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Biomedical subjects

T G Pretlow

Publications and source records attributed to T G Pretlow.

At least 109 records · Page 6Linked to original sources
PubMed1978

Velocity sedimentation of organelles at low centrifugal force in an isokinetic gradient.

Mast-cell granules and polystyrene microspheres (0.600 and 1.011 micrometer in diameter) were sedimented in a previously described [Pretlow (1971) Anal. Biochem. 41, 248--255] isokinetic gradient in a low-speed centrifuge. For the analytical velocity sedimentation of organelles, this gradient offers several advantages over gradients that are commonly used for the sedimentation of organelles: (a) the density gradient (0.0008 g.ml-1.cm-1) is small, and the effective densities of organelles will change relatively little during sedimentation; (b) the densities at all points in the gradient (1.017--1.027 g/ml) are less than those in gradients commonly used for the sedimentation of organelles, the effective densities of sedimenting organelles are consequently relatively large, and the effect of density as a determinant of velocity of sedimentation is less limiting than in conventional gradients; (c) the small slope of the gradient is associated with a relatively slow increase in the viscosity encountered by the sedimenting organelle; (d) the iso-osmotic gradient is not significantly affected by the gradient medium (Ficoll), and the osmolarity can be adjusted to the desired value by the selection of an appropriate salt solution as the solvent for the Ficoll; (e) the gradient will be isokinetic for particles of densities similar to most organelles. An ultracentrifuge is not required for work with this gradient.

Centrifugation, Density Gradient↗
PubMed1978

Subpopulation of human tonsillar lymphocytes with mitochondria visible by light microscopy.

We observed a subpopulation of human tonsillar lymphocytes with cytoplasmic inclusions that stained orthochromatically blue with Wright stain. These lymphocytes represented fewer than 2% of cells in suspensions of cells from most tonsils; however, suspensions of tonsillar cells from 4 to 18 studied patients contained more than 2% (2.8%--6.4%) lymphocytes with cytoplasmic inclusions. These cells sedimented less rapidly than the modal population of lymphocytes in a previously described isokinetic gradient. Histochemical and ultrastructural studies suggested that the stained inclusions were prominent mitochondria stained by the Wright stain.

Cell Separation↗
PubMed1978

Cytologic appearance of cells dissociated from rat colon and their separation by isokinetic and isopyknic sedimentation in gradients of Ficol.

Several methods for obtaining colon epithelial cells in suspension were compared. The largest number of epithelial cells per gram of colon was obtained in suspension when the colon was dissociated with 0.1% Pronase. Fivefold more cells per gram of tissue were obtained from the proximal 5 cm of colon than from the terminal 5 cm of colon. Several well-defined types of cells were observed in low frequency. They have not been described previously in normal colon. Suspensions of cells from colons of F344 rats always contained copious mucoid gel that was partially eliminated by washing the cells three times in culture medium with 10% fetal calf serum. Velocity sedimentation in a previously described isokinetic gradient of Ficoll in tissue culture medium permitted the separation of epithelial cells from lymphocytes and red blood cells. Type 2 cells, believed to be a subpopulation of epithelial cells, were obtained in a maximum purity of 95.7 +/- 1.2%. The frequency of 8 other cell types and their purification, when possible, are presented. Epithelial cells were separated from most bacteria and could be cultured with antibiotics after separation in the density gradient.

Animals↗
PubMed1978

Isolation of lymphocyte populations.

Lymphocytes are smaller than most mammalian cells. Although their densities are heterogeneous and overlap those of other mammalian cells, the average lymphocyte is less dense than the vast majority of mammalian cells. Because of these physical properties, lymphocytes sediment less rapidly than most mammalian cells during velocity sedimentation in the isokinetic gradient. Our separations of lymphocytes from several kinds of tissues including human blood, transplantable tumors, and the tumor of Hodgkin's disease are described.

Animals↗
PubMed1978

Prostate cancer: normal prostate from human and hamster.

"Prostatic acid phosphatase" is a term that has been used widely and ambiguously to refer to acid phosphatase, which 1) is elevated in the sera of patients with various diseases of the prostate, 2) is inhibited by one or more specific inhibitors, 3) attacks one or more specific substrates, 4) has certain unique antigenic properties, 5) is extracted from homogenates of prostate, and 6) is obtained from prostate secretions, etc. Most of the data adduced to justify this term is indirect. We have purified specific kinds of cells from prostates and other tissues. These purified cells have served as sources of enzymes known to be derived from particular kinds of cells. We studied several substrates and one inhibitor that have been claimed useful for the measurement of prostatic acid phosphatase. None of the substrates or inhibitors studied appeared to offer much "specificity," which would allow us to distinguish acid phosphatase activity from prostatic epithelial cells from acid phosphatase activities from several other kinds of purified cells.

Acid Phosphatase↗
PubMed1977

The interaction of N-acetylhexosaminidase with insolubilized concanavalin A.

The specific interaction between human N-acetylhexosaminidase and concanavalin A was evaluated with respect to temperature, time, pH and concentration of specific ligand in incubation mixtures containing the enzyme and insolubilized lectin. Elution of the enzyme from insolubilized concanavalin A is dependent on both temperature and concentration of alpha-methyl mannoside. Conditions for a high yield of the enzyme from chromatography on insolubilized concanavalin A are described.

Chemical Phenomena↗
PubMed1977

Separation of proximal tubule cells from suspensions of rat kidney cells by free-flow electrophoresis.

Suspensions of rat kidney cells obtained by disaggregation of the kidney with 0.25% trypsin were separated by electrophoresis. Previously, we found a correlation between cells with histochemically demonstrable alkaline phosphatase (HDAP) and cells with brush borders which established that HDAP is a useful marker for rat proximal tubule cells (Kreisberg et al., '77). The starting suspension of cells for electrophoresis consisted of 38.4 +/- 5.7% nucleated cells with HDAP, 39.8 +/- 5.7% nucleated cells without HDAP, and 20.8 +/- 9.2% red blood cells. After electrophoresis, the purest fraction contained 85.8 +/- 3.5% nucleated cells with HDAP, 8.4 +/- 2.2% nucleated cells lacking HDAP, and 5.8 +/- 2.8% red blood cells; 91.9 +/- 2.4% of the nucleated cells in the purest fractions had HDAP.

Alkaline Phosphatase↗
PubMed1977

Partial purification of human colonic carcinoma cells by sedimentation.

We have purified epithelial cells from human colonic tumours by velocity sedimentation in an isokinetic density gradient of Ficoll in tissue culture medium. In frozen sections of colonic carcinoma, histochemically demonstrable N-acetyl-beta-D-glucosaminidase (HDAG) was observed primarily in epithelial cells. We used this enzyme as a histochemical marker of epithelial cells. Initial suspensions of cells from colonic tumours suspended with 0-25% trypsin contained an average of 24% of the nucleated cells with HDAG. In the purest fraction obtained from gradient centrifugations, an average of 74% of the nucleated cells contained HDAG. After centrifugation, the quarter of the density gradient which contained the most rapidly sedimenting cells was purified 2-4-fold over that in the initial suspension. Cells in this zone of the gradient also gave rise to colonies in soft agar. Cells from initial suspension resulted in 15-25% as many colonies of 7 or more cells in cultures inoculated with the same number of nucleated cells. For the most part, cells obtained from the other zones of the gradient did not give rise to colonies in soft agar.

Acetylglucosaminidase↗
PubMed1977

Separation of lymphocytes and mast cells from the Furth transplantable mast cell tumor in an isokinetic gradient of Ficoll in tissue culture medium.

Cell suspensions of the transplantable Furth murine mast cell tumor were separated both by velocity sedimentation in an isokinetic gradient and by isopyknic sedimentation. Prior to separation, the suspension of tumor cells contained 60.3+/-13.1% (S.D.) malignant mast cells, 9.8+/-10.4% lymphocytes, 4.3+/-2.1% granulocytes, 1.7+/-1.9% macrophages, 0.6+/-0.4% unidentified cells, and 22.8+/-8.5% red blood cells. After either isokinetic or isopyknic sedimentation, more than 97% of the nucleated cells in the purest modal fraction were malignant mast cells. Velocity sedimentation in the isokinetic gradient offered several advantages over isopyknic separation of this tumor; namely, in isokinetic sedimentation, the cells are exposed to a lower centrifugal force for a shorter period of time; a much larger proportion of mast cells were in the highly purified zone of the gradient following velocity sedimentation; and lymphocytes were more highly purified (88.9+/-10.1% of the nucleated cells) following velocity sedimentation. Granulocytes and macrophages were purified more than 8-fold over the nucleated cells in the starting sample suspension. The purified cells from this tumor offer the opportunity to study the interactions between highly purified, easily identified, malignant cells and cells that may participate in the defense of the host against cancer.

Cell Separation↗
PubMed1977

Glucose-6-phosphate dehydrogenase isoenzymes in acinar epithelial cells separated from the mammary gland of the rat at intervals during the course of lactation.

Acinar epithelial cells were purifed from suspensions of cells from the lactating mammary glands of rats. As described previously, this purififaction was accomplished by sedimentation in an isokinetic gradient of Ficoll (polysucrose) in tissue culture medium, and the purity of the separated cells was established by electron microscopy and by histochemical markers. Isoenzymes of glucose-6-phosphate dehydrogenase were investigated at various intervals during lactation in separated populations of stromal and acinar cells. Acinar cells contained three- to eightfold more glucose-6-phosphate dehydrogenase activity than did stromal cells. The proportions of the respective isoenzymes varied during the course of lactation, and the observed changes were parallel in purified acinar cells and in the lactating mammary glands from which the cell suspensions were obtained. The availability of purified acinar cells in the investigation of interactions between hormones and cells from the mammary gland permits a greater degree of specificity than has been possible in the study of mammary cell suspensions which contain myoepithelial cells, duct cells, acinar cells, endothelial cells, fibroblasts, fat cells, mast cells, plasma cells, and blood cells.

Animals↗
PubMed1977

"Prostatic acid phosphatase?" A comparison of acid phosphatase activities in epithelial cells, granulocytes, monocytes, lymphocytes, and platelets purified by velocity sedimentation in isokinetic gradients of Ficoll in tissue culture medium.

Numerous investigators have found several substrates and inhibitors to be particularly suited for the demonstration of acid phosphatase of prostatic origin. There has been much controversy over the specificity or lack of specificity of several substrates and inhibitors. We have investigated acid phosphatase activities obtained from several kinds of purified cells. None of the substrates or inhibitors which we studied permitted us to discriminate "prostatic" acid phosphatase from acid phosphatase activities obtained from other kinds of cells.

Acid Phosphatase↗
PubMed1977

Studies on human hyperplastic prostates maintained in organ culture.

Prostates from patients with prostatic hyperplasia were maintained in organ culture for periods up to 8 days. Explants were disaggregated and studied after 2, 4, and 8 days of culture, as well as before culture. Although the data varied from patient to patient, short periods of culture (2 to 4 days) resulted in increased numbers of cells with histochemically demonstrable acid phosphatase; however, by 8 days of culture, cells with histochemically demonstrable acid phosphatase decreased in number. The ratio of acid phosphatase to beta-glucuronidase expressed per cell or per milligram of protein decrease; with time in culture.

Acid Phosphatase↗
PubMed1977

Separation of proximal tubule cells from suspensions of rat kidney cells in density gradients of Ficoll in tissue culture medium.

Rat kidneys were disaggregated with 0.25% trypsin. Cell were separated by velocity sedimentation in a previously described isokinetic gradient, by isopycnic sedimentation, and by velocity sedimentation followed by isopycnic sedimetation. In some fractions from the isokinetic gradient, 71.8+/-2.4+ of the nucleated cells contained histochemically demonstrable alkaline phosphatase (HDAP); in semithin sections, 62.7+/-2.3% of these cells had brush borders. The correspondence between fractions enriched for cells with HDAP and fractions enriched for brush border suggested that HDAP might be a suitable marker for rat proximal tubule cells. These cell constituted 46.5+/-2.6% of the nucleated cells in the starting sample suspension of kidney cells, and 81.9+/-2.7% of nucleated cells in the purified fractions from the gradients. More than 98% of nucleated cells in these fractions excluded typan blue. Following isopycnin centrifugation, the purest fractions contained 87.3+/-1.5% nucleated cells with HDAP, 9.6+/-2.5% nucleated cells iwithout HDAP, and 3.1+/-2.5% red blood cells. These proximal tubule cells had densities of 1.036 to 1.052 g/ml. With rate-zonal separation followed by isopycnic separation, the purest gradient fraction contained 93.0+/-1.9% nucleated cell with HDAP, 6.0+/-2.3% nucleated cells with HDAP, and 1.0+/-0.9% red blood cells. These proximal tubule cells sedimented a density of 1.041 g/ml. More than 98% of these cells excluded trypan blue.

Animals↗
PubMed1977

Functional characterization of cells separated from suspensions of Hodgkin diseases tumor cells in an isokinetic gradient.

Suspensions of cells from Hodgkin disease tumors were separated by velocity sedimentation in an isokinetic Ficall gradient. The separated cells were cultured with mitogens. The modal population of lymphocytes from the tumor contained 50%-75% of all lymphocytes separated from the tumor of each studied patient. Despite the fact that these cells appeared to be mature lymphocytes, the majority of which formed nonimmune E rosettes with sheep red blood cells, they showed a lack of responsiveness to phytohemogglutinin which was almost complete in all but one patient. Lymphocytes which had been obtained in suspension and purified by identical methods from tonsils and spleens without tumors did respond to stimulation by phytohemagglutinin.

Cell Separation↗
PubMed1976

Separation of megakaryocytes from rat bone marrow cells using velocity sedimentation in an isokinetic gradient ficoll in tissue culture medium.

A technique for the purification of rat megakaryocytes is described. Velocity sedimentation in a previously described isokinetic gradient of Ficoll (polysucrose) in tissue culture medium was more effective than isopycnic sedimentation for the purification of megakaryocytes and resulted in preparations of magekaryocytes which contained 2.4 +/- 0.8% (range 1.85-3.60%) megakaryocytes. Megakaryocytes exhibited a broad range of density between 1.06 and 1.15 gm/ml. The inaccuracy which is inherent in the use of velocity sedimentation without isopycnic sedimentation as a means of particle size analysis is discussed.

Animals↗