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Priscilla A Schaffer - One of the best experts on this subject based on the ideXlab platform.
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glutamine deprivation causes enhanced Plating Efficiency of a herpes simplex virus type 1 icp0 null mutant
Journal of Virology, 2008Co-Authors: Ryan M Bringhurst, Antonia A Dominguez, Priscilla A SchafferAbstract:Isoleucine deprivation of cellular monolayers prior to infection has been reported to result in partial complementation of a herpes simplex virus type 1 (HSV-1) ICP0 null (ICP0 − ) mutant. We now report that glutamine deprivation alone is able to enhance the Plating Efficiency of an ICP0 − virus and that isoleucine deprivation has little or no effect. Because a low glutamine level is associated with stress and because stress is known to induce reactivation, low levels of glutamine may be relevant to the reactivation of HSV-1 from latency. Additionally, we demonstrate that arginine and methionine deprivation result in partial complementation of the ICP0 − virus.
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Cellular Stress Rather than Stage of the Cell Cycle Enhances the Replication and Plating Efficiencies of Herpes Simplex Virus Type 1 ICP0− Viruses
Journal of Virology, 2006Co-Authors: Ryan M Bringhurst, Priscilla A SchafferAbstract:This lab reported previously that the Plating Efficiency of a herpes simplex virus type 1 ICP0-null mutant was enhanced upon release from an isoleucine block which synchronizes cells to G 1 phase (W. Cai and P. A. Schaffer, J. Virol. 65:4078-4090, 1991). Peak Plating Efficiency occurred as cells cycled out of G 1 and into S phase, suggesting that the enhanced Plating Efficiency was due to cellular activities present in late G 1 /early S phase. We have found, however, that the enhanced Plating Efficiency did not occur when cells were synchronized by alternative methods. We now report that the Plating Efficiency of ICP0 − viruses is not enhanced at a particular stage of the cell cycle but rather is enhanced by specific cellular stresses. Both the Plating and replication efficiencies of ICP0 − viruses were enhanced as much as 25-fold to levels similar to that of wild-type virus when monolayers were heat shocked prior to infection. In addition to heat shock, UV-C irradiation but not cold shock of monolayers prior to infection resulted in enhanced Plating Efficiency. We further report that the effect of cellular stress is transient and that cell density rather than age of the monolayers is the primary determinant of ICP0 − virus Plating Efficiency. As both cell stress and ICP0 are required for efficient reactivation from latency, the identification of cellular activities that complement ICP0 − viruses may lead to the identification of cellular activities that are important for reactivation from neuronal latency.
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a cellular function can enhance gene expression and Plating Efficiency of a mutant defective in the gene for icp0 a transactivating protein of herpes simplex virus type 1
Journal of Virology, 1991Co-Authors: Priscilla A SchafferAbstract:Abstract ICP0 transactivates herpes simplex virus type 1 genes of all classes as well as a number of heterologous viral and cellular genes, yet it is not essential for virus replication in vitro or in vivo. Stocks of ICP0 deletion mutants, however, exhibit significantly lower Plating efficiencies on standard 24-h-old Vero cell monolayers than do stocks of wild-type virus. In an attempt to determine whether the growth status of cells in the monolayer affects the ability of ICP0 mutants to initiate plaque formation, the Plating efficiencies and abilities of an ICP0 null mutant (7134) and of wild-type virus (KOS) to express selected viral proteins were determined on Vero cell monolayers whose growth had been arrested either by contact inhibition-trypsinization or by isoleucine deprivation and had then been released from growth arrest. The proportion of cells cycling synchronously after release from growth arrest was assessed by flow cytometry. The results of these studies indicate that the Plating Efficiency of 7134 was greatest on Vero cell monolayers 8 h after release from growth arrest induced by either treatment. Monolayers of both types released from growth arrest at other times supported 7134 plaque formation less efficiently. In contrast, the Plating Efficiency of KOS was nearly equal on monolayers at all times after release from growth arrest. Notably, both KOS and 7134 were equally efficient in entering cells and inducing expression of the immediate-early protein ICP4 in either 8- or 24-h monolayers. Relative to KOS, however, 7134 was significantly impaired in the expression of selected early and late genes in cells at 24 h postrelease. When the Plating efficiencies of 7134 and KOS were examined in 0-28 cells (Vero cells that are stably transformed with the ICP0 gene) whose growth had been arrested and then released, no differences in the Plating efficiencies of the two viruses as a function of growth status were noted. These findings suggest that a cellular function expressed maximally in cells 8 h after release from growth arrest can substitute operationally for ICP0 to enhance plaque formation and viral gene expression by 7134. They further suggest that one role of ICP0 in viral infection is to facilitate virus replication in cells that do not express this function.
Dong-wook Ryang - One of the best experts on this subject based on the ideXlab platform.
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Plating Efficiency of Single CD34+ Cell from Bone Marrow, Peripheral Blood and Cord Blood
Blood Research, 2020Co-Authors: Myung-geun Shin, Hyun-woo Choi, Bong-joon Oh, Jong-hee Shin, Dong-wook RyangAbstract:Background : Although bone marrow (BM) CD34+ cells and peripheral blood (PB) CD34+ cells are developmentally and functionally related, the recent data suggested that they may have different functional and clinical capabilities. Moreover, they have differential gene expression underlying the functional distinctions of primary human CD34+ hematopoietic stem and progenitor cells from BM and PB. The aim of this study was to investigate the Plating Efficiency of single CD34+ progenitor cell from BM, PB and umbilical cord blood (UCB). Methods : After sorting, single CD34+ cells were cultured in individual wells of 96-well plates in serum-free medium containing selected hematopoietic growth factors, with or without G-CSF. Plating Efficiency was microscopically determined by the presence of clusters of viable cells: [the number of positive (cells were present) wellstotal wells]100. CD34+ cell-derived colonies were classified according to the cell number per well. Results : Although there was some variation of Plating Efficiency of CD34+ cells among six normal BMs, six PBs and five UCBs, overall average Plating Efficiency of single CD34+ cells from BM, PB and UCB was 30% (30.011.7, meanSD), 79% (78.611.7) and 45% (45.39.3) respectively. As expected, the colony size was increased in the presence of G-CSF. Conclusion : The results of this study clearly showed that the different ex vivo expansion of single CD34+ progenitor cells from BM, PB and UCB. These might be an important data for understanding stem cell expansion in vivo and designing clinical application.
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direct confirmation of quiescent property of aml stem cells by showing markedly decreased Plating Efficiency in single leukemic stem cell culture
Blood, 2008Co-Authors: Myung-geun Shin, Jong-hee Shin, Hoon Kook, Dong-wook RyangAbstract:Cancer stem cell, recently discovered to exist in colon cancer and brain tumor, is resistant to anti-cancer drugs and radiotherapy, demanding the development of new drugs and treatment strategies targeting tumor stem cells. Leukemic stem cell (LSC) has been accused to play a pivotal role in pathogenesis of hematological malignancy such as acute myeloblastic leukemia (AML). Various anti-cancer medicines, particularly anti-proliferative agents, have been ineffective in treating LSC due to its slower division process and longer interphase compared to normal stem cell and hematopoietic cell. This study comparatively examined growth and proliferation capacity (Plating Efficiency) of clonogenic hematopoietic progenitors and LSC from healthy donors and AML patients using single cell sorting and culture system (BD FACS Aria cell sorter; BD Biosciences, San Jose, CA). A total of 384 normal hematopoietic stem cells (CD34+CD38+/CD38−) were obtained from peripheral bloods and cord bloods donated by four donors using single cell sorter, and individual single cells were cultured in 96-well plates with each well containing 100ul of serum media, 100ng/ml of stem cell factor, 100ng/ml of Flt-3, 100ng/ml of thrombopoietin and 50ng/ml of G-CSF for five days. 768 single LSC (CD34+CD38−) and 384 single CD34+CD38+ cells were obtained from three AML patients. Growth and proliferation capacities of normal hematopoietic stem cell and LSC were determined in terms of Plating Efficiency (number of the wells in which more than two cells grew/total number of cells in 96-well plate culture × 100). Plating Efficiency of individual normal single hematopoietic stem cells varied between samples. Eighty eight out of 192 single stem cells originated from cord blood grew into more than two cells, yielding Plating Efficiency of 45.8% and cells from the peripheral blood of two healthy donors 30.2% (58/192). In contrast, single LSC originated from the AML patients showed significantly lower Plating Efficiency with 14.6% (42/288), 3.6% (7/192) and 8.0% (23/288). These results directly confirmed quiescent and slowly dividing properties of LSC. In addition, Plating Efficiency of normal hematopoietic stem cells was shown to vary between their originating locations in healthy donors.
George Q Daley - One of the best experts on this subject based on the ideXlab platform.
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enhanced Plating Efficiency of trypsin adapted human embryonic stem cells is reversible and independent of trisomy 12 17
Cloning and Stem Cells, 2008Co-Authors: Elayne M Chan, Frank Yates, Leah F Boyer, Thorsten M Schlaeger, George Q DaleyAbstract:Human embryonic stem cells (hESCs) can be cultured abundantly and indefinitely, but are subject to accumulations of chromosomal aberrations. To preserve their genetic integrity, hESCs are commonly maintained as cell aggregates or clumps during passaging. However, clump passaging hinders large-scale culture and complicates the isolation of single cell clones. To facilitate the isolation of genetically modified clones of hESCs while preserving their genetic integrity, we employed trypsin single-cell passaging for brief periods before returning to clump passaging for long-term maintenance. We observed that accommodation to trypsin passage as single cells is an adaptive process where over three to four passages considerably increases the Plating Efficiency. However, trypsin passage was associated with abnormalities of chromosomes 12 and 17. Nevertheless, the high Plating Efficiency of trypsin passaged hESCs is a reversible phenotype, regardless of chromosomal abnormalities, suggesting that epigenetic events a...
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Enhanced Plating Efficiency of trypsin-adapted human embryonic stem cells is reversible and independent of trisomy 12/17.
Cloning and Stem Cells, 2008Co-Authors: Elayne M Chan, Frank Yates, Leah F Boyer, Thorsten M Schlaeger, George Q DaleyAbstract:Human embryonic stem cells (hESCs) can be cultured abundantly and indefinitely, but are subject to accumulations of chromosomal aberrations. To preserve their genetic integrity, hESCs are commonly maintained as cell aggregates or clumps during passaging. However, clump passaging hinders large-scale culture and complicates the isolation of single cell clones. To facilitate the isolation of genetically modified clones of hESCs while preserving their genetic integrity, we employed trypsin single-cell passaging for brief periods before returning to clump passaging for long-term maintenance. We observed that accommodation to trypsin passage as single cells is an adaptive process where over three to four passages considerably increases the Plating Efficiency. However, trypsin passage was associated with abnormalities of chromosomes 12 and 17. Nevertheless, the high Plating Efficiency of trypsin passaged hESCs is a reversible phenotype, regardless of chromosomal abnormalities, suggesting that epigenetic events a...
Ryan M Bringhurst - One of the best experts on this subject based on the ideXlab platform.
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glutamine deprivation causes enhanced Plating Efficiency of a herpes simplex virus type 1 icp0 null mutant
Journal of Virology, 2008Co-Authors: Ryan M Bringhurst, Antonia A Dominguez, Priscilla A SchafferAbstract:Isoleucine deprivation of cellular monolayers prior to infection has been reported to result in partial complementation of a herpes simplex virus type 1 (HSV-1) ICP0 null (ICP0 − ) mutant. We now report that glutamine deprivation alone is able to enhance the Plating Efficiency of an ICP0 − virus and that isoleucine deprivation has little or no effect. Because a low glutamine level is associated with stress and because stress is known to induce reactivation, low levels of glutamine may be relevant to the reactivation of HSV-1 from latency. Additionally, we demonstrate that arginine and methionine deprivation result in partial complementation of the ICP0 − virus.
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Cellular Stress Rather than Stage of the Cell Cycle Enhances the Replication and Plating Efficiencies of Herpes Simplex Virus Type 1 ICP0− Viruses
Journal of Virology, 2006Co-Authors: Ryan M Bringhurst, Priscilla A SchafferAbstract:This lab reported previously that the Plating Efficiency of a herpes simplex virus type 1 ICP0-null mutant was enhanced upon release from an isoleucine block which synchronizes cells to G 1 phase (W. Cai and P. A. Schaffer, J. Virol. 65:4078-4090, 1991). Peak Plating Efficiency occurred as cells cycled out of G 1 and into S phase, suggesting that the enhanced Plating Efficiency was due to cellular activities present in late G 1 /early S phase. We have found, however, that the enhanced Plating Efficiency did not occur when cells were synchronized by alternative methods. We now report that the Plating Efficiency of ICP0 − viruses is not enhanced at a particular stage of the cell cycle but rather is enhanced by specific cellular stresses. Both the Plating and replication efficiencies of ICP0 − viruses were enhanced as much as 25-fold to levels similar to that of wild-type virus when monolayers were heat shocked prior to infection. In addition to heat shock, UV-C irradiation but not cold shock of monolayers prior to infection resulted in enhanced Plating Efficiency. We further report that the effect of cellular stress is transient and that cell density rather than age of the monolayers is the primary determinant of ICP0 − virus Plating Efficiency. As both cell stress and ICP0 are required for efficient reactivation from latency, the identification of cellular activities that complement ICP0 − viruses may lead to the identification of cellular activities that are important for reactivation from neuronal latency.
Myung-geun Shin - One of the best experts on this subject based on the ideXlab platform.
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Plating Efficiency of Single CD34+ Cell from Bone Marrow, Peripheral Blood and Cord Blood
Blood Research, 2020Co-Authors: Myung-geun Shin, Hyun-woo Choi, Bong-joon Oh, Jong-hee Shin, Dong-wook RyangAbstract:Background : Although bone marrow (BM) CD34+ cells and peripheral blood (PB) CD34+ cells are developmentally and functionally related, the recent data suggested that they may have different functional and clinical capabilities. Moreover, they have differential gene expression underlying the functional distinctions of primary human CD34+ hematopoietic stem and progenitor cells from BM and PB. The aim of this study was to investigate the Plating Efficiency of single CD34+ progenitor cell from BM, PB and umbilical cord blood (UCB). Methods : After sorting, single CD34+ cells were cultured in individual wells of 96-well plates in serum-free medium containing selected hematopoietic growth factors, with or without G-CSF. Plating Efficiency was microscopically determined by the presence of clusters of viable cells: [the number of positive (cells were present) wellstotal wells]100. CD34+ cell-derived colonies were classified according to the cell number per well. Results : Although there was some variation of Plating Efficiency of CD34+ cells among six normal BMs, six PBs and five UCBs, overall average Plating Efficiency of single CD34+ cells from BM, PB and UCB was 30% (30.011.7, meanSD), 79% (78.611.7) and 45% (45.39.3) respectively. As expected, the colony size was increased in the presence of G-CSF. Conclusion : The results of this study clearly showed that the different ex vivo expansion of single CD34+ progenitor cells from BM, PB and UCB. These might be an important data for understanding stem cell expansion in vivo and designing clinical application.
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direct confirmation of quiescent property of aml stem cells by showing markedly decreased Plating Efficiency in single leukemic stem cell culture
Blood, 2008Co-Authors: Myung-geun Shin, Jong-hee Shin, Hoon Kook, Dong-wook RyangAbstract:Cancer stem cell, recently discovered to exist in colon cancer and brain tumor, is resistant to anti-cancer drugs and radiotherapy, demanding the development of new drugs and treatment strategies targeting tumor stem cells. Leukemic stem cell (LSC) has been accused to play a pivotal role in pathogenesis of hematological malignancy such as acute myeloblastic leukemia (AML). Various anti-cancer medicines, particularly anti-proliferative agents, have been ineffective in treating LSC due to its slower division process and longer interphase compared to normal stem cell and hematopoietic cell. This study comparatively examined growth and proliferation capacity (Plating Efficiency) of clonogenic hematopoietic progenitors and LSC from healthy donors and AML patients using single cell sorting and culture system (BD FACS Aria cell sorter; BD Biosciences, San Jose, CA). A total of 384 normal hematopoietic stem cells (CD34+CD38+/CD38−) were obtained from peripheral bloods and cord bloods donated by four donors using single cell sorter, and individual single cells were cultured in 96-well plates with each well containing 100ul of serum media, 100ng/ml of stem cell factor, 100ng/ml of Flt-3, 100ng/ml of thrombopoietin and 50ng/ml of G-CSF for five days. 768 single LSC (CD34+CD38−) and 384 single CD34+CD38+ cells were obtained from three AML patients. Growth and proliferation capacities of normal hematopoietic stem cell and LSC were determined in terms of Plating Efficiency (number of the wells in which more than two cells grew/total number of cells in 96-well plate culture × 100). Plating Efficiency of individual normal single hematopoietic stem cells varied between samples. Eighty eight out of 192 single stem cells originated from cord blood grew into more than two cells, yielding Plating Efficiency of 45.8% and cells from the peripheral blood of two healthy donors 30.2% (58/192). In contrast, single LSC originated from the AML patients showed significantly lower Plating Efficiency with 14.6% (42/288), 3.6% (7/192) and 8.0% (23/288). These results directly confirmed quiescent and slowly dividing properties of LSC. In addition, Plating Efficiency of normal hematopoietic stem cells was shown to vary between their originating locations in healthy donors.