Biomedical subjects

G J Czarnota

Publications and source records attributed to G J Czarnota.

13 recordsLinked to original sources

PubMed2005

Monitoring structural changes in cells with high-frequency ultrasound signal statistics.

We investigate the use of signal envelope statistics to monitor and quantify structural changes during cell death using an in vitro cell model. Using a f/2.35 transducer (center frequency 20 MHz), ultrasound backscatter data were obtained from pellets of acute myeloid leukemia cells treated with a DNA-intercolating chemotherapy drug, as well as from pellets formed with mixtures of treated and untreated cells. Simulations of signals from pellets of mixtures of cells were generated as a summation of point scatterers. The signal envelope statistics were examined by fitting the Rayleigh and generalized gamma distributions. The fit parameters of the generalized gamma distribution showed sensitivity to structural changes in the cells. The scale parameter showed a 200% increase (p<0.05) between untreated and cells treated for 24 h. The shape parameter showed a 50% increase (p<0.05) over 24 h. Experimental results showed reasonable agreement with simulations. The results indicate that high-frequency ultrasound signal statistics can be used to monitor structural changes within a very low percentage of treated cells in a population, raising the possibility of using this technique in vivo.

Antineoplastic Agents↗

PubMed2005

High-frequency ultrasound scattering from microspheres and single cells.

Assessing the proportion of biological cells in a volume of interest undergoing structural changes, such as cell death, using high-frequency ultrasound (20-100 MHz), requires the development of a theoretical model of scattering by any arbitrary cell ensemble. A prerequisite to building such a model is to know the scattering by a single cell in different states. In this paper, a simple model for the high-frequency acoustic scattering by one cell is proposed. A method for deducing the backscatter transfer function from a single, subresolution scatterer is also devised. Using this method, experimental measurements of backscatter from homogeneous, subresolution polystyrene microspheres and single, viable eukaryotic cells, acquired across a broad, continuous range of frequencies were compared with elastic scattering theory and the proposed cell scattering model, respectively. The resonant features observed in the backscatter transfer function of microspheres were found to correspond accurately to theoretical predictions. Using the spacing of the major spectral peaks in the transfer functions obtained experimentally, it is possible to predict microsphere diameters with less than 4% error. Such good agreement was not seen between the cell model and the measured backscatter from cells. Possible reasons for this discrepancy are discussed.

Apoptosis↗

PubMed2002

Ultrasonic spectral parameter characterization of apoptosis.

Ultrasound (US) spectral analysis methods are used to analyze the radiofrequency (RF) data collected from cell pellets exposed to chemotherapeutics that induce apoptosis and other chemicals that induce nuclear transformations. Calibrated backscatter spectra from regions-of-interest (ROI) were analyzed using linear regression techniques to calculate the spectral slope and midband fit. Two f/2 transducers, with operating frequencies of 30 and 34 MHz (relative bandwidths of 93% and 78%, respectively) were used with a custom-made imaging system that enabled the collection of the raw RF data. For apoptotic cells, the spectral slope increased from 0.37 dB/MHz before drug exposure to 0.57 dB/MHz 24 h after, corresponding to a change in effective scatterer radius from 8.7 to 3.2 microm. The midband fit increased in a time-dependent fashion, peaking at 13dB 24 h after exposure. The statistical deviation of the spectral parameters was in close agreement with theoretical predictions. The results provide a framework for using spectral parameter methods to monitor apoptosis in in vitro and in in vivo systems and are being used to guide the design of system and signal analysis parameters.

Apoptosis↗

PubMed1999

Ultrasound imaging of apoptosis: high-resolution non-invasive monitoring of programmed cell death in vitro, in situ and in vivo.

A new non-invasive method for monitoring apoptosis has been developed using high frequency (40 MHz) ultrasound imaging. Conventional ultrasound backscatter imaging techniques were used to observe apoptosis occurring in response to anticancer agents in cells in vitro, in tissues ex vivo and in live animals. The mechanism behind this ultrasonic detection was identified experimentally to be the subcellular nuclear changes, condensation followed by fragmentation, that cells undergo during apoptosis. These changes dramatically increase the high frequency ultrasound scattering efficiency of apoptotic cells over normal cells (25- to 50-fold change in intensity). The result is that areas of tissue undergoing apoptosis become much brighter in comparison to surrounding viable tissues. The results provide a framework for the possibility of using high frequency ultrasound imaging in the future to non-invasively monitor the effects of chemotherapeutic agents and other anticancer treatments in experimental animal systems and in patients.

Animals↗

PubMed1997

Three-dimensional structure of myelin basic protein. I. Reconstruction via angular reconstitution of randomly oriented single particles.

Myelin basic protein (MBP) plays an integral role in the structure and function of the myelin sheath. In humans and cattle, an 18.5-kDa isoform of MBP predominates and exists as a multitude of charge isomers resulting from extensive and varied post-translational modifications. We have purified the least modified isomer (named C1) of the 18.5-kDa isoform of MBP from fresh bovine brain and imaged this protein as negatively stained single particles adsorbed to a lipid monolayer. Under these conditions, MBP/C1 presented diverse projections whose relative orientations were determined using an iterative quaternion-assisted angular reconstitution scheme. In different buffers, one with a low salt and the other with a high salt concentration, the conformation of the protein was slightly different. In low salt buffer, the three-dimensional reconstruction, solved to a resolution of 4 nm, had an overall "C" shape of outer radius 5.5 nm, inner radius 3 nm, overall circumference 15 nm, and height 4.7 nm. The three-dimensional reconstruction of the protein in high salt buffer, solved to a resolution of 2.8 nm, was essentially the same in terms of overall dimensions but had a somewhat more compact architecture. These results are the first structures achieved directly for this unusual macromolecule, which plays a key role in the development of multiple sclerosis.

Animals↗

PubMed1997

Ultrasonic biomicroscopy of viable, dead and apoptotic cells.

Ultrasonic imaging is frequently used in medical diagnosis to differentiate normal and tumour tissues. Here we investigate if distinct types of cell death can be discriminated through the use of ultrasound biomicroscopy. By using a well-controlled system in vitro, we demonstrate that this imaging modality can be used to differentiate living cells, dead cells and cells that have died by programmed cell death or apoptosis. The results indicate a greater than twofold ultrasound backscatter signal from apoptotic cells in comparison to viable cells, whereas heat-killed cells exhibit an intermediate level of ultrasound backscatter. The results have potential implications in the study of disease-related biological processes involving apoptosis.

Antineoplastic Agents↗

PubMed1997

High resolution microanalysis and three-dimensional nucleosome structure associated with transcribing chromatin.

The nucleosome is the ubiquitous and fundamental DNA-protein complex of the eukaryotic chromosome, participating in the packaging of DNA and in the regulation of gene expression. Biophysical studies have implicated changes in nucleosome structure from chromatin that is quiescent to active in transcription. Since DNA within the nucleosome contains a high concentration of phosphorus whereas histone proteins do not, the nucleosome structure is amenable to microanalytical electron energy loss mapping of phosphorus to delineate the DNA within the protein-nucleic acid particle. Nucleosomes associated with transcriptionally active genes were separated from nucleosomes associated with quiescent genes using mercury-affinity chromatography. The three-dimensional image reconstruction methods for the total nucleosome structure and for the 3D DNA-phosphorus distribution combined quaternion-assisted angular reconstitution of sets of single particles at random orientations and electron spectroscopic imaging. The structure of the active nucleosome has the conformation of an open clam-shell, C- or U-shaped in one view, elongated in another, and exhibits a protein asymmetry. A three-dimensional phosphorus map reveals a conformational change in nucleosomal DNA compared to DNA in the canonical nucleosome structure. It indicates an altered superhelicity and is consistent with unfolding of the particle. The results address conformational changes of the nucleosome and provide a direct structural linkage to biochemical and physiological changes which parallel gene expression.

Chromatin↗

PubMed1997

The in situ architecture of Escherichia coli ribosomal RNA derived by electron spectroscopic imaging and three-dimensional reconstruction.

The structures of the large and small ribosomal subunits of Escherichia coli were reconstructed using spectroscopic electron microscopy and quaternion-assisted angular reconstitution to resolutions of better than 4 nm. In addition, the distributions of phosphorus within these complexes were reconstructed. The three-dimensional reconstruction of the distribution of this atomic element is an extension of microanalysis (in two dimensions) for phosphorus identification and mapping, as a signature of the arrangement of the phosphate backbones of the constituent ribosomal RNAs. The results on both the phosphorus reconstructions and the total reconstructions (protein and ribosomal RNA) reveal several passageways through both subunits. The structures correspond favourably with other independent reconstructions of the whole E. coli ribosome from cryoelectron micrographs and their accompanying models of translation (Frank et al., Nature, 376, 441-444, 1995; Stark et al., Structure, 3, 815-821, 1995). The overall reconstructions in conjunction with the phosphorus (rRNA) distributions are the first to be achieved synchronously for this nucleoprotein complex.

Electron Probe Microanalysis↗

PubMed1996

Structural states of the nucleosome.

The nucleosome is the fundamental component of the eukaryotic chromosome, participating in the packaging of DNA and in the regulation of gene expression. Its numerous interactions imply a structural dynamism. Previous biophysical studies under limited sets of conditions have not been able to reconcile structural differences and transitions observed. We have determined a series of nucleosome conformations over a >10,000-fold range in salt concentration using a combination of biochemical methods, spectroscopic electron microscopy, and three-dimensional reconstruction techniques for randomly oriented single particles. This study indicates several ionic strength-dependent nucleosome conformations and also reconciles the differences between currently existing divergent models for the nucleosome. At low ionic environments, the particle appears highly elongated, becoming more compact and prolate ellipsoidal as ionic strength is increased to 10 mm NaCl. At 30 mM NaCl, the particle exhibits a spheroidal conformation. As ionic strength is increased to 150 mM NaCl, the nucleosome conformation changes and becomes oblate. Above 450 mM NaCl, the structure becomes highly elongated again. The result of this study is a unifying concept in which the three-dimensional structure of the nucleosome is inferred to be dynamic in response to ionic interactions and in accord with biochemical and genetic studies.

Animals↗

PubMed1996

Visualization and analysis of unfolded nucleosomes associated with transcribing chromatin.

We have characterized the structure of transcriptionally active nucleosome subunits using electron spectroscopic imaging. Individual nucleosomes were analyzed in terms of total mass, DNA and protein content, while the ensemble of images of active nucleosomes was used to calculate a three-dimensional reconstruction. Transcriptionally active nucleosomes were separated from inactive nucleosomes by mercury-affinity chromatography thus making it possible to compare their structures. The chromatographic results combined with electron spectroscopic imaging confirm that active nucleosomes unfold to form extended U-shaped particles. Phosphorus mapping indicated that the nucleosomal DNA also underwent a conformational change consistent with particle unfolding. The three-dimensional structure of the Hg-affinity purified nucleosomes determined using quaternion-assisted angular reconstitution methods unites and resolves the different electron microscopic views of the particle and is concordant with a sulphydryl-exposing disruption of the H3-H4 tetramer.

Chromatin↗

PubMed1993

Conformational characterization of nucleosomes by principal component analysis of their electron micrographs.

Optimized fixation conditions were determined for protein-protein and protein-DNA crosslinking within calf-thymus nucleosomes in low monovalent salt concentrations. Nucleosomes were examined without heavy-atom staining by darkfield electron microscopy. The dimensions of these macromolecular complexes and those of HeLa core particles optimally fixed in divalent salt were analysed using principal component analysis. According to this analysis the structure of the calf-thymus nucleosomes was best presented by a prolate ellipsoid. Particle images had average major and minor axis lengths of 14.1 and 10.5 nm, respectively. In contrast, the HeLa nucleosomes were best modelled by an oblate ellipsoid from the analysis of their images, which had average major and minor axes of 13.3 and 11.5 nm. The applicability of this multivariate statistical analysis to the interpretation of macromolecular images is illustrated and discussed.

Animals↗

PubMed1992

The role of the N region in signal sequence and signal-anchor function.

To determine the role of sequences other than the hydrophobic core in mediating signal sequence function, we examined the behavior of fusion proteins and deletion mutants in cell-free systems. We demonstrate that neither the N nor the C region of the preprolactin signal sequence is necessary for translocation. However, insertion of sequences with either a net charge of +2.5 or -6.0 between the N region and the hydrophobic core of the signal converted it into a signal-anchor. The topologies adopted (types I and II, respectively) were opposite those predicted from the distribution of charges surrounding the hydrophobic core of the signals. When these mutant signals were located in the interior of an otherwise secreted protein, both sequences functioned as stop-transfer sequences. Related mutations were assayed in fusion proteins in which the IgM transmembrane domain functioned as an amino-terminal signal-anchor. For these molecules, the distribution of charged residues surrounding the hydrophobic core had no influence on the topology adopted. Our results suggest that features other than simple charge distribution play an important role in determining membrane topology in vitro.

Amino Acid Sequence↗

PubMedDate not supplied

A structure for the signal sequence binding protein SRP54: 3D reconstruction from STEM images of single molecules.

The 54-kDa subunit SRP54 of the signal recognition particle in eukaryotic cells is responsible for the recognition of nascent proteins destined for secretion or membrane integration. The three-dimensional structure of this protein was determined using computational techniques applied to images of the molecule obtained via high-resolution, low-dose, scanning transmission electron microscopy at low temperature. The reconstructions at spatial resolutions between 12 and 15 A feature two unequal domains joined by a slender linker. The two-domain structure is in agreement with genetic and biochemical data indicating organization of SRP54 into a larger N-terminal GTP-binding region and a smaller C-terminal peptide-binding region. The structure has similarities to other protein domains with related functions and similar amino acid sequences. The larger domain of the 3D reconstruction is consistent in shape and size with the GTP-binding domains of EF-Tu and p21-RAS, while the smaller domain is compatible in structure with part of the peptide-binding protein calmodulin. The overall shape of SRP54 and the deduced location of critical functional regions of the molecule provide a structural framework for its known biochemical properties in the targeting cycle of the signal recognition particle.

Amino Acid Sequence↗