bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurosecretory identity conferred by the apterous gene: Lateral horn leucokinin neurons in Drosophila
Authors: Herrero Pilar , Magariños Marta , Torroja Laura , Canal Inmaculada
Published: January 2003

The LIM‐HD protein Apterous has been shown to regulate expression of the FMRFamide neuropeptide in Drosophila neurons (Benveniste et al. [1998] Development 125:4757–4765). To test whether Apterous has a broader role in controlling neurosecretory identity, we analyzed the expression of several neuropeptides in apterous (ap) mutants. We show that Apterous is necessary for expression of the Leucokinin neuropeptide in a pair of brain neurons located in the lateral horn region of the protocerebrum (LHLK neurons). ap null mutants are depleted of Leucokinin in these cells, whereas hypomorphic mutants show reduced Leucokinin expression. Other Leucokinin‐containing neurons are not affected by mutations in ap gene. Co‐expression of apterous and Leucokinin is observed exclusively in the LHLK neurons, from larval stages to adulthood. Rescue assays performed in null ap mutants, by expressing Apterous protein under apGAL4 and elavGAL4 drivers, demonstrate the recovery of Leucokinin in the LHLK neurons. These results reinforce the emerging role of the LIM‐HD proteins in determining neuronal identity. They also clarify the neuroendocrine phenotype of apterous mutants. J. Comp. Neurol. 457:123–132, 2003. © 2003 Wiley‐Liss, Inc.
Expression of vesicular glutamate transporters, VGluT1 and VGluT2, in axon terminals of nociceptive primary afferent fibers in the superficial layers of the medullary and spinal dorsal horns of the rat
Authors: Li Jin‐Lian , Fujiyama Fumino , Kaneko Takeshi , Mizuno Noboru
Published: January 2003

We examined immunohistochemically whether the vesicular glutamate transporters (VGluTs), VGluT1 and VGluT2, might be expressed in synaptic terminals of nociceptive primary afferent fibers within laminae I and II of the medullary and spinal dorsal horns of the rat. VGluT1 immunoreactivity (IR) was intense in the inner part of lamina II but weak in lamina I and the outer part of lamina II. VGluT2‐IR was most intense in lamina I and the outer part of lamina II. Expression of VGluTs in synaptic terminals was confirmed by dual immunofluorescence histochemistry for VGluTs and synaptophysin. Expression of VGluTs in axon terminals of primary afferent fibers terminating in laminae I and II was also confirmed immunohistochemically after unilateral dorsal rhizotomy. The dual immunofluorescence histochemistry indicated expression of VGluTs in substance P (SP)‐containing axon terminals in lamina I and the outer part of lamina II. Electron microscopy confirmed the coexpression of VGluTs and SP in axon terminals within laminae I and II; VGluTs was associated with round synaptic vesicles at the asymmetric synapses. It was further observed that isolectin IB4, a marker for unmyelinated axons, often bound with VGluT2‐immunopositive structures but rarely with VGluT1‐immunopositive structures in lamina II. Thus, the results indicated in laminae I and II of the medullary and spinal dorsal horns that both VGluT1 and VGluT2 were expressed in axon terminals of primary afferent fibers, including SP‐containing nociceptive fibers and that VGluT in unmyelinated primary afferent fibers terminating in lamina II was primarily VGluT2. J. Comp. Neurol. 457:236–249, 2003. © 2003 Wiley‐Liss, Inc.
Fiber connections of the torus longitudinalis in a teleost: Cyprinus carpio re‐examined
Authors: Ito Hironobu , Yamamoto Naoyuki , Yoshimoto Masami , Sawai Nobuhiko , Yang Chun‐Ying , Xue Hao‐Gang , Imura Kosuke
Published: January 2003

Fiber connections of the carp torus longitudinalis were re‐examined by means of tract‐tracing methods. The torus longitudinalis projected mainly to the stratum marginale of the optic tectum, area pretectalis, and corpus cerebelli. The stratum marginale was anterogradely labeled only by biocytin, but not by horseradish peroxidase. Because the stratum is composed of extremely fine axons of the small toral neurons, this may be ascribed to different molecular weights of the tracers. The main afferent sources to the torus longitudinalis were the nucleus subvalvularis, which was located beneath the nucleus lateralis valvulae, the nucleus subeminentialis pars magnocellularis, and neurons along the posterior mesencephalo‐cerebellar tract. Some labeled cells also appeared in the area pretectalis, nucleus paracommissuralis, optic tectum, and torus semicircularis. In a previous paper, it was incorrectly reported that the valvula cerebelli was the main source of afferents to the torus longitudinalis. Here we report the reason for the previous mistake in relation to the techniques employed. J. Comp. Neurol. 457:202–211, 2003. © 2003 Wiley‐Liss, Inc.
Identification and localization of a neprilysin‐like activity that degrades tachykinin‐related peptides in the brain of the cockroach, Leucophaea maderae, and locust, Locusta migratoria
Authors: Isaac R. Elwyn , Nässel Dick R.
Published: January 2003

Neprilysin (NEP) is an endopeptidase, which has an important role in the inactivation of mammalian tachykinins. NEP‐like activity has also been found in the brain of several insects; however, the lack of information about the cellular localization of this peptidase has hindered our understanding of its role in peptidergic signaling in insects. We now provide evidence that membrane‐bound NEP is involved in the inactivation of tachykinin‐related peptides in the brain of the cockroach, Leucophaea maderae, and the locust, Locusta migratoria. The L. maderae enzyme cleaved the cockroach peptide LemTRP‐1 and the mammalian NEP substrate [DAla2,Leu5]enkephalin at the Gly‐Phe peptide bond. The enzyme was acted upon by the NEP inhibitors phosphoramidon (IC50, 0.64 μM) and thiorphan (IC50, 1.23 μM), and the detergent‐solubilized enzyme had an Mr of approximately 300,000 and a neutral pH optimum. This endopeptidase cleaved another insect tachykinin‐related peptide, CavTK‐II, in a predictable manner at the Ala‐Phe peptide bond, suggesting that the peptidase can hydrolyse tachykinin‐related peptides with different structures. NEP activity was histochemically localized in several, but not all, regions of neuropil in the brain of L. maderae, including the central body, the lobula of the optic lobe, and the tritocerebrum. All of these regions are known to receive neuronal processes containing tachykinin‐related peptides. A slightly different distribution pattern for NEP was observed in the brain of L. migratoria. Again, NEP was localized to regions of the neuropil that also display tachykinin‐related peptide immunoreactivity. The data reported provide evidence for an evolutionary conserved role for NEP in the inactivation of tachykinin‐related peptides in the brain. J. Comp. Neurol. 457:57–66, 2003. © 2003 Wiley‐Liss, Inc.
Cerebrospinal fluid‐contacting neurons in the rat spinal cord, a γ‐aminobutyric acidergic system expressing the P2X2 subunit of purinergic receptors, PSA‐NCAM, and GAP‐43 immunoreactivities: Light and electron microscopic study
Authors: Stoeckel Marie‐Elisabeth , Uhl‐Bronner Sandra , Hugel Sylvain , Veinante Pierre , Klein Marie‐Jeanne , Mutterer Jerome , Freund‐Mercier Marie‐José , Schlichter Rémy
Published: January 2003

Cerebrospinal fluid‐contacting neurons (CSFcNs) occur in various brain regions of lower vertebrates. In mammals, they are restricted to medullospinal areas, and little is known about their projection sites. In the present work, we investigated some morphofunctional characteristics of such neurons in the rat spinal cord by light and electron microscopic immunocytochemistry. CSFcNs expressing the P2X2 subunit of purinergic receptors were present throughout the spinal cord, though more numerous at lower thoracolumbar and sacral levels. These neurons coexpressed GAD and the polysialylated neural cell adhesion molecule (PSA‐NCAM), a marker of cellular plasticity. From low thoracic levels downward, tiny amyelinic axons (less than 200 nm in diameter) were tightly packed in bundles, which ran along the ependyma and extended ventrally, eventually concentrating against the walls of the ventral median fissure. In addition to P2X2, GAD, γ‐aminobutyric acid (GABA), and PSA, these axons expressed GAP‐43 immunoreactivity. Moreover, they were labelled along their entire lengths with antibodies against synaptotagmin and synaptophysin, but these failed to reveal intraspinal terminal fields. Taken together, our observations indicate the presence in the rat spinal cord of a highly plastic system of GABAergic CSFcNs that express the P2X2 subunit of purinergic receptors. The function of this original system remains open to question. In these neurons, the P2X2 receptors may confer a sensitivity to ATP either present in the CSF or released by nearby neurons of the central autonomic area. J. Comp. Neurol. 457:159–174, 2003. © 2003 Wiley‐Liss, Inc.
Critical calpain‐dependent ultrastructural alterations underlie the transformation of an axonal segment into a growth cone after axotomy of cultured Aplysia neurons
Authors: Spira Micha E. , Oren Ruthi , Dormann Ada , Gitler Daniel
Published: January 2003

The transformation of a stable axonal segment into a motile growth cone is a critical step in the regeneration of amputated axons. In earlier studies we found that axotomy of cultured Aplysia neurons leads to a transient and local elevation of the free intracellular Ca2+ concentration, resulting in calpain activation, localized proteolysis of submembranal spectrin, and, eventually, growth cone formation. Moreover, inhibition of calpain by calpeptin prior to axotomy inhibits growth cone formation. Here we investigated the mechanisms by which calpain activation participates in the transformation of an axonal segment into a growth cone. To that end we compared the ultrastructural alterations induced by axotomy performed under control conditions with those caused by axotomy performed in the presence of calpeptin, using cultured Aplysia neurons as a model. We identified the critical calpain‐dependent cytoarchitectural alterations that underlie the formation of a growth cone after axotomy. Calpain‐dependent processes lead to restructuring of the neurofilaments and microtubules to form an altered cytoskeletal region 50–150 μm proximal to the tip of the transected axon in which vesicles accumulate. The dense pool of vesicles forms in close proximity to a segment of the plasma membrane along which the spectrin membrane skeleton has been proteolyzed by calpain. We suggest that the rearrangement of the cytoskeleton forms a transient cellular compartment that traps transported vesicles and serves as a locus for microtubule polymerization. We propose that this cytoskeletal configuration facilitates the fusion of vesicles with the plasma membrane, promoting the extension of the growth cone's lamellipodium. The growth process is further supported by the radial polymerization of microtubules from the growth cone's center. J. Comp. Neurol. 457:293–312, 2003. © 2003 Wiley‐Liss, Inc.
Reelin expression during embryonic brain development in Crocodylus niloticus
Authors: Tissir F. , Lambert De Rouvroit C. , Sire J.‐Y. , Meyer G. , Goffinet A.M.
Published: January 2003

The expression of reelin mRNA and protein was studied during embryonic brain development in the Nile crocodile Crocodylus niloticus, using in situ hybridization and immunohistochemistry. In the forebrain, reelin was highly expressed in the olfactory bulb, septal nuclei, and subpial neurons in the marginal zone of the cerebral cortex, dorsal ventricular ridge, and basal forebrain. At early stages, reelin mRNA was also detected in subventricular zones. In the diencephalon, the ventral lateral geniculate nuclei and reticular nuclei were strongly positive, with moderate expression in the habenula and focal expression in the hypothalamus. High expression levels were noted in the retina, the tectum, and the external granule cell layer of the cerebellum. In the brainstem, there was a high level of signal in cochleovestibular, sensory trigeminal, and some reticular nuclei. No expression was observed in the cortical plate or Purkinje cells. Comparison with reelin expression during brain development in mammals, birds, turtles, and lizards reveals evolutionarily conserved, homologous features that presumably define the expression profile in stem amniotes. The crocodilian cortex contains subpial reelin‐positive cells that are also p73 positive, suggesting that they are homologous to mammalian Cajal‐Retzius cells, although they express the reelin gene less intensely. Furthermore, the crocodilian cortex does not contain the subcortical reelin‐positive cells that are typical of lizards but expresses reelin in subventricular zones at early stages. These observations confirm that reelin is prominently expressed in many structures of the embryonic brain in all amniotes and further emphasize the unique amplification of reelin expression in mammalian Cajal‐Retzius cells and its putative role in the evolution of the cerebral cortex. J. Comp. Neurol. 457:250–262, 2003. © 2003 Wiley‐Liss, Inc.
Reflectivity of shock compressed xenon plasma
Authors: Reinholz H. , Röpke G. , Wierling A. , Mintsev V. , Gryaznov V.
Published: January 2003

Experimental results [1] for the reflection coefficient of shock‐compressed dense xenon plasmas at pressures of 1.6 – 17 GPa and temperatures around 30 000 K using a laser beam with λ = 1.06 μm are compared with calculations based on different theoretical approaches to the dynamical collision frequency. It is found that a reasonable description can be given assuming a spatial electron density profile corresponding to a finite width of the shock wave front of about 2 · 10–6 m.
Experimental study of the creation of a fire‐rod II: Emissive probe measurements
Authors: Gyergyek T. , Čerček M. , Schrittwieser R. , Ionita C. , Popa G. , Pohoata V.
Published: January 2003

A planar electrode (collector) is immersed into a weakly magnetized discharge plasma column. Positive voltage steps are applied to the collector and time resolved measurements of the plasma response are performed by emissive probes using standard boxcar technique. On the emissive probe characteristics an interesting phenomenon is observed: a few μs after the application of the voltage step to the collector (time depends on the distance between the collector and the probe), the emissive probe starts to collect additional positive current in the potential region between plasma potentials of the high and low potential plasma. This phenomenon is investigated experimentally. We give a possible qualitative explanation that additional positive current collection is due to a combination of an interaction between the collector and the probe and space charge effects of positive ions around the probe.
Stability criteria of Rayleigh ‐ Taylor modes in magnetized plasmas
Authors: Al–Khateeb A.M. , Laham N.M.
Published: January 2003

The Rayleigh ‐ Taylor (RT) instability is investigated analytically in an inhomogeneous plasma in an external magnetic field. For the case of two distinct fluid layers separated by a sharp boundary and for a fluid of a continuously varying density, RT dispersion relations have been obtained and analyzed. Stability criteria of the excited modes are disscussed with respect to the mode propagation relative to the applied magnetic field. The magnetic field is found to act as a stabilizer up to a threshold value that can be determined from the dispersion relation.
Interpolation formula for the electrical conductivity of nonideal plasmas
Authors: Esser A. , Redmer R. , Röpke G.
Published: January 2003

On the basis of a quantum‐statistical approach to the electrical conductivity of nonideal plasmas we derive analytical results in the classical low‐density regime, in the degenerate Born limit, and for the contribution of the Debye‐Onsager relaxation effect. These explicit results are used to construct an improved interpolation formula of the electrical conductivity valid in a wide range of temperature and density which allows to compare with available experimental data of nonideal plasmas.
Scattering processes and electrical conductivity of partially ionized hydrogen plasma
Authors: Ramazanov T. , Galiyev K. , Dzhumagulova K. N. , Röpke G. , Redmer R.
Published: January 2003

We consider partially ionized hydrogen plasma for the density region ne = (1018 ÷ 1022) cm—3. The cross sections for scattering processes between the particles are calculated within the partial wave method. Charged particles in the system (electrons, protons) interact via an effective potential that takes into account threeparticle correlations. The Buckingham polarization potential is used to describe electron‐atom and proton‐atom interactions. The electrical conductivity is determined using the Chapman‐Enskog method. The results are compared with other available data.
Flow cytometric quantification of apoptosis and proliferation in mixed lymphocyte culture
Authors: Marín Luis , Minguela Alfredo , Torío Alberto , Moya‐Quiles María R. , Muro Manuel , Montes‐Ares Olga , Parrado Antonio , Álvarez‐López D. María Rocío , García‐Alonso Ana M.
Published: January 2003

Background

The one‐way mixed lymphocyte culture (MLC) is the classic culture used for studying the allogenic immunoresponse in vitro, but stimulator and responder cell identifications and quantification of apoptotic or proliferative responder cells are unreliable.

Methods

Peripheral blood mononuclear cells were labeled with 5‐ (and 6‐) carboxy fluorescein diacetate succinimidyl ester (CFSE) and stimulated with allogenic unlabeled irradiated cells in unidirectional cultures. Apoptosis was determined by the 7‐aminoactinomycin D technique, and the absolute number of each cell population was calculated by adding a fixed number of cells stained with propidium iodide as the reference standard for each test.

Results

CFSE labeling of cells under different cultures did not affect the results of proliferation or apoptosis. Data of apoptosis obtained with this method were comparable to those of the monoclonal antibody technique, and the proliferation level determined by [3H]‐thymidine incorporation or counting the number of proliferative living cells, as proposed in this method, showed a good correlation.

Conclusions

The method presented in this report allows the simultaneous determination of apoptosis and proliferation in MLCs and the analysis of cell phenotype, thereby avoiding the use of radioactivity. This assay opens new perspectives for a better understanding of the mechanisms implied in the establishment or break of tolerance to the graft in solid organ transplants. Cytometry Part A 51A: 107–118, 2003. © 2003 Wiley‐Liss, Inc.
Analysis‐only detection of Giardia by combining immunomagnetic separation and two‐color flow cytometry
Authors: Ferrari Belinda C. , Veal Duncan
Published: January 2003

Background

Giardia is a protozoan parasite of concern to water utilities. Giardia detection relies on cyst isolation and confirmation with the use of fluorescence microscopy. It is of interest to develop a flow cytometric (FCM) method that reliably detects one cyst in 10 L of water. To date all available antibodies have targeted the same epitope on the cyst wall. To achieve a reliable method, two independent probes are required.

Methods

Giardia cysts were spiked into a backwash water sample with and without prior hybridization to peptide nucleic acid (PNA) probes. Immunomagnetic separation (IMS) as a pre‐enrichment step was compared with filtration of the water sample. Cysts were recovered with two‐color FCM. Those cysts hybridized with PNA and fluorescein isothiocyanate (FITC) were dual stained with monoclonal antibody (mAb) conjugated to phycoerythrin (PE); those not hybridized to PNA were dual stained with mAb‐FITC and mAb‐PE.

Results

A fourfold increase in fluorescent signal intensity was obtained when combining the mAb‐PE and PNA probe compared with two‐color antibody staining. When combined with IMS, Giardia was successfully identified by FCM, with no false positives detected.

Conclusions

Analysis‐only FCM detection of Giardia in water is feasible. Further method development incorporating PNA probe hybridization after IMS is necessary. Cytometry Part A 51A:79–86, 2003. © 2003 Wiley‐Liss, Inc.
Quantifying cell scattering: The blob algorithm revisited
Authors: Kort Eric J. , Jones Ashley , Daumbach Michael , Hudson Eric A. , Buckner Bree , Resau James H.
Published: January 2003

Background

A method to objectively quantify cell scattering would permit quantitative evaluation of therapies and compounds intended to affect this physiologic process, which has relevance to normal (e.g., development) and pathologic (e.g., metastasis) events.

Methods

A grid‐based modified blob analysis was performed on a set of images of Madin‐Darby Canine Kidney (MDCK) cells to quantify the following parameters: the number of cellular clusters in each image, the size of the clusters in terms of pixel counts, and the number of cells in each cluster. These parameters were used as measures of cell scattering and were compared with subjective assessments of scattering made by three experienced examiners.

Results

The quantitative parameters correlated strongly to subjective assessments. The algorithm displayed a different concept of “clustering” than the examiners and consistently identified more clusters than did the examiners. There was close agreement in the number of cells counted. All three quantitative parameters correlated strongly to the subjective scattering scores, as follows: cluster count (rs = −0.765 to −0.789, P < 0.0001), cluster size in pixels (rs = 0.838 to 0.845, P < 0.0001), and cluster size in cells (rs = 0.758 to 0.804, P < 0.0001). The parameters were continuous, providing greater resolving power than ordinal subjective scores.

Conclusions

The findings confirmed that our algorithm reproduces the traditional classification of scattering with improved resolution, quantification, and objectivity. Cytometry Part A 51A:119–126, 2003. © 2003 Wiley‐Liss, Inc.
Rapid expression cloning of receptors using epitope‐tagged ligands and high‐speed cell sorting
Authors: Robeva Anna S. , Yan‐Neale Yan , Burfeind Patricia , Bodian Dale L. , Chirn Gung‐Wei , Kolbinger Frank , Labow Mark , Vallon Rüdiger D. W.
Published: January 2003

Background

In this study we describe a new approach for expression cloning of receptors.

Methods

Our approach was based on highly efficient transfer of retroviral cDNA libraries into target cells and detection of receptor–ligand interaction with the use of an antibody directed against an epitope tag on recombinant ligands. Detection of the complex and isolation of receptor‐transduced cells were achieved by flow cytometry and rare event high‐speed cell sorting. Recovery of the cDNA coding for the receptor(s) was achieved by polymerase chain reaction.

Results

As a proof‐of‐concept study we set out to clone the receptor for B‐lymphocyte stimulator protein (BlyS), not known at the start of the project but reported while this work was in progress. First, we detected binding of epitope‐tagged BlyS to IM9 cells. Second, human T‐lymphoblasts (CEM cells), which do not bind BlyS, were transduced with a retroviral cDNA library generated from IM9 cells. Transduced CEM cells binding epitope‐tagged BlyS protein were identified by flow cytometry. After three sequential rounds of cell sorting, transduced CEM cell populations with high binding capacity for BlyS were identified. To determine the cDNAs conferring binding to the transduced CEM cells, the integrated proviral DNAs were amplified by polymerase chain reaction and analyzed by DNA sequencing. Rescued cDNAs contained Transmembrane Activator and calcium‐modulator and cyclophilin ligand (CAML) Interactor (TACI) and B‐Cell Maturation factor (BCMA) sequences, representing two published receptors of BlyS.

Conclusions

Our data demonstrated that flow cytometry and high‐speed cell sorting combined with transduction of retroviral cDNA libraries and binding of epitope‐tagged orphan ligands as a selectable phenotype can be used efficiently for expression cloning of receptors. Of particular interest was our finding that apparently it is not necessary to purify the ligand but that conditioned medium containing the ligand can be used instead. Thus we concluded that our approach shortens the time to identify receptors for many orphan ligands and helps to exploit these receptors as drug targets. Cytometry Part A 51A:59–67, 2003. © 2003 Wiley‐Liss, Inc.
Automated flow cytometry for acquisition of time‐dependent population data
Authors: Abu‐Absi Nicholas R. , Zamamiri Abdelqader , Kacmar James , Balogh Steven J. , Srienc Friedrich
Published: January 2003

Background

The implementation of flow cytometry in many experimental settings can be limited by the extensive amount of sample handling and preparation required for analysis. We describe a system that automatically performs sample handling and flow cytometric analysis, thus allowing one to construct detailed pictures of changes in cell population distributions as a function of time.

Methods

Cell samples from bioreactors were loaded into a microchamber designed to perform all sample preparation steps including washing, fixation, staining, and dilution. The sample was then transported into a sample loop of known volume from which it was injected into the flow cell for determination of cell counts and measurement of scattering and fluorescence parameters. The apparatus was fully automated and controlled with a personal computer equipped with a data acquisition card. An inexpensive mechanism that continuously replenished the sheath fluid was implemented to ensure continuous and uninterrupted operation of the flow cytometer for several days. The device was interfaced with a FACSCalibur equipped with CellQuest software for data acquisition and analysis.

Results

The set‐up was tested with batch cultures of Saccharomyces cerevisiae expressing the green fluorescent protein (GFP). On‐line cell counts showed close agreement with off‐line measurements throughout the exponential growth of a yeast culture. The time course of light scattering, GFP fluorescence, and viability distributions provided a detailed description of changes occurring in growing cell cultures based on sampling approximately every 15 min for more than 40 consecutive hours. Therefore, the device could be used to obtain descriptions of the dynamic behavior of cell populations with no user intervention required for several days.

Conclusions

The system significantly expanded the utility of flow cytometry by eliminating cumbersome and time‐consuming steps that make the application of flow cytometry impractical in certain situations. It is anticipated that the described set‐up will find utility in biotechnology applications such as monitoring of cell cultures, screening of biologically active compounds, and in functional genomics efforts for phenotypic characterizations of cells. Cytometry Part A 51A:87–96, 2003. © 2003 Wiley‐Liss, Inc.
Analysis of the distribution of MRI contrast agents in the livers of small animals by means of complementary microscopies
Authors: Kahn E. , Tessier C. , Lizard G. , Petiet A. , Bernengo J.‐C. , Coulaud D. , Fourré C. , Frouin F. , Clément O. , Jourdain J.‐R. , Delain E. , Guiraud‐Vitaux F. , Colas‐Linhart N. , Siauve N. , Cuenod C.‐A. , Frija G. , Todd‐Pokropek A.
Published: January 2003

Background

Magnetic resonance imaging (MRI) contrast agents contain magnetic molecules such as iron (Fe) or gadolinium (Gd) that are injected in vivo into rats or mice to study their distribution inside the liver. Fluorescent europium (Eu) can be used as a model of Gd to obtain comparable information of this distribution of corresponding contrast agents. In a similar approach, Fe can be attached to Texas Red and used as a model of ferumoxides and be detected by fluorescence.

Methods

To combine and compare the advantages of different microscopic imaging modes, characterization studies were carried out by means of a confocal laser scanning microscope (CLSM), a secondary ion mass spectrometric (SIMS) microscope, and an electron energy loss spectrometric (EELS) microscope. In the case of CLSM, the locations of fluorescent signals inside preparations were determined by factor analysis of biomedical image sequences (FAMIS) and selection of image sequences at emission.

Results

By CLSM and FAMIS, we distinguished chelated Eu and Texas Red attached to Fe. By SIMS microscopy, we distinguished Eu and Gd of chlorides and chelates and Fe of a ferumoxide. By EELS microscopy, we distinguished Eu and Gd of chlorides.

Conclusions

Analysis of compounds inside correlative specimens by means of CLSM, SIMS, and EELS microscopes provided complementary results. Cytometry Part A 51A:97–106, 2003. © 2003 Wiley‐Liss, Inc.
Detection of β‐lactamase reporter gene expression by flow cytometry
Authors: Knapp Tom , Hare Eric , Feng Luxin , Zlokarnik Gregor , Negulescu Paul
Published: January 2003

Background:

Flow cytometry of gene expression in living cells requires accurate, sensitive, nontoxic fluorescent indicators capable of detecting transcription of specific genes. This is typically achieved by using genes that encode fluorescent proteins or enzymes coupled to promoters of interest. The most commonly used reporters are green fluorescent protein and β‐galactosidase (lacZ). In this study, we characterized the performance of a cell‐permeant, ratiometric, β‐lactamase substrate, coumarin cephalosporin fluorescein (CCF2/AM). We compared its characteristics with that of the β‐galactosidase/fluorescein di‐β‐D‐galactopyranoside reporter system.

Methods:

Jurkat cell lines were generated for β‐lactamase and β‐galactosidase reporters with the use of similar plasmid constructs. Rare event flow cytometric detection for the β‐galactosidase and β‐lactamase reporters were assayed by using mixed populations of negative (WT) and positive (constitutively expressing) cells for each reporter. To determine sensitivity at low reporter copy number, we measured the activity of an unstimulated inducible promoter and detected positive events as a function of substrate incubation time. Technical issues related to data processing and optical configuration are also presented.

Results:

The low population coefficients of variation afforded by ratiometric detection of the β‐lactamase system improved the statistical performance of the assay in comparison with a single‐dye, intensity‐based assay, leading to markedly improved detection for low copy number and rare events. At low levels of gene expression, β‐lactamase was detected with approximately 10‐fold higher confidence than was β‐galactosidase. In rare event detection experiments, cells expressing high levels of β‐lactamase proteins were reliably detected at frequencies of 1:106 compared with about 1:104 for β‐galactosidase.

Conclusion:

The ratiometric fluorescence readout of the β‐lactamase system based on fluorescence resonance energy transfer allowed more sensitive and accurate detection of gene expression than the currently available β‐galactosidase substrates. Further, the cell‐permeant nature of the substrate improved experimental convenience. These properties facilitated cell engineering and enabled a variety of applications including selection of rare cells from large populations and measurement of low‐expressing or downregulated genes. Cytometry Part A 51A:68–78, 2003. © 2003 Wiley‐Liss, Inc.
Involvement of serotonin mechanisms in the antinociceptive effect of S(+)‐ketoprofen
Authors: Díaz‐Reval Ma. Irene , Ventura‐Martínez Rosa , Déciga‐Campos Myrna , Terrón José A. , Cabré Francesc , López‐Muñoz Francisco J.
Published: January 2003

Serotonin (5‐hydroxytryptamine; 5‐HT) plays a role in the modulation and processing of pain and evidence has been provided that some nonsteroidal anti‐inflammatory drugs (NSAIDs) act, at least in part, through this system. The present study was designed to investigate the possible participation of 5‐HT1, 5‐HT2, 5‐HT3, and 5‐HT7 receptor subtypes in the antinociceptive effect of S(+)‐ketoprofen (S‐KP) at spinal and supraspinal level using the “pain induced functional impairment model in rat” (PIFIR model). S‐KP was administered orally (p.o.) and antagonist drugs for 5‐HT receptors (5‐HT1/5‐HT2, and 5‐HT3) were administered intrathecally (i.t.) or intracerebroventricularly (i.c.v.) 15 min before S‐KP. S‐KP (3.4 mg/kg p.o.) produced a significant antinociceptive effect in this model. Pre‐treatment with the 5‐HT1/5‐HT2/5‐HT7 receptor antagonist, methiothepin (1.5 μg, i.c.v.), significantly reversed the antinociceptive effect of S‐KP. In contrast, no significant differences were observed following i.t. administration of methiothepin. Pre‐treatment i.t., but not i.c.v., with the 5‐HT3/5‐HT4 receptor antagonist, tropisetron (0.9 μg), on the other hand, significantly reversed S‐KP‐induced antinociception. These results indicate that serotonin mechanisms are involved in the antinociceptive effect of S‐KP. 5‐HT1/5‐HT2/5‐HT7 receptors participate at the supraspinal level and 5‐HT3/5‐HT4 receptors participate at spinal level. Drug Dev. Res. 57:187–192, 2002. © 2003 Wiley‐Liss, Inc.
Regeneration or scarring: An immunologic perspective
Authors: Harty Mark , Neff Anton W. , King Michael W. , Mescher Anthony L.
Published: January 2003

Complete regeneration of complex tissues and organs is usually precluded by fibrotic reactions that lead to scarring. Fish, salamanders, and larval anurans are among the few vertebrates capable of regenerating lost appendages, and this process seems to recapitulate ontogenic development of the structure in most respects. Recent work has revealed a capacity for excellent regeneration in certain mammalian tissues: embryonic or fetal skin and the ear of the MRL mouse. Analyses of these two systems suggest that processes of regenerative growth and patterning for the formation of new structures such as hair follicles may involve modulation of the inflammatory response to the injury in a way that reduces fibrosis and formation of scar tissue. We review evidence that this modulation includes changes in cytokine signaling and may involve properties of the extracellular matrix mediated by factors that include hyaluronic acid and “anti‐adhesive substrates” such as tenascin‐C. New studies and classic work on the capacity for limb regeneration in amphibians are then reviewed, focusing on the loss of this ability in prometamorphic anuran hindlimbs and the view that changing properties of the immune system may also underlie the declining regenerative potential in this system. Finally, we review recent work in comparative and developmental immunology, which raises the possibility that phylogenetic changes in regenerative capacity may be the result of evolutionary changes in cellular activities of the immune system. Developmental Dynamics 226:268–279, 2003.© 2003 Wiley‐Liss, Inc.
Dyslexia and music: measuring musical timing skills
Authors: Overy Katie , Nicolson Roderick I. , Fawcett Angela J. , Clarke Eric F.
Published: January 2003

Over the last few decades, a growing amount of research has suggested that dyslexics have particular difficulties with skills involving accurate or rapid timing, including musical timing skills. It has been hypothesised that music training may be able to remediate such timing difficulties, and have a positive effect on fundamental perceptual skills that are important in the development of language and literacy skills (Overy, 2000). In order to explore this hypothesis further, the nature and extent of dyslexics' musical difficulties need to be examined in more detail. In the present study, a collection of musical aptitude tests (MATs) were designed specifically for dyslexic children, in order to distinguish between a variety of musical skills and sub‐skills. 15 dyslexic children (age 7–11, mean age 9.0) and 11 control children (age 7–10, mean age 8.9) were tested on the MATs, and their scores were compared. Results showed that the dyslexic group scored higher than the control group on 3 tests of pitch skills (possibly attributable to slightly greater musical experience), but lower than the control group on 7 out of 9 tests of timing skills. Particular difficulties were noted on one of the tests involving rapid temporal processing, in which a subgroup of 5 of the dyslexic children (33%) (mean age 8.4) was found to account for all the significant error. Also, an interesting correlation was found between spelling ability and the skill of tapping out the rhythm of a song, which both involve the skill of syllable segmentation. These results support suggestions that timing is a difficulty area for dyslexic children, and suggest that rhythm skills and rapid skills may need particular attention in any form of musical training with dyslexics. Copyright © 2003 John Wiley & Sons, Ltd.
The Chemistry of Borylene Complexes
Authors: Braunschweig Holger , Colling Miriam
Published: January 2003

Over the past seven years, complexes of various transition metals with bridged or terminal borylene ligands BR have for the first time been reported and fully characterised. Being closely related to important organometallic compounds, such as carbene, vinylidene, and carbonyl complexes, they have already attracted considerable interest both from experimental and computational points of view. Those studies have provided some detailed information about the nature of the metal−boron bond and about the nature of metal and borylene moieties required for the formation of stable complexes. Various methods for their preparation have been developed and the first studies on their reactivity have demonstrated an interesting chemistry. (© Wiley‐VCH Verlag GmbH, 69451 Weinheim, Germany, 2003)
3,6‐Di(2‐pyridyl)‐1,4,2,5‐dioxadiazine and a Silver Coordination Polymer with an Unprecedented Metallosupramolecular Topology
Authors: Richardson Chris , Steel Peter J.
Published: January 2003

The new ligand 3,6‐di(2‐pyridyl)‐1,4,2,5‐dioxadiazine (6) has been prepared and reacted with silver tetrafluoroborate to form a novel coordination polymer in which adjacent chains are interdigitated and π‐stacked. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Direct Synthesis of Enantiopure Tripod Ligands from C2‐Symmetric Precursors
Authors: Hegelmann Ina , Burzlaff Nicolai
Published: January 2003

A new enantiopure chiral tripod ligand is obtained from C2‐symmetric bis(camphorpyrazol‐1‐yl)methane by introducing a carboxylate group at the bridging carbon atom. A prochiral centre is formed rather than an additional stereocentre. Therefore, a homochiral tripod ligand is achieved without separation of the stereoisomers. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
A New Porous 3‐D Framework Constructed From Fivefold Parallel Interpenetration of 2‐D (6,3) Nets: A Mixed‐Valence Copper(I,II) Coordination Polymer [CuI2CuII(4,4′‐bpy)2(pydc)2]·4H2O
Authors: Zhang Xian‐Ming , Chen Xiao‐Ming
Published: January 2003

A mixed‐valence copper(I,II) coordination polymer [CuI2CuII(4,4′‐bpy)2(pydc)2]·4H2O (4,4′‐bpy = 4,4′‐bipyridine and pydc = pyridine‐2,4‐dicarboxylate) featuring the “metal‐complex ligands” [CuII(pydc)2]2− was designed and synthesized hydrothermally from Cu(MeCO2)2, pyridine‐2,4‐dicarboxylate, 4,4′‐bipyridine and NaOH. This compound was shown by single‐crystal structure analysis to be the first porous 3‐D framework constructed from fivefold parallel interpenetration of 2‐D (6,3) nets. The one‐dimensional channels occupying 17.4% of the crystal volume are occupied by guest water molecules and the porous network is stable after removal of the lattice water molecules, as indicated by TGA and PXRD measurements. The high stability of the 3‐D framework may be attributed to the multiple entanglement of the 2‐D nets. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Synthesis, Structure and Reactivity of Sulfur‐Rich [Fe(L1)(L2)(‘tpS4’)] Complexes with Rigid [Fe(‘tpS4’] Cores and σ−π Co‐Ligands (‘tpS4’2− = 1,2‐Bis(2‐mercaptophenylthio)phenylene(2−); L1, L2 = CO, PR3, NO)
Authors: Sellmann Dieter , Blum David C. F. , Heinemann Frank W. , Sutter Jörg
Published: January 2003

Optimisation of synthesis and purification steps has made the new ligand ‘tpS4’2− available in preparative amounts. Treatment of FeCl2·4H2O with ‘tpS4’‐Li2 or ‘tpS4’‐Na2 yielded [Fe(‘tpS4’)] (1), which in the solid state and in the absence of co‐ligands probably trimerises to give paramagnetic [Fe(‘tpS4’)]3. The [Fe(‘tpS4’)] fragment displays helical coordination of the ‘tpS4’2− ligand and binds σ−π ligands such as CO, NO and phosphanes to give diamagnetic 18‐valence‐electron complexes of general formula [Fe(L1)(L2)(‘tpS4’)]. The cis‐dicarbonyl complex [Fe(CO)2(‘tpS4’)] (2) is labile and readily dissociates CO to give dinuclear [Fe(CO)(‘tpS4’)]2 (3). Simultaneous coordination of CO and phosphanes yielded [Fe(CO)(PR3)(‘tpS4’)] with R = Me (4), Et (5), Pr (6) and Bu (7), but it proved impossible to obtain analogous complexes with bulky PPh3 or PCy3 ligands. Bis(phosphane) complexes could be obtained only with PMe3 and the bidentate dppe, which yielded [Fe(PMe3)2(‘tpS4’)] (8) and [Fe(dppe)(‘tpS4’)] (11). Alkylphosphanes with longer alkyl substituents, such as PnPr3 and PnBu3, gave the labile derivatives [Fe(PR3)2(‘tpS4’)] with R = nPr (9) and nBu (10). Complexes 9 and 10 reversibly dissociate one PR3 ligand, yielding diamagnetic [Fe(PR3)(‘tpS4’)] fragments. These findings explain the decisive and hitherto inexplicable influence of PPr3 and PBu3 ligands upon the stabilisation of N2H2 in the diazene complexes [μ‐N2H2{Fe(PR3)(‘tpS4’)}2] with R = Pr, Bu. Treatment of [Fe(‘tpS4’)] (1) or [Fe(CO)(‘tpS4’)]2 (3) with NO or NO+ yielded [Fe(NO)2(‘tpS4’)] (12). The molecular structures of 3, 4, 7 and 8 were determined by X‐ray structure analysis. Spectroscopic and structural results indicate that the differences in reactivity between [Fe(L1)(L2)(‘tpS4’)] and homologous [Fe(L1)(L2)(‘S4’)] complexes can be traced back to subtle electronic and structural effects. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Chloro‐ and Alkyltitanium Complexes of a New Dianionic Ancillary Ligand: A Linked Amidinate−Amide
Authors: Meerendonk Wouter J. van , Schröder Kai , Brussee Edward A. C. , Meetsma Auke , Hessen Bart , Teuben Jan H.
Published: January 2003

The lithium amidinate [Me3SiNC(Ph)N(CH2)3N(Me)SiMe3]Li(THF) (2), with a pendant methyl(trimethylsilyl)amine functionality, was prepared and found to react with [TiCl4(THF)2] to give the titanium amidinate−amide dichloride complex [η2,η1‐Me3SiNC(Ph)N(CH2)3NMe]TiCl2 (3) by elimination of LiCl and Me3SiCl. The elimination of Me3SiCl from the cyclopentadienyl derivative Cp[η2‐Me3SiNC(Ph)N(CH2)3N(SiMe3)Me]TiCl2 (5) to give Cp[η2,η1‐Me3SiNC(Ph)N(CH2)3NMe]TiCl (4) is much less favorable, and was found to be readily reversible. Dialkyltitanium complexes [η2,η1‐Me3SiNC(Ph)N(CH2)3NMe]Ti(CH2R)2 [R = Ph (6), SiMe3 (7)] were prepared, but could not be activated for catalytic ethene polymerization. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Synthesis and Structure of the First Holmium and Erbium Diiodide Complexes of Composition LLnI2 (Ln = Ho, Er)
Authors: Nikiforov Grigori B. , Roesky Herbert W. , Labahn Thomas , Vidovic Denis , Neculai Dante
Published: January 2003

Two new diiodide complexes of stoichiometry LLnI2 [Ln = Ho (1), Er (2)] with the β‐diketiminato ligand 2‐{[2‐(diethylamino)ethyl]amino}‐4‐{[2‐(diethylamino)ethyl]imino}pent‐2‐ene have been synthesized by the reaction of the lithium salt of the ligand with the corresponding triiodides. The complexes are thermally stable and soluble in common organic solvents. The X‐ray structural and elemental analysis show that LLnI2 is neutral, and as well free of solvent and lithium salt. The complexes adopt a pseudo‐octahedral geometry with the two iodine atoms arranged in trans position to each other. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Synthesis and Structure of the First Non‐Metallocene TiIII Fluoride Complex LTiF2·2Me3SnCl Supported by a β‐Diketiminato Ligand
Authors: Nikiforov Grigori B. , Roesky Herbert W. , Magull Jörg , Noltemeyer Mathias , Schmidt Hans‐Georg , Ilyin Evgeny G. , Kokunov Yuri B. , Demsar Alojz
Published: January 2003

The TiIII dichloride complex LTiCl2 (1) and the first non‐metallocene TiIII fluoride complex LTiF2·2Me3SnCl (2) supported by the β‐diketiminato ligand 2‐{[2‐(diethylamino)ethyl]amino}‐4‐{[2‐(diethylamino)ethyl]imino}pent‐2‐ene have been synthesized. Elemental analysis, mass spectrometry and X‐ray structural analysis show that 1 is monomeric, neutral, and free of solvent and lithium salt. The complex adopts a pseudo‐octahedral geometry with the two chlorine atoms arranged in trans position to each other. Compound 1 is soluble in common organic solvents and thermally surprisingly robust. Complex 2 was prepared using Me3SnF as a fluorinating agent. X‐ray structural analysis revealed that complex 2 consists of the LTiF2 unit and two molecules of Me3SnCl coordinated through a fluorine bridge to the titanium center. The fluorine atoms in 2 are located in trans positions to each other and the geometry around the titanium atom is distorted octahedral. Elemental analysis and mass spectrometry proved that 2 releases the coordinated Me3SnCl under vacuum or during sublimation. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Synthesis of a Hexadentate Hexameric Aluminum Imide and Its Metathesis Reactions
Authors: Reddy N. Dastagiri , Kumar S. Shravan , Roesky Herbert W. , Vidovic Denis , Magull Jörg , Noltemeyer Mathias , Schmidt Hans‐Georg
Published: January 2003

The reaction of AlH3·NMe3 with one equivalent of 2‐cyanothiophene in toluene afforded [HAlNCH2(C4H3S)]6 (2) in good yield. Treatment of 2 with SiMe3Br and SiMe3Cl in toluene under refluxing conditions resulted in the formation of compounds [BrAlNCH2(C4H3S)]6 (3) and [ClAlNCH2(C4H3S)]6 (4), respectively. In a similar way [PhC≡CAlNCH2(C4H3S)]6 (5) was readily obtained from the reaction between 2 and PhC≡CH. When 2 was treated with PhSH the Al‐N cluster core dissociated and a four‐membered ring compound [(PhS)2AlNHCH2(C4H3S)]2 (6) was formed. In contrast, a similar hexameric aluminum imide (HAlNCH2Ph)6 (1) retains its Al‐N network when treated with PhSH to yield (PhSAlNCH2Ph)6 (7). An exchange of ethyl groups and hydrides occurred when 2 was treated with excess of ZnEt2, forming [EtAlNCH2(C4H3S)]6 8. Compounds 2−4 and 6−8 were characterized by X‐ray single‐crystal analysis. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
4‐Ethynylpyridine as a Bridging Moiety in Mixed Rh/Re Complexes
Authors: Zuo Jing‐Lin , Biani Fabrizia Fabrizi de , Santos Ana M. , Köhler Klaus , Kühn Fritz E.
Published: January 2003

A paramagnetic, dimeric RhIIIRhII complex of the formula [Rh2(form)4(C≡CC5H4N)] (1) [form = N,N′‐di‐p‐tolylformamidinate; C5H4N = pyridyl (py)] has been synthesized and used as starting material to prepare heterotrimetallic complexes of composition [Rh2(form)4(C≡Cpy){Re(CO)3(bipy)}][OS(=O)2CF3] (2) and [Rh2(form)4(C≡Cpy){Re(CO)3(tbu2bipy)}][OS(=O)2CF3] (3). These complexes have been examined by vibrational spectroscopy, EPR spectroscopy, thermogravimetry and cyclic voltammetry. They are heat‐stable and can be handled in air without problems. The observed interactions between the Rh2 and the Re subunits, however, are weak, and a significant charge transfer does not seem to take place. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)