bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric characterization of perfused human bone marrow cultures: Identification of the major cell lineages and correlation with the CFU‐GM assay
Authors: Brott D. A. , Maher R. J. , Parrish C. R. , Richardson R. J. , Smith A. K. 
Published: April 2003

AbstractBackgroundProlific cultures of human bone marrow mononuclear cells (BM MNCs) were recently developed that include a full spectrum of hematopoietic and accessory cells, with the presence of autofluorescent cells indicating adequate cell expansion. However, phenotypic and functional clonogenic characterizations of the autofluorescent cells and the various other subpopulations present in these cultures have not been carried out.MethodsCells from a continuously perfused bioreactor inoculated with BM MNCs and cultured for 12 days in serum‐containing medium with PIXY321, erythropoietin, and with or without FLT3‐L were evaluated by using flow cytometry.ResultsTwo antibodies, CD71 and CD13, allowed the separation of the autofluorescent cells into two distinct populations. The CD71+CD13++ autofluorescent population contained the colony‐forming unit (CFU) fibroblast, and the CD71++CD13++ autofluorescent population contained macrophage/dendritic like cells. The CFU‐granulocyte/macrophage (CFU‐GM) could not be thoroughly evaluated with CD71 and CD13. However, the number of CD13+/++Lin− cells correlated with the number of CFU‐GM (r = 0.83), with approximately 1 CFU‐GM for every 30 CD13+/++Lin− cells.ConclusionsThe data showed that CD71 and CD13 antibodies separate the autofluorescent cells into two populations but do not separate hematopoietic cells into specific phenotypic populations. The data also showed that the number of CD13+/++Lin− cells correlated with the number of CFU‐GM. These data present the initial step toward detailed phenotypic analysis of ex vivo expanded human BM MNC cultures. Cytometry Part A 53A:22–27, 2003. © 2003 Wiley‐Liss, Inc.
High‐throughput flow cytometry: Validation in microvolume bioassays
Authors: Ramirez Sergio , Aiken Charity T. , Andrzejewski Brett , Sklar Larry A. , Edwards Bruce S. 
Published: April 2003

AbstractBackgroundWe recently reported an automated sample handling system, designated HyperCyt, by which samples are aspirated from microplate wells and delivered to the flow cytometer for analysis at rates approaching 100 samples per minute. In this approach, an autosampler and peristaltic pump introduce samples into a tubing line that directly connects to the flow cytometer. Air bubbles are inserted between samples to prevent sample dispersion. In the present work, we compare results of HyperCyt with those of conventional manual flow cytometric analysis in representative flow cytometric bioassays and describe a cell suspension method in which HyperCyt exploits the use of microvolume wells.MethodsHuman eosinophils and neutrophils were treated with trypsin to generate a wide (>25‐fold) range of membrane P‐selectin glycoprotein ligand‐1 (PSGL‐1) expression and then stained with fluorescent anti–PSGL‐1 antibodies. Human peripheral blood mononuclear cells were stained with fluorescein isothiocyanate‐ and phycoerythrin‐conjugated monoclonal antibodies for multiparameter immunophenotype analysis. U937 cells labeled with PKH62GL were used to assess cell settling in microplate wells.ResultsDifferences in PSGL‐1 expression levels were detected by HyperCyt autosampling of leukocytes from 96‐well plates at an analysis rate of approximately 1.5 s/well. HyperCyt measurements linearly correlated with parallel manual measurements (r2 = 0.98). Lymphocyte subpopulations were accurately distinguished and reproducibly quantified in multiparameter immunophenotyping assays performed over a range of HyperCyt analysis rates (1.4–5.5 s/sample). When assay volumes were reduced to 10 μl/well in 60‐well Terasaki plates, cells could be maintained in uniform suspension for up to 30 min by periodically inverting plates on a rotating carousel before HyperCyt analysis. HyperCyt analysis of five fluorescence‐level Cyto‐Plex beads sampled from Terasaki plate microwells at 2.5 s/well produced highly reproducible results over a wide range of input bead concentrations (from 7 × 105 to 20 × 106 beads/ml) that linearly correlated with manual analysis results.ConclusionsThe HyperCyt autosampling system enabled a 10‐fold or greater increase in sample thoughput compared with conventional manual flow cytometric sample analysis, with comparable analysis results. Assays were performed efficiently in 10‐μl volumes to enable significant reagent cost savings, use of quantity‐limited reagents at otherwise prohibitive concentrations, and maintenance of uniform suspensions of cells for prolonged periods. Cytometry Part A 53A:55–65, 2003. © 2003 Wiley‐Liss, Inc.
On synchronization techniques: performance and impact on time metrics monitoring
Authors: Adami Davide , Garroppo Rosario G. , Giordano Stefano , Lucetti Stefano 
Published: April 2003

AbstractThe paper presents an experimental comparison of device synchronization strategies to monitor quality of service (QoS) time metrics, such as the one‐way delay and the delay variation. The compared strategies are based on global positioning system (GPS), public Internet network time protocol (NTP) servers and ad hoc GPS‐based NTP servers with different treatments of the NTP traffic. The presented experimental results allow to quantitatively evaluate the level of performance achievable using each synchronization technique. Finally, considering a satellite test bed, we focus our analysis on two relevant aspects of time metrics monitoring: the impact of the synchronization errors on the metrics observation and the different values that can be measured when a metric is monitored at the application or data link level. Copyright © 2003 John Wiley & Sons, Ltd.
Translation of specification units between IP and ATM quality of service declarations
Authors: Schmitt Jens 
Published: April 2003

AbstractAn integral part of any network quality of service (QoS) system are its QoS declarations. QoS declarations consist of service classes, parameters, and specification units. QoS declarations are a component of the QoS architecture, as such they are a source of heterogeneity stemming from the fact that different QoS systems may be based on different QoS architectures and thus use different QoS declarations. A particular problem in that domain is thetranslationof specification units for QoS systems that are based on different forwarding technologies with respect to variable vs fixed packet sizes, i.e. packet vs cell switching. This is a problem that can be dealt with generically such that its solution can be applied to several situations of technically heterogeneous QoS systems like an RSVP/IntServ—or DiffServ—over an ATM‐based system. While straightforward translations have been proposed, we investigate how more efficient translations can be achieved by using a slight but effective modification of existing AAL framing schemes as well as making use of statistical knowledge about packet size distributions. Copyright © 2003 John Wiley & Sons, Ltd.
Cardiac muscle cell formation after development of the linear heart tube
Authors: Kruithof Boudewijn P.T. , van den Hoff Maurice J.B. , Wessels Andy , Moorman Antoon F.M. 
Published: April 2003

AbstractAfter the development of the linear heart tube, additional myocardium is formed leading to the muscular mantle around the caval and pulmonary veins and the muscular septa in the embryonic heart. Here, we report the results of our in vivo and in vitro studies of this late myocardium‐generating process in the mouse. By using an immunohistochemical approach, we determined that myocardium formation starts around embryonic day 12 in the dorsal mesocardium. In subsequent stages of development, the process extends downstream into the intracardiac mesenchymal tissues of the atrioventricular canal and outflow tract and upstream into the extracardiac mediastinal mesenchyme embedding the pulmonary and caval veins. Given the spatiotemporal pattern of myocardium formation, we applied a three‐dimensional in vitro explant culture assay to investigate the myocardium‐generating potential of the different cardiac compartments. We determined that this potential is stage‐ and mesenchyme‐dependent. This latter finding suggests an important role for mesenchyme in myocardium formation after the development of the linear heart tube. Developmental Dynamics 227:1–13, 2003 © 2003 Wiley‐Liss, Inc.
Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the mylz2 promoter
Authors: Ju Bensheng , Chong Shang Wei , He Jiangyan , Wang Xukun , Xu Yanfei , Wan Haiyan , Tong Yan , Yan Tie , Korzh Vladimir , Gong Zhiyuan 
Published: April 2003

AbstractA 1,934‐bp muscle‐specific promoter from the zebrafish mylz2 gene was isolated and characterized by transgenic analysis. By using a series of 5′ promoter deletions linked to the green fluorescent protein (gfp) reporter gene, transient transgenic analysis indicated that the strength of promoter activity appeared to correlate to the number of muscle cis‐elements in the promoter and that a minimal −77‐bp region was sufficient for a relatively strong promoter activity in muscle cells. Stable transgenic lines were obtained from several mylz2‐gfp constructs. GFP expression in the 1,934‐bp promoter transgenic lines mimicked well the expression pattern of endogenous mylz2 mRNA in both somitic muscle and nonsomitic muscles, including fin, eye, jaw, and gill muscles. An identical pattern of GFP expression, although at a much lower level, was observed from a transgenic line with a shorter 871‐bp promoter. Our observation indicates that there is no distinct cis‐element for activation of mylz2 in different skeletal muscles. Furthermore, RNA encoding a dominant negative form of cAMP‐dependent protein kinase A was injected into mylz2‐gfp transgenic embryos and GFP expression was significantly reduced due to an expanded slow muscle development at the expense of GFP‐expressing fast muscle. The mylz2‐gfp transgene was also transferred into two zebrafish mutants, spadetail and chordino, and several novel phenotypes in muscle development in these mutants were discovered. Developmental Dynamics 227:14–26, 2003. © 2003 Wiley‐Liss, Inc.
Analysis of hindbrain patterning defects caused by the kreislerenu mutation reveals multiple roles of Kreisler in hindbrain segmentation
Authors: Sadl Virginia S. , Sing Angela , Mar Lynn , Jin Fuzi , Cordes Sabine P. 
Published: April 2003

AbstractThe embryonic hindbrain is subdivided into eight subunits, termed rhombomeres (r1–r8). The Kreisler (Krml1/MafB/val) transcription factor is expressed in and essential for patterning rhombomeres 5 and 6. Here, we have shown that in the chemically induced kreislerenu (krenu) allele, a point mutation in the DNA binding domain abolishes or severely reduces Kreisler‐dependent transcription. Comparison of krenu/krenu embryos with those homozygous for the classic kreisler (kr) mutation has reconciled past discrepancies and revealed multiple roles of Kreisler in hindbrain segmentation. These analyses demonstrate that Kreisler is required for maintenance and expansion but not initiation of the Krox20 expressing r5 domain. The differences in the “r5‐like” phenotype of krenu/krenu and kr/kr mouse embryos, and zebrafish carrying mutations in the Kreisler orthologue valentino (val) suggest that Kreisler performs many of its r5‐specific functions by associating with other proteins. By contrast, kr/kr and krenu/krenu mouse and val‐/‐ zebrafish embryos all exhibit indistinguishable defects in r6 specification. Thus, transcriptionally active Kreisler is required for r6 specification. Unlike mouse krenu/krenu and zebrafish val‐/‐ embryos, kr/kr embryos exhibited anterior defects. We determined that the kr chromosomal inversion caused ectopic Kreisler expression in r3 of kr/kr and kr/+ embryos. Hence, Kreisler regulates maintenance and expansion of r5 and specification of r6 but is not required for r3 development. Developmental Dynamics 227:134–142, 2003. © 2003 Wiley‐Liss, Inc.
Soluble and bound forms of GFRα1 elicit different GDNF‐independent neurite growth responses in primary sensory neurons
Authors: Mikaels‐Edman Åsa , Baudet Christel , Ernfors Patrik 
Published: April 2003

AbstractWe have investigated the role of glial cell‐line derived neurotrophic factor (GDNF) and the effect of soluble or immobilized localization of its GDNF family receptor α1 (GFRα1) on neurite growth in cultured embryonic Bax−/− dorsal root ganglion neurons, which survive in the absence of trophic support. Whereas GDNF alone has a moderate effect on neurite growth, soluble and immobilized GFRα1 elicit opposing and GDNF‐independent effects on neurite growth by a phospholipase C (PLC) γ‐dependent mechanism. Thus, GFRα1 elicits nerve growth responses independent of GDNF. However, GDNF in the presence of soluble or immobilized GFRα1 reverse the GDNF‐independent GFRα1 modulation of neurite growth. The different outcome of soluble and bound GFRα1 combined with our previous immunohistochemical data showing GFRα1‐protein in Schwann cells but not axons suggest terminal Schwann cells as a source of locally administered target‐derived GFRα1 and place this receptor in the path of axonal growth and guidance. Thus, target‐derived GFRα1 play opposing roles when presented alone and with GDNF and, therefore, can function as a nerve growth cue that both can promote and prevent growth in the developing peripheral nervous system. Developmental Dynamics 227:27–34, 2003 © 2003 Wiley‐Liss, Inc.
Loss of maternal Smad5 in zebrafish embryos affects patterning and morphogenesis of optic primordia
Authors: Hammerschmidt Matthias , Kramer Carina , Nowak Matthias , Herzog Wiebke , Wittbrodt Joachim 
Published: April 2003

AbstractThe mechanisms of patterning and morphogenesis of vertebrate eye primordia are heavily debated. Taking advantage of the maternal effect of a zebrafish smad5 null mutation (Mm169), we investigate the effect of early signaling by members of the bone morphogenetic proteins (Bmps) on eye field patterning and optic vesicle morphogenesis. In contrast to previous Xenopus and chick studies demonstrating a late dorsalizing effect of Bmp4 expressed in the dorsal neural retina itself, we show that patterning of the eye primordia already starts during blastula and early gastrula stages. At these stages, bmps expressed on the ventrolateral side of the embryo promote dorso‐distal fates in the entire neuroectoderm, including the eye primordia. Despite a normal split of the eye field in the midline, the eye primordia of Mm169 embryos fail to evaginate laterally. They display a concentric pattern with retinal cells in the center and optic stalk cells in the periphery, representing a flattened version of the topologic relationships present in the mature wild‐type eye. Different interpretations of these latter findings are presented. They can be best explained with a model according to which zebrafish eye morphogenesis occurs as a telescopic extension of disc‐like, concentric primordia, similar to the development of appendages from imaginal discs in Drosophila. Developmental Dynamics 227:128–133, 2003. © 2003 Wiley‐Liss, Inc.
Localization of aggrecan and versican in the developing rat central nervous system
Authors: Popp Susanna , Andersen Julie S. , Maurel Patrice , Margolis Richard U. 
Published: April 2003

AbstractThe localization of aggrecan and mRNA splice variants of versican in the developing rat central nervous system has been examined by using specific polyclonal antibodies to the nonhomologous glycosaminoglycan attachment regions of these hyaluronan‐binding chondroitin sulfate proteoglycans. At embryonic day 16 (E16), aggrecan and versican splice variants containing either or both the α‐and β‐domains are present in the marginal zone and subplate of the cerebral cortex and in the amygdala, internal capsule, and the optic and lateral olfactory tracts. There is strong staining of versican but not of aggrecan in the hippocampus and dentate gyrus by E19, whereas both aggrecan and α‐versican are present in the fimbria. At E19, aggrecan is seen throughout the cerebral cortex, whereas the distribution of versican is considerably more limited, being confined essentially to the marginal zone and subplate. At 1 week postnatal, both aggrecan and versican are present in the prospective white matter and in the molecular and granule cell layers of the cerebellum, but neither proteoglycan is seen in the external granule cell layer. α‐ but not β‐versican staining is seen in Purkinje cells, and aggrecan staining of Purkinje cells is also rather minimal. In the spinal cord at E13, aggrecan is present in the dorsal root entry zone, ventral funiculus, mantle layer, and floor plate, as well as in the dorsal root ganglia and ventral roots. However, α‐versican is confined to the dorsal root entry zone and the ependyma surrounding the spinal canal, and β‐versican is not present in spinal cord parenchyma at this developmental stage, being limited to the surrounding connective tissue. By E19, there are significant amounts of all three proteoglycans in the spinal cord. Aggrecan staining is most intense in the lateral funiculus and the fasciculi gracilis and cuneatus, where α‐versican staining is also strong. In contrast, β‐versican is seen predominantly in the motor columns. Differences in the localization and temporal expression patterns of these chondroitin sulfate proteoglycans suggest that, like neurocan and phosphacan, they have partially complementary roles during central nervous system development. Development Dynamics 227:143–149, 2003. © 2003 Wiley‐Liss, Inc.
RhoA is highly up‐regulated in the process of early heart development of the chick and important for normal embryogenesis
Authors: Kaarbø Mari , Crane Denis I. , Murrell Wayne G. 
Published: April 2003

AbstractWe have used molecular techniques, combined with classic embryological methods, to identify up‐regulated genes associated with early heart development. One of the cDNAs identified and isolated by screening a chick lambda cDNA library was the small guanosine triphosphatase RhoA. RhoA has at least three different length mRNA species, each varying in the length of the 3′ untranslated region. In situ hybridisation and immunocytochemistry analysis of RhoA expression show marked up‐regulation in the heart‐forming region. In other systems, RhoA signalling has been shown to be important for both gene expression and morphology. To investigate the function of RhoA in early heart development, we used small interfering RNAs (siRNA) in early chick embryos. Disruption of RhoA expression by siRNA treatment resulted in lack of heart tube fusion and abnormal head development. These data indicate that RhoA is important for normal embryogenesis. Developmental Dynamics 227:35–47, 2003. © 2003 Wiley‐Liss, Inc.
Epicardial‐like cells on the distal arterial end of the cardiac outflow tract do not derive from the proepicardium but are derivatives of the cephalic pericardium
Authors: Pérez‐Pomares José M. , Phelps Aimée , Sedmerova Martina , Wessels Andy 
Published: April 2003

AbstractA series of recent studies strongly suggests that the myocardium of the cardiac distal outflow tract (d‐OFT) does not derive from the original precardiac mesoderm but, instead, differentiates from a so‐called anterior heart field. Similar findings were also reported for the endocardium of the d‐OFT. However, very little information is available on the origin of the epicardium of the OFT. To address this issue, we have performed a study in which we have combined experimental in vivo and in vitro techniques (construction of proepicardial chimeras, proepicardial ablation, OFT insertion of eggshell membrane pieces, and culture on collagen gels) with molecular characterization techniques to determine this origin and define the properties of d‐OFT epicardium compared with proepicardially derived epicardium. Our results demonstrate that the coelomic/pericardial epithelium in the vicinity of the aortic sac (and not the proepicardium) is the origin of d‐OFT epicardium. This “pericardially” derived epicardium and the proepicardially derived epicardial tissues differ in their morphologic appearance, gene‐expression profile, and in their ability to undergo epithelial‐to‐mesenchymal transformation. We conclude that the heterogeneity in the epicardial cell population of the OFT could be a factor in the complex developmental remodeling events at the arterial pole of the heart. Developmental Dynamics 227:56–68, 2003. © 2003 Wiley‐Liss, Inc.
TALE class homeodomain gene Irx5 is an immediate downstream target for Hoxb4 transcriptional regulation
Authors: Theokli Christopher , Morsi El‐Kadi Ali S. , Morgan Richard 
Published: April 2003

AbstractThe Hox genes are a family of homeodomain‐containing transcription factors that determine anteroposterior identity early on in development. Although much is now known about their regulation and function, very little is known of their effector (downstream target) genes. Here, we show that the TALE class homeodomain transcription factor Irx5 is a direct, positively regulated target of Hoxb4. Developmental Dynamics 227:48–55, 2003. © 2003 Wiley‐Liss, Inc.
Loss of the Tg737 protein results in skeletal patterning defects
Authors: Zhang Qihong , Murcia Noel S. , Chittenden Laura R. , Richards William G. , Michaud Edward J. , Woychik Richard P. , Yoder Bradley K. 
Published: April 2003

AbstractTg737 mutant mice exhibit pathologic conditions in numerous tissues along with skeletal patterning defects. Herein, we characterize the skeletal pathologic conditions and confirm a role for Tg737 in skeletal patterning through transgenic rescue. Analyses were conducted in both the hypomorphic Tg737orpk allele that results in duplication of digit one and in the null Tg737Δ2‐3βGal allele that is an embryonic lethal mutation exhibiting eight digits per limb. In early limb buds, Tg737 expression is detected throughout the mesenchyme becoming concentrated in precartilage condensations at later stages. In situ analyses indicate that the Tg737orpk mutant limb defects are not associated with changes in expression of Shh, Ihh, HoxD11–13, Patched, BMPs, or Glis. Likewise, in Tg737Δ2‐3βGal mutant embryos, there was no change in Shh expression. However, in both alleles, Fgf4 was ectopically expressed on the anterior apical ectodermal ridge. Collectively, the data argue for a dosage effect of Tg737 on the limb phenotypes and that the polydactyly is independent of Shh misexpression. Developmental Dynamics 227:78–90, 2003. © 2003 Wiley‐Liss, Inc.
Retinoic acid signalling centres in the avian embryo identified by sites of expression of synthesising and catabolising enzymes
Authors: Blentic Aida , Gale Emily , Maden Malcolm 
Published: April 2003

AbstractRetinoic acid is an important signalling molecule in the developing embryo, but its precise distribution throughout development is very difficult to determine by available techniques. Examining the distribution of the enzymes by which it is synthesised by using in situ hybridisation is an alternative strategy. Here, we describe the distribution of three retinoic acid synthesising enzymes and one retinoic acid catabolic enzyme during the early stages of chick embryogenesis with the intention of identifying localized retinoic acid signalling regions. The enzymes involved are Raldh1, Raldh2, Raldh3, and Cyp26A1. Although some of these distributions have been described before, here we assemble them all in one species and several novel sites of enzyme expression are identified, including Hensen's node, the cardiac endoderm, the presumptive pancreatic endoderm, and the dorsal lens. This study emphasizes the dynamic pattern of expression of the enzymes that control the availability of retinoic acid as well as the role that retinoic acid plays in the development of many regions of the embryo throughout embryogenesis. This strategy provides a basis for understanding the phenotypes of retinoic acid teratology and retinoic acid–deficiency syndromes. Developmental Dynamics 227:114–127, 2003. © 2003 Wiley‐Liss, Inc.