bluuebunny/crossref_metadata_2025_split huggingface dataset:
Caspase‐3 and caspase‐9 mediate developmental apoptosis in the mouse olfactory system
Authors: Cowan Catherine M. , Roskams A. Jane 
Published: May 2004

AbstractNeuronal 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

AbstractWe 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

AbstractUsing 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

AbstractWe 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

AbstractBackgroundMolecular 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.ProgressThe 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.ChallengeIt 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

AbstractBackgroundHere 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.MethodsLive, 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.ResultsLive (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.ConclusionsUsing 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

AbstractBackgroundCytomics 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.MethodsWe 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.ResultsWe 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.ConclusionsResults 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

AbstractBackgroundElasticity 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.MethodsThe 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.ResultsRudimentary 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.ConclusionsPreliminary 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

AbstractBackgroundSince 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.MethodsDifferent 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.ResultsThese 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.ConclusionsStudies 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

AbstractCytomics 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

AbstractBackgroundSpecific 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.MethodsRecent 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.ResultsIn 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.ConclusionsThis 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

AbstractBackgroundIn 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.MethodsWe 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.ResultsPercentage 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.ConclusionsWe 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

AbstractBackgroundMost 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?MethodsModel 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.ResultsGEPs 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.ConclusionsPurification 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

AbstractBackgroundThe 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.MethodsAptamers 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.ResultsWe 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.ConclusionsCurrently, 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.