bluuebunny/crossref_metadata_2025_split huggingface dataset:
The dynamic process of apoptosis analyzed by flow cytometry using Annexin‐V/propidium iodide and a modified in situ end labeling technique
Authors: Span L.F.R. , Pennings A.H.M. , Vierwinden G. , Boezeman J.B.M. , Raymakers R.A.P. , de Witte T. 
Published: October 2004

AbstractBackgroundTo study the apoptotic process in time, we used the following flow cytometric (FCM) techniques: phosphatidylserine (PS) translocation by Annexin‐V (AnV), DNA fragmentation by in situ end labeling (ISEL), and propidium iodide (PI) staining. Because PS translocation is assumed to be an early feature of programmed cell death (PCD), we questioned if AnV positivity implies inevitable cell death.MethodsApoptosis was induced in Jurkat cells by γ‐irradiation, incubation with camptothecin (CPT), or cytosine β‐D‐arabinofuranoside (Ara‐C). At different time intervals, PCD was quantified by AnV/PI and ISEL. To analyze the influence of cell handling procedures on PCD, we applied these three FCM techniques on CD34+ bone marrow (BM) stem cells after selection and after a freeze‐thaw procedure. Various AnV/PI− CD34+ fractions were cultured in a single‐cell single‐well (SCSW) assay.ResultsJurkat cells under three different detrimental conditions showed essentially the same pattern of apoptosis in time. Initially developed AnV+/PI− cells subsequently (within 1 h) showed ISEL positivity, after which they turned into AnV+/PI++ cells with even higher levels of ISEL positivity (80–90%). Eventually, they lost some of their PI and ISEL positivity and formed the AnV+/PI+ fraction. Cell handling of CD34+ cells caused high and variable AnV+/PI− fractions (overall range 23–62%). Within total AnV+ and AnV+/PI− populations, only a minority of CD34+ cells showed ISEL positivity (range 4–8% and 0.8–6%, respectively). Different fractions of AnV+/PI− CD34+ cells did have clonogenic capacity.ConclusionsPCD of cell suspensions in vitro can be followed accurately in time by these three FCM techniques. PS translocation is followed rapidly (within 1 h) by oligo‐nucleosomal DNA fragmentation, after which cell (and nuclear) membrane leakage occurs. Detection of PS asymmetry by AnV‐fluorescein isothiocyanate (FITC) is not always associated with (inevitable) apoptosis, as can be concluded from the proliferative capacity of AnV+ /PI− CD34+ cells in the SCSW assay. Cytometry 47:24–31, 2002. © 2001 Wiley‐Liss, Inc.
Clinical utility of CD23 and FMC7 antigen coexistent expression in B‐cell lymphoproliferative disorder subclassification
Authors: Ahmad Ejaz , Garcia Diana , Davis Bruce H. 
Published: October 2004

AbstractBackground: CD23 and FMC7 are normal B‐cell antigens utilized during diagnostic immunophenotyping of suspected lymphoproliferative disorders. However, the diagnostic utility of coexistent antigenic expression patterns with simultaneous two‐color staining and flow cytometric analysis has not been studied extensively. Methods: Using multiparameter flow cytometry, we evaluated the expression pattern of FMC7 and CD23 in 218 cases of B‐cell lymphoma from blood and bone marrow specimens. Results: The CD23(+)/FMC7(‐) pattern was the most common pattern in patients with chronic lymphocytic leukemia and related variants. The widest variation of patterns was found in patients with follicular cell lymphoma, large cell lymphoma, and Waldenström's macroglobulinemia, a lymphoplasmacytoid disorder, although most cases expressed the CD23‐/FMC7(+) pattern. The CD23 and FMC7 antigen, along with the CD5 coexpression pattern, provides critical adjunctive data. These data allow accurate classification of the majority of cases, thereby providing a key aspect of a reliable diagnostic algorithm. The CD23 and FMC7 antigen expression pattern, along with selected other antigens, was predictive of subtypes in >95% of lymphoproliferative cases and narrowed the differential diagnosis in the remaining cases. Conclusion: The flow cytometric CD23/FMC7 expression pattern achieved by multicolor immunophenotyping facilitates accurate and reproducible classification of B‐cell lymphomas and has diagnostic utility. Cytometry (Clin. Cytometry) 50:1–7, 2002. © 2002 Wiley‐Liss, Inc.
A novel cell surface antigen, 4C8, is expressed on human eosinophils
Authors: Aoyama Yasutaka , Yamane Takahisa , Hino Masayuki , Ohta Kensuke , Suzuki Kenichi , Kitagawa Seiichi , Masuyama Jun‐ichi , Tatsumi Noriyuki 
Published: October 2004

AbstractBackground: A novel monoclonal antibody, anti‐4C8, reacted with human peripheral lymphocytes and monocytes but not with neutrophils. In this study, we investigated whether the 4C8 antigen is expressed on human peripheral eosinophils. Methods: Expression of the 4C8 antigen on eosinophils was analyzed by flow cytometry and molecular analysis of the antigen was performed with eosinophils by Western blotting. Results: Among human peripheral granulocytes, the 4C8 antigen was expressed on CD16‐negative cells but not on CD16‐positive cells. The 4C8 antigen also appeared to be expressed on eosinophils. To confirm the latter finding, eosinophils were purified from peripheral blood. On flow cytometric analysis, anti‐4C8 antibody reacted with purified eosinophils. On Western blotting analysis, anti‐4C8 reacted with a single band of 80 kDa in lysates from purified eosinophils. The correlation between the percentage of eosinophils determined by May‐Giemsa staining and the percentage of 4C8‐positive/CD16‐negative cells among granulocytes was good (r = 0.91, P < 0.0001). Conclusions: Only a few cell surface antigens are available to distinguish human peripheral eosinophils from neutrophils. The novel cell surface antigen, 4C8, is a useful new marker of human eosinophils. Cytometry (Clin. Cytometry) 50:8–13, 2002. © 2002 Wiley‐Liss, Inc.
Influence of EDTA and heparin on lipopolysaccharide binding and cell activation, evaluated at single‐cell level in whole blood
Authors: Coló Brunialti Milena Karina , Kallás Esper Georges , Freudenberg Marina , Galanos Chris , Salomao Reinaldo 
Published: October 2004

AbstractBackground: The use of whole blood (WB) in studying lipopolysaccharide (LPS)‐induced cellular activation preserves the milieu in which LPS‐cell interaction occurs in vivo. However, little information is available on using such a system at a single‐cell level. We evaluated LPS binding and cell activation in WB by using flow cytometry. The influence of heparin or EDTA as anticoagulants was also addressed. Methods: Blood was obtained from healthy donors in EDTA and/or heparin tubes. Biotinylated LPS (LPSb) was used to evaluate cell binding of LPS in WB. Cells were surface stained with appropriate antibodies and LPSb was detected by adding streptavidin‐allophycocyanin (APC). LPS‐induced activation was evaluated by the expression of surface activation markers and by the detection of intracellular tumor necrosis factor‐alpha (TNF‐α). Results: LPSb bound promptly to monocytes in EDTA‐ and heparin‐treated blood. In EDTA‐treated blood, membrane‐bound LPSb decreased after 60 min of incubation, whereas it remained detectable in heparinized blood during the 6 h of incubation. LPS induced TNF‐α and enhanced the expression of HLA‐DR in monocytes, as well as the expression of CD69 in T and B lymphocytes. Induction of both TNF‐α in monocytes and CD69 in lymphocytes was more efficient in heparinized blood. Conclusion: Detection of membrane‐bound LPSb on monocytes differed in EDTA or heparin‐treated blood, and cell activation was better obtained in heparinized blood. Cytometry (Clin. Cytometry) 50:14–18, 2002. © 2002 Wiley‐Liss, Inc.
Walker tumor cells express larger amounts of the antiapoptotic protein Bcl‐2 and presents higher resistance to toxic concentrations of Ca2+ than the tumor cells K 562
Authors: Milani Graziela , Kowaltowski Alicia J. , Saad Sara T.O. , Metze Konradin , Vercesi Anibal E. 
Published: October 2004

AbstractCa2+ homeostasis was studied in two tumor cell lines (Walker 256 and K 562) previously shown to exhibit different mitochondrial Ca2+ accumulation capacity. When intact, both cells present cytosolic Ca2+ concentrations within the range expected for mammalian cells, as determined through fura‐2 fluorescence ratios. In order to study intracellular Ca2+ distribution, digitonin was used to permeabilize the plasma membrane without affecting intracellular organelle structure, as assessed using electron microscopy. Digitonin‐permeabilized Walker 256 cells incubated with Ca2+ presented uptake of the cation exclusively through mitochondrial activity. In addition, very large Ca2+ loads were necessary to promote a disruption of Walker 256 mitochondrial membrane potential. K 562 cells presented active Ca2+ uptake through both nonmitochondrial and mitochondrial compartments and suffered disruption of mitochondrial membrane potential at lower Ca2+ loads than Walker 256 mitochondria. The higher Ca2+ resistance in Walker 256 cells could be attributed to Bcl‐2 overexpression, as evidenced by immunocytochemical staining. Thus, we correlate natural Bcl‐2 overexpression, observed in Walker 256 cells, with higher resistance to mitochondrial Ca2+ overload, as was shown previously in mitochondria from cells transfected with the bcl‐2 gene. Drug Dev. Res. 52:508–514, 2001. © 2001 Wiley‐Liss, Inc.
Radioresistant Burkitt’s lymphoma cells exhibit defective MAPK signalling
Authors: Michael‐Robinson Julie M. , Spring Kevin J. , Lavin Martin F. , Watters Dianne J. 
Published: October 2004

AbstractUsing a pair of isogenic Burkitt’s lymphoma cell lines, one of which is sensitive (BL30A) and the other resistant (BL30K) to apoptosis induced by ionising radiation and exogenous ceramide, we investigated mitogen‐activated protein kinase (MAPK) signalling to determine which members of this kinase family are involved in the apoptotic process in these cells. We have previously shown that BL30A cells produce ceramide after irradiation and that this does not occur in BL30K cells (Michael et al. [1997] Cancer Res 57:3600–3605). We show that p38 MAPK is activated transiently in both cells after ionising radiation. On the other hand, although JNK is rapidly activated in both cells, this activation is only transient in the resistant cells, whereas in the sensitive cells the activation is sustained. Addition of exogenous ceramide resulted in only a transient activation of JNK in both cells. Interestingly, ERK activity was decreased in BL30A cells after ceramide treatment, whereas no such decrease occurred in the resistant cells. Treatment of BL30A cells with phorbol ester before irradiation, which blocks the increase in ceramide and apoptosis, also prevents the sustained increase in JNK activity. At the same time, ERK activity is increased. Our results suggest that p38 MAPK is not required for apoptosis signalling in response to ionising radiation in Burkitt’s lymphoma cells and that sustained activation of JNK is necessary for apoptosis in these cells. These results also support the hypothesis that a balance between JNK and ERK activity determines cell fate after exposure to ceramide or ionising radiation. In addition, our results suggest different signalling pathways from exogenous ceramide and radiation, supporting the concept of different intracellular pools of active ceramide. Drug Dev. Res. 52:534–541, 2001. © 2001 Wiley‐Liss, Inc.
Dissecting the apoptotic mechanisms of chemotherapeutic drugs and lymphocytes to design effective anticancer therapies
Authors: Ruefli Astrid A. , Davis Joanne E. , Sutton Vivien R. , Trapani Joseph A. , Smyth Mark J. , Johnstone Ricky W. 
Published: October 2004

AbstractIt has now been demonstrated that chemotherapeutic drugs and killer cells can activate both common and unique programmed cell death pathways to effectively eliminate tumor cells. Although chemotherapy and radiation treatments are effective methods of reducing tumor burden, minimal residual disease often enables the recurrence and emergence of more resistant and aggressive tumors. Immune cells have evolved over millions of years to discriminate self from nonself and to destroy cells that harbor pathogens. Therefore, enhancing innate immune response in combination with chemotherapy or irradiation may offer improved treatment of many cancers—in particular, hematological malignancies. Better understanding and dissection of cell death pathways may advance the development of more effective chemotherapeutic and immunotherapeutic therapies that can activate specific apoptotic pathways to overcome the antiapoptotic effects of tumorigenic genetic mutations. Drug Dev. Res. 52:549–557, 2001. © 2001 Wiley‐Liss, Inc.
Proapoptotic plasma membrane pore: P2X7 receptor
Authors: Morelli Anna , Ferrari Davide , Bolognesi Giorgio , Rizzuto Rosario , Virgilio Francesco Di 
Published: October 2004

AbstractATP is not only the universal currency of energy metabolism but also a ubiquitous extracellular messenger. Among its many effects the ability to trigger cell death has attracted considerable interest. The plasma membrane receptor responsible for this effect has been cloned and shown to belong to the P2X family (P2X7). The P2X7 receptor is a plasma membrane ion channel that, under certain conditions, can grow to form a nonselective pore. Prolonged opening of this receptor is a potent apoptotic stimulus for several cell types. The main mechanism responsible for this deadly effect seems to reside in the drastic upset of the intracellular concentration of Ca2+ as well as K+. The loss of intracellular K+ has a permissive effect on the progression of apoptosis by causing the activation of key proapoptotic enzymes such as caspase 3 and NUC18. The P2X7 receptor could be an interesting model for the study of apoptotic pathways triggered by plasma membrane channels. Drug Dev. Res. 52:571–578, 2001. © 2001 Wiley‐Liss, Inc.
Long‐term protection of brain tissue from cerebral ischemia by peripherally administered peptidomimetic caspase inhibitors
Authors: Deckwerth Thomas L. , Adams Lisa M. , Wiessner Christoph , Allegrini Peter R. , Rudin Markus , Sauter Andre , Hengerer Bastian , Sayers Robert O. , Rovelli Giorgio , Aja Teresa , May Rachel , Nalley Kip , Linton Steve , Karanewsky Donald S. , Wu Joe C. , Roggo Silvio , Schmitz Albert , Contreras Patricia C. , Tomaselli Kevin J. 
Published: October 2004

AbstractApoptotic cell death occurs in the injured and diseased central nervous system. It is mediated by a family of caspases, which are activated by the lethal stimulus and cleave multiple protein substrates that are critical for cell viability. Previous studies demonstrated that caspase‐mediated apoptotic cell death contributes to the loss of brain tissue after experimental cerebral ischemia and that peptidic caspase inhibitors can be efficacious in reducing infarct size after icv administration. Here we present the novel small molecule peptidomimetic caspase inhibitor IDN5370/CGP82630, which belongs to the structural class of oxoazepinoindoline caspase inhibitors. It is 10–100‐fold more potent than current peptidic inhibitors in inhibiting multiple caspases in vitro and promoting neuronal survival. IDN5370 and a derivative, IDN7866, were tested for their ability to reduce infarct size after permanent and transient cerebral ischemia. When administered icv to rats subjected to permanent middle cerebral artery occlusion (MCAO), IDN5370 significantly reduced cortical infarct as measured by magnetic resonance imaging at 2 days after artery occlusion. Protection of brain tissue persisted for 28 days after artery occlusion. To determine whether compounds of this structural class could reduce infarct size after peripheral administration, IDN7866, which penetrates the blood–brain barrier and inhibits caspase 3 in situ in the hippocampus after kainate‐induced seizures, was administered iv in both permanent and transient MCAO models. Infarct size was reduced significantly in both models 24 h after artery occlusion. These results demonstrate that peripherally administered peptidomimetic caspase inhibitors can attenuate brain injury after cerebral ischemia and confer a long‐lasting protective effect on the infarcted brain tissue. Drug Dev. Res. 52:579–586, 2001. © 2001 Wiley‐Liss, Inc.
Griseofulvin: Interaction with normal and subtilisin‐treated tubulin
Authors: Chaudhuri Asish Ray , Ludueña Richard F. 
Published: October 2004

AbstractGriseofulvin, an antifungal drug, possesses antimitotic activity by inhibiting microtubule assembly. The interaction of griseofulvin with tubulin is unique in that it increases the sulfhydryl titer of tubulin without affecting the hydrophobic areas. Because the C‐terminal regions of both α‐ and β‐tubulin influence the conformational and the drug‐binding properties of tubulin, we decided to study the interaction of griseofulvin with αβ tubulin and tubulin treated with subtilisin to selectively remove the C‐terminal regions from both the α‐ and β‐subunits (αsβs). We found that the apparent Kd of griseofulvin for αsβs tubulin (83 μM) was virtually unaltered compared to its Kd for αβ tubulin (91 μM) as determined by the tryptophan fluorescence quenching assay. The increment of the sulfhydryl titer (0.47 mol/mol) by griseofulvin was not affected by removing the C‐termini from the α‐ and β‐subunits. Moreover, the lack of effect of griseofulvin on the hydrophobic areas was not changed after the cleavage of the C‐termini of the α‐ and β‐subunits. These data therefore strongly suggest that griseofulvin binds at a certain region of tubulin distant from the C‐terminal domains of the α‐ and β‐subunits and that the C‐terminal domains of both subunits do not have any conformational influence over the binding site of griseofulvin. Determining the binding site of griseofulvin will help in understanding its role in regulating microtubule assembly and dynamics. Drug Dev. Res. 53:44–49, 2001. © 2001 Wiley‐Liss, Inc.
Two Na,K‐ATPase β2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis
Authors: Rajarao Johannes R. , Canfield Victor A. , Loppin Benjamin , Thisse Bernard , Thisse Christine , Yan Yi‐Lin , Postlethwait John H. , Levenson Robert 
Published: October 2004

AbstractWe have identified cDNAs encoding a second zebrafish ortholog of the human Na,K‐ATPase β2 subunit. The β2b cDNA encodes a 292 amino acid‐long polypeptide with 74% identity to the previously characterized zebrafish β2a subunit. By using a zebrafish meiotic mapping panel, we determined that the β2b gene (atp1b2b) was tightly linked to markers on linkage group 5, whereas the β2a gene was located on linkage group 23. In situ hybridization analysis shows that in developing zebrafish embryos, atp1b2a and atp1b2b are predominantly expressed in the nervous system. β2a transcripts were abundantly expressed throughout brain as well as spinal cord neurons and lateral line ganglia. In contrast, β2b mRNA expression was primarily detected in sensory organs, including retina, otic vesicles, and lateral line neuromast cells. These results suggest that the β2a and β2b genes play distinct roles in developing brain and sensory organs, and raise the possibility that the functions encoded by the single mammalian β2 gene may be partitioned between the two zebrafish β2 orthologs. © 2002 Wiley‐Liss, Inc.
Temporal sequence of gene expression leading caudal prosencephalon to develop a midbrain/hindbrain phenotype
Authors: Hidalgo‐Sánchez Matías , Alvarado‐Mallart Rosa‐Magda 
Published: October 2004

AbstractTransplantation of prosomeres 1‐2 into the cerebellar plate were used, by using chick/quail chimeras, to analyse the temporal sequence of the genetic cascade leading the graft to develop a midbrain/hindbrain phenotype. Our results show that (1) at Hamburger and Hamilton (HH) stage 13, Pax2 and En2 are already induced within the graft, before all other genes of the cascade, whereas misexpression of Fgf8 is also observed within the contiguous host cerebellar plate; (2) within the graft, Otx2 repression and Gbx2 induction (see Hidalgo‐Sánchez et al. [1999] Development 126:3191–3203) are secondary events that affect, from stages HH14–15, the areas in contact with the host Gbx2/Fgf8‐expressing cerebellar plate; (3) at these stages, the repressed Otx2 territory extends beyond the areas induced to express Gbx2, with the two territories not abutting before HH17–18; (4) Fgf8 expression becomes progressively induced within the Otx2‐repressed/Gbx2‐induced territory, starting at HH15–16. Our results support the hypothesis that the host‐Gbx2/graft‐Otx2 interface could trigger the genetic cascade induced within the graft and that the Gbx2‐induced domain could play a key role during the establishment of the induced intragraft midbrain/hindbrain boundary. © 2001 Wiley‐Liss, Inc.
PDGF‐a/PDGF alpha‐receptor signaling is required for lung growth and the formation of alveoli but not for early lung branching morphogenesis
Authors: Boström Hans , Gritli‐Linde Amel , Betsholtz Christer 
Published: October 2004

AbstractPlatelet‐derived growth factors (PDGF) constitute a family of four gene products (PDGF‐A–D) acting by means of two receptor tyrosine kinases, PDGFRα and β. Three of the ligands (PDGF‐A, ‐B, and ‐C) bind to PDGFRα with high affinity. Knockout of pdgf‐a in mice has demonstrated a role for PDGF‐A in the recruitment of smooth muscle cells to the alveolar sacs and their further compartmentalization into alveoli. Although this is a late, postnatal step in lung development, pdgf‐a antisense oligonucleotides were previously shown to inhibit epithelial branching in rat lung explants in vitro, which reflects an early embryonic process. These conflicting results may be explained by substitution of genetic loss of pdgf‐a by maternal transfer of PDGF‐A to the knockout embryo or the presence of other PDGFRα agonists (PDGF‐B and ‐C) in vivo, potentially masking an effect of PDGF‐A on branching morphogenesis. Alternatively, the administration of pdgf‐a antisense oligonucleotides affected other processes than the intended. To discriminate between these opposing possibilities, we have analyzed lung development in pdgfrα −/− embryos and lung primordia grown in vitro. Our analysis shows that, while the pdgfrα −/− lungs and explanted lung rudiments were smaller than normal, branching morphogenesis appears qualitatively intact and proceeds until at least embryonic day 15.5, generating both prospective conducting and respiratory airways. We conclude that, although PDGF‐AA signaling over PDGFRα may have direct or indirect roles in overall lung growth, it does not specifically control early branching of the lung epithelium. © 2002 Wiley‐Liss, Inc.
Importance of size in defining binge eating episodes in bulimia nervosa
Authors: Keel Pamela K. , Mayer Stephanie A. , Harnden‐Fischer Jennifer H. 
Published: October 2004

AbstractObjectiveThis study sought to determine if amount of food consumed is important in defining binge eating episodes in individuals with bulimia nervosa (BN).MethodWomen (N = 30) with DSM‐IV BN (OBN) and women (N = 25) who would have met DSM‐IV criteria for BN except that their binge episodes were not objectively large (SBN) were recruited from the community. Subjects completed telephone interviews and questionnaires.ResultsResults demonstrated no significant differences between women with OBN and SBN in levels of dietary restraint, disinhibition, or hunger; no significant differences in general psychopathology; and significant differences in frequency of binge/purge episodes and impulsiveness. Differences in impulsiveness remained after controlling for frequency of binge/purge episodes.DiscussionThese results partially validate current diagnostic criteria for bulimia nervosa and elucidate one factor, impulsiveness, that may be important in understanding objective binge episodes in bulimia nervosa. © 2001 by John Wiley & Sons, Inc. Int J Eat Disord 29: 294–301, 2001.