subpopulations of HSPC and differentiated cells from UCB of those healthy newborns, who have previously been tested positive for BCR/ABL by screening of their UCB mononuclear cells using RT-qPCR and FISH methods. We isolated cells from human UCB samples positive for BCR/ABL and negative controls. The isolated cells were sorted into 5 hematopoietic and progenitor cell subpopulations. We analyzed BCR/ABL in sorted and expanded subpopulations of UCB using FISH and RT-qPCR. We found that the number of BCR/ABL positive cells was similar in each studied subpopulation and the same as in differentiated lymphocytes. Our data showed that there is no specific subpopulation of hematopoietic and progenitor stem cells with an increased leukemogenic potential due to the presence of higher copies of BCR/ABL.
Tumor necrosis factor-alpha (TNF) is a major mediator of apoptosis as well as immunity and inflammation. Inappropriate production of TNF or sustained activation of TNF signaling has been implicated ...in the pathogenesis of a wide spectrum of human diseases, including cancer, osteoporosis, sepsis, diabetes, and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. TNF binds to two specific receptors, TNF-receptor type I (TNF-R1, CD120a, p55/60) and TNF-receptor type II (TNF-R2, CD120b, p75/80). Signaling through TNF-R1 is extremely complex, leading to both cell death and survival signals. Many findings suggest an important role of phosphorylation of the TNF-R1 by number of protein kinases. Role of TNF-R2 phosphorylation on its signaling properties is understood less than TNF-R1. Other cellular substrates as TRADD adaptor protein, TRAF protein family and RIP kinases are reviewed in relation to TNF receptor-mediated apoptosis or survival pathways and regulation of their actions by phosphorylation.
Abstract Cytotoxicity is one of the major defence mechanisms against both virus-infected and tumor cells. Radioactive51 chromium (51 Cr) release assay is a “gold standard” for assessment of natural ...killer (NK) cytolytic activity in vitro . Several disadvantages of this assay led us to design alternative tools based on flow cytometry analysis. Four different fluorescent dyes, calcein acetoxymethyl ester (CAM), carboxyfluorescein succinimidyl ester (CFSE), Vybrant DiO (DiO) and MitoTracker Green (MTG) were tested for labeling of NK target K-562 cells. Target staining stability, spontaneous release of fluorochromes and subsequent accumulation in bystander unstained cells were measured using fluorimetry and flow cytometry. Healthy donor peripheral blood mononuclear cells and affinity column purified NK cells were used as effectors coincubated with target K-562 cells at different E:T ratios for 3 h and 90 min, respectively. Fluorescent probe 7-amino-actinomycin D was used for live and dead cell discrimination. Bland–Altman statistical method was applied to measure true agreement for all CAM–51 Cr, CFSE–51 Cr, DiO–51 Cr and MTG–51 Cr pairs analyzed. Based on the data, none of the four proposed methods can be stated equivalent to the standard51 Cr release assay. Considering linear relationships between data obtained with four fluorochromes and51 Cr release assay as well as linear regression analysis with R2 =0.9393 value for CAM–51 Cr pair, we found the CAM assay to be the most closely related to the51 Cr assay.
Childhood leukemia arises from hematopoietic stem cells by induction of mutations. Quite often chromosomal translocations arise prenatally as first key event in multistage process of leukemogenesis. ...These translocations result in so called preleukemic gene fusions (PGFs), such as BCR-ABL and TEL-AML1, which generate hybrid proteins with altered properties. Critical DNA damage resulting in translocations are DNA double-strand breaks (DSBs). BCR-ABL and TEL-AML1 were shown to be associated with increased constitutive DSBs in various model systems. We analyzed cells from peripheral blood and CD34-/CD34+ cells from bone marrow of pediatric acute lymphoblastic leukemia (ALL) patients harboring BCR-ABL or TEL-AML1. We used sensitive technique that is based on automated enumeration of DSB co-localizing proteins γH2AX and 53BP1, which form so called DNA repair foci. We found that level of constitutive γH2AX/53BP1 foci is significantly higher in cells of ALL pediatric patients than in healthy subjects. There was also significant increased level of constitutive γH2AX/53BP1 foci in cells from ALL patients harboring BCR-ABL or TEL-AML1 compared to patients without PGFs. The same increase was observed regardless cell type for both PGFs. Our data on increased DSB levels in the BCR-ABL/TEL-AML1 patient's cells support a model where BCR-ABL/TEL-AML1 induces DNA instability through facilitating mutagenesis and appearance of additional genetic alterations driving leukemogenesis.
Intercellular flow of the phytohormone auxin underpins multiple developmental processes in plants. Plant-specific pin-formed (PIN) proteins and several phosphoglycoprotein (PGP) transporters are ...crucial factors in auxin transport-related development, yet the molecular function of PINs remains unknown. Here, we show that PINs mediate auxin efflux from mammalian and yeast cells without needing additional plant-specific factors. Conditional gain-of-function alleles and quantitative measurements of auxin accumulation in Arabidopsis and tobacco cultured cells revealed that the action of PINs in auxin efflux is distinct from PGP, rate-limiting, specific to auxins, and sensitive to auxin transport inhibitors. This suggests a direct involvement of PINs in catalyzing cellular auxin efflux.
Adequate storage compounds are a prerequisite for successful development during the later stages of somatic embryogenesis; however, the critical amount of reserves below which somatic embryos fail to ...mature and germinate has not been determined. We analyzed storage lipids during Norway spruce (Picea abies (L.) Karst.) somatic embryogenesis. As maturation progressed, lipids, which were stored as lipid bodies in the cytoplasm, were localized first in suspensor cells of the early embryos, and later in the embryonic root pole, superficial layers of the hypocotyl and in cotyledons. The concentration of total lipids exhibited marked variation, with values peaking during cotyledon development and then decreasing during maturation. Linoleic (18:2), oleic (18:1), palmitic (16:0) and 5,9-octadecenic (5,9-18:2) acids were the most abundant fatty acids in embryos. As embryos developed, linoleic acid concentration increased slightly, whereas oleic acid concentration decreased. Oleic acid was the most prominent component of the fatty acid spectrum in isolated dormant zygotic embryos and megagametophytes. Addition of 5% polyethylene glycol to the medium during somatic embryo maturation caused a shift in the fatty acid spectrum toward that of zygotic embryos. During maturation, changes in the exogenous carbohydrate supply had no significant effect on total lipid concentration in mature embryos. A marked decrease in lipid concentration was detected during desiccation, indicating the importance of adequate lipid reserves during this developmental stage. The lipid content of zygotic embryos differed considerably with harvest year and location, suggesting that zygotic embryo data cannot be an indicator of somatic embryo quality.
Objectives: Patients presenting with ST-elevation myocardial infarction (STEMI) benefit from rapid cardiac reperfusion therapy. Emergency medical service (EMS) agencies can improve patient outcomes ...by calling STEMI alerts to the receiving facility. The aim of this study was to evaluate the use of pre-hospital activation systems for suspected ST-elevation myocardial infarctions (STEMI) throughout Colorado.
Methods: A cross sectional, survey design was utilized to collect all data from EMS agencies in Colorado. A univariable logistic regression model was used to identify factors predictive of an agency reporting that they utilize a STEMI activation protocol.
Results: 84.5% 95% CI: 78.3 to 90.7% of agencies included indicate that they utilize a STEMI activation protocol. Based on the logistic regression analysis, the number of EMT employees was significantly associated with whether or not an agency indicates that they utilize a STEMI activation protocol. For every 10% increase in the number of EMTs employed by an EMS agency, there was a 3.0 95% CI: 1.5 to 6.0, p = 0.0012 fold increase in the odds of the agency indicating they utilize a STEMI activation protocol.
Conclusions: Our study provides evidence that larger agencies are more likely to utilize a STEMI activation protocol. In areas without a STEMI system of care, improvements in smaller agencies that cover more ground (with longer transport times) should be the focus for protocol implementation. Based on the current prevalence of such training, competency based training in reading ST-elevations on ECG should be considered by EMS agencies.
This study was performed to test a new technique for treatment of chronic non-healing wound (diabetic ulcer) using autologous biograft composed of autologous skin fibroblasts on biodegradable ...collagen membrane (Coladerm) in combination with autologous mesenchymal stem cells (MSC) derived from the patient's bone marrow.
The bone marrow aspirate of the patient with diabetic foot was applied directly to the wound and injected into the edges of the wound, finally covered with prepared autologous biograft. The patient received two additional treatments with cultured MSC on day 7 and 17.
The wound showed a steady overall decrease in wound size and an increase in the vascularity of the dermis and in the dermal thickness of the wound bed after 29 days of combined treatment.
Closing and healing of the non-healing diabetic ulcer was achieved by using the given combined therapy.
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