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Biomedical subjects

P Todd

Publications and source records attributed to P Todd.

At least 91 records · Page 5Linked to original sources
PubMed1989

Investigations on gel forming media for use in low gravity bioseparations research.

Microgravity research includes investigations designed to gain insight on methods of separating living cells. During a typical separation certain real-time measurements can be made by optical methods, but some materials must also be subjected to subsequent analyses, sometimes including cultivation of the separated cells. In the absence of on-orbit analytical or fraction collecting procedures, some means is required to "capture" cells after separation. The use of solutions that form gels was therefore investigated as a means of maintaining cells and/or macromolecules in the separated state after two types of simple ground-based experiments. Microgravity electrophoresis experiments were simulated by separating model cell types (rat, chicken, human and rabbit erythrocytes) in a vertical density gradient containing low-conductivity buffer, 1.7%-6.5% Ficoll, 6.8-5.0% sucrose, and 1% SeaPrep low-melting temperature agarose and demonstrating that, upon cooling, a gel formed in the column, and cells could be captured in the positions to which they had migrated. Two-phase extraction experiments were simulated by choosing two-polymer solutions in which phase separation occurs in normal saline at temperatures compatible with cell viability and in which one or both phases form a gel upon cooling. Suitable polymers included commercial agaroses (1-2%), maltodextrin (5-7%) and gelatin (5-20%).

Animals↗
PubMed1989

Stochastics of HZE-induced microlesions.

Fundamental biological experiments with bacteria, yeast, and mammalian cells irradiated with ions heavier than helium indicate that maximal probability of single-hit inactivation does not occur when the ion has LET below about 100-200 keV/micrometer. Theoretical treatments of cell inactivation data and the radiation chemistry in particle tracks are consistent with this finding. If a "microlesion" is defined as a linear array, within a tissue, of cells inactivated with maximum probability, surrounded by non-lethally damaged cells, then, by this definition, there must be an LET below which "microlesion" damage cannot be expected. In a retrospective survey of experimental literature in which single-particle effects in tissues were sought, it was found that little or no evidence has been reported supporting single-particle effects in tissues when LET was below 200 keV/micrometer, while some experimenters who irradiated tissues with particles having LET greater than 200 keV/micrometer reported effects that could be attributed to single-particle tracks.

Animals↗
PubMed1989

Gravity-dependent phenomena at the scale of the single cell.

Progress in gravitational cell biology research will depend on the continuing evaluation of a wide variety of physical phenomena affected by gravity and their roles in extracellular, intercellular, and intracellular processes. This paper examines those responses of organisms to gravity which depend on functions at the single cell level. Single cell functions are affected by perturbations in their internal and external environment by a variety of factors, one of which is the effect of gravity. Physical phenomena that could influence cell function include sedimentation, buoyancy-driven convection, streaming potential, hydrostatic pressure, and interactions among physical transport processes. Thermal motion and fluid viscosity play a significant role in all transport processes at the cellular level. The sedimentation of intracellular organelles tends to be counteracted by the cytoskeleton. Intracellular convective transport may be possible in large cells. In a microgravity environment extracellular solutes must be transported by diffusion or active circulatory processes in the absence of density gradient-driven convection, and flocculation and coalescence are reduced by the lack of motion of aggregates.

Animals↗
PubMed1988

Flow cytometric detection of herpes simplex virus type 2 infected and transformed cells by immunoenzymatic and by indirect immunofluorescence staining.

The glucose oxidase antiglucose oxidase (GAG) immunoenzymatic staining procedure has been used to detect herpes simplex virus (HSV) antigens microscopically. In this study, the GAG procedure was adapted to cells in suspension, and its potential usefulness in flow cytometry was examined. HSV-2 infected monkey kidney and HSV-2 transformed mouse cells were stained using antisera to HSV-2 or to an HSV-2 specific protein with a molecular weight of 38 Kd, respectively, with the GAG procedure. Flow cytometric analysis of the GAG stained cells was then performed by the measurement of scattered light intensity in the angular intervals 1 degree-2 degrees, 2.5 degrees-19 degrees, and 3 degrees-6 degrees. The greatest scattered light intensity decrement caused by staining occurred in the 3 degrees-6 degrees angular interval, as predicted by previous work. In infected cells, which stain intensely by immunofluorescence, the difference between positively and negatively stained cells was adequate for detecting infected cells using the GAG method; however, this was not the case for the lightly staining transformed cells. The indirect immunofluorescence method of analysis of the same populations was superior to the scattered light method of analysis of the GAG stained infected and transformed cells.

Animals↗
PubMed1988

Density gradient electrophoresis of cells in a reversible gel.

Density gradient electrophoresis permits the separation of cell types according to surface charge density with high resolution. Any source of flow compromises the resolving power of density gradient electrophoresis. Although procedures have been devised to successfully counteract electroosmotic and convective flows, the final collection of separands requires that they be pumped out of the electrophoresis column. Experiments were therefore designed to test the hypothesis that this flow could also be eliminated by trapping the separated bands in a gel, from which they could be collected by slicing the gel cylinder. Glutaraldehyde-fixed rat and rabbit erythrocytes were used as test particles in a phosphate-buffered isotonic Ficoll-sucrose density gradient in a 2.2 cm diameter, thermostated vertical glass column that could be opened at both ends. Two types of agarose were used as gel polymers: Electrophoresis grade agarose (J.T. Baker Chemical Co.) at final concentrations of 0.1 to 0.25% and SeaPrep ultralow gelling agarose (Marine Colloids Div., FMC Corp.) at a final concentration of 1.0%. Electrophoretic separability of the test particles and fluid stability were tested independently at 55 degrees C and 32 degrees C at which the two agaroses were, respectively, liquid. The experiments demonstrated that the higher temperatures required and the presence of agarose compromised neither the stability of the density gradient nor the migration properties of the cells, and cells can be separated in a sol at a temperature that is compatible with cell viability.

Animals↗
PubMed1988

Simulation of launch and re-entry acceleration profiles for testing of shuttle and unmanned microgravity research payloads.

Microgravity experiments designed for execution in Get-Away Special canisters, Hitchhiker modules, and Reusable Re-entry Satellites will be subjected to launch and re-entry accelerations. Crew-dependent provisions for preventing acceleration damage to equipment or products will not be available for these payloads during flight; therefore, the effects of launch and re-entry accelerations on all aspects of such payloads must be evaluated prior to flight. A procedure was developed for conveniently simulating the launch and re-entry acceleration profiles of the Space Shuttle (3.3 and 1.7 x g maximum, respectively) and of two versions of NASA's proposed materials research Re-usable Re-entry Satellite (8 x g maximum in one case and 4 x g in the other). By using the 7 m centrifuge of the Gravitational Plant Physiology Laboratory in Philadelphia it was found possible to simulate the time dependence of these 5 different acceleration episodes for payload masses up to 59 kg. A commercial low-cost payload device, the "Materials Dispersion Apparatus" of Instrumentation Technology Associates was tested for (1) integrity of mechanical function, (2) retention of fluid in its compartments, and (3) integrity of products under simulated re-entry g-loads. In particular, the sharp rise from 1 g to maximum g-loading that occurs during re-entry in various unmanned vehicles was successfully simulated, conditions were established for reliable functioning of the MDA, and crystals of 5 proteins suspended in compartments filled with mother liquor were subjected to this acceleration load.

Acceleration↗
PubMed1988

Cell bioprocessing in space: applications of analytical cytology.

Cell bioprocessing in space consists of the preparation, cultivation, purification and investigation of cells and their products in the microgravity environment of orbital space flight. Inertial acceleration is used as an independent variable to explore the limits of specific bioprocessing functions, such as cell growth and secretion, gravity-dependent phenomena in cell bioreactors, cell fusion, the influence of thermal convection on processes at cellular dimensions, the electrophoretic separation of cell subpopulations and subcellular particles, and two-phase partitioning of cells, bioparticles, and macromolecules. Analytical cytology techniques are under development for on-orbit application to future cell growth and separation experiments, such as those anticipated in the Space Station era.

Acceleration↗
PubMed1986

Electrophoretic separation and analysis of living cells from solid tissues by several methods. Human embryonic kidney cell cultures as a model.

Preparative electrophoresis of living cells has been considered for some time as a potential tool for isolating, from heterogeneous mixtures, subpopulations of cells according to function. Such a purification depends upon the retention of electrophoretic heterogeneity and the retention of function. Human embryonic kidney cells that had been in monolayer culture for 1-5 subcultivations were resuspended by treatment with trypsin and/or EDTA and suspended in a variety of electrophoresis buffers, ranging in ionic strength from 0.0015 to 0.15 M. Analytical electrophoresis with a Zeiss Cytopherometer or Pen Kem 3000 automated light-scattering electrophoretic analyzer indicated that electrophoretic heterogeneity was retained under the full range of conditions tested. Preparative electrophoresis by three methods--in a density gradient, with continuous flow, and in microgravity--indicated that electrophoretic heterogeneity coincided with functional heterogeneity; for example, some electrophoretically isolated subpopulations produced increased levels of urokinase while others produced increased level of tissue plasminogen activator.

Buffers↗
PubMed1986

The evolving microlesion concept.

The "microlesion concept", introduced by D. Grahn in the 1970's, was further explored using published quantitative carcinogenesis data. Laboratory experiments performed by T. C. H. Yang and co-workers and by R. J. M. Fry and co-workers using the Fe ion beam of the Lawrence Berkeley Laboratory Bevalac have resulted in quantitative data on in vitro and in vivo (respectively) carcinogenesis in mouse systems. These data sets were interpreted on the basis of track calculations, and it was found that the action cross section for tumor induction in cultured cells is approximately 0.032 micrometer2 and that it is about 1/1000th as great in mouse Harderian gland cells. This great difference in carcinogenic sensitivity is a reflection of the biological differences between these two highly promoted systems, neither of which may be quantitatively applicable to humans in space.

Animals↗
PubMed1986

Chemical competition in target radical reactions: numerical simulation of the theory and comparison with measured oxygen effect on DNA damage in cells.

An intracellular radiation-chemical reaction scheme is tested in which solute and solvent radicals R. react with non-target molecules Sa (scavengers) or with target molecules (presumed to be DNA) to produce target radicals T., which may also be produced by direct ionization of DNA. The rate of target radical decomposition to become 'uncommitted damage' that the cell may repair is affected by the concentration of oxygen (O2), thiols (S) and electronaffinic sensitizers (F), which compete with one another to form, respectively, target products TO2, TS and TF. This uncommitted damage is then subject to biochemical modification, including molecular repair, by the cell. The rate equations for this competing reaction scheme were written and programmed for computer simulations of changes in oxygen, thiol and electronaffinic sensitizer concentrations. A reaction scheme that also includes some non-radical target damage was also simulated. Simulations were made using available experimental data concerning intranuclear concentrations and reaction rate constants, respectively, ko, ks and k1 for the reactions T. + O2----TO2, T. + S----TS and T. + F----TF, which produce uncommitted chemical damage. Experimental data on strand-break induction in glutathione-proficient and glutathione-deficient cells, in cells treated with thiol active agents, and in cells treated with hypoxic sensitizers, along with the computer simulations, generally agree that thiol molecules can react with target radicals to reverse T. in competition with O2 and/or electronaffinic sensitizers. Forward reaction rate constants ko, ks (dithiothreitol), ks (glutathione) and k1 (misonidazole) in the approximate ratio 10:0.3:0.02:0.4 satisfied the above reaction scheme, and approximately 5 per cent non-radical target molecule damage could be included with satisfactory agreement with experimental data.

DNA↗
PubMed1985

Lethal, potentially lethal, and nonlethal damage induction by heavy ions in cultured human cells.

In the fields of high-LET radiotherapy and space radiation safety it is important to know the relative probabilities with which a cell whose nucleus is struck by a heavy ion will be damaged or killed. Experiments were performed in which synchronous cultured human T-1 cells (presumptive HeLa) were irradiated with natural alpha particles of energy approximately 3.5 MeV at various times after mitotic selection up to the middle of S phase. Nuclear-area histograms were determined as a function of time after mitosis under conditions identical to those used for irradiation. The efficiency with which one particle passing through the nucleus killed a cell was found to be 0.14-0.20. This value was extrapolated to experimental cell survival data obtained when asynchronous cultured human cells were irradiated with He, Li, B, C, N, O, Ne, Ar ions of energy 6.58 or 5.5 MeV/amu, and the cell killing efficiency was found to be in the broad range of 0.5-1.0 under single-hit conditions. Similarly irradiated cells were examined for colony-size distribution by an image analysis technique, and it was found that the loss of large colonies was dose and LET-dependent in a systematic way. Dose-response data suggest two predominant subpopulations, resistant and sensitive cells, and it appears that the sensitive population is affected by single-hit kinetics. The single-hit coefficient for the induction of inherited slow growth varied with LET in a similar way to that for survival. The action cross section for this form of heritable damage appears to be comparable to the geometric cross section of the cell nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha Particles↗
PubMed1984

Alteration by prolactin of surface charge and membrane fluidity of rat 13762 mammary ascites tumor cells.

Intraperitoneal injection of ovine prolactin (100 micrograms/d) in Fischer 344 rats bearing transplantable 13762 mammary ascites tumor (MAT) cells modifies the surface charge density and membrane fluidity of the tumor cells. In each of five experiments the mean electrophoretic mobility (epm) of MAT cells taken from prolactin-treated rats was significantly lower than that of cells from nonhormone-treated controls. Prolactin concentrations were increased in vivo by (a) direct intraperitoneal injection of ovine prolactin; (b) subcutaneous implantation of diethylstilbestrol-containing silastic capsules to produce pituitary prolactin secreting tumors; or (c) a single subcutaneous injection of polyestradiol phosphate, a long-acting estrogen. In an effort to establish that the prolactin effect was a direct one, two in vivo protocols were used: (a) MAT cells were coincubated with anterior pituitary halves obtained from nontumor-bearing littermates; or (b) rat or ovine prolactin was added to serum-free culture media containing MAT cells. In both protocols, the epm of the prolactin-treated cells was significantly lower. The isoelectric focusing pH of whole cells was increased by prolactin treatment from 4.93 to 5.12, consistent with a reduction in the number of surface carboxyl groups. The fluidity of membranes of treated cells was drastically increased, as measured by spin-label probe rotation rates. These combined results imply that the hormone exerts its effect by stimulating events in the cell that lead to a reduction of the average density of carboxylic acid residues on the tumor cell surface.

Animals↗
PubMed1984

The microlesion concept in HZE particle dosimetry.

High energy, high-Z (HZE) particles are present in high-altitude and high-inclination satellite orbits. Most of the HZE dose above LET = 200 keV/micrometer is due to Fe nuclei. Individual HZE particles can damage several cells adjacent to one another along the particle track in tissue. The outcome has been described as a "microlesion" by D. Grahn. The present study attempts to define conditions for microlesions in specific tissues, to seek biological evidence that microlesions are produced, and to evaluate the microlesion as a potentially useful unit of dose in assessing hazards to spaceworkers. Microlesions in individuals cells and hair follicles have been described. Microbial studies have provided some evidence for independent secondary electron action. Whether or not a few hundred microlesions would be damaging to the whole organism depends upon the nature of damage to critical tissues. For example, cancer may occur if microlesions kill several cells in a straight line and mutate other cells alongside the particle track. Fe particle irradiation of the mouse Harderian gland (Fry et al., this issue) produces tumors efficiently. Microlesions in the lens, cornea, and retina need to be considered. Further dialogue is required before a final decision can be made concerning the most appropriate way to assess the HZE hazard.

Animals↗