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Biomedical subjects

D C Dooley

Publications and source records attributed to D C Dooley.

31 records · Page 2Linked to original sources
PubMed1983

Studies of cell separation: a comparison of the osmotic response of human lymphocytes and granulocyte-monocyte progenitor cells.

The feasibility of using hypo- or hypertonic stress to selectively destroy lymphocytes while sparing stem cells was investigated. Lymphocytes were isolated from peripheral blood and exposed to Hanks' balanced salt solutions ranging in concentration from 66 to 2700 mOsm. The Boyle-van't Hoff plot of cell volume versus reciprocal osmolality was linear. Following osmotic stress, viabilities of the lymphocytes and the granulocyte-monocyte progenitor cells (CFUc) were determined. Lymphocyte viability was assessed by tritiated thymidine incorporation following mitogen stimulation. CFUc viability was measured with the soft agar colony assay. Both types of cells were found to possess high osmotic tolerances compared to other blood cells. While progenitor cells in general appeared to survive anisotonic exposure somewhat better than lymphocytes, significant statistical differences were not established for most situations. The highest degree of CFUc enrichment was twofold, but there was a concomitant 50% drop in CFUc survival. These results suggest that osmotic stress is not a useful procedure for the separation of peripheral blood lymphocytes and stem cells.

Bone Marrow↗
PubMed1982

Isolation of large numbers of fully viable human neutrophils: a preparative technique using percoll density gradient centrifugation.

Techniques were developed for the isolation of human neutrophils from large volumes of human blood. This preparative procedure avoids exposure to Ficoll/Hypaque and offers a low cost alternative to centrifugal elutriation. First, leukocyte-enriched suspensions were obtained by treating buffy coats with 2% dextran. The leukocyte suspensions were then fractionated on three steps Percoll density gradients. The gradients were designed to carry large numbers of cells while maintaining resolution of the cell peaks. As a result, at least 320 X 10(6) leukocytes could be processed on a single 12 ml gradient. Extensive testing of the procedure showed that the purity of the neutrophil band was 96.5 +/- 3.16% (n = 50). The band contained 82.4 +/- 14.6% (n = 39) of the neutrophils applied to the gradient. Viability assays (bacterial killing, chemotaxis) demonstrated that neutrophils isolated on Percoll gradients were just as active as those harvested by centrifugal elutriation. Recovery data are presented and compared to the results obtained by others.

Blood Bactericidal Activity↗
PubMed1982

Glycerol permeation of the human granulocyte.

This communication presents a study of the glycerol permeability of the human granulocyte. The study was undertaken to facilitate the use of glycerol as a cryoprotective agent. Granulocytes were purified by centrifugation-elutriation and permeability measured both in monolayer and suspension. Cell permeability was determined by following the passage of 3H-glycerol across the cell membrane. Glycerol efflux was measured at concentrations ranging from 1 microM to 1750 mM (2100 mOsm) under isosmotic and hypotonic conditions. Influx was determined at concentrations between 1 and 100 mM. The monolayer and suspension assays yielded similar kinetics for efflux. Both influx and efflux kinetics were biphasic. The permeability coefficient for efflux was independent of concentration. Since the kinetics were curvilinear, they could not be completely described by a single permeability coefficient. However, "average" values were computed and found to be 1.7 x 10(-5) cm/min for efflux, and 2.8 x 10(-5) cm/min for influx.

Animals↗
PubMed1982

Glycerolization of the human neutrophil for cryopreservation: osmotic response of the cell.

The human neutrophil probably requires a very high intracellular concentration of cryoprotectant to avoid cellular injury during cryopreservation. Accordingly, new techniques were developed so that high concentrations of protectant could be safely used. To accomplish that, it was necessary to overcome the osmotic limitations of the neutrophil as well as problems with cytotoxicity and membrane permeability. To reduce complications from toxicity, glycerol was selected as the protective agent. To minimize osmotic stress, the protectant concentration was changed slowly and gently by a system combining cross-flow filtration and exponential gradient glycerolization. With that system, it was possible to introduce and remove high concentrations of glycerol with little loss of neutrophil viability. For both ascending and descending gradients, calculations were used to follow the changes in intracellular glycerol concentration. Similar calculations were used to determine the relationship between cell volume, gradient slope, and medium osmolality. From those data, guidelines were formulated for the glycerolization and deglycerolization of the human neutrophil.

Cell Membrane Permeability↗
PubMed1981

The effect of osmotic stress on the function of the human granulocyte.

Clinical investigations have shown that the transfusion of human granulocytes can protect the neutropenic patient against acute bacterial infection. The increasing utilization of granulocyte therapy plus an inability to store these cells in liquid media have created a requirement for a cryopreservation technique. However, the cryopreservation of human granulocytes has proven quite difficult to achieve. It is likely that one of the contributing factors is the lability of these cells under osmotic stress. The purpose of this paper was to precisely define the osmotic tolerance limits of the human granulocyte and thereby provide guidelines for the laboratory manipulation of this cell. To accomplish this, cells were exposed to hypertonic or hypotonic media and then tested for viability under isotonic conditions. Function tests included phagocytosis of particles, bacterial killing, migration in the Boyden chamber, and migration under agarose. The experiments revealed that granulocytes will tolerate a narrow range of osmolalities extending from 200 mOsm to 600-700 mOsm. The osmotic damage occurs in less than 30 seconds and appears to be irreversible. The hypertonic limit of the cell cannot be extended by reducing the temperature to 0 degrees C during the osmotic challenge.

Blood Bactericidal Activity↗
PubMed1980

Permeation of Chinese hamster ovary cells by glycerol: mechanism and kinetics.

When monolayer cultures of Chinese hamster ovary cells were exposed to 3H-glycerol ranging in concentration from 0.1 microM to 200 mM, glycerol in flux was found to increase in direct proportion to the extracellular concentration of glycerol. Other experiments indicated that the same relationship existed at concentrations in excess of 1.0 M. Similarly, glycerol efflux was found to vary in direct proportion to the intracellular concentration of glycerol. In neither case could influx or efflux be saturated. Glycerol influx was not affected by depletion of ATP, alkylation by parachloromercuribenzene sulfonic acid, or exposure to persantine. Altering the pH or temperature also had little effect. Attempts at demonstrating countertransport of glycerol were negative. These data indicate that glycerol probably passes through the membrane by a non-mediated process. For cells in monolayer, the kinetics of influx and efflux are biphasic. Similar biphasic kinetics are observed with cells in suspension culture. A close fit to the data may be obtained by adding together two first-order curves. The pair of curves for influx are clearly different from the pair for efflux. The fit provided by the two pairs of first-order functions suggested that glycerol might diffuse into and out of two intracellular compartments. However, the experimental data do not agree with the predicted behavior of a two-compartment system. As a result, the exact nature of the diffusion limiting steps which are described by the first-order equations remains undefined.

Animals↗
PubMed1979

Ordered replication of DNA sequences: synthesis of mouse satellite and adjacent main band sequences.

The replication of mouse satellite DNA was delayed when synchronized 3T3 cells were exposed to low concentrations of hydroxyurea during S phase, It appears that the onset of satellite replication is not a time dependent event, but instead requires that a certain amount of main band DNA be synthesized first. Using hydroxyapatite chromatography and S1 nuclease digestion, a procedure was developed to quantitate the synthesis of both satellite and neighboring main band sequences. The replication kinetics of satellite determined by this method agree with previous estimates. Main band sequences adjacent to satellite appear to replicate in concert with satellite DNA. The results are discussed and related to the limitations of the techniques utilized.

Animals↗
PubMed1977

Replication kinetics of three DNA sequence families in synchronized mouse cells.

Balb/C3T3 cells entered the quiescent Go state following serum deprivation. On addition of fresh serum, more than 95% of the culture resumed growth, but with asynchronous kinetics. If hydroxyurea were added just before the first cells reached S phase, at at least 90% of the cells accumulated at the Gl/S border over the next ten hours. When the block was removed, the culture moved synchronously into S phase. As the cells traversed S, the replication kinetics of three classes of rapidly renaturing DNA were analyzed. Main band highly repeated DNA and foldback DNA replicated continuously, In contrast, satellite DNA replication did not commence until three hours into S, whereupon its rate of synthesis increased ver rapidly, reaching a maximum with the next two hours. These results are discussed in the light of earlier work utilizing other methods of cell synchronization.

Animals↗
PubMed1976

Endonucleases and simian virus 40 virions: origin of a virion-associated endonuclease.

The origin and role of the endonuclease activity associated with purified virions of simian virus 40, previously described by this and other laboratories, have been further investigated. We found that the enzymatic activity from virions of temperature-sensitive (ts) mutants is not more heat labile than that from wild-type virions. This result was obtained for a variety of ts mutants, including three of the tsA class, and in experiments in which the enzyme was tested in both the presence and absence of viral particles. Comparison of the viron enzyme with endonucleases prepared from either serum or nuclei of uninfected cells reveals a similarity between the viron and serum enzymes based on chromatographic behavior and relative activity with different cations. Virus particles prepared free of this endonuclease were still infectious. We were unsuccessful in uncovering endonuclease in such preparations upon disruption. These data emphasize the necessity for caution in interpreting the role of particle-associated enzymes.

Animals↗
PubMed1973

Deoxyribonucleic acid-membrane complexes in the Bacillus subtilis transformation system.

The fate of transforming deoxyribonucleic acid (DNA) in Bacillus subtilis was studied by isolating the DNA-membrane complex on Renografin gradients. Soon after uptake, transforming DNA binds to the cell membrane and displays a greater resistance to shear than the recipient genome-membrane complex. Ten minutes after uptake, a portion of the donor DNA is released from the membrane. Most of the released donor radioactivity represents unintegrated, biologically inactive DNA. Recombinant or integrated DNA is enriched 1.5- to 1.7-fold in the membrane. This enrichment last at least 30 min after termination of DNA uptake, and probably much longer. The data suggest that transforming DNA may be integrated into the recipient genome on, or close to, the cell membrane.

Bacillus subtilis↗
PubMed1971

Macromolecular synthesis in Bacillus subtilis during development of the competent state.

Rates of deoxyribonucleic acid, ribonucleic acid, and protein synthesis were examined in purified competent cells of Bacillus subtilis during the development of the transformable state. To become competent, a cell must depart from the normal course of vegetative growth and pass through a precompetent phase beginning as early as 90 to 180 min before the appearance of transformability. While in the precompetent state, the cell decreases its rate of deoxyribonucleic acid synthesis and lowers its ratio of ribonucleic acid synthesis to protein synthesis. This altered pattern of synthesis eventually leads to a decreased buoyant density of precompetent cells. Once a cell has become both precompetent and low in density, it can be converted to a competent (transformable) cell. The early alterations in macromolecular synthesis were found in two competence regimens, one utilizing a nutritional step-down and one free of such a shift. The data imply that the precompetent state is a generalized characteristic of the B. subtilis transformation system and is not specific to the procedure used to allow competence development. Since precompetence-specific events occur very early in a competence regimen, we conclude that the induction of precompetence is unrelated to sporulation or a nutritional shift.

Bacillus subtilis↗
PubMedDate not supplied

Density gradient isolation of peripheral blood mononuclear cells using a blood cell processor.

Large numbers of mononuclear cells (MNC) are needed for hematologic reconstitution using peripheral blood stem cells. The possibility of isolating those cells by discontinuous Ficoll-diatrizoate density gradient centrifugation in two blood cell processors (the Haemonetics V50 [V50] and the Cobe 2991 [2991]) were examined. Buffy coats from peripheral blood containing 6.23 X 10(8) MNC were separated in the V50, resulting in a recovery of 75 percent. The purity of the cells, defined as the percentage of lymphocytes and monocytes among all leukocytes, was 95 percent. With larger cell loads (3 to 7 X 10(9) MNC), the yield was higher in the V50 than in the Cobe 2991 (92 versus 75%). After separation in the V50 or the 2991, the cloning efficiencies of hematopoietic progenitor cells (CFU-GM and BFUe) were not different from those of cells isolated on 5 ml Ficoll-diatrizoate gradients in centrifuge tubes. Both leukapheresis and MNC separation can be carried out with the same bowl and tubing set in the V50. With that approach, an average of 6 X 10(9) MNC were processed in 16 experiments. An average recovery of 82 percent with 95 percent purity was achieved. The authors conclude that, in terms of simplicity of operation, cost effectiveness, and maintenance of sterility, the V50 may be better suited than the 2991 for the purification of MNC from peripheral blood.

Cell Separation↗