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PubMed2002

Flow cytometry analysis of changes in the DNA content of the polychlorinated biphenyl degrader Comamonas testosteroni TK102: effect of metabolites on cell-cell separation.

Flow cytometry was used to monitor changes in the DNA content of the polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 during growth in the presence or absence of PCBs. In culture medium without PCBs, the majority of stationary-phase cells contained a single chromosome. In the presence of PCBs, the percentage of cells containing two chromosomes increased from 12% to approximately 50%. In contrast, addition of PCBs did not change the DNA contents of three species that are unable to degrade PCBs. In addition, highly chlorinated PCBs that are not degraded by TK102 did not result in a change in the DNA content. These results suggest that PCBs did not affect the DNA content of the cells directly; rather, the intermediate metabolites resulting from the degradation of PCBs caused the increase in DNA content. To study the effect of intermediate metabolites on the DNA content of the cells, four bph genes, bphA1, bphB, bphC, and bphD, were disrupted by gene replacement. The resulting mutant strains accumulated intermediate metabolites when they were grown in the presence of PCBs or biphenyl (BP). When the bphB gene was disrupted, the percentage of cells containing two chromosomes increased in cultures grown with PCBs or BP. When grown with BP, cultures of this mutant accumulated two intermediate metabolites, 2-hydroxybiphenyl (2-OHBP) and 3-OHBP. Addition of 2- or 3-OHBP to a wild-type TK102 and non-PCB-degrading species culture also resulted in an increase in the percentage of cells containing two chromosomes. Electron microscopy revealed that cell-cell separation was inhibited in this culture. This is the first report that hydroxy-BPs can inhibit bacterial cell separation while allowing continued DNA replication.

Biodegradation, Environmental↗
PubMed1987

Cytosolic free calcium levels in cultured pituitary cells separated by centrifugal elutriation: effect of gonadotropin-releasing hormone.

The cytosolic concentration of free Ca2+ ([Ca2+]i) in normal rat pituitary cells separated by centrifugal elutriation was monitored with the fluorescent Ca2+ indicator Quin 2. GnRH (10(-7) M) induced a rapid rise (6-8 sec) in the gonadotroph's [Ca2+]i, followed by a plateau phase of prolonged elevated [Ca2+]i which lasted about 15 min. The stimulatory effect of GnRH was dose dependent, with an ED50 of 10(-9) M, and was blocked by the potent antagonist [Dp-Glu1,pclPhe2,DTrp3.6]GnRH. GnRH elevated [Ca2+]i only in gonadotroph-enriched cell fractions, whereas TRH and GH-releasing factor (GRF) elevated [Ca2+]i in mammotroph- and somatotroph-enriched cells fractions, respectively. A rapid increase (first phase) in [Ca2+]i induced by GnRH was observed in Ca2+-free medium containing EGTA, but this rapid phase was terminated within 2 min. Readdition of Ca2+ to the medium induced a second slower rise in [Ca2+]i (plateau phase). Addition of K+ caused a rapid rise in [Ca2+]i, which was dependent on extracellular Ca2+, but was not affected by prior stimulation with GnRH. On the other hand, stimulation of gonadotroph's [Ca2+]i response by GnRH desensitized the cells to a subsequent GnRH challenge within the time frame studied. These findings indicate an elevation of [Ca2+]i induced by GnRH, TRH, and GRF in their respective separated target cells in the rat pituitary. The rise in [Ca2+]i in GnRH-stimulated gonadotrophs originates partly from intracellular Ca2+ pools and partly from influx of Ca2+ across the cell membrane.

Aminoquinolines↗
PubMed1992

Evaluation of a monocyte enrichment procedure using the blood cell separator Fresenius AS 104.

The small number of circulating monocytes in peripheral blood makes it difficult to collect enough cells for studies on the metabolism and analysis of receptor functions of this cell type for diagnostic aims and treatment. Therefore, a new procedure to enrich monocytes from peripheral blood using the cell separator Fresenius AS 104 was developed and evaluated. The separation is done using the tubing set 'P1Y-Gran-collect', which is a closed system with a single-stage separation chamber. The procedure is running continuously, while the cells are harvested cyclically. After processing of 4.9 l whole blood an average yield of (2.47 +/- 2.05) x 10(9) monocytes could be collected with an efficiency of 46 +/- 20%. The monocyte enrichment procedure using the Fresenius AS 104 provides a sufficient number of cells for the next steps: purification and concentration of the cells for drug monitoring, cell culturing, receptor analysis and further in vitro diagnostics.

Blood Cell Count↗
PubMed1986

Serum ferritin estimation in blood donors subject to apheresis on IBM 2997 cell separator.

The serum ferritin concentration was tested in blood donors selected for apheresis on IBM Cell Separator. Of 41 donors, 28 were males and 13 females. All donors exhibited normal hemoglobin and RBC. The mean value of serum ferritin was 90.93 ng/ml in males and 48.38 ng/ml in females. In males--often with repeated blood donations--a low value of serum ferritin suggesting reduced Fe stores in organism was found in one individual only. In contrast, reduced serum ferritin levels were observed in 4 females who, before apheresis, regularly donated blood or had several pregnancies in their anamnesis. The obtained results point to prelatent or latent sideropenia. Serum ferritin concentration was measured in 18 donors selected for apheresis. The examination of ferritin was performed prior to, immediately after, and one week post separation. No significant changes in serum ferritin concentrations due to the separation procedure were observed. Preventive tests of serum ferritin in multiple blood donations and in women with previous pregnancies are recommended.

Blood Component Removal↗
PubMed1979

DNA polymerases in mouse spermatogenic cells separated by sedimentation velocity.

Activity levels of DNA polymerase alpha and DNA polymerase beta have been measured in mouse spermatogenic cells separated by sedimentation velocity. Testes from prepuberal (17 day old) and sexually mature mice were dissociated and separated by unit gravity sedimentation into 6 populations of cells. Phase contrast microscopy and [3H]thymidine labeling kinetics revealed that at least 85% of the cells in fraction A were pachytene-stage primary spermatocytes, fraction B was enriched for primary spermatocytes and round spermatids, fraction C contained spermatogonia and/or pre-leptotene primary spermatocytes and later stages of spermatids (no spermatids were present in fraction C from the testes of 17 day old mice) and fractions D to F contained mixed populations of cells, many in later stages of spermiogenesis. When expressed as activity in 10(6) cells or as a specific activity, fractions A, B, and C from mature animals population initially loaded onto the gradient while fractions D, E and F had activity levels similar to or below the population of dissociated cells. The ratio of activity between the DNA polymerases was constant in fractions A, B, and C, but in fractions D, E, and F, the ratio decreased due to a more rapid decline of activity of polymerase alpha. A comparison of activity levels in fraction C from prepuberal and sexually mature mice revealed an increase in DNA polymerase alpha activity and a decrease in the activity of DNA polymerase beta in the cells from the 17 day old animals.

Animals↗
PubMed1985

Repopulating potential of canine bone marrow cells: differences between large and small cells separated by velocity sedimentation.

This study compares the pattern of haemopoietic recovery in dogs after total-body irradiation and transfusion of different populations of cryopreserved autologous bone marrow cells. Dogs in group 1 received unseparated marrow cells. Group-II dogs were transfused with small (less than 5.1 mm/h) and group-III dogs with large (greater than 7.1 mm/h) bone marrow cells, separated by velocity sedimentation. Myeloid progenitor cells (CFU-GM) present in slowly sedimenting cell fractions were characterized by a higher radiosensitivity in vitro and a lower proportion of cells in S-phase as compared to rapidly sedimenting CFU-GM. Although autografts in all groups contained comparable numbers of CFU-GM, transfusions resulted in different patterns of recovery. Fractions of small bone marrow cells contained most of the pluripotent stem cells, leading to speedy haematological reconstitution and long-term survival. The pattern of early recovery was similar in dogs of group I and of group II. In group III, the recovery in all cell lineages was delayed, going along with marked extramedullary haemopoiesis. The data may indicate the limits of committed progenitors in reflecting pluripotential stem cells, in particular, if bone marrow grafts have been modified in vitro. Differences in the repopulating potential of the graft might be reflected by distinct physical and biological properties of the CFU-GM.

Animals↗
PubMed1989

Two populations of mouse lymphokine-activated killer cells separated by use of soybean agglutinin.

We separated lymphokine-activated killer (LAK) cells induced from spleen cells of BALB/c mice by culturing with recombinant interleukin-2 (rIL-2) into soybean agglutinin-positive (SBA+) and soybean agglutinin-negative (SBA-) fractions with a cell sorter at the time when LAK activity reached a maximum (day 3). We found that the cells with LAK activity were enriched in the SBA+ fraction. Analysis of cell surface phenotypes revealed that the SBA+ cells are of non-T cell origin, while the SBA- fraction consists of T cells. We also found that the large granular lymphocyte (LGL) fraction of spleen cells obtained with a Percoll gradient became SBA+ after culture for 3 days with rIL-2, whereas cells of high density did not. The change in SBA binding sites was also examined on C57BL/6 mouse spleen cells and we found that NK1.1+ non-T cells selectively acquire SBA binding sites at an early stage of activation with rIL-2. On the other hand, sorted SBA- cells gradually acquired SBA binding sites on extended culturing with rIL-2. These results suggest that the expression of SBA binding sites is related to the stage of cell activation with rIL-2, though cells of the NK-lineage became SBA+ at an earlier period of a culture with rIL-2 than cells of the T-lineage. Utilizing these findings, we separated NK-derived LAK cells by means of SBA-binding, and used the separated cells for adoptive immunotherapy for experimental pulmonary metastasis. We found that SBA(+)-NK cell-derived LAK cells showed stronger activity for the inhibition of experimental pulmonary metastasis than SBA(-)-T cell-derived LAK cells.

Animals↗
PubMed2006

The fission yeast Rpb4 subunit of RNA polymerase II plays a specialized role in cell separation.

RNA polymerase II is a complex of 12 subunits, Rpb1 to Rpb12, whose specific roles are only partly understood. Rpb4 is essential in mammals and fission yeast, but not in budding yeast. To learn more about the roles of Rpb4, we expressed the rpb4 gene under the control of regulatable promoters of different strength in fission yeast. We demonstrate that below a critical level of transcription, Rpb4 affects cellular growth proportional to its expression levels: cells expressing lower levels of rpb4 grew slower compared to cells expressing higher levels. Lowered rpb4 expression did not affect cell survival under several stress conditions, but it caused specific defects in cell separation similar to sep mutants. Microarray analysis revealed that lowered rpb4 expression causes a global reduction in gene expression, but the transcript levels of a distinct subset of genes were particularly responsive to changes in rpb4 expression. These genes show some overlap with those regulated by the Sep1-Ace2 transcriptional cascade required for cell separation. Most notably, the gene expression signature of cells with lowered rpb4 expression was highly similar to those of mcs6, pmh1, sep10 and sep15 mutants. Mcs6 and Pmh1 encode orthologs of metazoan TFIIH-associated cyclin-dependent kinase (CDK)-activating kinase (Cdk7-cyclin H-Mat1), while Sep10 and Sep15 encode mediator components. Our results suggest that Rpb4, along with some other general transcription factors, plays a specialized role in a transcriptional pathway that controls the cell cycle-regulated transcription of a specific subset of genes involved in cell division.

Cell Division↗
PubMedDate not supplied

In vitro response to hyperthermia or X-irradiation of diploid and tetraploid RIF-1 cells separated by centrifugal elutriation.

The RIF-1 cell line at the University of Utah is comprised of approximately 65 per cent diploid and 35 per cent tetraploid cells. Because sensitivity to heat cell killing has been shown to be ploidy dependent (Lucke-Huhle 1978), the responses of these subpopulations were examined independently. Diploid and tetraploid cells were separated from stock in vitro cultures by centrifugal elutriation and maintained entirely in vitro. No influence of ploidy on Do or Dq of heat dose survival curves could be detected. Neither did ploidy affect sensitivity to X-irradiation. However, separation of the diploid and tetraploid subpopulations was imperfect. The ratio of diploid to tetraploid cells in the separated and parent (mixed ploidy) lines was therefore monitored at each passage. Tetraploid contamination of the diploid cell subline was undetectable at the time of separation but regrew to 35 per cent by 40 days after separation. Diploid contamination of the tetraploid subline was initially less than 5 per cent and remained quite low until it became undetectable at 74 days. Differences in regrowth of the contaminating subline could not be accounted for by differences in plating efficiency or doubling time, but might result from subpopulation interactions. If so, such phenomena should be considered when using ploidy-dependent cytotoxic treatments.

Cell Line↗
PubMed1975

[Immunological activities of rat lymphocytes. III. --Isolation of differing subpopulations of lymphocytes by two techniques of T cell separation].

In previous studies we have isolated by density gradient separation a population of relatively dense T lymphocytes capable of inhibiting the mitogenic response to concanavalin A (ConA) and phytohaemagglutinin. In this study we compared two current methods of cell separation for their relative yield in T lymphocytes able or not to respond to Con A. The present results show that cells passed through nylon wool columns--in contrast to those obtained by removal of lymphocytes which bind erythrocyte-antibody-complement complex-partially lose their ability to respond to ConA. The response lost by passage through nylon wool columns can be restored by centrifugation of the T cell suspension on a gradient of "Ficoll-Hypaque". This suggests that methods of T cells purification using density gradient separation may remove the more dense suppressor cells.

Animals↗
PubMed1987

Human peripheral blood and bone marrow cell separation using density gradient centrifugation on Lymphoprep and Percoll in haematological diseases.

Density gradient centrifugation on Lymphoprep as described by Böyum [1] was used for mononuclear cell separation of peripheral blood (PB) and bone marrow (BM) samples in healthy subjects and in haematological diseases. The cells were analysed morphologically from cytocentrifuge preparations stained with May-Grünwald-Giemsa. At least 200 cells per preparation were counted manually. The separation results of PB samples in healthy subjects were comparable with those described by Böyum. This method was also suitable for the separation of lymphocytes and blast cells in chronic lymphoproliferative diseases and in acute leukaemias, respectively. In other haematological diseases, however, the mononuclear cell fractions of PB samples and especially of BM samples were contaminated with myeloid and erythroid cells. Percoll gradients were used with success for the enrichment of myeloid BM cells and PB reticulocytes. However, absolutely pure myeloid cell fractions from different maturation stages could not be separated. The results indicate that cells to be investigated should be morphologically examined, to avoid erroneous interpretation of biochemical and functional activity of the cells.

Blood Cells↗
PubMed1995

Plateletpheresis-induced increase in platelet reactivity using different cell separators.

OBJECTIVE: Since plateletpheresis is being used increasingly, it is important to regard quality control to check health risks for donors and to exclude these. DESIGN: Controlled randomised prospective open comparative study. SETTING: Department of Transfusion Medicine of a University Clinic. PARTICIPANTS: 112 platelet donors were examined. INTERVENTIONS: Prior to and after plateletpheresis platelet reactivity was determined. The platelet concentrates in the two groups of 56 donors each were produced using either the cell separator 'CS-3000' and the collecting chamber PLT 30TM with the Omnix system (group I) or the cell separator 'AS-104' (group II). RESULTS: In group I five donors showed a pathologically increased platelet reactivity (p = 0.1297) after plateletpheresis. In group II there were 10 donors with a pathologically increased platelet reactivity (p = 0.0046) after plateletpheresis. The mean concentration of platelets was reduced by separation using the CS-3000 Omnix from 238 +/- 49 x 10(3)/microliters to 172 +/- 32 x 10(3)/microliters (68 +/- 27 x 10(3)/microliters) and from 243 +/- 53 x 10(3)/microliters to 180 +/- 31 x 10(3)/microliters (63 +/- 33 x 10(3)/microliters) using the AS-104. In the first case the platelet yield was 3.9 x 10(11) platelets/concentrate, in the latter case it was 2.9 x 10(11) platelets/concentrate. The 'CS-3000 Omnix' is significantly more effective in separating (58.4 +/- 15.5%) than the 'AS-104' with 44.2 +/- 7.2% (p < 0.0001). CONCLUSIONS: Since both donor groups were comparable regarding all factors recorded--especially the cardiovascular risk factors--the separation process could be responsible for the different traumatisation of platelets.

Adult↗
PubMed2001

Concentration of progenitor cells collected from bone marrow fluid using a continuous flow cell separator.

In ABO major incompatibility on bone marrow transplantation (BMT), red cells must be removed from collected marrow fluid to prevent hemolysis. We report the concentration of progenitor cells collected using a continuous flow cell separator (Cobe Spectra). The average volume of concentrated bone marrow was 132 +/- 47 ml and that of red cells included was 5.1 +/- 2.4 ml. The red cell removal rate was 97.6%. The recovery rate was 40.6% for total nuclear cells, 77.9% for mononuclear cells, 100% for CD34+ cells, and 93.9% for colony forming unit granulocyte-macrophage. Eighteen patients undergoing allogeneic BMT showed no signs of fever or hemolysis during concentrated marrow fluid transfusion. The recovery rate of progenitor cells was high, indicating sufficient recovery of hemopoiesis. This technique is applicable in ABO-incompatible BMT and in frozen-storage stem cells.

ABO Blood-Group System↗
PubMed1980

Control of thrombocytosis by plateletpheresis using a cell separator.

Three patients with marked thrombocythaemia secondary to myeloproliferative disorders were treated with plateletpheresis using an Aminco cell separator and myelosuppressive agents. Immediate control of the raised platelet count was achieved by plateletpheresis and long-term control by the concomitant cytotoxic therapy. The indications for plateletpheresis in patients with marked thrombocytosis are discussed.

Adult↗
PubMed1984

Granulocyte collection: a comparison of Fenwal CS 3000, IBM 2997, and haemonetics cell separators.

With the advent of sophisticated automated blood processors, the collection of large numbers of granulocytes for transfusion has been made more practical in the past ten years. Harvesting granulocytes by filtration leukapheresis has been abandoned in most centers because of adverse reactions in both donors and recipients. Currently, both continuous and discontinuous flow centrifugation leukapheresis techniques are available. However, both corticosteroids and hydroxyethyl starch are required for optimal granulocyte collection. In this paper, we critically compare the three major cell separators used for the collection of granulocytes by centrifugation leukapheresis. All three instruments separate blood cells by centrifugation; the IBM-2997 and the Fenwal CS-3000 function by continuous-flow centrifugation, and the Haemonetics Model 30 by discontinuous-flow centrifugation. Factors such as the donor preapheresis white blood cell count, the blood flow rate through the machine and the centrifuge speed effect granulocyte collection. Comparisons will be made of the cost of the software for each machine, the time required for granulocyte collection, the convenience of set-up and tear-down, the amount of skill and experience demanded of the operator. Donor factors will be discussed. Since the blood processor may be used for other procedures including plateletpheresis, therapeutic plasma exchange, lymphocytapheresis and erthrocytapheresis, the pros and cons of each machine as used for some of these procedures will be included in the discussion. In our experience, total leukocytes and granulocytes (neutrophils) collected with the three different instruments varied only slightly when unstimulated donors were studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Centrifugation↗
PubMed2005

Myoepithelial cells: pathology, cell separation and markers of myoepithelial differentiation.

Until recently the myoepithelial cell has been studied relatively little in terms of its role in breast cancer. A number of malignancies showing myoepithelial differentiation have been reported in the literature, although they are still thought to be relatively rare and only limited studies are published. As a result of recent expression profiling experiments, one type of tumor with myoepithelial features, the so-called 'basal' breast cancer, has received a renewed interest, although it has been known to pathologists for more than two decades. These tumors, which express markers of both luminal and myoepithelial cells, are now being studied using antibodies against some new molecules that have emerged from studies of sorted normal luminal and myoepithelial cells. These immunohistochemical data, combined with genomic studies, may lead to better identification and management of patients with 'basal' tumors.

Animals↗