bluuebunny/crossref_metadata_2025 huggingface dataset:
Discrete expression of TRPV2 within the hypothalamo‐neurohypophysial system: Implications for regulatory activity within the hypothalamic‐pituitary‐adrenal axis
Authors: Wainwright Anna , Rutter A. Richard , Seabrook Guy R. , Reilly Kathryn , Oliver Kevin R.
Published: May 2004

Transient receptor potential channel proteins (TRPs) constitute a steadily growing family of ion channels with a range of purported functions. It has been demonstrated that TRPV2 is activated by moderate thermal stimuli and, in the rat, is expressed in medium to large diameter dorsal root ganglion neurons. In this study, antisera specific for the human TRPV2 homologue were raised and characterized for immunohistochemical use. Subsequently, thorough investigation was made of the localization of this cation channel in the macaque primate brain. TRPV2‐immunoreactive material was highly restrictively localized to hypothalamic paraventricular, suprachiasmatic, and supraoptic nuclei. Confocal double‐ and triple‐labeling studies demonstrated that TRPV2 immunoreactivity is preferentially localized to oxytocinergic and vasopressinergic neurons. Few, if any, cells in these regions expressed TRPV2 immunoreactivity in the absence of oxytocin immunoreactivity or vasopressin immunoreactivity. Expression in the paraventricular and supraoptic nuclei suggests that TRPV2 is likely to play a fundamental role in mediating cation transport in neurohypophysial neurons. TRPV2 has been shown to be translocated upon cell activation and neurons expressing TRPV2 immunoreactivity in vivo are among those known to engage in sporadic, intense activity. Taken together, these data suggest that this channel may play a vital role in mediating physiological activities associated with oxytocin and vasopressin release such as parturition, lactation, and diuresis. These data may also implicate the involvement of TRPV2 in disorders of the hypothalamic‐pituitary‐adrenal axis, including anxiety, depression, hypertension, and preterm labor. J. Comp. Neurol. 474:24–42, 2004. © 2004 Wiley‐Liss, Inc.
Type 1 nitrergic (ND1) cells of the rabbit retina: Comparison with other axon‐bearing amacrine cells
Authors: Vaney David I.
Published: May 2004

NADPH diaphorase (NADPHd) histochemistry labels two types of nitrergic amacrine cells in the rabbit retina. Both the large ND1 cells and the small ND2 cells stratify in the middle of the inner plexiform layer, and their overlapping processes produce a dense plexus, which makes it difficult to trace the morphology of single cells. The complete morphology of the ND1 amacrine cells has been revealed by injecting Neurobiotin into large round somata in the inner nuclear layer, which resulted in the labelling of amacrine cells whose proximal morphology and stratification matched those of the ND1 cells stained by NADPHd histochemistry. The Neurobiotin‐injected ND1 cells showed strong homologous tracer coupling to surrounding ND1 cells, and double‐labelling experiments confirmed that these coupled cells showed NADPHd reactivity. The ND1 amacrine cells branch in stratum 3 of the inner plexiform layer, where they produce a sparsely branched dendritic tree of 400–600 μm diameter in ventral peripheral retina. In addition, each cell gives rise to several fine beaded processes, which arise either from a side branch of the dendritic tree or from the tapering of a distal dendrite. These axon‐like processes branch successively within the vicinity of the dendritic field before extending, with little or no further branching, for 3–5 mm from the soma in ventral peripheral retina. Consequently, these cells may span one‐third of the visual field of each eye, and their spatial extent appears to be greater than that of most other types of axon‐bearing amacrine cells injected with Neurobiotin in this study. The morphology and tracer‐coupling pattern of the ND1 cells are compared with those of confirmed type 1 catecholaminergic cells, a presumptive type 2 catecholaminergic cell, the type 1 polyaxonal cells, the long‐range amacrine cells, a novel type of axon‐bearing cell that also branches in stratum 3, and a type of displaced amacrine cell that may correspond to the type 2 polyaxonal cell. J. Comp. Neurol. 474:149–171, 2004. © 2004 Wiley‐Liss, Inc.
Cholinergic elements in the zebrafish central nervous system: Histochemical and immunohistochemical analysis
Authors: Clemente Diego , Porteros Ángel , Weruaga Eduardo , Alonso José Ramón , Arenzana Francisco Javier , Aijón José , Arévalo Rosario
Published: May 2004

Recently, the zebrafish has been extensively used for studying the development of the central nervous system (CNS). However, the zebrafish CNS has been poorly analyzed in the adult. The cholinergic/cholinoceptive system of the zebrafish CNS was analyzed by using choline acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) histochemistry in the brain, retina, and spinal cord. AChE labeling was more abundant and more widely distributed than ChAT immunoreactivity. In the telencephalon, ChAT‐immunoreactive (ChAT‐ir) cells were absent, whereas AChE‐positive neurons were observed in both the olfactory bulb and the telencephalic hemispheres. The diencephalon was the region with the lowest density of AChE‐positive cells, mainly located in the pretectum, whereas ChAT‐ir cells were exclusively located in the preoptic region. ChAT‐ir cells were restricted to the periventricular stratum of the optic tectum, but AChE‐positive neurons were observed throughout the whole extension of the lamination except in the marginal stratum. Although ChAT immunoreactivity was restricted to the rostral tegmental, oculomotor, and trochlear nuclei within the mesencephalic tegmentum, a widespread distribution of AChE reactivity was observed in this region. The isthmic region showed abundant AChE‐positive and ChAT‐ir cells in the isthmic, secondary gustatory and superior reticular nucleus and in the nucleus lateralis valvulae. ChAT immunoreactivity was absent in the cerebellum, although AChE staining was observed in Purkinje and granule cells. The medulla oblongata showed a widespread distribution of AChE‐positive cells in all main subdivisions, including the octavolateral area, reticular formation, and motor nuclei of the cranial nerves. ChAT‐ir elements in this area were restricted to the descending octaval nucleus, the octaval efferent nucleus and the motor nuclei of the cranial nerves. Additionally, spinal cord motoneurons appeared positive to both markers. Substantial differences in the ChAT and AChE distribution between zebrafish and other fish species were observed, which could be important because zebrafish is widely used as a genetic or developmental animal model. J. Comp. Neurol. 474:75–107, 2004. © 2004 Wiley‐Liss, Inc.
Postsynaptic localization of γ‐aminobutyric acid transporters and receptors in the outer plexiform layer of the goldfish retina: An ultrastructural study
Authors: Klooster Jan , Nunes Cardozo Bob , Yazulla Stephen , Kamermans Maarten
Published: May 2004

The γ‐aminobutyric acid (GABA)‐ergic system in the outer plexiform layer (OPL) of the goldfish retina was studied via light and electron immunohistochemistry. The subcellular distributions of immunoreactivity (‐IR) of plasma membrane GABA transporters GAT2 and GAT3, the α1 and α3 subunits of the ionotropic GABAA receptor, and the ρ1 subunit of the ionotropic GABAC receptor were determined. The localization of the GAT2‐IR and GAT3‐IR to horizontal cell dendrites at the base of the cone synaptic complex was the main characteristic at the ultrastructural level. Very rarely, GAT2‐IR and GAT3‐IR were found in horizontal cell dendrites innervating rod spherules. α1‐IR and α3‐IR were seen in wide bands in the OPL, whereas ρ1‐IR appeared as a narrow band in the OPL. Most α1‐IR was intracellular in rod and cone terminals. Membrane‐associated α1‐IR was observed in cone pedicles but not in rod spherules; postsynaptic elements were also labeled. α3‐IR was concentrated in the lateral elements of horizontal cell dendrites in cone pedicles. In contrast, ρ1‐IR was found mainly on the spinules of the horizontal cell dendrites in cone pedicles. In addition, in another type of cone pedicle, ρ1‐IR was found at the position of OFF‐bipolar cell dendrites. α3‐IR and ρ1‐IR were rarely found in horizontal cell dendrites innervating rods. We suggest that two GABAergic pathways exist in the outer retina— first, a GABAergic positive loop with GABA receptors mainly on the horizontal cell dendrites and spinules and, second, a GABAergic feedback pathway involving GABA receptors on cone pedicles and GABA transporters on horizontal cells and that this pathway presumably modulates feedback strength from horizontal cells to cones. J. Comp. Neurol. 474:58–74, 2004. © 2004 Wiley‐Liss, Inc.
Caspase‐3 and caspase‐9 mediate developmental apoptosis in the mouse olfactory system
Authors: Cowan Catherine M. , Roskams A. Jane
Published: May 2004

Neuronal apoptosis is a key component in the sculpting of tissues during embryonic and postnatal development and is driven largely by the action of caspases. In the mouse olfactory system, caspase‐3 and ‐9 are expressed in olfactory receptor neurons (ORNs) of adult mice, and their selective retrograde activation drives ORN apoptosis following ablation of their target, the olfactory bulb (OB). Here, we show that both of these caspases are expressed at the earliest stages of ORN embryonic development, and their expression is concentrated in outgrowing ORN axons. The retention, in null mice for both caspases, of a population of ORNs that would normally undergo developmental apoptosis beginning at E13 of development, results in a permanently expanded population of ORNs. In turn, in some caspase‐3 null mice, the ORN target organ, the OB, also develops abnormally, resulting in the formation of secondary, apparently functional, extracranial ectopic OBs. J. Comp. Neurol. 474:136–148, 2004. © 2004 Wiley‐Liss, Inc.
Induction of enhanced green fluorescent protein expression in response to lesions in the nervous system
Authors: Dubois‐Dauphin Michel , Eder‐Colli Lorenza , Vallet Philippe , Stutz André , Nef Serge , Vassalli Jean‐Dominique
Published: May 2004

We have generated a mouse strain carrying a transgene driven by a strong and ubiquitous promoter (human cytomegalovirus hCMV/β‐actin) and containing an enhanced green fluorescent protein (eGFP) coding sequence upstream of the 3′ untranslated region (3′UTR) of tissue‐type plasminogen activator (t‐PA) mRNA. The 3′UTR of t‐PA mRNA is known to be involved in the reversible deadenylation and translational repression of transcripts in mouse oocytes. hCMV/β‐actin‐eGFP‐3′UTR t‐PA transgenic mice express eGFP mRNA in all brain structures analyzed but lack eGFP fluorescence, with the exception of blood vessels, choroid plexus, and Purkinje cells. Taking advantage of these features, we tested whether certain pathological conditions, in particular injuries of the nervous system, might trigger eGFP fluorescence in traumatized cells or neurons. From this perspective, we analyzed eGFP mRNA expression and eGFP fluorescence in experimental models of nervous system lesions, such as motoneuron axotomy and cerebral stroke induced by middle cerebral artery occlusion. We found an increase in eGFP fluorescence in specific brain areas in cells suffering or reacting to these injuries. This increased fluorescence is correlated with an increased transcription of eGFP in lesioned cells, presumably enhanced by a release of the translational silencing mediated by the 3′UTR region of the t‐PA mRNA. This transgenic mouse model may prove useful to study the development of neurodegenerative lesions. J. Comp. Neurol. 474:108–122, 2004. © 2004 Wiley‐Liss, Inc.
Distribution of glutamatergic immunoreactive neurons in the terminal abdominal ganglion of the crayfish
Authors: Nagayama Toshiki , Kimura Ken‐Ichi , Araki Makoto , Aonuma Hitoshi , Newland Philip L.
Published: May 2004

Using an antiserum directed against glutamate, we have analyzed the distribution of glutamate‐like immunoreactive neurons in the terminal abdominal ganglion of the crayfish Procambarus clarkii. Approximately 160 central neurons (157 ± 8; mean ± SEM, n = 8) showed positive glutamate‐like immunoreactivity, which represents approximately 25% of the total number of neurons in the terminal ganglion. Using a combination of intracellular staining with the marker Lucifer yellow and immunocytochemical staining has shown that most excitatory motor neurons are glutamatergic and that glutamate acts as an excitatory transmitter at peripheral neuromuscular junctions. Seven of 10 identified spiking local interneurons and only 2 of 19 identified ascending interneurons, showed positive immunoreactivity. Our observation that inhibitory spiking interneurons were immunopositive, whereas excitatory ascending interneurons were immunonegative, indicates that glutamate is likely to act as an inhibitory neurotransmitter within the central nervous system. Local pressure injection of L‐glutamate into the neuropil of the ganglion caused a hyperpolarization of the membrane potentials of many interneurons. γ‐Aminobutyric acid (GABA)ergic posterolateral nonspiking interneurons and the bilateral nonspiking interneuron LDS showed no glutamate‐like immunoreactivity, whereas non‐GABAergic anterolateral III nonspiking interneurons showed glutamate‐like immunoreactivity. Thus, not only GABA but also glutamate are used in parallel as inhibitory neurotransmitters at central synapses. J. Comp. Neurol. 474:123–135, 2004. © 2004 Wiley‐Liss, Inc.
Nonlinear instability of periodic BGK waves for Vlasov‐Poisson system
Authors: Lin Zhiwu
Published: May 2004

We investigate the nonlinear instability of periodic Bernstein‐Greene‐Kruskal (BGK) waves. Starting from an exponentially growing mode to the linearized equation, we proved nonlinear instability in the L1‐norm of the electric field. © 2004 Wiley Periodicals, Inc.
Cytomics—New technologies: Towards a human cytome project
Authors: Valet G. , Leary J.F. , Tárnok A.
Published: May 2004

Background

Molecular cell systems research (cytomics) aims at the understanding of the molecular architecture and functionality of cell systems (cytomes) by single‐cell analysis in combination with exhaustive bioinformatic knowledge extraction. In this way, loss of information as a consequence of molecular averaging by cell or tissue homogenisation is avoided.

Progress

The cytomics concept has been significantly advanced by a multitude of current developments. Amongst them are confocal and laser scanning microscopy, multiphoton fluorescence excitation, spectral imaging, fluorescence resonance energy transfer (FRET), fast imaging in flow, optical stretching in flow, and miniaturised flow and image cytometry within laboratories on a chip or laser microdissection, as well as the use of bead arrays. In addition, biomolecular analysis techniques like tyramide signal amplification, single‐cell polymerase chain reaction (PCR), and the labelling of biomolecules by quantum dots, magnetic nanobeads, or aptamers open new horizons of sensitivity and molecular specificity at the single‐cell level. Data sieving or data mining of the vast amounts of collected multiparameter data for exhaustive multilevel bioinformatic knowledge extraction avoids the inadvertent loss of information from unknown molecular relations being inaccessible to an a priori hypothesis.

Challenge

It seems important to address the challenge of a human cytome project using hypothesis‐driven molecular information collection from disease associated cell systems, supplemented by systematic and exhaustive knowledge extraction. This will allow the description of the molecular setup of normal and abnormal cell systems within a relational knowledge system, permitting the standardised discrimination of abnormal cell states in disease. As one of the consequences, individualised predictions of further disease course in patients (predictive medicine by cytomics) by characteristic discriminatory data patterns will permit individualised therapies, identification of new pharmaceutical targets, and establishment of a standardised framework of relevant molecular alterations in disease. This special issue of Cytometry, on new technologies in cytomics, focuses on prominent examples of this presently fast‐moving scientific field, and represents one of the preconditions for the formulation of a human cytome project. © 2004 Wiley‐Liss, Inc.
Distinguishing modes of cell death using the ImageStream® multispectral imaging flow cytometer
Authors: George Thaddeus C. , Basiji David A. , Hall Brian E. , Lynch David H. , Ortyn William E. , Perry David J. , Seo Michael J. , Zimmerman Cathleen A. , Morrissey Philip J.
Published: May 2004

Background

Here we demonstrate the ability of the ImageStream® 100 Multispectral Imaging Cytometer to discriminate between live, necrotic, and early and late apoptotic cells, using unique combinations of photometric and morphometric features.

Methods

Live, necrotic, and early and late apoptotic cells were prepared and analyzed by immunofluorescence microscopy, conventional flow cytometry, and imaging flow cytometry, both as single populations and as a heterogeneous mixture of cells.

Results

Live (annexin V−, 7‐AAD−) and early apoptotic (annexin V+, 7‐AAD−) cells were readily identifiable using either conventional or ImageStream based flow cytometric methods. However, inspection of multispectral images of cells demonstrated that the annexin V+, 7‐AAD+ population contained both necrotic and late‐stage apoptotic cells. Although these cells could not be distinguished using conventional flow cytometric techniques, they were separable using unique combinations of photometric and morphometric measures available using ImageStream technologies.

Conclusions

Using multispectral imagery, morphologically distinct cell populations can be distinguished using features not available with conventional flow cytometers. In particular, the ability to couple morphometric with photometric measures makes it possible to distinguish live cells from cells in the early phases of apoptosis, as well as late apoptotic cells from necrotic cells. © 2004 Wiley‐Liss, Inc.
Single‐cell analysis of yeast, mammalian cells, and fungal spores with a microfluidic pressure‐driven chip‐based system
Authors: Palková Zdena , Váchová Libuse , Valer Marc , Preckel Tobias
Published: May 2004

Background

Cytomics aims at understanding the function of cellular systems by analysis of single cells. Recently, there has been a growing interest in single cell measurements being performed in microfluidic systems. These systems promise to integrate staining, measurement, and analysis in a single system. One important aspect is the limitation of allowable cell sizes due to microfluidic channel dimensions. Here we want to demonstrate the broad applicability of microfluidic chip technology for the analysis of many different cell types.

Methods

We have developed a microfluidic chip and measurement system that allows flow cytometric analysis of fluorescently stained cells from different organisms. In this setup, the cells are moved by pressure‐driven flow inside a network of microfluidic channels and are analyzed individually by fluorescence detection.

Results

We have successfully applied the system to develop a methodology to detect viable and dead cells in yeast cell populations. Also, we have measured short interfering RNA (siRNA) mediated silencing of protein expression in mammalian cells. In addition, we have characterized the infection state of Magnaportae grisea fungal spores.

Conclusions

Results obtained with the microfluidic system demonstrate a broad applicability of microfluidic flow cytometry to measurement of various cell types. © 2004 Wiley‐Liss, Inc.
Deformability‐based flow cytometry
Authors: Lincoln Bryan , Erickson Harold M. , Schinkinger Stefan , Wottawah Falk , Mitchell Daniel , Ulvick Sydney , Bilby Curt , Guck Jochen
Published: May 2004

Background

Elasticity of cells is determined by their cytoskeleton. Changes in cellular function are reflected in the amount of cytoskeletal proteins and their associated networks. Drastic examples are diseases such as cancer, in which the altered cytoskeleton is even diagnostic. This connection between cellular function and cytoskeletal mechanical properties suggests using the deformability of cells as a novel inherent cell marker.

Methods

The optical stretcher is a new laser tool capable of measuring cellular deformability. A unique feature of this deformation technique is its potential for high throughput, with the incorporation of a microfluidic delivery of cells.

Results

Rudimentary implementation of the microfluidic optical stretcher has been used to measure optical deformability of several normal and cancerous cell types. A drastic difference has been seen between the response of red blood cells and polymorphonuclear cells for a given optically induced stress. MCF‐10, MCF‐7, and modMCF‐7 cells were also measured, showing that while cancer cells stretched significantly more (five times) than normal cells, optical deformability could even be used to distinguish metastatic cancer cells from nonmetastatic cancer cells. This trimodal distribution was apparent after measuring a mere 83 cells, which shows optical deformability to be a highly regulated cell marker.

Conclusions

Preliminary work suggests a deformability‐based cell sorter similar to current fluorescence‐based flow cytometry without the need for specific labeling. This could be used for the diagnosis of all diseases, and the investigation of all cellular processes, that affect the cytoskeleton. © 2004 Wiley‐Liss, Inc.
Microgenomics: Identification of new expression profiles via small and single‐cell sample analyses
Authors: Taylor Theresa B. , Nambiar Prashant R. , Raja Rajiv , Cheung Evelyn , Rosenberg Daniel W. , Anderegg Birgit
Published: May 2004

Background

Since the sequencing of the human genome has been finished, microgenomics has been booming, employing highly sophisticated, high‐throughput platforms. But these mainly chip‐based methods can only generate biologically relevant data if the samples investigated consist of homogenous cell populations, in which no unwanted cells of different specificity and/or developmental stage obscure the results.

Methods

Different sampling methods have been routinely applied to overcome the problem presented by heterogeneous samples, e.g., global surveys, cell cultures, and microdissection. Various methods of laser‐assisted microdissection, employing either positive or negative selection of tissue areas or even single cells, are available.

Results

These laser‐assisted microdissection methods allow for fast and precise procurement of extremely small samples. Through subsequent application of recently developed methods of linear mRNA amplification in a pool of isolated total RNA, it has now become possible to perform complex high‐throughput RNA expression profiling by microdissecting and processing even single‐cell samples.

Conclusions

Studies using the tools and methods of microgenomics have shed light on how those new approaches will eventually aid in the development of a new generation of diagnostics, e.g., leading to new patient‐specific drugs tailored to the requirements assessed by assaying only a few biopsy cells. © 2004 Wiley‐Liss, Inc.
Microscopy‐based multicolor tissue cytometry at the single‐cell level
Authors: Ecker Rupert C. , Steiner Georg E.
Published: May 2004

Cytomics is a novel perspective from which to look at life. As with genomics and proteomics before, this discipline requires novel and innovative techniques and technologies to focus on its substrate of research—the cytome. With cytomics being the discipline that analyzes cellular systems and their interdependencies, advanced microscopy represents a key technology in cytomics research. Yet, conventional microscopy‐based investigations, i.e., “look and conclude” analyses, do not meet the major cytomics criteria of 1) relating multiple parameters to each other, 2) within large populations of cells, 3) on a single‐cell basis, and 4) in a quantitative and observer‐independent manner. However, emerging improvements in the fields of fluorophore technology, sensitive fluorescence detection devices, and sophisticated image analysis procedures, are important and necessary steps into the cytomics era. Tissue represents an important class of cytomes, hence tissue cytometry—on the single cell level—can be expected to become an important cytomics technology. In this report, the techniques and technologies of microscopy‐based multicolor tissue cytometry (MMTC) are outlined and applications are discussed, including the phenotypic characterization of tissue infiltrating leukocytes, in situ quantification of proliferation markers and tumor suppressors, and in situ quantification of apoptosis. © 2004 Wiley‐Liss, Inc.
Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis
Authors: Ecker Rupert C. , de Martin Rainer , Steiner Georg E. , Schmid Johannes A.
Published: May 2004

Background

Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence.

Methods

Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single‐pixel basis and a mathematical procedure called “linear unmixing” of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed.

Results

In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight‐color immunofluorescence protocol for tissue sections was successfully established. Moreover, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated.

Conclusions

This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET. © 2004 Wiley‐Liss, Inc.
Quantitative histology by multicolor slide‐based cytometry
Authors: Gerstner Andreas O. H. , Trumpfheller Christine , Racz Paul , Osmancik Pavel , Tenner‐Racz Klara , Tárnok Attila
Published: May 2004

Background

In lymphatic organs, the quantitative analysis of the spatial distribution of leukocytes by tissue cytometry would give relevant information about alterations during diseases (leukemia, HIV, AIDS) and their therapeutic regimen, as well as in experimental settings.

Methods

We have developed a semiautomated analysis method for laser scanning cytometry (LSC) termed “multiple thresholding,” which is suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for nuclear DNA and up to four antigens using direct or indirect immunofluorescence staining. Measurement is triggered on DNA‐fluorescence (argon laser, Ar) or on specific cell labeling. Due to the heterogeneity of cell density, measurements are performed repeatedly at different threshold levels (low threshold: regions of low cellular density, germinal center; high threshold: dense regions, mantle zone). Data are acquired by single‐ (Ar) or dual‐laser excitation (Ar‐HeNe) in order to analyze single‐ (FITC) up to four‐color (FITC/PE/PECy5/APC) stained specimen.

Results

Percentage and cellular density of cell‐subsets is quantified in different microanatomical regions of the specimen. These data were highly correlated with manual scoring of identical specimens (r2 = 0.96, P < 0.0001). With LSC, semiautomated operator‐independent immunophenotyping in tissue sections of lymphatic organs with up to three antibodies simultaneously is possible.

Conclusions

We expect this tissue cytometric approach to yield new insight into processes during diseases and help to quantify the success of therapeutic interventions. © 2004 Wiley‐Liss, Inc.
Getting the right cells to the array: Gene expression microarray analysis of cell mixtures and sorted cells
Authors: Szaniszlo Peter , Wang Nan , Sinha Mala , Reece Lisa M. , Van Hook James W. , Luxon Bruce A. , Leary James F.
Published: May 2004

Background

Most biological samples are cell mixtures. Some basic questions are still unanswered about analyzing these heterogeneous samples using gene expression microarray technology (MAT). How meaningful is a cell mixture's overall gene expression profile (GEP)? Is it necessary to purify the cells of interest before microarray analysis, and how much purity is needed? How much does the purification itself distort the GEP, and how well can the GEP of a small cell subset be recovered?

Methods

Model cell mixtures with different cell ratios were analyzed by both spotted and Affymetrix MAT. GEP distortion during cell purification and GEPs of purified cells were studied. CD34+ cord blood cells were purified and analyzed by MAT.

Results

GEPs for mixed cell populations were found to mirror the cell ratios in the mixture. Over 75% pure samples were indistinguishable from pure cells by their overall GEP. Cell purification preserved the GEP. The GEPs of small cell subsets could be accurately recovered by cell sorting both from model cell mixtures and from cord blood.

Conclusions

Purification of small cell subsets from a mixture prior to MAT is necessary for meaningful results. Even completely hidden GEPs of small cell subpopulations can be recovered by cell sorting. © 2004 Wiley‐Liss, Inc.
RNA and DNA aptamers in cytomics analysis
Authors: Ulrich Henning , Martins Antonio Henrique B. , Pesquero João Bosco
Published: May 2004

Background

The systematic evolution of ligands by exponential enrichment (SELEX) technique is a combinatorial library approach in which DNA or RNA molecules (aptamers) are selected by their ability to bind their protein targets with high affinity and specificity, comparable to that of monoclonal antibodies. In contrast to antibodies conventionally selected in animals, aptamers are generated by an in vitro selection process, and can be directed against almost every target, including antigens like toxins or nonimmunogenic targets, against which conventional antibodies cannot be raised.

Methods

Aptamers are ideal candidates for cytomics, as they can be attached to fluorescent reporters or nanoparticles in order to study biological function by fluorescence microscopy, by flow cytometry, or to quantify the concentration of their target in biological fluids or cells using ELISA, RIA, and Western blot assays.

Results

We demonstrate the in vitro selection of anti‐kinin B1 receptor aptamers that could be used to determine B1 receptor expression during inflammation processes. These aptamers specifically recognize their target in a Northern‐Western blot assay, and bind to their target protein whenever they are exposed in the membrane.

Conclusions

Currently, aptamers are linked to fluorescent reporters. We discuss here the present status and future directions concerning the use of the SELEX technique in cytomics. © 2004 Wiley‐Liss, Inc.
Data pattern analysis for the individualised pretherapeutic identification of high‐risk diffuse large B‐cell lymphoma (DLBCL) patients by cytomics
Authors: Valet Günter K. , Hoeffkes Heinz Gert
Published: May 2004

Background

Clinical outcome predictions in phase III studies are mostly derived for patient groups, but not for individual patients, although individualised predictions are an ultimate goal to permit a personalised fine tuning of therapy. This may permit earlier application of target therapies, minimise general damage to the organism, and result in improved complete remission rates in malignant diseases.

Methods

In this study, Lymphochip cDNA microarray gene expression results of DLBCL patients, from a published prospective meta‐analysis study on the prediction of group prognosis, were analysed for individualised predictions using a nonstatistical data pattern classification approach. The training set was comprised of the same 160 DLBCL patients as in the prognosis study, with the validation set of 80 patients remaining unknown to the learning process. This permits the assessment of prospective classifier performance towards unknown patients.

Results

Pretherapeutic predictions for the training and validation set patients were correct in 98.1% and 78.3% of the cases for nonsurvival and in 67.3% and 45.3% for survival. The discriminatory data pattern consisted of 14 known and 10 unknown gene products.

Conclusions

The better than 95% correct pretherapeutic prediction for about one‐half of the ultimately nonsurviving high‐risk patients of the training set is promising for clinical considerations about individualised therapy in such cases. Reliable individualised survival predictions are not possible with the information content of the present dataset. It seems necessary to investigate additional gene products, since survival may significantly depend on non–lymphocyte‐associated genes that escape to the lymphocyte‐oriented Lymphochip gene activation analysis. © 2004 Wiley‐Liss, Inc.
The CD200 and CD200 receptor cell surface proteins interact through their N‐terminal immunoglobulin‐like domains
Authors: Hatherley Deborah , Barclay A. Neil
Published: May 2004

CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a receptor on myeloid cells (CD200R) involved in regulation of macrophage function. Both CD200 and CD200R contain two Ig superfamily domains like many other leukocyte membrane proteins. Site‐directed mutagenesis of CD200R showed that, like CD200, it interacted through its N‐terminal domain. This indicated that the cell‐cell interaction spans four Ig superfamily domains and this distance is similar to many interactions found between T cells and antigen‐presenting cells. This suggests that this topology is also important in interactions of CD200 on a variety of cells with CD200R on myeloid cells, and comparable contact sites may be important mediating regulation in other cell‐cell interactions. The mutagenesis showed that the binding involved the predicted GFCC′ face of its N‐terminal domain, like that of CD200, suggesting that the interaction evolved from a homotypic interaction.
Activated T killer cells induce apoptosis in lung epithelial cells and the release of pro‐inflammatory cytokine TNF‐α
Authors: Shinbori Toshifumi , Walczak Henning , Krammer Peter H.
Published: May 2004

Apoptosis is thought to be involved in lung epithelial cell damage in acute respiratory distress syndrome and interstitial pneumonia. Both the role of apoptosis and its underlying molecular mechanisms in human lung tissue remain unclear. To address these issues, we developed an in vitro assay in which a human lung epithelial cell line and a staphylococcal enterotoxin B (SEB)‐reactive human CD8+ CTL line were co‐cultured in the presence of SEB. SEB‐stimulated CD8+ CTL induced apoptosis in the lung epithelial cell line primarily through the perforin/granzyme‐mediated pathway. In these cells, apoptosis was initially independent of death receptor pathways. We also tested the effect of IFN‐γ on modulation of apoptosis in lung epithelial cells. In IFN‐γ‐pretreated lung epithelial cells, CD95 (APO‐1/Fas) activation as well as TNF‐related apoptosis‐inducing ligand (TRAIL) receptor and TNFR activation led to apoptosis. Furthermore, we found that the interaction of SEB‐stimulated CD8+ CTL with lung epithelial cells induced an increase in TNF‐α secretion. These results suggest an important role for bacterial superantigen‐reactive CD8+ CTL in induction of lung epithelial cell apoptosis and in modulation of inflammatory processes in lung tissue.
Allylsilanes in Organic Synthesis − Recent Developments
Authors: Chabaud Laurent , James Philippe , Landais Yannick
Published: May 2004

Allylsilanes have been used extensively over the last 30 years. A survey of the most recent advances in this field is described, including transformations of allylsilanes through electrophilic, radical and organometallic processes. Particular emphasis will be placed on the stereocontrol arising from these processes. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Nested lumped‐parameter models for foundation vibrations
Authors: Wu Wen‐Hwa , Lee Wen‐How
Published: May 2004

The concept of polynomial‐fraction approximation is explored in this article to develop a nested type of systematic lumped‐parameter model for unbounded soil. Based on the optimal coefficients determined from the flexibility formulation, the reciprocal of the polynomial‐fraction is first taken to represent the dynamic stiffness function of foundation and then decomposed into a linear polynomial and another polynomial‐fraction. The nested division introduced in this study is operated to generate a nested form for this decomposed polynomial‐fraction, which directly corresponds to a nested discrete‐element model. The nested type of lumped‐parameter model is then easily constructed by connecting this nested discrete‐element model in series with another simple discrete‐element model corresponding to the linear polynomial. Copyright © 2004 John Wiley & Sons, Ltd.
A mathematical basis for the convergence of the capacity spectrum method
Authors: Lin Yu‐Yuan , Chuang Tsai‐Fu , Chang Kuo‐Chung
Published: May 2004

The capacity spectrum method is adopted by the ATC‐40 document for evaluating the inelastic deformation demands of reinforced concrete structures. Several studies have shown that the iterative procedure needed in the method may not give convergent outcomes in some cases. This paper focuses on the convergence of the capacity spectrum method in the constant velocity region of the response spectrum. The results obtained from the examples discussed in this study show that the convergent characteristics of this method depend on the elastic period, the hysteretic damping model, the yield displacement and the ductility ratio of the system analyzed. The capacity spectrum method can converge only for the case that the absolute value of the first derivative of the government equation derived from the demand and capacity diagrams of structures is smaller than 1.0. Copyright © 2004 John Wiley & Sons, Ltd.
An envelope for Mohr's circle in seismically excited three‐dimensional structures
Authors: Menun Charles
Published: May 2004

A previously developed response‐spectrum‐based procedure for computing the envelope that bounds the time‐varying realizations of Mohr's circle at any prescribed location within a two‐dimensional structure is extended for use with three‐dimensional structures subjected to as many as three translational components of ground acceleration. The proposed envelope, which is completely defined by quantities that are routinely used and calculated in conventional response spectrum analyses, is developed for the general case in which the principal directions of the earthquake, along which the ground accelerations are uncorrelated, are unknown. The accuracy of the proposed envelope is evaluated by comparing it to the results of an ensemble of time‐history analyses performed on a concrete arch dam using simulated accelerograms. It is found that the proposed envelope has a level of accuracy that is suitable for structural design and analysis. The largest observed difference between the simulated and predicted mean envelopes is less than 5%. Copyright © 2004 John Wiley & Sons, Ltd.
Approximate modal decomposition of inelastic dynamic responses of wall buildings
Authors: Sangarayakul C. , Warnitchai P.
Published: May 2004

Two approximate methods for decomposing complicated inelastic dynamic responses of wall buildings into simple modal responses are presented. Both methods are based on the equivalent linear concept, where a non‐linear structure is represented by a set of equivalent linear models. One linear model is used for representing only one vibration mode of the non‐linear structure, and its equivalent linear parameters are identified from the inelastic response time histories by using a numerical optimizer. Several theoretical relations essential for the modal decomposition are derived under the framework of complex modal analysis. Various numerical examinations have been carried out to check the validity of the proposed modal decomposition methods, and the results are quite satisfactory in all cases. Fluctuating bending moment and shear at any location along the wall height contributed by each individual vibration mode can be obtained. Modal contributions to shear and flexural strength demands, as well as the corresponding modal properties, under various seismic loading conditions can also be identified and examined in detail. Furthermore, the effects of higher vibration modes on seismic demands of wall buildings are investigated by using the modal decomposition methods. Several new insights into the complicated inelastic dynamics of multi‐story wall buildings are presented. Copyright © 2004 John Wiley & Sons, Ltd.
Near‐fault ground motions, and the response of elastic and inelastic single‐degree‐of‐freedom (SDOF) systems
Authors: Mavroeidis G. P. , Dong G. , Papageorgiou A. S.
Published: May 2004

In order to investigate the response of structures to near‐fault seismic excitations, the ground motion input should be properly characterized and parameterized in terms of simple, yet accurate and reliable, mathematical models whose input parameters have a clear physical interpretation and scale, to the extent possible, with earthquake magnitude. Such a mathematical model for the representation of the coherent (long‐period) ground motion components has been proposed by the authors in a previous study and is being exploited in this article for the investigation of the elastic and inelastic response of the single‐degree‐of‐freedom (SDOF) system to near‐fault seismic excitations. A parametric analysis of the dynamic response of the SDOF system as a function of the input parameters of the mathematical model is performed to gain insight regarding the near‐fault ground motion characteristics that significantly affect the elastic and inelastic structural performance. A parameter of the mathematical representation of near‐fault motions, referred to as ‘pulse duration’ (TP), emerges as a key parameter of the problem under investigation. Specifically, TP is employed to normalize the elastic and inelastic response spectra of actual near‐fault strong ground motion records. Such normalization makes feasible the specification of design spectra and reduction factors appropriate for near‐fault ground motions. The ‘pulse duration’ (TP) is related to an important parameter of the rupture process referred to as ‘rise time’ (τ) which is controlled by the dimension of the sub‐events that compose the mainshock. Copyright © 2004 John Wiley & Sons, Ltd.
Distinct types of astroglial cells in the hippocampus differ in gap junction coupling
Authors: Wallraff Anke , Odermatt Benjamin , Willecke Klaus , Steinhäuser Christian
Published: May 2004

Previous studies have shown that two subpopulations of cells with astrocytic properties coexist in the mouse hippocampus, which display distinct morphological and functional characteristics, specifically a nonoverlapping expression of either AMPA‐type glutamate receptors (GluR cells) or glutamate transporters (GluT cells). Use of transgenic mice with hGFAP promoter‐controlled EGFP expression and patch‐clamp recordings allow reliable identification of the two cell types in hippocampal slices. Extending functional characterization, we report here the complete lack of gap junctional tracer coupling in GluR cells, while GluT cells are shown to be extensively coupled. This distinction is valid in immature as well as adult animals. Analysis of transgenic mice expressing β‐Gal under regulatory elements of the Cx43 promoter revealed the absence of Cx43 in GluR cells. Experiments using gap junction blockers demonstrated that passive currents, displayed primarily by GluT cells, do not reflect intercellular coupling but are attributable to intrinsic membrane properties of individual cells. This study supports the notion that the two subpopulations of hGFAP‐EGFP‐positive cells represent distinct cell types with contrasting physiological properties. Since GluR cells do not participate in the astrocytic gap junctional network, their functional role must be different from spatial buffering of ions or signaling molecules, i.e., properties generally assigned to astrocytes. © 2004 Wiley‐Liss, Inc.
Role of ammonia in reversal of glutamate‐mediated Müller cell swelling in the rat retina
Authors: Izumi Yukitoshi , Matsukawa Mio , Benz Ann M. , Izumi Masayo , Ishikawa Makoto , Olney John W. , Zorumski Charles F.
Published: May 2004

Glutamate is thought to participate in a variety of retinal degenerative disorders. However, when exposed to glutamate at concentrations up to 1 mM, ex vivo rat retinas typically exhibit Müller cell swelling, but not excitotoxic neuronal damage. This Müller cell swelling is reversible following glutamate washout, indicating that the glial edema is not required for glutamate‐induced neuronal injury. It is unclear whether glutamate directly induces the Müller cell swelling or whether a metabolite of glutamate such as glutamine acts as an osmolyte to generate the cellular edema. To examine this issue, ex vivo rat retinas were exposed to 1 mM glutamate or 1 mM glutamine and were evaluated histologically. Glutamate was also combined with 1 mM ammonia or with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase, the enzyme that catalyzes the synthesis of glutamine from glutamate and ammonia. Glutamate‐mediated Müller cell swelling was blocked by co‐administration of ammonia and the reversibility of Müller cell swelling was inhibited by MSO administered following glutamate exposure. Glutamine alone failed to induce Müller cell swelling. These results indicate that glutamate‐mediated Müller cell swelling is unlikely to result from glutamine accumulation. Rather, conversion of glutamate to glutamine in a reaction involving ammonia helps reverse Müller cell swelling following exposure to exogenous glutamate. © 2004 Wiley‐Liss, Inc.
Review of donepezil, rivastigmine, galantamine and memantine for the treatment of dementia in Alzheimer's disease in adults with Down syndrome: implications for the intellectual disability population
Authors: Prasher V. P.
Published: May 2004

The management of dementia in Alzheimer's disease has dramatically changed since the development of anti‐dementia drugs. However, there is limited information available regarding the bio‐medical aspects of the differing drugs; particularly relating to adults with intellectual disability. Indeed the information available for the intellectual disabled population is limited to adults with Down syndrome. This review highlights the important pharmacological and clinical aspects of donepezil, rivastigmine, galantamine and memantine and supports the view that such drugs play an important part in the management of dementia in adults with intellectual disability. Future clinical and research issues are discussed. Copyright © 2004 John Wiley & Sons, Ltd.