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At least 127 records · Page 7Linked to original sources
PubMed2001

The Cbk1p pathway is important for polarized cell growth and cell separation in Saccharomyces cerevisiae.

During the early stages of budding, cell wall remodeling and polarized secretion are concentrated at the bud tip (apical growth). The CBK1 gene, encoding a putative serine/threonine protein kinase, was identified in a screen designed to isolate mutations that affect apical growth. Analysis of cbk1Delta cells reveals that Cbk1p is required for efficient apical growth, proper mating projection morphology, bipolar bud site selection in diploid cells, and cell separation. Epitope-tagged Cbk1p localizes to both sides of the bud neck in late anaphase, just prior to cell separation. CBK1 and another gene, HYM1, were previously identified in a screen for genes involved in transcriptional repression and proposed to function in the same pathway. Deletion of HYM1 causes phenotypes similar to those observed in cbk1Delta cells and disrupts the bud neck localization of Cbk1p. Whole-genome transcriptional analysis of cbk1Delta suggests that the kinase regulates the expression of a number of genes with cell wall-related functions, including two genes required for efficient cell separation: the chitinase-encoding gene CTS1 and the glucanase-encoding gene SCW11. The Ace2p transcription factor is required for expression of CTS1 and has been shown to physically interact with Cbk1p. Analysis of ace2Delta cells reveals that Ace2p is required for cell separation but not for polarized growth. Our results suggest that Cbk1p and Hym1p function to regulate two distinct cell morphogenesis pathways: an ACE2-independent pathway that is required for efficient apical growth and mating projection formation and an ACE2-dependent pathway that is required for efficient cell separation following cytokinesis. Cbk1p is most closely related to the Neurospora crassa Cot-1; Schizosaccharomyces pombe Orb6; Caenorhabditis elegans, Drosophila, and human Ndr; and Drosophila and mammalian WARTS/LATS kinases. Many Cbk1-related kinases have been shown to regulate cellular morphology.

Amino Acid Sequence↗
PubMed2003

The endo-beta-1,3-glucanase eng1p is required for dissolution of the primary septum during cell separation in Schizosaccharomyces pombe.

Schizosaccharomyces pombe cells divide by medial fission throughout contraction of an actomyosin ring and deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomoysin ring and septum assembly, little information is available concerning the mechanism of cell separation. Here we describe the characterization of eng1+, a new gene that encodes a protein with detectable endo-beta-1,3-glucanase activity and whose deletion is not lethal to the cells but does interfere in their separation. Electron microscopic observation of mutant cells indicated that this defect is mainly due to the failure of the cells to degrade the primary septum, a structure rich in beta-1,3-glucans, that separates the two sisters cells. Expression of eng1+ varies during the cell cycle, maximum expression being observed before septation, and the protein localizes to a ring-like structure that surrounds the septum region during cell separation. This suggests that it could also be involved in the cleavage of the cylinder of the cell wall that covers the division septum. The expression of eng1+ during vegetative growth is regulated by a C2H2 zinc-finger protein (encoded by the SPAC6G10.12c ORF), which shows significant sequence similarity to the Saccharomyces cerevisiae ScAce2p, especially in the zinc-finger region. Mutants lacking this transcriptional regulator (which we have named ace2+) show a severe cell separation defect, hyphal growth being observed. Thus, ace2p may regulate the expression of the eng1+ gene together with that of other genes whose products are also involved in cell separation.

Base Sequence↗
PubMed2001

Ex vivo evaluation of PBMNCs collected with a new cell separator.

BACKGROUND: This study reports on an evaluation of the ability of a cell separator (Amicus, Baxter Healthcare) and the integral MNC computer software program to collect a variety of MNC subsets. The collection efficiency (CE) of the Amicus for these MNC subsets was compared to that of another cell separator (CS-3000 Plus, Baxter). The collected MNCs were also assayed ex vivo to determine if these cells remained functional. STUDY DESIGN AND METHODS: Healthy volunteer blood donors were recruited to provide PBMNCs for the isolation of CD3+, CD4+, CD8+, CD19+, NK, and gammadelta+ cells and monocytes. Cells were collected with an Amicus (test arm; n = 16) or a CS-3000 Plus (control arm; n = 11) cell separator. Cells were counted on a flow cytometer and CEs were calculated. For functional studies, the Amicus-collected MNC data were compared to CS-3000 Plus historical data. Functional studies performed included surface antigen expression assays (CD8+), proliferation assays (CD4+ and CD8+ cells), NK cytotoxicity assays for K562 and HUVE cells, and E-selectin induction on endothelial cells through NK+ contact dependency. Dendritic cells (DCs) were generated from CD34+ cells collected on the Amicus, positively selected by the use of antibody-bound, magnetic bead technology, and then cultured ex vivo with a combination of growth factors to generate the DCs. RESULTS: CEs were higher on the Amicus than on the CS-3000 Plus for CD3+ (68 vs. 54%), CD4+ (70 vs. 56%), CD8+ (68 vs. 52%), and CD19+ (60 vs. 48%) cells (p<0.05). For the two separators, CEs were equivalent for monocytes, NK+, and gammadelta+ cells. The Amicus separator collected significantly fewer platelets than did the CS-3000 Plus (p<0.00001). CD4+, CD8+, and NK cells proliferated normally. NK cells appropriately stimulated E-selectin expression on endothelial cells. Culture-generated DCs obtained by using Amicus-collected CD34+ cells expressed appropriate cell surface markers. CONCLUSION: The Amicus separator is acceptable for the collection of PBMNC subsets. The device collects CD3+, CD4+, CD8+, and CD19+ T- and B-cell subsets with greater efficiency and collects MNCs with significantly fewer contaminating platelets than does the CS-3000 Plus. Cells collected on the Amicus are suitable for use in a variety of research and clinical immunobiologic studies.

Blood Donors↗
PubMed1978

In situ lymphoid cells of mouse mammary tumors. III. In vitro stimulation of tumor cell survival by lymphoid cells separated from mammary tumors.

Lymphoid cells were isolated by isokinetic gradient centrifugation from mouse mammary tumors and evaluated for their ability to affect tumor cell survival with the microcytotoxicity assay. In all experiments stimulation of the growth or survival of tumor cells was seen. This stimulation was not seen with separated lymphoid cells treated with anti-lymphocyte serum and complement. The lymphoid cells separated from tumors were more stimulatory than were lymph node cells; even toxic to the tumor cells, the separated tumor-associated lymphoid cells were markedly stimulatory. The gradient procedure had no effect on the activity of sensitized lymph node cells, but it did separate cytotoxicity and stimulation into different fractions.

Animals↗
PubMed1974

Cell separation in blue-green bacteria.

Autolysin-like enzymes appear to be responsible for cell separation in Agmenellum quadruplicatum. Mutants that are impaired in cell separation and grow as chains exhibit reduced cell lytic activity. Lysozyme, extracted autolysin, and antibiotics that affect peptidoglycan synthesis phenotypically suppress chain formation. Various aspects of the regulation of the cell separation process were also examined. Studies involving antibiotic inhibitors of macromolecular synthesis and general growth inhibitors provided no evidence for the active regulation of the cell separation process during the latter portion of the division cycle. Evidence was obtained, however, for the partial restriction of peptidogly-can hydrolysis by unknown secondary modifications. The thin electron-dense layer of peptidoglycan along the sides of cells was much more resistant to hydrolysis by egg-white lysozyme than was the septum between daughter cells. The middle portion of the septum was more sensitive than was the layer immediately adjacent to the cytoplasmic membrane. Under conditions that would not osmotically stabilize spheroplasts, lysozyme facilitates rapid cell separation in chain-forming mutants with little leakage of cellular protein or loss of viability.

Anti-Bacterial Agents↗
PubMed1997

Two alternative mechanisms of cell separation in staphylococci: one lytic and one mechanical.

Electron microscopy studies revealed two different mechanisms of cell separation in Staphylococcus aureus. Both mechanisms were initiated by the centrifugal lytic action (directed outward from the center) of murosomes, which perforated the peripheral cell wall. Thereafter, during the first type of cell separation, murosomes also lysed large parts of the cross wall proper in the opposite, i.e., centripetal direction, forming spokelike lytic lesions ("separation scars") next to the most prominent structure of the cross wall, the splitting system. This bidirectional lytic action of murosomes revealed that the staphylococcal cross wall is composed of permanent and transitory parts; transitory parts constituted about one-third of the volume of the total cross wall and seemed to be digested during cell separation. The second mechanism of cell separation was encountered within the splitting system, which has been regarded as the main control unit for lytic cell separation for more than 25 years. The splitting system, however, represents mainly a mechanical aid for cell separation and becomes effective when cell-wall autolytic activities are insufficient.

Microscopy, Electron↗
PubMed1979

An immunological approach to cell separation of the central nervous system.

In this paper I review the various steps of development required for an immunological approach to cell separation. Key points include choice of brain region, isolation procedure, ligand selection, functional assays for separated cell population, and selection of separation procedure. I review briefly some results we have obtained using cerebellar cell populations isolated from 10-day-mice.

Animals↗
PubMed1984

Surface histamine receptors in rat peritoneal mast cells. Separation of receptor-bearing cells via binding to a histamine-protein conjugate, Cell-ect.

The Seragen Cell-ect Total Histamine KitTM was used to separate histamine receptor-bearing rat peritoneal mast cells from cells lacking these receptors. It was found that approximately 75% of the peritoneal mast cell population carried cell surface histamine receptors. The results further suggest that the mast cell histamine receptors present are mainly of an H1-type, as judged by the capacity of a specific H1-antagonist to reduce the histamine receptor-dependent cell adhesion. Moreover, an H1-agonist is less efficient in this respect and an H2-antagonist does not affect the cell adhesion at all. A possible functional role for these receptors, however, remains to be clarified.

Animals↗
PubMed2006

A new D,L-endopeptidase gene product, YojL (renamed CwlS), plays a role in cell separation with LytE and LytF in Bacillus subtilis.

A new peptidoglycan hydrolase, Bacillus subtilis YojL (cell wall-lytic enzyme associated with cell separation, renamed CwlS), exhibits high amino acid sequence similarity to LytE (CwlF) and LytF (CwlE), which are associated with cell separation. The N-terminal region of CwlS has four tandem repeat regions (LysM repeats) predicted to be a peptidoglycan-binding module. The C-terminal region exhibits high similarity to the cell wall hydrolase domains of LytE and LytF at their C-terminal ends. The C-terminal region of CwlS produced in Escherichia coli could hydrolyze the linkage of d-gamma-glutamyl-meso-diaminopimelic acid in the cell wall of B. subtilis, suggesting that CwlS is a d,l-endopeptidase. beta-Galactosidase fusion experiments and Northern hybridization analysis suggested that the cwlS gene is transcribed during the late vegetative and early stationary phases. A cwlS mutant exhibited a cell shape similar to that of the wild type; however, a lytE lytF cwlS triple mutant exhibited aggregated microfiber formation. Moreover, immunofluorescence microscopy showed that FLAG-tagged CwlS was localized at cell separation sites and cell poles during the late vegetative phase. The localization sites are similar to those of LytF and LytE, indicating that CwlS is involved in cell separation with LytF and LytE. These specific localizations may be dependent on the LysM repeats in their N-terminal domains. The roles of CwlS, LytF, and LytE in cell separation are discussed.

Bacillus subtilis↗
PubMed1998

Platelet collection with the Amicus and the AS.TEC 204 blood cell separators.

BACKGROUND: Platelet collection with two new blood cell separators was compared with respect to platelet yield, separation efficiency, white cell (WBC) contamination, and measures of component quality. Both machines offer a menu for the collection of a desired platelet yield. The harvesting of a definite platelet yield is a means of collecting a standardized therapeutic platelet dose. STUDY DESIGN AND METHODS: One hundred seventy-two platelet collections were carried out with different protocols in the two devices (Amicus and AS.TEC 204). For that reason, healthy donors underwent plateletpheresis procedures using the continuous- and discontinuous-flow systems. Separations with a definite endpoint volume and the option of collecting a desired platelet yield of 5.5 x 10(11) in the Amicus and 3.3 x 10(11) in the AS.TEC 204 machine were investigated. RESULTS: With the Amicus blood cell separator, platelet yield (5.02+/-0.46 x 10[11]) and separation efficiency (66.9+/-6.2%) were significantly higher than those with the AS.TEC 204 (3.02+/-0.62 x 10[11] and 46.4+/-6.7%, respectively). There was less deviation between the platelet yield and the desired platelet yield in the AS.TEC 204 machine than in the Amicus device. WBC contamination was 2.06+/-2.91 x 10(6) in components obtained from the AS.TEC 204 machine, and 87.5 percent of the platelet concentrates had a WBC content <5 x 10(6). None of the components from the Amicus device contained more than 5 x 10(6) WBCs. Except for platelet morphology score, measures of component quality were almost equal in the platelet concentrates obtained from the studied machines. CONCLUSION: Platelet yield and separation efficiency were significantly higher in the Amicus blood cell separator. The collection of a desired platelet yield was better achieved with the AS.TEC 204 device.

Blood Component Removal↗
PubMed1975

Simple method for a medium-cell separation.

The present method makes possible a quick separation of cells from the medium with the aid of strips of filter paper and physiological solution. Cell suspension is put on the strip and all water soluble components are washed away by soaking in saline solution, while cells remain on the spot. The experiment on 14-C-uridine uptake proved the suitability of the method for membrane-transport studies.

Animals↗
PubMed1979

Electrophoretic cell separation by means of microspheres.

The electrophoretic mobility of fixed human erythrocytes immunologically labeled with poly(vinylpyridine) or poly(glutaraldehyde) microspheres was reduced by approximately 40%. This observation was utilized in preparative scale electrophoretic separations of fixed human and turkey erythrocytes, the mobilities of which under normal physiological conditions do not differ sufficiently to allow their separation by continuous flow electrophoresis. We suggest that resolution in the electrophoretic separation of cell subpopulations, currently limited by finite and often overlapping mobility distributions, may be significantly enhanced by immunospecific labeling of target populations using microspheres.

Animals↗
PubMed1983

[Thrombopoiesis, thrombocyte count and thrombocyte function before and following cell separation].

About 50% of the circulating platelets can be taken from healthy donors by means of the cell separation technique. In order to investigate the effect of cell separation on thrombopoesis bone marrow samples were taken from 12 voluntary donors up to 5 times. The megacaryocytes were analyzed before and after cell separation by morphometric measures and determinations of platelet count and platelet function (adhesion, aggregation, spreading, retraction) were carried out. Removing of large amounts of platelets form the circulation by stimulation of thrombopoesis resulted in an increase of the peripherical platelet count already 48 h after cell separation. Of the platelet functions tested only aggregation induced by collagen was found to be increased after cell separation as compared to that before the procedure.

Blood Platelets↗
PubMed1993

A soap technique for cell separation to study the seed coat of Sesbania punicea.

A technique is described for separating plant cells used for morphological studies. The plant material is placed in a concentrated solution of olive oil castile soap for 1-2 days or more. The material is then thoroughly washed and placed between two glass slides. The upper glass slide is lifted from the lower one, then gently pressed down several times. Through this procedure Malpighian cells of the seed coat of Sesbania punicea, mesophyll cells of Euphorbia peplus and of Trifolium pratense and cortical cells of the aerial roots of Monstera deliciosa have been separated. Various shapes of the Malpighian cells of the Sesbania punicea seed coat can be observed along with intermediates.

Cell Separation↗
PubMed1990

[Comparison of 3 types of cell separators].

The aim of the present investigation was to compare the efficiency of three commercially available cell separators with respect to thrombocyte yields. In addition, advantages and disadvantages of the different cell separator types were studied. There was no difference in the total number of platelets harvested by the different cell separators. In contrast the thrombocyte count of packages from the V 50 device was significantly lower as compared with the others (p = 0.05). We conclude that the cell separators tested are comparable with respect to the efficiency of processing platelet concentrates. However, the larger amount of plasma of the V 50 might be a disadvantage in some circumstances. The AS 104 has advantages in the handling and the duration of cell separation. In addition it offers technical improvements such as control of the ACD flow by a separate adjustable pump and a hemolysis detector.

Blood Transfusion↗