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Biomedical subjects

J R Mourant

Publications and source records attributed to J R Mourant.

15 recordsLinked to original sources
PubMed2002

Angular dependent light scattering from multicellular spheroids.

We demonstrate that the effects of cell-cell contact and of changes in cell shape have only a minor effect on the angular distribution of light scattering from mammalian fibroblast cells. This result is important for the development of light scattering as a noninvasive tool for tissue diagnostics such as cancer detection. Changes in cell organization that are not accompanied by changes in internal cellular structure may not be measurable. On the other hand, changes in internal cellular structure should be measurable without interference from changes in overall cellular organization. The second major result of this work is that there are small but significant differences between light scattering of tumorigenic and nontumorigenic cells grown in a three-dimensional culture system. The cause of the differences in light scattering are likely due to the nontumorigenic cells arresting in the G1 phase of the cell cycle, while the tumorigenic cells continue to proliferate.

Animals↗
PubMed2002

Polarized angular dependent spectroscopy of epithelial cells and epithelial cell nuclei to determine the size scale of scattering structures.

An understanding of the relationship between tissue structures and light scattering from tissue will help facilitate the development and acceptance of noninvasive optical diagnostics including elastic scattering spectroscopy, diffuse reflectance, and optical coherence tomography. For example, a quantitative model of the structures that scatter light in epithelial cells would allow determination of what structures control the characteristics of in vivo light transport measurements and subsequently could provide a detailed relationship between cellular structures and optical measurements. We have determined the size distribution of refractive index structure variations in epithelial cells as well as in nuclei isolated from epithelial cells from measurements of the angular dependence of polarized light scattering. The quantitative size distributions we obtained for both whole cells and isolated nuclei include particles with effective radii of 2 microm to 10 nm or less and contain orders of magnitude more small particles than large particles. These results demonstrate that not only are biological cells very heterogeneous, but so are the nuclei within them. Light scattering is likely sensitive to structures smaller than those commonly investigated by standard pathology methods.

Animals↗
PubMed2000

High-angle scattering events strongly affect light collection in clinically relevant measurement geometries for light transport through tissue.

Measurement of light transport in tissue has the potential to be an inexpensive and practical tool for non-invasive tissue diagnosis in medical applications because it can provide information on both morphological and biochemical properties. To capitalize on the potential of light transport as a diagnostic tool, an understanding of what information can be gleaned from light transport measurements is needed. We present data concerning the sensitivity of light transport measurements, made in clinically relevant geometries, to scattering properties. The intensity of the backscattered light at small source-detector separations is shown to be sensitive to the phase function, and furthermore the collected light intensity is found to be correlated with the amount of high-angle scattering in the medium.

Biophysical Phenomena↗
PubMed2000

Light scattering from cells: the contribution of the nucleus and the effects of proliferative status.

As part of our ongoing efforts to understand the fundamental nature of light scattering from cells and tissues, we present data on elastic light scattering from isolated mammalian tumor cells and nuclei. The contribution of scattering from internal structures and in particular from the nuclei was compared to scattering from whole cells. Roughly 55% of the elastic light scattering at high-angles (> 40 degrees) comes from intracellular structures. An upper limit of 40% on the fractional contribution of nuclei to scattering from cells in tissue was determined. Using cell suspensions isolated from monolayer cultures at different stages of growth, we have also found that scattering at angles greater than about 110 degrees was correlated with the DNA content of the cells. Based on model calculations and the relative size difference of nuclei from cells in different stages of growth, we argue that this difference in scattering results from changes in the internal structures of the nucleus. This interpretation is consistent with our estimate of 0.2 micron as the mean size of the scattering centers in cells. Additionally, we find that while scattering from the nucleus accounts for a majority of internal scattering, a significant portion must result from scattering off of cytoplasmic structures such as mitochondria.

Animals↗
PubMed1999

Non-invasive measurement of chemotherapy drug concentrations in tissue: preliminary demonstrations of in vivo measurements.

Measurements of the tissue concentrations of two chemotherapy agents have been made in vivo on an animal tumour model. The method used is based on elastic scattering spectroscopy (ESS) and utilizes a fibre-optic probe spectroscopic system. A broadband light source is used to acquire data over a broad range of wavelengths and, therefore, to facilitate the separation of absorptions from various chromophores. The results of the work include measurements of the time course of the drug concentrations as well as a comparison of the optical measurements with high performance liquid chromatography (HPLC) analysis of the drug concentrations at the time of sacrifice. It is found that the optical measurements correlate linearly with HPLC measurements, but give lower absolute values.

Animals↗
PubMed1999

Noninvasive, in-situ measurement of drug concentrations in tissue using optical spectroscopy.

A small, lightweight system capable of noninvasive measurement of drug concentrations in tissue, without the use of reagents, would be advantageous for the study of the drug pharmacokinetics in space-travel applications. We have applied elastic-scattering spectroscopy (ESS) for noninvasive, real-time in vivo measurement of the concentrations of certain drugs in tissue, utilizing a simple fiber-optic-probe spectroscopic system. The system uses a broadband light source, enabling the detection of compounds with absorption bands in most regions of the visible, and potentially in the near-infrared (NIR) to 1700 nm. Subcutaneous tumors were grown in 4 Nude mice; the mice were treated with one of two chemotherapy agents, and the ESS system was used to perform pharmacokinetic measurements on the tumors following drug administration. Time histories of the drug concentrations in the tumors agreed with the known pharmacokinetics of the two drugs, and HPLC assays following sacrifice showed good relative correlation with the ESS values. Most photodynamic therapy agents and many chemotherapy drugs, including some that are not fluorescent, are ideal candidates for the ESS system. Importantly, the method can provide absolute measurements of compound concentrations.

Animals↗
PubMed1998

Evidence of intrinsic differences in the light scattering properties of tumorigenic and nontumorigenic cells.

BACKGROUND: The objective of this study was to determine whether there are intrinsic differences in the light scattering properties of tumorigenic and nontumorigenic cells from a multistep carcinogenesis model. METHODS: Wavelength-dependent and polarization-dependent light scattering properties of cell suspensions were measured. RESULTS: Statistically significant differences were found between the tumorigenic and nontumorigenic cells. CONCLUSIONS. Differences in the light scattering properties of tumorigenic and nontumorigenic cells are attributed to a change in the average size of the scattering centers on the order of a few ten of nanometers. This work is relevant to the development of noninvasive optical methods for cancer diagnosis.

Animals↗
PubMed1995

Spectroscopic diagnosis of bladder cancer with elastic light scattering.

BACKGROUND AND OBJECTIVES: Recently, significant progress has been made toward the development of optical, noninvasive medical diagnostics. The goal of this study was to evaluate elastic scatter measurements as a tool for diagnosing bladder cancer. STUDY DESIGN/MATERIALS AND METHODS: In vivo measurements on 10 patients with suspected bladder cancer were made with the optical biopsy system (OBS) developed at Los Alamos National Laboratory. Elastic-scatter spectra over the wavelength range 250-800 nm were obtained using a fiber-optic probe through one of the lumens of a urological cystoscope. Measurements were made on putatively normal areas and areas of uncertain abnormality, as well as those suspected to be cancerous. After measurements were made with the OBS, biopsy samples were taken at the measurement sites. Comparisons of the histopathology and the optical spectra were then made. RESULTS AND CONCLUSIONS: A diagnostic algorithm for distinguishing malignant from nonmalignant tissue based on the values of the slopes over the wavelength range 330-370 nm has a sensitivity of 100% and specificity of 97% for the limited number of patients in this study.

Absorption↗
PubMed1994

Ligand binding to heme proteins: the effect of light on ligand binding in myoglobin.

Extended illumination slows the rebinding of CO to myoglobin after photodissociation at cryogenic temperatures. Two types of models have been put forward to explain the effect: motions of the CO within the heme pocket or conformational transitions of the protein. To resolve this ambiguity, we have studied the effect of extended illumination on ligand binding to horse and sperm whale myoglobin (hMb and swMb) with temperature-derivative spectroscopy, monitoring the reaction in the CO stretch bands in the infrared and the conformation-sensitive band III near 760 nm. The experiments show that the stretch frequency of the photodissociated CO does not change upon illumination, implying that the slowing of the CO rebinding is caused by conformational relaxation of Mb from the bound state toward the deoxy structure. The light-induced relaxation (LIR) depends on the number of photons absorbed but not on the light intensity or duration separately. LIR occurs on photon absorption in either the bound or photodissociated state and depends on the temperature at which the MbCO is illuminated. The LIR proceeds in jumps through a small number of conformational substates. The effective barrier for rebinding increases with each step. The substates populated are similar to those found in the thermally-induced relaxation (TIR) that is observed above 160 K. LIR depends markedly on the structural details; it differs for swMbCO and hMbCO and even for the three A substates of swMbCO. Pronounced differences exist between the effects in MbCO and MbO2. The similarity of LIR and TIR leads to a revised model for ligand binding to swMbCO and hMbCO, in which the relaxation is crucial for the escape of the ligand from the pocket, as was first suggested by Friedman [Friedman, J. M. (1985) Science 228, 1273-1280].

Animals↗
PubMed1993

Ligand binding to heme proteins: II. Transitions in the heme pocket of myoglobin.

Phenomena occurring in the heme pocket after photolysis of carbonmonoxymyoglobin (MbCO) below about 100 K are investigated using temperature-derivative spectroscopy of the infrared absorption bands of CO. MbCO exists in three conformations (A substrates) that are distinguished by the stretch bands of the bound CO. We establish connections among the A substates and the substates of the photoproduct (B substates) using Fourier-transform infrared spectroscopy together with kinetic experiments on MbCO solution samples at different pH and on orthorhombic crystals. There is no one-to-one mapping between the A and B substates; in some cases, more than one B substate corresponds to a particular A substate. Rebinding is not simply a reversal of dissociation; transitions between B substates occur before rebinding. We measure the nonequilibrium populations of the B substates after photolysis below 25 K and determine the kinetics of B substate transitions leading to equilibrium. Transitions between B substates occur even at 4 K, whereas those between A substates have only been observed above about 160 K. The transitions between the B substates are nonexponential in time, providing evidence for a distribution of substates. The temperature dependence of the B substate transitions implies that they occur mainly by quantum-mechanical tunneling below 10 K. Taken together, the observations suggest that the transitions between the B substates within the same A substate reflect motions of the CO in the heme pocket and not conformational changes. Geminate rebinding of CO to Mb, monitored in the Soret band, depends on pH. Observation of geminate rebinding to the A substates in the infrared indicates that the pH dependence results from a population shift among the substates and not from a change of the rebinding to an individual A substate.

Animals↗
PubMed1993

Ligand binding to heme proteins: III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin.

Fouier-transform infrared (FTIR) difference spectra of several His-E7 and Val-E11 mutants of sperm whale carbonmonoxymyoglobin were obtained by photodissociation at cryogenic temperatures. The IR absorption of the CO ligand shows characteristic features for each of the mutants, both in the ligand-bound (A) state and in the photodissociated (B) state. For most of the mutants, a single A substate band is observed, which points to the crucial role of the His-E7 residue in determining the A substrate spectrum of the bound CO in the native structure. The fact that some of the mutants show more than one stretch band of the bound CO indicates that the appearance of multiple A substates is not exclusively connected to the presence of His-E7. In all but one mutant, multiple stretch bands of the CO in the photodissociated state are observed; these B substates are thought to arise from discrete positions and/or orientations of the photodissociated ligand in the heme pocket. The red shifts of the B bands with respect to the free-gas frequency indicate weak binding in the heme pocket. The observation of similar red shifts in microperoxidase (MP-8), where there is no residue on the distal side, suggests that the photodissociated ligand is still associated with the heme iron. Photoselection experiments were performed to determine the orientation of the bound ligand with respect to the heme normal by photolyzing small fractions of the sample with linearly polarized light at 540 nm. The resulting linear dichroism in the CO stretch spectrum yielded angles alpha > 20 degrees between the CO molecular axis and the heme normal for all of the mutants. We conclude that the off-axis position of the CO ligand in the native structure does not arise from steric constraints imposed by the distal histidine. There is no clear correlation between the size of the distal residue and the alpha of the CO ligand.

Amino Acid Sequence↗
PubMed1992

Spectroscopic evidence for conformational relaxation in myoglobin.

The time and temperature dependencies of the line area (M0) and position (M1) of band III at approximately 760 nm have been measured with Fourier-transform infrared spectroscopy in deoxymyoglobin (Mb) and continuously photolyzed carbon monoxide myoglobin (MbCO). Below 200 K, the area of band III in the photoproduct Mb* increases with time even on time scales of hours. This behavior indicates changes in the distribution of activation enthalpy barriers for ligand rebinding under extended illumination. The band position of Mb* shifts to higher wavenumbers with increasing temperature up to 100 K owing to kinetic hole burning; the same protein coordinate that controls the position of band III also determines the rebinding barrier height. The shift ceases above 100 K, implying that more than one protein coordinate affects the height of the rebinding barrier. Above 160 K, the line position in Mb* shifts again and coalesces with the value of Mb for temperatures above 200 K. The shift is accompanied by an increase of the line area, reflecting a slowing of rebinding kinetics. Both effects are explained in the framework of the model introduced by Steinbach et al. [(1991) Biochemistry 30, 3988-4001]. Above approximately 160 K, the conformational relaxation Mb*----Mb simultaneously shifts the line position of band III and increases the enthalpy barrier for ligand rebinding. Furthermore, equilibrium fluctuations lead to an averaging of the band position and the rebinding enthalpy.

Carbon Monoxide↗
PubMed1990

Conformational substates and motions in myoglobin. External influences on structure and dynamics.

Myoglobin, a simppe dioxygen-storage protein, is a good laboratory for the investigation of the connection between protein structure, dynamics, and function. Fourier-transform infrared spectroscopy on carbon-monoxymyoglobin (MbCO) shows three major CO bands. These bands are excellent probes for the investigation of the structure-function relationship. They have different CO binding kinetics and their CO dipoles form different angles with respect to the heme normal, implying that MbCO exists in three major conformational substates, A0, A1, and A3. The entropies and enthalpies of these substates depend on temperature above approximately 180 K and are influenced by pH, solvent, and pressure. These results suggest that even a protein as simple as Mb can assume a small number of clearly different structures that perform the same function, but with different rates. Moreover, protein structure and dynamics depend strongly on the interaction of the protein with its environment.

Fourier Analysis↗
PubMed1988

Electroporation: high frequency of occurrence of a transient high-permeability state in erythrocytes and intact yeast.

We present the first determinations of population distributions of macromolecule uptake due to electroporation, the percentage of cells which participate and, for the yeast, the subpopulation of cells whose membranes exhibit significant recovery following macromolecule uptake. Flow cytometry is used to measure the uptake of a first test molecule (green fluorescence, FITC-dextran; 70 kDa) and also, for the yeast, the subsequent uptake of a second, much smaller, test molecule (red fluorescence, propidium iodide; 660 Da), which provides a measure of membrane recovery. A dramatic 20% (erythrocytes) to 75% (intact Schizosaccharomyces pombe) of cells can take up the first test molecule within 5 min of a pulse.

Cell Membrane Permeability↗