bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation
Authors: Lorenz Mike , DesMarais Vera , Macaluso Frank , Singer Robert H. , Condeelis John
Published: January 2004

Motility is associated with the ability to extend F‐actin‐rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live‐cell time‐lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP‐β‐actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F‐actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F‐actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti‐cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured. Cell Motil. Cytoskeleton 57:207–217, 2004. © 2004 Wiley‐Liss, Inc.
The roadblock light chains are ubiquitous components of cytoplasmic dynein that form homo‐ and heterodimers
Authors: Nikulina Karina , Patel‐King Ramila S. , Takebe Sachiko , Pfister K. Kevin , King Stephen M.
Published: January 2004

The Roadblock/LC7 class of light chains associate with the intermediate chains at the base of the soluble dynein particle. In mammals, there are two Roadblock isoforms (Robl1 and Robl2), one of which (Robl2) is differentially expressed in a tissue‐dependent manner and is especially prominent in testis. Here we define the α helical content of Robl and demonstrate using both the yeast two‐hybrid system and in vitro biochemistry that Robl1 and Robl2 are capable of forming homo‐ and heterodimers. This is the first report of heterodimer formation by any cytoplasmic dynein component, and it further enlarges the number of potential cytoplasmic dynein isoforms available for binding specific cellular cargoes. In addition, we have generated an antibody that specifically recognizes Robl light chains and shows a 5–10 fold preference for Robl2 over Robl1. Using this antibody, we show that Robl is a ubiquitous cytoplasmic dynein component, being found in samples purified from brain, liver, kidney, and testis. Immunofluorescence analysis reveals that Robl is present in punctate organelles in rat neuroblastoma cells. In testis, Robl is found in Leydig cells, spermatocytes, and sperm flagella. Cell Motil. Cytoskeleton 57:233–245, 2004. © 2004 Wiley‐Liss, Inc.
Conductor geometry and capacitor quality for performance optimization of low‐frequency birdcage coils
Authors: Giovannetti Giulio , Francesconi Raffaello , Landini Luigi , Santarelli Maria Filomena , Positano Vincenzo , Viti Vittorio , Benassi Antonio
Published: January 2004

Birdcage coils are used widely in nuclear magnetic resonance (NMR) applications for their ability to operate in transmit/receive mode with high signal‐to‐noise ratio (SNR) and wide field homogeneity. When used at low frequencies, a number of drawbacks arise that drastically reduce their overall performances. In this article we show how the choice of the conductors cross‐geometry and capacitor quality affects the coil overall performance. Several birdcage coils, tuned at 7.66 MHz, were designed and built, using conductors with different shapes and capacitors with different quality factors. Realized coils were tested and characterized by laboratory workbench in order to extract several quality indices. In addition, experiments were done in a real magnetic resonance (MR) scanner to evaluate the field homogeneity. We show that an optimized choice of conductor shape and capacitors allows us to obtain high‐quality birdcage coils tuned at frequencies in the megahertz range. Our results are applicable in several situations as MR imaging (MRI) in low‐field scanners, imaging of nuclear species at intermediate fields, Overhauser systems, and prepolarized NMR. © 2004 Wiley Periodicals, Inc. Concepts Magn Reson Part B (Magn Reson Engineering) 20B: 9–16, 2004.
Functional compartmentalization in the flocculus and the ventral dentate and dorsal group y nuclei: An analysis of single olivocerebellar axonal morphology
Authors: Sugihara Izumi , Ebata Shigeto , Shinoda Yoshikazu
Published: January 2004

The cerebellar cortex consists of multiple longitudinal bands defined by their olivocerebellar projection. Single olivocerebellar axons project to a narrow longitudinal band in the cerebellar cortex and to the cerebellar nucleus with their axon collaterals. This olivocortical and olivonuclear organization is related to the functional compartmentalization of the cerebellar system. To reveal the detailed morphologic organization in the flocculus and the cerebellar and vestibular nuclei, we examined olivocerebellar projection by reconstructing the entire trajectories of 19 single olivofloccular axons and by anterograde mapping with biotinylated dextran in the rat. The flocculus was composed of 12 longitudinal band‐shaped compartments that subdivided 5 previously described zones. These longitudinal bands were innervated differentially by the caudal and rostral portions of the dorsal cap (DC) and the ventrolateral outgrowth (VLO) and the rostral pole of the medial accessory olive. Single olivofloccular axons with an average of 5.1 climbing fibers usually projected to a single longitudinal band in the flocculus and to the ventral dentate or dorsal group y nucleus with their collaterals. DC neurons projected moderately to the rostrolateral portion of the ventral dentate nucleus, whereas VLO neurons projected densely to the medial portion of the ventral dentate nucleus and the dorsal group y nucleus with rostrocaudal topography. DC and VLO neurons did not project to the vestibular nuclei, although floccular Purkinje cells projected to the vestibular, ventral dentate, and dorsal group y nuclei. The fine morphologically identified longitudinal bands and topographic olivonuclear projections were correlated with previous electrophysiologically defined functional zones in the flocculus and inferior olive. J. Comp. Neurol. 470:113–133, 2004. © 2004 Wiley‐Liss, Inc.
Evolutionary changes in the olfactory projection neuron pathways of eumalacostracan crustaceans
Authors: Sullivan Jeremy M. , Beltz Barbara S.
Published: January 2004

Output from the olfactory lobe (primary olfactory center) of eumalacostracan crustaceans is transmitted to the medulla terminalis (MT) and hemiellipsoid body (HB) in the lateral protocerebrum (higher order center) by a large population of projection neurons. In eureptantian crustaceans (lobsters, crayfish, and crabs), these projection neurons also form the output pathway from an additional neuropil, the accessory lobe (higher order center), which appears to have arisen de novo in these animals. In a previous study of lobsters and crayfish we showed that whereas projection neurons innervating the olfactory lobe project primarily to the MT, those innervating the accessory lobe project exclusively to the HB (Sullivan and Beltz [ 2001a] J. Comp. Neurol. 441:9–22). In the present study, we used focal dye injections to examine the olfactory projection neuron pathways of representatives of four eumalacostracan taxa (Stomatopoda, Dendrobranchiata, Caridea, and Stenopodidea) that diverged from the eureptantian line prior to the appearance of the accessory lobe. These experiments were undertaken both to examine the evolution of the olfactory pathway in the Eumalacostraca and to provide insights into the changes in this pathway that accompanied the appearance of the accessory lobe. The innervation patterns of the olfactory projection neurons of the species examined were found to differ markedly, varying from that observed in the most basal taxon examined (Stomatopoda), in which the neurons primarily project to the MT, to those observed in the two highest taxa examined (Caridea and Stenopodidea), in which they primarily target the HB. These results suggest that substantial changes in the relative importance of the MT and HB within the olfactory pathway have occurred during the evolution of the Eumalacostraca. J. Comp. Neurol. 470:25–38, 2004. © 2004 Wiley‐Liss, Inc.
Sema3A regulates the timing of target contact by cranial sensory axons
Authors: Dillon Thomas E. , Saldanha Jason , Giger Roman , Verhaagen Joost , Rochlin M. William
Published: January 2004

The trigeminal ganglion provides the somatosensory innervation for the anterior rat tongue. At early embryonic stages (embryonic day [E] 12–13) pre‐tongue explants repel trigeminal axon outgrowth, and this is mediated by Sema3A (Rochlin and Farbman [1998] J. Neurosci. 18:6840–6852; Rochlin et al. [2000] J. Comp. Neurol. 422:579–593). Despite a decrease in repulsion by E14 and older tongue explants, Sema3A mRNA persists throughout the dorsal epithelium through E18, after axons have begun to penetrate papilla epithelium. We investigated the hypothesis that Sema3A continues to act as a repellent and that subpopulations of trigeminal axons that penetrate the epithelium become unresponsive to Sema3A. Sema3A repelled trigeminal axons in vitro regardless of the neurotrophic factor used to stimulate axon outgrowth, but the minimum level of Sema3A required to repel depended on the neurotrophic factor. Thus, in vitro, trigeminal axons are repelled by Sema3A when they would be penetrating the Sema3A‐mRNA rich epithelium in vivo. Whereas dorsal epithelium on tongue explants dissected at stages preceding target contact (E15) repelled trigeminal axons in vitro, explants dissected at later stages (E18), after axons would have penetrated the epithelium in vivo, were not repellent. To determine whether Sema3A prevents premature target penetration in vivo, we assessed the timing of target contact by sensory axons in Sema3A−/minus; and +/+ mice. Contact of the epithelium occurs prematurely in Sema3A−/minus; mice, but not penetration. Taken together, our data imply that Sema3A acts as a short‐range repellent that regulates the timing of target contact by trigeminal axons. J. Comp. Neurol. 470:13–24, 2004. © 2004 Wiley‐Liss, Inc.
Primary afferent terminal sprouting after a cervical dorsal rootlet section in the macaque monkey
Authors: Darian‐Smith Corinna
Published: January 2004

We examined the role of primary afferent neurons in the somatosensory cortical “reactivation” that occurs after a localized cervical dorsal root lesion (Darian‐Smith and Brown [2000] Nat. Neurosci. 3:476–481). After section of the dorsal rootlets that enervate the macaque's thumb and index finger (segments C6–C8), the cortical representation of these digits was initially silenced but then re‐emerged for these same digits over 2–4 postlesion months. Cortical reactivation was accompanied by the emergence of physiologically detectable input from these same digits within dorsal rootlets bordering the lesion site. We investigated whether central axonal sprouting of primary afferents spared by the rhizotomy could mediate this cortical reactivation. The cortical representation of the hand was mapped electrophysiologically 15–25 weeks after the dorsal rootlet section to define this reactivation. Cholera toxin subunit B conjugated to horseradish peroxidase was then injected into the thumb and index finger pads bilaterally to label the central terminals of any neurons that innervated these digits. Primary afferent terminal proliferation was assessed in the spinal dorsal horn and cuneate nucleus at 7 days and 15–25 postlesion weeks. Labeled terminal bouton distributions were reconstructed and the “lesion” and control sides compared within each monkey. Distributions were significantly larger on the side of the lesion in the dorsal horn and cuneate nucleus at 15–25 weeks after the dorsal rootlet section, than those mapped only 7 days postlesion. Our results provide direct evidence for localized sprouting of spared (uninjured) primary afferent terminals in the dorsal horn and cuneate nucleus after a restricted dorsal root injury. J. Comp. Neurol. 470:134–150, 2004. © 2004 Wiley‐Liss, Inc.
Multipotency of purified, transplanted globose basal cells in olfactory epithelium
Authors: Chen Xueyan , Fang Hengsheng , Schwob James E.
Published: January 2004

By comparison with the rest of the nervous system, the olfactory epithelium has an unparalleled ability to renew and repair itself throughout life. However, the identity and capacity of the various types of progenitor cells that underlie that ability are not well established. We used selective isolation, transplantation, and engraftment of various types of marker‐labeled cells into the epithelium of methyl bromide‐lesioned, unmarked host mice to dissect progenitor cell capacity. Globose basal cells were purified from other potential progenitors using the monoclonal antibody GBC‐2 (GBC, globose basal cell) and fluorescence activated cell sorting. Transplanted globose basal cells engraft and, in aggregate, give rise to globose basal cells, neurons, sustentacular cells, and several other kinds of non‐neuronal cells. Individual clones, derived from single engrafted globose basal cells, can consist of a mixture of neurons and non‐neuronal cells, only neurons, or only non‐neuronal cells. Neurons that arise after transplantation mature to the point of expressing odorant receptors and olfactory marker protein and of projecting axons to the olfactory bulb. In contrast, other kinds of epithelial cells are neither neurogenic nor multipotent. For example, sustentacular and duct cells give rise only to themselves after transplantation. Furthermore, horizontal basal cells do not engraft in mice, in which the endogenous population is spared after lesion. Thus, some subtype(s) of GBC is a multipotent progenitor cell, whose multipotency is activated after destruction of both neurons and non‐neuronal cells. The results suggest that progenitor cell transplantation may prove useful as a therapeutic modality as well as an analytical tool. J. Comp. Neurol. 469:457–474, 2004. © 2004 Wiley‐Liss, Inc.
Development of GABA‐immunoreactive cells in the spinal cord of the sea lamprey, P. marinus
Authors: Ruiz Y. , Pombal M.A. , Megías M.
Published: January 2004

The lamprey spinal cord increases in length and size during all its life cycle; thus, it is expected that new cells will be generated. This expectation suggests that the locomotor circuits must be continuously remodeled. Key elements in the cellular network controlling locomotor behavior are inhibitory cells. Here, we studied the γ‐aminobutyric acid–immunoreactive (GABA‐ir) cells in the lamprey spinal cord during postembryonic development. Three major populations of GABA‐ir cells were identified according to their distribution: those located in the gray matter, those contacting the cerebrospinal liquid (LC cells), and those located in the white matter. The results show (1) the number of GABA‐ir cells per segment increase from prolarvae (<10 mm) to adulthood; (2) the lower number of GABA‐ir cells in 100 μm of spinal cord is 66 ± 7, found in premetamorphic larvae, and the highest is 107 ± 6, found in postmetamorphic animals; (3) the gray matter and LC GABA‐ir cells show different variations in number depending on the developmental period. Thus, in the 10‐mm larvae, the gray matter GABA‐ir cells are more abundant than LC cells, whereas in the young postmetamorphic specimens, the contrary occurs. Most of the GABA‐ir cells located in the white matter were classified as edge cells. They increase in number from the beginning of the prolarval period, where there are not white matter–positive cells, to the middle larval period, where there are 9 ± 4 GABA‐ir edge cells per segment. This value was unaltered in later periods, where GABA‐ir edge cells represent 20–30% of the total number of edge cells per segment. The increase in number of GABA‐ir cells in these populations during a specific point of the lamprey life cycle may indicate different inhibitory requirements of the locomotor circuit at different developmental periods. J. Comp. Neurol. 470:151–163, 2004. © 2004 Wiley‐Liss, Inc.
Axonal projections from the parasubthalamic nucleus
Authors: Goto Marina , Swanson Larry W.
Published: January 2004

The parasubthalamic nucleus (PSTN) is a differentiation of the lateral hypothalamic area, characterized by a distinct population of neurons expressing β‐preprotachykinin (β‐PPT) mRNA. The axonal projections from the PSTN have been analyzed with the PHAL anterograde tract tracing method in rats. The results indicate that the cell group is distinguished by massive projections to parasympathetic preganglionic neurons in the brainstem (especially in the salivatory nuclei and dorsal motor nucleus of the vagus nerve) and to parts of the parabrachial nucleus and nucleus of the solitary tract that relay viscerosensory and gustatory information. In addition, the PSTN projects to cortical parts of the cerebral hemisphere (infralimbic, agranular insular, postpiriform transition and lateral entorhinal areas, and posterior basolateral amygdalar nucleus)—directly and also indirectly via thalamic feedback loops involving the paraventricular and mediodorsal nuclei—and to nuclear parts of the cerebral hemisphere (central amygdalar nucleus, striatal fundus, rhomboid nucleus of the bed nuclei of the stria terminalis, and substantia innominata). The PSTN is thus positioned to influence directly many cerebral hemisphere and hindbrain components of the central parasympathetic control network that is active, for example, during feeding behavior and cardiovascular regulation. J. Comp. Neurol. 469:581–607, 2004. © 2004 Wiley‐Liss, Inc.
Immunohistochemistry and neural connectivity of the Ov shell in the songbird and their evolutionary implications
Authors: Zeng Shaoju , Zhang Xinwen , Peng Weimin , Zuo Mingxue
Published: January 2004

The neuropeptide immunohistochemistry and neural connectivity of areas surrounding the thalamic auditory nucleus (the nucleus ovoidalis [Ov]), as well as the areas to which it is connected, were investigated in a songbird, the Bengalese finch. The results showed that met‐enkephalin was present in the Ov shell and most of the areas connected to it, but not in the Ov core. Anterograde and retrograde tracing studies showed that the Ov shell was more widely connected than the Ov core. The Ov shell was mainly connected to: 1) areas flanking the primary telencephalic auditory field (i.e., fields L2b, L1, and L3) and areas surrounding the robust nucleus of the archistriatum (RA); 2) several hypothalamic areas such as the nucleus ventromedialis hypothalami (VMN) and the nucleus anterior medialis hypothalami (AM). Some of these areas connected to the Ov shell are thought to be involved in auditory mediated neurosecretory activities. These results, which are similar to those reported previously in non‐songbirds, suggest that the Ov shell and other surrounding areas of auditory and song‐control nuclei are conserved in birds. These findings are discussed in terms of the evolution of the core‐and‐surround organization of auditory and song‐control nuclei. J. Comp. Neurol. 470:192–209, 2004. © 2004 Wiley‐Liss, Inc.
Organization of parabrachial projections from the spinal trigeminal nucleus oralis: An anterograde tracing study in the rat
Authors: Dallel Radhouane , Ricard Olivier , Raboisson Patrick
Published: January 2004

In recent years, we have accumulated data showing that the spinal trigeminal nucleus oralis (Sp5O) contributes to the processing of somatosensory inputs from the orofacial region. Although the parabrachial area (PB) represents the main brainstem relay for autonomic, nociceptive, and gustatory afferents, few data are available regarding the topographical distribution of the efferent projections from the Sp5O to the PB. We have addressed this question with the rat, by using the anterograde tracer Phaseolus vulgaris leucoagglutinin. A dense trigeminoparabrachial pathway from the Sp5O toward, predominantly, the ipsilateral PB was revealed. Projections come mainly from the dorsal part of the Sp5O that was found to innervate densely the medial, external medial, and ventral lateral subnuclei. In contrast, the ventral part of the Sp5O projected almost exclusively to an as yet not formally described region, located dorsally and laterally to the lateral tip of the brachium conjunctivum, close to the Kölliker‐Fuse nucleus. These results suggest that distinct regions within the Sp5O may be involved in the processing of gustatory and nociceptive information. J. Comp. Neurol. 470:181–191, 2004. © 2004 Wiley‐Liss, Inc.
OL‐protocadherin expression in the visual system of the chicken embryo
Authors: Müller Karina , Hirano Shinji , Puelles Luis , Redies Christoph
Published: January 2004

The expression of OL‐protocadherin, a homotypically binding cell adhesion molecule, was mapped in the visual system of the chicken embryo at intermediate to late stages of development (11–19 days of incubation). The expression was compared with that of four classic cadherins, described previously. OL‐protocadherin is expressed by the isthmooptic nucleus, its retinopetal projection, and possibly its retinal target neurons, the amacrine cells. Ganglion cells begin to express OL‐protocadherin at relatively late stages of development. The layers of the optic tectum, the projection neurons in the stratum griseum centrale, and the tectofugal pathways show differential OL‐protocadherin immunoreactivity. Several of the diencephalic target nuclei of the tectothalamic projection, such as the principal pretectal nucleus, subpretectal nucleus, and nucleus rotundus, contain distinct subregions or populations of neurons expressing OL‐protocadherin. In these centers, the expression pattern of OL‐protocadherin differs from that of the four classic cadherins, though it shows partial overlap with them. Other retinorecipient and/or tectorecipient nuclei (ventral geniculate nucleus, lateral dorsolateral nucleus, superficial synencephalic nucleus, pretectal area, and griseum tectale) also show a differential immunoreactivity for OL‐protocadherin and other cadherins. Some of these nuclei and the optic tectum display a similar sequence of cadherin expression from superficial to deep layers, in a pattern that may reflect mutual interconnections. This result indicates a partial conservation of cadherin expression across interconnected embryonic divisions, from the mesencephalon to the ventral thalamus. In conclusion, OL‐protocadherin is a marker for specific functional gray matter structures and neural circuits in the chicken visual system. J. Comp. Neurol. 470:240–255, 2004. © 2004 Wiley‐Liss, Inc.
Electron microscopy of degenerative changes in the chick basilar papilla after gentamicin exposure
Authors: Hirose Keiko , Westrum Lesnick E. , Cunningham Dale E. , Rubel Edwin W
Published: January 2004

We present a sequential study of the substructural alterations in the chick basilar papilla at the earliest signs of hair cell degeneration. Three‐day posthatch chicks received a single injection of gentamicin (300 mg/kg) and were killed at 6, 8, 12, 15, 18, 21, and 24 hours after the injection. The basilar papillae were studied by conventional transmission electron microscopy. Examination was limited to the basal region, where all hair cells are eliminated by this treatment. As early as 8 hours and clearly by 12 hours, altered fine structure was seen in hair cells. Changes included rounding and swelling of the hair cells, condensation of nuclear chromatin, dissolution of ribosomes, dilatation of the mitochondria, and accumulation of inclusion bodies and lysosomes. By 15–18 hours, lysosomes increased and became denser, afferent terminals appeared swollen, and the first cell extrusion was seen. Efferents were unaffected, and supporting cells, though having inclusion bodies now, retained normal intercellular junctions. By 21–24 hours, large regions of complete hair cell loss were composed of expanded supporting cell processes with normal‐appearing intercellular junctions and portions of extruded hair cells, partially attached to the supporting cell surface. These observations demonstrate that auditory hair cells undergo a rapid and controlled process of hair cell extrusion that allows preservation of the reticular lamina and minimal contamination of surrounding structures by intracytoplasmic contents of the damaged hair cells. J. Comp. Neurol. 470:164–180, 2004. © 2004 Wiley‐Liss, Inc.
Components of the Reelin signaling pathway are expressed in the spinal cord
Authors: Yip Yee Ping , Capriotti Christine , Magdaleno Susan , Benhayon David , Curran Tom , Nakajima Kazunori , Yip Joseph W.
Published: January 2004

The Reelin signaling pathway in the brain involves the binding of Reelin to very‐low‐density lipoprotein receptors (VLDLR) and apolipoprotein E receptor 2 (ApoER2). After Reelin binds the lipoprotein receptors on migrating neurons, the intracellular adaptor protein Disabled‐1 (Dab1) becomes phosphorylated, ultimately resulting in the proper positioning of cortical neurons. Previous work showed that Reelin also affects the positioning of sympathetic preganglionic neurons (SPN) in the spinal cord (Yip et al. [2000] Proc Natl Acad Sci USA 97:8612–8616). We asked in the present study whether components of the Reelin signaling pathway in the brain also function to control SPN migration in developing spinal cord. Results showed that Reelin and reelin mRNA are found adjacent to migrating SPN. In addition, dab1 mRNA and protein are expressed by migrating SPN, and dab1‐null mice show abnormal SPN migration similar to that seen in reeler. Finally, vldlr and apoER2 are also expressed in migrating SPN, and mice lacking both vldlr and apoER2 show aberrant SPN location that is identical to that of reeler and dab1‐null mice. Because molecules known to be involved in Reelin signaling in the brain are present in the developing spinal cord, it is likely that the Reelin signaling pathways in the brain and spinal cord function similarly. The relative simplicity of the organization of the spinal cord makes it a potentially useful model system with which to study the molecular and cellular function of the Reelin signaling pathway in control of neuronal migration. J. Comp. Neurol. 470:210–219, 2004. © 2004 Wiley‐Liss, Inc.
Retinoic acid signaling in the brain marks formation of optic projections, maturation of the dorsal telencephalon, and function of limbic sites
Authors: Luo Tuanlian , Wagner Elisabeth , Grün Felix , Dräger Ursula C.
Published: January 2004

As retinoic acid (RA) is known to regulate the expression of many neuronal proteins, it is likely to influence overall development and function of the brain; few particulars, however, are available about its role in neurobiological contexts due mainly to problems in RA detection. To ask whether the function of RA in the rostral brain is concentrated in particular neurobiological systems, we compared sites of RA synthesis and actions, as detected by RA signaling in reporter mice, for embryonic and adult ages. We found that most sites of RA actions in the forebrain do not colocalize with RA synthesis, consistent with a dominant RA supply by diffusion and the circulation. The changing RA patterns distinguish preferentially two complex functional schemes. (1) Within the visual system when the first optic axons grow toward their targets, RA signaling delineates the topographical adjustment of the retinal map, which is encoded in the coordinates of the visual world, to central visual maps, which are formed in the segmental brain coordinates. (2) The second scheme begins early in forebrain morphogenesis as a distinction of the dorsal telencephalon. With progressing development, and in the adult, the RA patterns then focus on widely distributed structures, most of which belong to the limbic system. These are sites in which emotional perception is combined with higher cognitive processes and in which normal function requires ongoing remodeling of synaptic connections, indicating that the developmental role of RA in promotion of neuronal differentiation programs continues in the adult brain for highly flexible neural circuits. J. Comp. Neurol. 470:297–316, 2004. © 2004 Wiley‐Liss, Inc.
Compiling data‐parallel programs for clusters of SMPs
Authors: Benkner Siegfried , Brandes Thomas
Published: January 2004

Clusters of shared‐memory multiprocessors (SMPs) have become the most promising parallel computing platforms for scientific computing. However, SMP clusters significantly increase the complexity of user application development when using the low‐level application programming interfaces MPI and OpenMP, forcing users to deal with both distributed‐memory and shared‐memory parallelization details. In this paper we present extensions of High Performance Fortran (HPF) for SMP clusters which enable the compiler to adopt a hybrid parallelization strategy, efficiently combining distributed‐memory with shared‐memory parallelism. By means of a small set of new language features, the hierarchical structure of SMP clusters may be specified. This information is utilized by the compiler to derive inter‐node data mappings for controlling distributed‐memory parallelization across the nodes of a cluster and intra‐node data mappings for extracting shared‐memory parallelism within nodes. Additional mechanisms are proposed for specifying inter‐ and intra‐node data mappings explicitly, for controlling specific shared‐memory parallelization issues and for integrating OpenMP routines in HPF applications. The proposed features have been realized within the ADAPTOR and VFC compilers. The parallelization strategy for clusters of SMPs adopted by these compilers is discussed as well as a hybrid‐parallel execution model based on a combination of MPI and OpenMP. Experimental results indicate the effectiveness of the proposed features. Copyright © 2004 John Wiley & Sons, Ltd.
A compiler for multiple memory models
Authors: Midkiff S. P. , Lee J. , Padua D. A.
Published: January 2004

The design of consistency models for both hardware and software is a difficult task. For a programming language, it is particularly difficult because the target audience for a high‐level programming language is much wider than the target audience for a machine language, making usability a more important criterion. Exacerbating this problem is the reality that the programming language community has little experience designing programming language consistency models, and therefore each new attempt is very much a voyage into uncharted territory. A concrete example of the difficulties of the task is the current Java Memory Model. Although designed to be easy to use by Java programmers, it is poorly understood and at least one common idiom (the ‘double check idiom’) to exploit the model is unsafe. In this paper, we describe the design of an optimizing Java compiler that will accept either as input or as an interface implementation a consistency model for the code to be compiled. The compiler will use Shasha and Snir's delay set analysis, and our CSSA program representation to provide a canonical representation for the effects of different consistency models on optimizations and analysis. The compiler will serve as a testbed to prototype new memory models, and to measure the effects of different memory models on program performance. Copyright © 2004 John Wiley & Sons, Ltd.
Space–time mapping and tiling: a helpful combination
Authors: Griebl Martin , Faber Peter , Lengauer Christian
Published: January 2004

Tiling is a well‐known technique for sequential compiler optimization, as well as for automatic program parallelization. However, in the context of parallelization, tiling should not be considered as a stand‐alone technique, but should be applied after a dedicated parallelization phase, in our case after space–time mapping. We show how tiling can benefit from space–time mapping, and we derive an algorithm for computing tiles which can minimize the number of communication startups, taking the number of physically available processors into account. We also present how the use of a simple cost model reduces real execution time. Copyright © 2004 John Wiley & Sons, Ltd.
Parallel object‐oriented framework optimization
Authors: Quinlan Daniel J. , Schordan Markus , Miller Brian , Kowarschik Markus
Published: January 2004

Sophisticated parallel languages are difficult to develop; most parallel distributed memory scientific applications are developed using a serial language, expressing parallelism through third party libraries (e.g. MPI). As a result, frameworks and libraries are often used to encapsulate significant complexities. We define a novel approach to optimize the use of libraries within applications. The resulting tool, named ROSE, leverages the additional semantics provided by library‐defined abstractions enabling library specific optimization of application codes. It is a common perception that performance is inversely proportional to the level of abstraction. Our work shows that this is not the case if the additional semantics can be leveraged. We show how ROSE can be used to leverage the semantics within the compile‐time optimization. Copyright © 2004 John Wiley & Sons, Ltd.
SWARP: a retargetable preprocessor for multimedia instructions
Authors: Pokam Gilles , Bihan Stéphane , Simonnet Julien , Bodin François
Published: January 2004

In this paper, we propose SWARP, a retargetable preprocessor for exploiting multimedia instructions. The system mixes loop distribution, unrolling and pattern matching to exploit complex multimedia instructions. Contrary to all available systems, it can be extended at the user level. Using with a TriMedia processor we show that our system achieves important good code quality with a set of frequently used loop kernels for multimedia applications. Copyright © 2004 John Wiley & Sons, Ltd.
Development of Herceptin resistance in breast cancer cells
Authors: Kute Timothy , Lack Christopher M. , Willingham Mark , Bishwokama Bimjhana , Williams Holly , Barrett Kathy , Mitchell Tanita , Vaughn James P.
Published: January 2004

Background

Herceptin, a humanized antibody to HER‐2, is now utilized in the clinic for metastatic breast cancer treatment. The response rate for HER‐2+ patients is only 30% and little is known as to mechanisms of resistance. The mechanism of Herceptin action is also unknown but has been related to cell cycle inhibition.

Methods

The effects of Herceptin and other antibody treatments were determined by cell counting and cell cycle analysis. HER‐2 and p27 expression levels were analyzed by flow cytometry and levels of activated AKT were compared by Western blot analysis. Cellular HER‐2 and p27 expression was measured by immunofluorescence.

Results

Herceptin treatment of BT‐474 cells results in inhibition of cell growth and arrest in the G1 phase. The efficacy of growth arrest was not directly correlated to the binding affinity of antibodies to Her‐2. Our laboratory has developed cell lines that are resistant to Herceptin treatment. In resistant cell lines, binding of antibodies is not hindered. However, Herceptin has completely lost the ability to inhibit cell proliferation. Yet, the mouse isotype 4D5 maintains significant inhibitory activity upon Herceptin‐resistant clones.

Conclusions

Herceptin binds effectively to Her‐2 on the cell surface of Herceptin‐resistant cell lines and the level of Her‐2 expression on the cell surface is not downregulated. Herceptin resistance is not due to downregulation of levels of AKT protein expression, although, phosphorylation of AKT is enhanced in resistant lines and could have a role in resistance. Resistance appears to correlate with the loss of nuclear expression of the cyclin‐dependent kinase inhibitor, p27, as defined by immunofluorescence and flow cytometry studies and cdk‐2 binding studies. © 2004 Wiley‐Liss, Inc.
Spatial pattern analysis of nitrergic neurons in the developing myenteric plexus of the human fetal intestine
Authors: Román V. , Bagyánszki M. , Krecsmarik M. , Horváth A. , Resch B. Á. , Fekete É.
Published: January 2004

Background

Enteric nervous system precursors derived from the neural crest migrate along defined pathways to colonize the bowel. The individual cells in different environments experience different growth, differentiation, and survival conditions. Hence, the spatial distribution of the neurons is determinant with regard to functional maturation. The question arises as to whether the distribution is random or nonrandom.

Methods

Nitrergic cells were visualized by means of nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. Stained specimens were photographed, and the borders of the myenteric plexus and the nuclei of the nitrergic neurons were digitalized. Plexus Pattern Analysis software was used to count the nuclei of nitrergic neurons, calculate the proportions of the areas covered by the plexus and the gut wall, and perform randomization analyses.

Results

The distribution pattern of the nitrergic neurons changed markedly between weeks 14 and 22 of gestation. The nitrergic neurons were randomly distributed at week 14 but were aggregated in the plexus and within the individual ganglia at week 19. The dynamics of these changes exhibited regional differences.

Conclusions

The results suggest that, in addition to the gut wall and the plexus, other intraganglionic constituents may contribute to the aggregation of nitrergic cells and such examinations should be extended to other cell types in the future. © 2004 Wiley‐Liss, Inc.
Measurement of basal, substrate induced and total P‐glycoprotein activity in bronchoalveolar lavage T‐cell subsets
Authors: Donnenberg V. S. , Wilson J. W. , Burckart G. J. , Zeevi A. , Iacono A. , Donnenberg A. D.
Published: January 2004

Background

P‐glycoprotein (P‐gp) is a member of the ABC transporter superfamily. P‐gp activity can be detected by measuring efflux of fluorescent substrates such as rhodamine 123 (R123). Our objectives were to evaluate P‐gp activity in T cells freshly isolated from bronchoalveolar lavage (BAL) and to develop a strategy to distinguish between basal, in vitro substrate‐induced, and total P‐gp activities.

Methods

Cells were obtained from blood (n = 44) and BAL (n = 34), stained for expression of CD3, CD4, CD8, and CD14, and incubated with R123 (0.13 μM) ± cyclosporine (5 μM), a specific P‐gp inhibitor. P‐gp activity was detected as median fluorescence intensity (MFI) and percentage of cells falling below a pre‐established cutoff.

Results

BAL T cells displayed significant basal P‐gp activity, which was most apparent when measured as percentage below the cutoff. Induced activity (difference between P‐gp activity measured after load and efflux) was determined equally well when using the MFI or the percentage below cutoff parameter. Total activity was represented by the efflux parameters (MFI or percentage below cutoff) or by the activity‐time area under the curve (AUC) method. The two efflux parameters correlated well but were insensitive to the time‐dependent nature of dye efflux. In the AUC method, two samples with identical R123 brightness or percentage below cutoff after dye efflux can have very different total activities, depending on their basal activity. The AUC method was also most sensitive in distinguishing between P‐gp activity in peripheral blood and resident lung T cells. Application of this methodology to the comparison of P‐gp activity in BAL and peripheral CD8+ T cells best revealed the elevated total P‐gp activity in BAL T cells.

Conclusions

We have systematically evaluated several methodologies for analysis of P‐gp activity and arrived at a novel and robust strategy amenable to standardization and evaluation of the effects of P‐gp modulators in vivo and in vitro. © 2004 Wiley‐Liss, Inc.
Flow cytometer for measurement of the light scattering of viral and other submicroscopic particles
Authors: Steen Harald B.
Published: January 2004

Background

Light scattering is an essential parameter in flow cytometry, facilitating functions such as size measurement, discrimination of cell types on the basis of shape and morphology, detection of fluorescence‐negative cells, and gating of fluorescence measurements. Light scattering measurement of viruses is generally not feasible with current flow cytometers due to their small size. The problem is aggravated by the fact that the light scattering of particles in this size range (<200 nm) falls off with roughly the sixth power of their linear dimensions.

Methods

A new optical layout using darkfield illumination and detection has been developed. A 532‐nm laser was used for excitation, and scattered light was collected with large aperture optics.

Results

Light scattering histograms of polymer particles with diameters of 70–300 nm were recorded without gating by other parameters. By extrapolation, a detection limit of about 50 nm was obtained. Different species of virus with sizes of approximately 100 nm also were recorded.

Conclusions

Flow cytometric light scattering measurement of submicroscopic particles, in a size range that includes many viral species, is now feasible. The results indicate that it may be practically impossible to measure by flow cytometry the light scattering of particles smaller than 40 nm. © 2004 Wiley‐Liss, Inc.
Quantitation of MHC tetramer‐positive cells from whole blood: Evaluation of a single‐platform, six‐parameter flow cytometric method
Authors: Wölfl Matthias , Schalk Stefan , Hellmich Martin , Huster Katharina M. , Busch Dirk H. , Berthold Frank
Published: January 2004

Background

Quantitation of antigen‐specific T cells provides an insight into the development and dynamics of T‐cell responses in tumor immunology and infectious diseases. Soluble major histocompatibility class I tetramers are widely used to monitor immune responses; however, variations due to handling and analysis are likely to confound comparisons between different experiments and laboratories.

Methods

Whole blood from healthy donors was stained with HLA‐A*0201/tetramers specific for an epitope of phosphoprotein 65, the immunodominant antigen in cytomegalovirus infection. With the help of Trucount tubes, a single‐platform, four‐color flow cytometric assay was established to obtain absolute counts of tetramer‐positive cells. Various staining and gating strategies were evaluated.

Results

The no‐wash method was a quick and straightforward procedure for the quantitation of tetramer‐positive events from whole blood. The level for background staining was low. This information about the intra‐assay–related variation and the physiologic variation will allow validation and interpretation of data in future studies.

Conclusions

The method is highly reliable and can be standardized for multiple experiments. It is therefore suitable for the direct ex vivo analysis of antigen‐specific T cells in a variety of clinical settings such as infectious, autoimmune, or neoplastic diseases and can be implemented as a tool for multicenter studies. © 2004 Wiley‐Liss, Inc.
New method for the analysis of cell cycle–specific apoptosis
Authors: Tao Deding , Wu Jianhong , Feng Yongdong , Qin Jichao , Hu Junbo , Gong Jianping
Published: January 2004

Background

In this study, a new method for the analysis of cell cycle specificity of apoptosis was designed by using a modified annexin V and propidium iodide (API) method.

Methods

Cells of the human promyelocytic HL‐60 line treated with camptothecin (CPT) or ultraviolet light (UV) were labeled with fluorescein isothiocyanate–conjugated annexin V and prefixed with 1% methanol‐free formaldehyde on ice, and their DNA was stained stoichiometrically with propidium iodide in the presence of digitonin. Cellular green and red fluorescences were measured by flow cytometry.

Results

Cell cycle specificity of apoptosis obtained by the API method and those analyzed for the presence of DNA strand breaks by using terminal deoxynucleotidyl transferase (TdT) assay were similar: CPT‐ or UV‐induced apoptosis preferentially in S‐ or G1‐phase cells, respectively. When the internucleosomal DNA degradation was prevented by the serine protease inhibitor N‐tosyl‐L‐phenylalanine chloromethyl ketone, apoptotic cells could not be detected by the TdT assay but were identified by the API method.

Conclusions

The API method, similar to the TdT assay, accurately detects the cell cycle phase specificity of apoptosis. Also, the API method appears to detect earlier stages of apoptosis than the TdT assay. © 2004 Wiley‐Liss, Inc.
Comparison of flow cytometry and laser scanning cytometry for the analysis of CD34+ hematopoietic stem cells
Authors: Oswald Joachim , Jørgensen Birgitte , Pompe Tilo , Kobe Fritz , Salchert Katrin , Bornhäuser Martin , Ehninger Gerhard , Werner Carsten
Published: January 2004

Background

Characterization of hematopoietic stem cells (HSCs) by laser scanning cytometry (LSC) was compared with conventional flow cytometry (FCM). The method was evaluated for application in the development of advanced cell culture substrates that were supposed to support the ex vivo expansion of HSC. For this purpose, adherent HSCs were grown in culture on thin polymer films coated with reconstituted collagen I fibrils and subsequently analyzed by LSC.

Methods

CD34+ HSCs were isolated from cord blood by immunomagnetic separation and cultivated on polymer films coated with reconstituted collagen I fibrils. Cell surface antigens (CD34, CD29) were stained with antibodies, and nuclei were labeled with a DNA stain (TO‐PRO‐3 iodide) that does not interfere with the fluorochromes of the antibodies. Fluorescence intensity of the adherent cells was measured by means of LSC. Before and after in vitro expansion for time periods of up to 7 days, suspension cells were analyzed with LSC and FCM.

Results

LSC‐based analysis enabled reliable quantification of CD34+ cells with bright antigen expression before cell culture. At this stage, LSC and FCM data for CD34 expression at given HSC samples largely coincided. After in vitro expansion, LSC data deviated from FCM data for cells with dim CD34 antigen expression, whereas the fluorescence intensity of the CD29 antigen remained comparable. The deviation between LSC and FCM data for CD34dim was attributed to the better resolution of weak fluorescence by FCM. Based on the preceding evaluation of the method, LSC analysis could be applied to characterize HSCs cultivated on collagen I–coated polymer films without detachment of the cells from the substrate.

Conclusions

LSC‐based analysis allows for the automated evaluation of adherent HSCs. Although resolution of weakly expressed antigens can be achieved more precisely with FCM, the method provides a valuable tool to study interactions of HSCs with bioartificial substrates. © 2004 Wiley‐Liss, Inc.