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PubMed1999

Methotrexate resistance conferred by transplantation of drug-resistant transgenic marrow cells fractionated bycounterflow elutriation.

Introduction of genes conferring drug resistance into hematopoietic cells may allow for improved chemotherapy by protection of normally drug-sensitive cells from the toxic side-effects of antitumor agents. We recently reported that transplantation of murine marrow transgenic for drug-resistant dihydrofolate reductase (DHFR) protected mice from lethal doses of methotrexate (MTX), demonstrating the feasibility of imparting drug resistance to recipients of marrow expressing drug-resistant DHFR activity. In order to optimize this strategy it is necessary to identify the hematopoietic cell populations which mediate drug resistance. For this purpose, we separated committed progenitor populations from primitive hematopoietic cells in DHFR transgenic marrow by counterflow elutriation (CE). As expected, supplementation with a fraction containing committed progenitors afforded protection from MTX-induced aplasia observed early after transplantion in animals administered MTX. In contrast, supplementation with a fraction containing primitive hematopoietic cells depleted of committed progenitors failed to provide immediate protection from early aplasia, but instead contributed to drug resistance 4 to 5 weeks after transplantation. The presence of primitive hematopoietic progenitors in both fractions was evident from Southern hybridization analysis for donor transgenic cells 2 months post-transplant. We conclude that protection from aplasia associated with MTX administration immediately after transplantation requires expression of drug-resistant DHFR activity in more differentiated, committed hematopoietic cells, while primitive DHFR transgenic progenitors contribute to long-term drug resistance. These results help to define the appropriate target cells for improved chemotherapy by protection of hematopoietic cells through the introduction of new genes conferring drug resistance.

Animals↗
PubMed1979

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction.

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: 1) In both cases the cells are arrested in the G1 portion of the cell cycle; 2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.

Animals↗
PubMed1985

Membrane characteristics and osmotic fragility of red cells, fractionated with anglehead centrifugation and counterflow centrifugation.

Red cell populations were separated on the basis of differences in density using anglehead centrifugation and on the basis of differences in mean cell volume using counterflow centrifugation. In the different fractions, mean surface area was calculated, phospholipid and cholesterol content determined as well as the osmotic behaviour in hypotonic salt solutions. Older red cells appeared to be more resistant to hypotonic salt solutions, due to favourable surface area to volume ratio.

Cell Separation↗
PubMed1966

Amino acid incorporation by cell fractions from the oviduct of the laying hen and the synthesis of egg-white proteins.

1. Homogenates of the magnum of the hen oviduct have been fractionated by differential centrifuging. 2. Centrifuging at 600g for 10min. gave a pellet containing most of the particulate material of the cell, but on washing this fraction some particles were removed from it. The washed 600g pellet contained most of the DNA of the homogenate. 3. Centrifuging the 600g supernatants at 10000g for 10min. gave particulate fractions (I particles) richer in RNA than other fractions which were active in incorporating amino acids into protein in isolation. When minced tissue was incubated with radioactive amino acids before homogenizing these particles were the most radioactive of the cell fractions. 4. The pellet obtained by centrifuging the 10000g supernatant at 105000g for 60min. was very small; its RNA/protein ratio was raised compared with that of the homogenate. It only incorporated amino acids in isolation to a small extent or not at all. 5. The 105000g supernatant contained a large proportion of the protein of the homogenate. 6. The I particles could be subfractionated by layering over denser sucrose to give a pellet with lower RNA content and incorporating activity, and also suspended material richer in both these properties. 7. Treatment of the I particles with deoxycholate gave rise to particles sedimenting at 105000g for 60min. containing most of the RNA of the original particles, but only about 34% of the protein, and with a high activity in incorporating amino acids. 8. The I particles, or particles derived from them by deoxycholate treatment, required GTP and phosphoenolpyruvate for the incorporation of free amino acids. The omission of ATP reduced the incorporation to varying extents. Chicken-liver cell sap stimulated the activity. 9. Radioactive amino acids linked to transfer RNA were transferred to protein by I particles. GTP and phosphoenolpyruvate were required for this transfer. The phosphoenolpyruvate requirement could not be replaced by increasing the GTP concentration. ATP partially inhibited the transfer. 10. After incorporation by the cell-free system, the hot-trichloroacetic acid extract, but not the lipid extract, was radioactive. Ribonuclease and puromycin inhibited at low concentrations. Lecithinase-C was much less inhibitory. Transfer of amino acid, from a radioactive lipid-amino acid complex prepared from hen oviduct, was not detected. 11. After short periods of incubation of minced tissue with [(14)C]lysine some of the radioactive protein of the isolated I particles behaved as ovalbumin. The distribution of radioactivity in the protein resembled that in ovalbumin in soluble extracts of the tissue obtained after longer periods of incubation.

Amino Acids↗
PubMed1995

Composition and immunoreactivity of the A60 complex and other cell fractions from Mycobacterium bovis BCG.

Surface static cultures of Mycobacterium bovis BCG contained cells embedded in an extracellular matrix, whose mechanical removal yielded free cells that were pressure disrupted and fractionated into cytoplasm and walls. Cell envelopes were either mechanically disrupted or extracted with detergents. Intracellular and extracellular fractions were analysed for proteins, polysaccharides, and antigen 6O (A60), a major complex immunodominant in tuberculosis. A60 was present in extracellular matrix, cytoplasm and walls: it represented a substantial portion of the proteins and polysaccharides of these fractions. While the protein/polysaccharide ratio varied according to the origin of A60 preparations, the electrophoretic patterns of A60 proteins (which accounted for the immunogenicity of the complex) remained unchanged. Western blots pointed to the proteins present within the 29-45 kDa range as the A60 components endowed with the highest immunogenicity level. Since the most heavily stained protein bands in SDS-PAGE patterns were located outside the region best recognized by antisera, a striking discordance was found between concentration and immunogenicity patterns of A60 proteins. The electrophoretic patterns of A60- and non-A60-proteins from cytoplasm were also different. A60 complexes in dot blots and some electrophoresed A60 proteins reacted with monoclonal antibodies directed against lipoarabinomannan (LAM), a highly immunogenic polymer of cell envelope. This contaminating compound was removed from A60 with organic solvents and detergents. SDS-PAGE and Western blot patterns of proteins from delipidated A60 were similar to those of native A60 proteins.

Antigens, Bacterial↗
PubMed1988

On the type of DNA polymerase activity in neuronal, astroglial, and oligodendroglial cell fractions from young, adult, and old rat brains.

DNA polymerase activity in isolated neuronal, astroglial, and oligodendroglial cell-enriched fractions from rat brains of different ages was measured. Attempts were made to distinguish the total activity into beta and alpha polymerase types making use of inhibitors like ddTTP and aphidicolin. The results indicate that at all the ages studied (16th day embryonic and 1, 225, and greater than 540 days postnatal), neurons possess the highest polymerase activity in comparison with other types of cells. Further, throughout the postnatal life the polymerase present in neuronal cells is of the beta type and this activity remains fairly constant from adult to old age. In contrast, both astroglial and oligodendroglial cells at adult and old stages of life appear to possess other type(s) of polymerase activity in addition to the predominant beta polymerase. It is inferred that neurons, being postmitotic, are equipped with efficient DNA-repair machinery throughout their life span.

Age Factors↗
PubMed2000

Detection of Chlamydia pneumoniae DNA and antigen in the circulating mononuclear cell fractions of humans and koalas.

Chlamydia pneumoniae is a common respiratory pathogen of humans which, in addition to causing disease at the respiratory site, has recently been linked to disease at other body sites. If C. pneumoniae does contribute to disease at nonrespiratory sites, then it must have a mechanism by which it reaches these sites. We analyzed the peripheral blood mononuclear cell (PBMC) fractions from 60 healthy human blood donors for the presence of C. pneumoniae DNA (by ompA PCR) and chlamydial antigens (by genus- and species-specific monoclonal antibody staining). Ten of the sixty (16. 7%) blood donors were C. pneumoniae positive by PCR, and all 10 of these PCR-positive individuals' samples demonstrated specific staining with anti-C. pneumoniae monoclonal antibodies. The only other host naturally infected with C. pneumoniae is the koala, in which the bacterium also causes respiratory infections. We demonstrated the presence of C. pneumoniae DNA and antigens in the PBMC fractions of 30% of 20 koalas tested. Our finding of C. pneumoniae-infected PBMCs in koalas as well as humans suggests that the ability to infect PBMCs and to disseminate from the respiratory site is not restricted to the human biovar of C. pneumoniae but is a general characteristic of this chlamydial species.

Animals↗
PubMed1980

High spontaneous thymidine incorporation into a non-T lymphocyte population in Hodgkin's disease unmasked after cell fractionation.

In order to study the nature of the spontaneously stimulated cells in Hodgkin's disease, lymphocytes from 10 patients with Hodgkin's disease and 10 healthy control subjects were separated into low- and high-density fractions. Both fractions were subdivided into T and non T cell populations by means of rosette sedimentation. In Hodgkin's disease patients, non-T cells with a high density showed a significantly increased spontaneous thymidine incorporation (mean, 5677 cpm; range, 405--26490 cpm) compared to corresponding cells from control subjects (mean, 484 cpm; range, 235--850 cpm), and to unseparated high-density lymphocytes in Hodgkin's disease patients. Evidence is presented that in Hodgkin's disease, the high-density lymphocyte fraction comprises non-T cells with an apparent potency for DNA synthesis which is, however, inhibited by T cells present in the same high-density fraction.

Adult↗
PubMed1985

Biogenesis of the polymeric IgA receptor in rat hepatocytes. II. Localization of its intracellular forms by cell fractionation studies.

In the companion paper (Sztul, E. S., K. E. Howell, and G. E. Palade, J. Cell Biol., 100:1248-1254), we have shown that pulse labeling of hepatic proteins with [35S]cysteine can be obtained in vivo in intact rats. Soluble label clears the plasma in approximately 5 min, and incorporated label reaches peak values in the liver approximately 20 min after injection. In the present study, we show that the 105,000-mol-wt protein (105K), kinetically the earliest intracellular form of secretory component (SC), is the predominant form found, between 5 and 20 min postinjection, in homogeneous rough microsomal fractions. The second kinetically defined form, i.e., 116K, is the predominant species present in relatively homogeneous, light Golgi fractions in which it appears at approximately 15 min, and peaks at approximately 25 min, postinjection. The third kinetically defined form, 120K, is found 30 min after injection as the major SC species (albeit still accompanied by its immediate precursor, 116K), in a sinusoidal plasmalemmal fraction isolated by immunoadsorption to anti-SC-coated Sepharose beads. These findings lead to the following conclusions: (a) SC is synthesized on polysomes attached to the rough endoplasmic reticulum (ER) membrane; (b) it is partially translocated across the ER membrane and core glycosylated co-translationally to give a 105K peptide; (c) 105K moves from the ER to the Golgi complex where it is terminally glycosylated to give the 116K form; (d) the latter moves to the sinusoidal plasmalemma where it appears together with the final mature form, 120K. Kinetic evidence indicates that the vesicular carriers involved in the transport of SC from the Golgi complex to the sinusoidal plasmalemma, and from the latter to the biliary front of the hepatocytes, are present in a Golgi heavy fraction and a crude carrier vesicle fraction from which they remain to be isolated, purified, and characterized.

Animals↗
PubMed1977

Detection of mitogen-activated T and non-T lymphocytes by virus plaque assay. Virus plaque assay on the cells fractionated by unit gravity sedimentation.

Virus plaque assay (VPA) was utilized for the quantitative evaluation of activated lymphocytes. We examined what types of cells, especially which of activated T and non-T lymphocytes, were detected as infective centres after infection with vesicular stomatitis virus. Marked increases in DNA synthesis and in virus-plaque forming cells (V-PFC) were observed not only during the activation of T lymphocytes with Con A, but also, though to a lesser extent, during the activation with lipopolysaccharide (LPS) of non-T lymphocyte preparations of nude spleen from which theta-positive lymphocytes and macrophages were completely depleted. The latter observation was further confirmed by the VPA on the populations enriched in LPS-activated non-T lymphocytes fractionated by the unit gravity sedimentation method. Fast sedimenting cells were found to be more active in DNA synthesis and contained more infective centres after infection than those sedimenting slowly and original unfractionated cells. Both the capacity for DNA synthesis and virus-replication were considered to be general properties accompanying lymphocyte activation.

Animals↗
PubMed1981

Quantitation of the B and A cell fractions in human pancreas from early fetal life to puberty.

The morphological development of B and A cells in human pancreas was studied with immunoperoxidase strains in 172 cases ranging in age from 12 weeks of fetal life to puberty. Tissue was defined as polypeptide rich or poor to take account of the known islet heterogeneity in the gland. Pancreatic cell density was measured and B and A cell development was calculated in terms of (a) volume fraction and (b) density (micron2/100 nuclei). B cell density increased throughout fetal life to reach a peak at the postnatal age of 2 months, whereas A cell density was greatest in the 6 month fetus. The ratio of B to A cell volume fractions is approximately 1.5 throughout most of fetal life and rises postnatally to a stable value of 5 by the age of 2.5 years.

Adolescent↗
PubMed1992

Rat bone marrow erythroid cell fractionation by counter current distribution in non-charge-sensitive two-phase systems.

Counter-current distribution in non charge-sensitive aqueous poly(ethylene glycol)-dextran two phase systems allows the fractionation of rat bone marrow cells into two broad cell subpopulations with different distribution coefficients in a relatively short time. Morphological identification and enzymatic studies suggest that erythroid cells are mainly present in the subpopulation with the higher distribution coefficient. The distribution coefficient and, therefore, surface hydrophobicity of these cells, apparently increase in parallel with an increase in their degree of differentiation and maturation.

Animals↗
PubMed2005

Aminopeptidase P isozyme expression in human tissues and peripheral blood mononuclear cell fractions.

Aminopeptidase P (APP) isoforms specifically remove the N-terminal amino acid from peptides that have a proline residue in the second position. The mRNA levels of three different isoforms, each coded by a different gene, were determined in 16 human tissues and in peripheral blood mononuclear cell (PBMC) fractions by RT-PCR. The cytosolic isoform, APP1, and the cell surface membrane-bound isoform, APP2, are expressed in all of the human tissues and PBMC fractions examined. The very high expression of APP2 mRNA in kidney compared to other tissues was confirmed by enzyme activity measurements. Among the PBMC fractions, APP2 expression is highest in resting CD8(+) T cells, but decreases in these cells following their activation with phytohemagglutinin; in contrast, expression of APP2 increases in CD4(+) T cells upon activation. The third isoform, APP3, is a hypothetical protein identified by nucleotide sequencing. A detailed analysis of its amino acid sequence confirmed that the protein is an aminopeptidase P-like enzyme with greater similarity to Escherichia coli APP than to either APP1 or APP2. Two splice variants of APP3 exist, one of which is predicted to have a mitochondrial localization (APP3m) while the other is cytosolic (APP3c). Both forms are variably expressed in all of the human tissues and PBMC fractions examined.

Alternative Splicing↗
PubMed1984

[Antibiotic concentration in kidney cell fractions].

A two-compartment model of kidney cells is designed. The first compartment is the cytoplasm, the other is a large organelle fraction consisting of lysosomes and mitochondria. Drugs easily accumulated in the second compartment are considered to be harmful to the kidney. Although small molecular antibiotics move across membranes, they reach a certain equilibrium within 30 minutes. Therefore reproducible values of antibiotic concentrations in both fractions can be obtained. The two-compartment model provides a new approach to pharmacokinetic study at a subcellular level.

Anti-Bacterial Agents↗