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At least 379 records · Page 21Linked to original sources
PubMed1989

Radiobiology of ultrasoft X rays. III. Normal human fibroblasts and the significance of terminal track structure in cell inactivation.

Ultrasoft characteristic X rays from carbon (0.28 keV) are severely attenuated as they pass through biological material, causing a nonuniform distribution of dose to cell nuclei. Complications of studying ultrasoft X rays can be minimized in this context by using cells with very thin cytoplasm and nuclei (e.g., less than the attenuation length of the X rays), and which exhibit a more nearly exponential dose response to cell killing, such as normal human fibroblasts compared with V79 cells. Using this cell system, we report the relative biological effectiveness (RBE) of A1-K and C-K X rays to be near unity. Previous studies of cell inactivation by characteristic carbon X rays gave RBEs of 3 to 4, supporting the idea that localized energy depositions from secondary electrons and primary track ends represent the principal mode of biological action for other low-LET radiations. In part, the reported high RBEs result from the use of mean dose to describe energy deposited within the cell nuclei by these poorly penetrating radiations. Implicit in the use of mean dose is that cellular damage varies linearly with dose within a critical target(s), an assumption that is of questionable validity for cells that exhibit pronounced curvilinear dose responses. The simplest interpretation of the present findings is that most energy depositions caused by track-end effects are not necessarily more damaging than the sparsely ionizing component.

Cell Survival↗
PubMed2000

Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells.

The ability to track the distribution and differentiation of progenitor and stem cells by high-resolution in vivo imaging techniques would have significant clinical and research implications. We have developed a cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles. The particles are efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10-30 pg of superparamagnetic iron per cell. Iron incorporation did not affect cell viability, differentiation, or proliferation of CD34+ cells. Following intravenous injection into immunodeficient mice, 4% of magnetically CD34+ cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance (MR) imaging in tissue samples. In addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. Localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells.

Animals↗
PubMed1988

Association of thrombospondin of endothelial cells with other matrix proteins and cell attachment sites and migration tracks.

Different biochemical and cytochemical techniques were applied to characterize the sites of localization of thrombospondin in cultured endothelial cells. The results obtained by [35S]methionine labeling, immunoblotting, immunoprecipitation, fluorescence microscopy, ultracytochemistry, immunogold labeling, and silver enhancement experiments revealed that thrombospondin secreted by endothelial cells is structurally organized together with proteoheparan sulfate in spherical granules at the cell surface. These granules are about 100 to 300 nm in size. Heparin or enzymatic degradation with heparitinase, but not with ABC lyase, release thrombospondin from the cell surface. Fibronectin is expressed in the extracellular matrix of endothelial cells in a fibrillar organization, clearly distinct from the punctate pattern of thrombospondin on the cell surface. Furthermore, secreted thrombospondin is highly enriched together with fibronectin and proteoheparan sulfate in cell attachment sites and in cell migration tracks. In cell migration tracks proteoheparan sulfate more clearly resembles the fibrillar distribution pattern of fibronectin, whereas thrombospondin reveals a rather monodisperse pattern. The obtained data suggest preferential sites of interaction between thrombospondin and heparan sulfate proteoglycans on the cell surface and a participation of thrombospondin in cell adhesion and cell migration.

Animals↗
PubMed2005

Optimizing techniques for tracking transplanted stem cells in vivo.

The potential for bone marrow-derived cells (BMDCs) to contribute to nonhematopoietic tissues has generated considerable debate in recent years. Causes for the controversies include disparities in the techniques used to track engraftment of BMDCs, inappropriate tissue preparation, a lack of appropriate positive and negative controls, and basic misunderstandings about how to properly collect and interpret images from epifluorescent and confocal microscopes. Our laboratory was among the first to use bone marrow transplants from transgenic mice constitutively expressing enhanced green fluorescent protein (GFP) to study the ability of BMDCs to give rise to nonhematopoietic tissue types, a system that is now in widespread use. During our 6 years of experience using GFP, as well as beta-galactosidase and the Y chromosome, to track BMDCs in vivo, we have identified many difficulties and have developed techniques to resolve them. We discuss several of these methods, and, in particular, we describe ratiometric analysis techniques for improving detection of transplanted cells derived from genetically modified bone marrow. Finally, to help resolve reported discrepancies regarding the frequency with which BMDCs contribute to skeletal myofibers, we demonstrate that the pattern of highly autofluorescent myofibers in skeletal muscle is clearly distinct from that of GFP-expressing myofibers and describe how unambiguous conclusions can be drawn from such data.

Animals↗
PubMed1973

Freeze-etching and x-ray diffraction of the isolated double-track layer from the cell wall of a gram-negative marine pseudomonad.

The isolated double-track layer of the cell wall of the gram-negative marine pseudomonad studied here contains a cleavage plane. This finding localizes the single cleavage plane of the cell wall and shows that the molecular architecture of this layer provides the lipid-enriched layer which cleaves preferentially in the frozen cell. The observation that the isolated double-track layer of the cell wall is sufficiently ordered at the molecular level to yield a well-defined X-ray diffraction pattern with a d-spacing of 0.44 nm shows that its molecular architecture is very similar to that of true membranes. This specific d-spacing is produced by the highly ordered packing of the hydrophobic portions of phospholipid molecules. Therefore, the double-track layer of the cell wall has been shown, by these two biophysical means, to have a molecular architecture which would allow it to function as the membrane-like "molecular sieve" layer, whose presence has been deduced from physiological data. This layer is important in the retention of cell wall-associated enzymes and in the control of the movement of large molecules through the cell wall.

Bacteria↗
PubMed2000

Tracking single proteins within cells.

We present experiments in which single proteins were imaged and tracked within mammalian cells. Single proteins of R-phycoerythrin (RPE) were imaged by epifluorescence microscopy in the nucleoplasm and cytoplasm at 71 frames/s. We acquired two-dimensional trajectories of proteins (corresponding to the projection of three-dimensional trajectories onto the plane of focus) for an average of 17 frames in the cytoplasm and 16 frames in the nucleus. Diffusion constants were determined from linear fits to the mean square displacement and from the mean displacement squared per frame. We find that the distribution of diffusion constants for RPE within cells is broader than the distributions obtained from RPE in a glycerol solution, from a Monte Carlo simulation, and from the theoretical distribution for simple diffusion. This suggests that on the time scales of our measurements, the motion of single RPE proteins in the cytoplasm and nucleoplasm cannot be modeled by simple diffusion with a unique diffusion constant. Our results demonstrate that it is possible to follow the motion of single proteins within cells and that the technique of single molecule tracking can be used to probe the dynamics of intracellular macromolecules.

Animals↗
PubMed1996

Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging.

The mobility of cell surface MHC molecules and their ability to form dynamic associations may be related to the physiological status of the cell and to the potential to bind effector T lymphocytes. To investigate these properties, we have prepared HLA DR specific monoclonal antibodies coupled in a 1:1 mole ratio to the fluorescent phycobiliprotein, R-phycoerythrin (PE). We show that these small particles can be sequentially imaged using a cooled slow-scan charge coupled device camera and hence can be used for single particle tracking experiments. We have applied this technique to investigate the movements of HLA DR molecules on fibroblasts transfected with human DR alpha and DR beta genes. PE-IgG was bound to the transfected fibroblasts and particle tracks were obtained by sequential imaging over a period of typically 30 minutes. Analysis of particle tracks revealed the presence of directed motion and domain-limited diffusion in addition to random diffusion. The contributions of these three types of motion showed cell to cell variability. Velocities of directed motion were of the order of 2 nm second-1 whilst domain diameters were in the range 200-800 nm. Diffusion coefficients for random diffusion were in the range 1 x 10(-13)-5 x 10(-12) cm2 second-1. The higher mobilities were observed for the lower intensity fluorescent spots, which possibly correspond to images of single particles. Much lower mobility was observed with a cell where the spot intensities were approximately double that of the lower intensity spots. These spots could be images of double particles implying the association of at least two HLA DR alpha beta dimers. These data are relevant to the study of MHC class II cell surface redistribution and antigen presentation in specific immunity.

Antibodies, Monoclonal↗
PubMedDate not supplied

Genetically engineered fluorescent cell marker for labeling CD34+ hematopoietic stem cells.

To address the challenge of labeling and tracking stem cells in vivo, we have engineered a fluorescent cell marker CD34EGFP by utilizing the mechanism of the cell-specific activity of CD34 promoter in CD34(+) stem cells. A retroviral vector derived from a murine stem cell virus was constructed to integrate the CD34EGFP gene into the genome of the cells for labeling. Our experiment demonstrates that the 454 bp segment upstream of the murine CD34 cDNA sequence has full function of promoter activity and can serve as a cell-specific promoter for driving the expression of EGFP in CD34(+) hematopoietic stem cells (HSC), providing a living color for labeling stem cells. The CD34EGFP marker was tested in various types of cells, including terminally differentiated cells, CD34(+) mouse myeloid leukemia progenitor cells, CD34(-) hematopoietic cells, and CD34(+) HSCs. We show that the engineered CD34EGFP cell marker is expressed in the CD34(+) stem or progenitor cells but not in CD34(-) or terminally differentiated cells. RT-PCR assay indicates that the transcription level of the CD34EGFP gene from CD34 promoter is almost the same as that from CMV promoter in CD34(+) progenitor cells. The approach we present here offers a framework for genetic engineering of fluorescent cell markers for labeling and tracking stem cells in vivo. We anticipate that a variety of cell markers could be generated by coupling variants of fluorescent proteins with various cell-specific promoters.

Animals↗
PubMed1999

Use of caged fluorochromes to track macromolecular movement in living cells.

One way to visualize and track the movement of macromolecules in the living cell is to follow their movement after tagging the molecule with a 'caged' or chemically masked fluorochrome. The fluorochrome does not fluoresce until the caging group is released by spot photoactivation, and the bright fluorescent signal can then be tracked as it moves into the dark surrounding area of the cell. When coupled with rapid imaging microscopy, it is possible to measure rates of movement as fast as macromolecular diffusion. This article describes the use of photoactivatable fluorochromes to track the intracellular movement of both proteins and nucleic acids and to track cell lineages.

Animals↗
PubMed1998

Identification of the adult resting progenitor cell by autoradiographic tracking of oligodendrocyte precursors in experimental CNS demyelination.

The new population of oligodendrocytes remyelinating an experimental demyelinating optic nerve lesion has been tracked backwards in time. Using autoradiography combined with electron microscopy and immunocytochemistry, serial sections of optic nerves from young adult cats were studied from 42 h (2 days) post-injection to 93 h (4 days) post-injection. The remyelinating oligodendrocyte lineage was found to commence with the single division of a resting progenitor cell residing in a central fascicular location outside the demyelinative lesion. The division of the founding progenitor cell occurs at 2 days post-lesion and results in two motile daughter cells. These cells, previously described by us as precursor cells, migrate to the lesion, closely appose demyelinated axons, produce axon wrapping processes and differentiate to become remyelinating oligodendrocytes. This study confirms that remyelinating oligodendrocytes originate from resting progenitor cells outside the lesion and not from mature oligodendrocytes, and implies that repeated demyelinative injury could exhaust the reparative capacity of such a region.

Animals↗
PubMed1992

Multiple cell hits by particle tracks in solid tissues.

Relative Biological Effectiveness (RBE) and Quality Factor (Q) at extreme values of Linear Energy Transfer (LET) have been determined on the basis of experiments with single-cell systems and specific tissue responses. In typical single cell systems, each heavy particle (Ar or Fe) passes through a single cell or no cell. In tissue end-point experiments each heavy particle passes through several cells, and the LET can exceed 200 keV/micrometer in every cell. In most laboratory animal tissue systems, however, only a small portion of the hit cells are capable of expressing the end-point of interest to the investigator, such as cell killing, mutation or carcinogenesis. The following question must therefore be addressed: Do RBE's and Q factors derived from single-cell experiments properly account for the increased probability of multiple-cell damage by HZE tracks? A model is offered in which measured radiation effects and known tissue properties are combined to estimate the value of a multiplier of damage effectiveness on the basis of number of cells at risk, p3n, per track containing a hit cell, where n is the number of cells per track, based on tissue and organ geometry, and P3 is the probability that a cell in the track is capable of expressing the experimental end-point.

Animals↗
PubMed2006

The MR tracking of transplanted ATDC5 cells using fluorinated poly-L-lysine-CF3.

Magnetic resonance (MR) imaging using super-paramagnetic iron oxides (SPIOs) is a powerful tool to monitor transplanted cells in living animals. However, since SPIOs are negative contrast agents it is difficult to track transplanted cells in bone and cartilage that originally display low signals. In this study, we examined the feasibility of tracking with fluorescein isothiocyanate (FITC)-labeled poly-L-lysine-CF(3) (PLK-CF(3)) using mouse ATDC5 cells, a stem cell line of bone and cartilage cells. FITC-labeled PLK-CF(3) was easily internalized by ATDC5 cells by adding it into culture medium. No acute or long-term toxicities were seen at less than 160 microg/ml. Labeled cells transplanted into the cranial bone of mice were detected for at least 7 days by MR images. FITC-labeled PLK-CF(3) is a useful positive contrast agent for MR tracking in bone and cartilage.

Animals↗
PubMed1995

In vivo characterization of two cell types in the rat globus pallidus which have opposite responses to dopamine receptor stimulation: comparison of electrophysiological properties and responses to apomorphine, dizocilpine, and ketamine anesthesia.

Extracellular single-unit recording techniques were used to examine the rat globus pallidus (GP). In both locally anesthetized, paralyzed rats and ketamine-anesthetized rats, we observed two distinct biphasic extracellular waveforms, which we have labeled Type I (negative/positive waveform) and Type II (positive/negative waveform). No significant differences were observed in the firing pattern or number of cells per track between these cell types, although the Type II neurons had a faster mean firing rate in the locally anesthetized animals. A portion of both cell types could be antidromically activated from the subthalamic nucleus, although Type II neurons had significantly slower conduction velocities. The most striking pharmacological difference between the two cell types was that Type I GP neurons were inhibited by systemic administration of the dopamine agonist apomorphine; previous studies have repeatedly shown that Type II GP cells are excited by this treatment. Pretreatment with a subthreshold dose of apomorphine reduced the responsiveness of Type I cells to a subsequent high dose of apomorphine, as has been shown for Type II cells. However, pretreatment with the NMDA antagonist dizocilpine (MK801) produced a significant change in the pattern of response to apomorphine for Type II GP neurons only. Relative to observations in locally anesthetized, paralyzed rats, ketamine anesthesia reduced the firing rate of both cell types, but did not significantly alter their direction of response to apomorphine. Thus, this study has confirmed the existence of two GP cell types with distinct extracellular waveforms and different responses to dopamine receptor stimulation. These data may necessitate a reevaluation of general theoretical models of basal ganglia function in order to account for these opposite effects of dopamine receptor stimulation on pallidal output.

Anesthesia↗
PubMed2005

Tracking movement in cell biology.

This article is an overview of techniques for measuring movement of proteins, vesicles and cells using digital image processing. Diverse techniques have been developed during the last decade. Both strong and weak points of each technique are discussed, together with short description on the actual application. Among these techniques, the optical flow estimation technique is explained in more detail and an example of protein movement analysis is described.

Algorithms↗
PubMed1982

Computer-based tracking of living cells.

A computer-based tracing technique has been developed to follow the movement of living cells and keep them centered in the field of view of an optical microscope. With the use of an image-processing system, the video image of a cell can be sufficiently processed to allow computer-recognition of the cell boundaries. Determination of the location of the center of the cell enables comparison of successive cell positions and correction for any cell movement. In order to illustrate the versatility of this technique, patterns of movement were obtained of cancerous and non-cancerous cells in an effort to determine the difference in motility between the two cell types. After examination of the data gathered, it was found that there is no difference in the motility between the two cell types over 1-h periods.

Animals↗