Red cell magnesium as a function of cell age

The variation in the magnesium content of human red cells as a function of cell age has been measured by atomic absorption spectrophotometry. The cell population was split into different age fractions using discontinuous density gradient centrifugation, since it is known that cell density increases with age. A mathematical model relating predicted cell age to cell density has been developed which allows the quantification of the observed fall-off in magnesium content with cell age. This model suggests that cells lose magnesium monoexponentially with age, the half-life being approximately 100 days. A previously proposed hypothesis that magnesium could enter the cells only at erythropoiesis and then decay monoexponentially predicted a half-life of 22.4 days and is therefore seen to be an oversimplification of magnesium kinetics in the red cell. The relevance of the present findings to pathologic conditions with abnormal red cell magnesium concentrations is discussed.     

The mysterious pulmonary brush cell: a cell in search of a function

Brush cells, also termed tuft, caveolated, multivesicular, and fibrillovesicular cells, are part of the epithelial layer in the gastrointestinal and respiratory tracts. The cells are characterized by the presence of a tuft of blunt, squat microvilli (approximately 120-140/cell) on the cell surface. The microvilli contain filaments that stretch into the underlying cytoplasm. They have a distinctive pear shape with a wide base and a narrow microvillous apex. The function of the pulmonary brush cell is obscure. For this reason, a working group convened on August 23, 2004, in Bethesda, Maryland, to review the physiologic role of the brush (microvillous) cell in normal airways and alveoli and in respiratory diseases involving the alveolar region (e.g., emphysema and fibrosis) and airway disease characterized by either excessive or insufficient amounts of airway fluid (e.g., cystic fibrosis, chronic bronchitis, and exercise-induced asthma). The group formulated several suggestions for future investigation. For example, it would be useful to have a panel of specific markers for the brush cell and in this way separate these cells for culture and more direct examination of their function (e.g., microarray analysis and proteomics). Using quantitative analysis, it was suggested to examine the number and location of the cells in disease models. Understanding the function of these cells in alveoli and airways may provide clues to the pathogenesis of several disease states (e.g., cystic fibrosis and fibrosis) as well as a key for new therapeutic modalities.     

Modulation of synovial cell products by a factor from a human cell line: T lymphocyte induction of a mononuclear cell factor.

A human cell line (U937) can be stimulated to produce a soluble factor (MCF) by either lectin-activated T lymphocytes or their soluble products. In prior studies, we showed that MCF produced by peripheral blood mononuclear cells can increase production of collagenase and prostaglandin E2 by adherent synovial cells obtained from enzymatically dispersed rheumatoid synovium. We show here that peripheral blood T lymphocytes or cloned human T lymphocyte lines are capable of inducing MCF production by the monocyte-like U937 cells. MCF can be demonstrated in the supernatant fluid from cocultures of U937 cells and T lymphocytes that have been stimulated with phytohemagglutinin or concanavalin A for 24-48 hr. In addition, the supernatant fluid from 24-hr lectin-stimulated T lymphocytes can be transferred onto the U937 cells and subsequently, MCF activity can be recovered from the U937 culture medium. The activity of the soluble T cell product on the U937 cells is both time- and dose-dependent. A human cell line capable of MCF production in continuous culture has not been previously available. The use of a monocyte-like cell line (U937) and cloned T lymphocytes now makes it possible to demonstrate the role of discrete cell populations in the production of MCF and other mediators.     

Establishment and characterization of a mantle cell lymphoma cell line

A mantle cell lymphoma (MCL) cell line (JeKo-1) was established from peripheral blood mononuclear cells of a patient with a large cell variant of MCL showing leukaemic conversion. JeKo-1 cells were Epstein-Barr virus negative and showed a B-cell phenotype with IgM⁺, IgD⁺, CD3^?, CD5⁺, CD10^?, CD19⁺, CD20⁺ and CD23^?; they overexpressed cyclin Dl, Bcl-2, c-Myc and Rb proteins. Bcl-1/J_H gene rearrangement was confirmed by polymerase chain reaction, although karyotypic analysis showed 40/41 chromosomes devoid of apparent t(11;14)(q13;q32) translocation. JeKo-1 cells were highly tumourigenic in SCID mice.     

Characterization of a new megakaryocytic cell line: the Dami cell

A new human megakaryocytic cell line (Dami) has been established from the blood of a patient with megakaryoblastic leukemia. The Dami cells grow primarily in suspension with a doubling time of 24 to 30 hours. By light and electron microscopy, the Dami cells range in size from 12 to 120 micron in diameter and have lobulated nuclei characteristic of megakaryocytes. At least 89% of the cells react with monoclonal antibodies against platelet glycoproteins (GP) Ib and IIB/IIIa, and glycophorin. The cells do not react with antibodies against lymphoid, monocyte, granulocyte, or macrophage antigens. Thirteen percent of the cells become polyploid, spontaneously achieving greater than 4N DNA ploidy levels. In response to phorbol myristate acetate (PMA), the proportion of cells with ploidy levels greater than 4N increased threefold and could be separated into discrete ploidy groups. PMA also increased the expression of GPIb, the GPIIb/GPIIIa complex,l and von Willebrand factor. Cytogenetic analysis revealed a human male hyperdiploid karyotype with a modal chromosome number of 54 to 64 and several consistent clonal chromosomal abnormalities. These included a partial deletion of chromosome 5 and a translocation involving chromosome 3. In contrast to other megakaryocytic cell lines in which only a small portion of the cells express the megakaryocytic phenotype, nearly all of the Dami cells express platelet glycoproteins. Thus, the Dami cells provide a superior model in which to study human megakaryocyte biochemistry and differentiation.     

Structure of a cholinergic cell membrane

Cell membranes are complex assemblies of proteins and lipids making transient or long-term associations that have yet to be characterized at a molecular level. Here, cryo-electron microscopy is applied to determine how phospholipids and cholesterol arrange between neighboring proteins (nicotinic acetylcholine receptors) of Torpedo cholinergic membrane. The lipids exhibit distinct properties in the two leaflets of the bilayer, influenced by the protein surfaces and by differences in cholesterol concentration. In the outer leaflet, the lipids show no consistent motif away from the protein surfaces, in keeping with their assumed fluidity. In the inner leaflet, where the cholesterol concentration is higher, the lipids organize into extensive close-packed linear arrays. These arrays are built from the sterol groups of cholesterol and the initial saturated portions of the phospholipid hydrocarbon chains. Together, they create an ordered ∼7 Å-thick “skin” within the hydrophobic core of the bilayer. The packing of lipids in the arrays appears to bear a close relationship to the linear cholesterol arrays that form crystalline monolayers at the air-water interface.

Cell membranes are fundamental components of all living organisms, and intense efforts have been made to understand how the constituent proteins and lipids build their complex bilayer structures (1, 2). While biochemical, biophysical, and X-ray–scattering studies (3–7) have elucidated some important underlying principles, the inherent mobility and wide-ranging heterogeneity of the lipids have limited our ability to determine their three-dimensional structures at a molecular level. Most membrane-directed research has therefore focused on extracted proteins (8), or proteoliposomes (9, 10), viewing lipid molecules immobilized against the protein surfaces or trapped within a protein complex. However, these approaches do not fully recapitulate, or inform on, the protein-lipid and lipid-lipid interplay that exists in cell membranes in situ.The present cryo-electron microscopy (cryo-EM) study exploits the regularity and high protein content of the postsynaptic cell membrane of Torpedo to determine its three-dimensional protein-lipid structure. This membrane has a relatively simple composition, most densely populated by a single protein (nicotinic acetylcholine receptor), embedded in a cholesterol-rich phospholipid bilayer (11–13). Moreover in tubular vesicles, which bud from the isolated membranes (Fig. 1A), the protein arranges on a regular but slightly varying surface lattice (15), as it does in vivo at the Torpedo synapse (14) and at the neuromuscular junction (18). Density maps obtained from such vesicles have shown that cholesterol attaches to specific sites on the protein in both leaflets of the bilayer and assembles into protein-bridging aggregates or microdomains (17, 19).Open in a separate windowFig. 1.Overview and density profile of the lipid bilayer. (A) Isolated cell membrane and budding acetylcholine receptor tubes. In intact tissue, the receptors typically form dimer ribbons packed tightly side to side (14), a regular arrangement that is lost during membrane extraction, but is restored in the budding tubes (15). Since the protein organizes the same way in both contexts, the tube membranes may recapitulate precisely the region of the cell membrane at the synapse where receptors are most densely packed (16). (B) Cross-section determined from segments of tubes having the same lattice dimensions and curvature. The phospholipid headgroup regions in the outer (O) and inner (I) leaflets give rise to a pair of parallel bands in the spaces between individual receptors. MX identifies a helix of the receptor (see C), which substitutes for some of the phospholipid headgroups (17); the arrow points to the receptor’s central water-filled pore. (C) Structure of receptor and cross-sectional 12 Å-thick slabs at the levels of the gray lines, which identify the peaks of density associated with the phospholipid headgroups (17). The receptor is a heteropentamer (stoichiometry: α_δ, α_γ, β, γ, δ), which includes four TM helices (M1-M4) and a transverse submembrane helix, MX, in each subunit. (D) Mean lipid densities in maps 1 and 2 at successive radii across the lipid bilayer. The shaded columns mark the positions of the two density peaks, which are at different distances from the central low-density trough.In earlier studies, the density maps were obtained from the tubes by helical reconstruction, using local averaging to combine the three-dimensional data from multiple subsets (17, 20). Here, the analysis is confined to single well-populated subsets, built from tube segments having the same lattice dimensions and curvature. Although requiring a much larger initial dataset, this approach succeeds in resolving longer-range details of the lipids. Thus, in the inner leaflet of the bilayer, which has the highest cholesterol content, the lipids are now seen to organize into close-packed linear arrays, building within the hydrophobic core a static sterol-hydrocarbon “skin.” Monolayer reconstitution experiments suggest that packing of lipids in these arrays bears a close relationship to the linear cholesterol arrays which form two-dimensional crystals at the air-water interface.     

Purification of a glycoprotein vascular endothelial cell mitogen from a rat glioma-derived cell line.

A growth factor that is mitogenic for vascular endothelial cells, with an ED50 of approximately 1 ng/ml, has been purified 170,000-fold to apparent homogeneity from tissue culture medium conditioned by a rat glioma-derived cell line. The pure protein is a 46-kDa dimer composed of two subunits of equivalent mass as established by comparison of migration in SDS/polyacrylamide gels with and without prior reduction. This glioma-derived growth factor is a glycoprotein and is not mitogenic for BALB/c 3T3 fibroblasts, properties that further distinguish it from other well-characterized vascular endothelial cell mitogens. In contrast to acidic and basic fibroblast growth factors and to platelet-derived endothelial cell growth factor, which have no secretory leader sequences and might only be released by leakage from damaged cells, the glycoprotein nature of this mitogen implies that it is processed through the glycosylating secretory pathway. This secretable growth factor could, therefore, be readily available in the extracellular space under normal physiological conditions in vivo to promote vascular endothelial cell proliferation associated with blood-vessel growth and maintenance.     

Resveratrol inhibits cell growth in a human cholangiocarcinoma cell line

Background/Aims: Cholangiocarcinoma is a devastating tumour with a poor prognosis. An efficient therapy is unavailable in unoperable patients and new drugs are widely sought for and required. Resveratrol (RES) is a natural molecule with a reported anticancer effect, evaluated on different tumour cell lines. We tested the efficacy of RES on a cholangiocarcinoma cell line for the first time. Methods: We used the human SK‐ChA‐1 cell line, cultured in the classical two‐dimensional model and in the three‐dimensional spheroids. After RES exposure morphology, cell viability (colony‐forming assay), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and cancer antigen (CA) 19‐9 medium releases, cellular transglutaminase activity, karyotype and cell cycle were evaluated. Results: Resveratrol inhibited cell growth in both the cell culture systems used (from ?15 to ?80% vs untreated controls) and induced a 40‐fold increase of LDH and ALP activities in the culture medium. Also, transglutaminase (TG) activity increased in the cell lysates, together with a cell cycle perturbation characterised by an accumulation in the G₁/S phase. Karyotype and CA 19‐9 expression were not influenced by the treatment. Conclusions: The observed cytotoxic effect of RES on the human cholangiocarcinoma SK‐ChA‐1 cell line cultured two‐ and three‐dimensionally suggests to further analyse its chemotherapic/chemopreventive possibilities for this kind of cancer.     

Capsaicin-induced cell death in a human gastric adenocarcinoma cell line

AIM: Capsaicin, a pungent ingredient found in red pepper, has long been used in spices, food additives, and drugs. Cell death induced by the binding of capsaicin was examined in a human gastric adenocarcinoma cell line (AGS cells). METHODS: By using XTT-based cytotoxicity assay, flow cytometry using the TUNEL method, and quantitation of DNA fragmentation, both cell death and DNA fragmentation were detected in AGS cells treated with capsaicin. By using Western blotting methods, capsaicin reduced the expression of Bcl-2, the antiapoptotic protein, in AGS cells in a concentration-dependent manner. RESULTS: After incubation of AGS cells with capsaicin for 24 h, cell viability decreased significantly in a dose-dependent manner. After incubation of AGS cells with capsaicin for 24 h, apoptotic bodies also significantly increased, and were again correlated with the dose of capsaicin. When the concentration of capsaicin was 1 mmol/L, the amount of DNA fragments also increased. Similar results were also in the lower traces. CONCLUSION: These results suggest that capsaicin-induced cell death might be via a Bcl-2 sensitive apoptotic pathway. Therefore, capsaicin might induce protection from gastric cancer.     

Isolation of a vascular cell wall-specific monoclonal antibody recognizing a cell polarity by using a phage display subtraction method

Using a strategy consisting of (i) the isolation of cell walls from synchronously differentiating cells of Zinnia, (ii) the generation of mAbs with an antibody phage display method, and (iii) screening with a subtraction method, we isolated mAbs recognizing vascular development-specific cell wall components without prior antigen identification. One of the isolated mAbs, designated CN 8, recognized a cell wall component contained in the hemicellulosic fraction. Immunohistochemical analyses showed that the CN 8 epitope was localized to the cell wall of immature tracheary elements and xylem parenchyma cells. In immature tracheary elements, the CN 8 epitope had a polarized localization pattern regardless of whether the cells are formed as parts of vessels in situ or as single tracheary elements in vitro, suggesting that cell polarity autonomously formed on the cell wall may function in tracheary element differentiation.     

Capsaicin-induced cell death in a human gastric adenocarcinoma cell line

AIM: Capsaicin, a pungent ingredient found in red pepper,has long been used in spices, food additives, and drugs.Cell death induced by the binding of capsaicin was examined in a human gastric adenocarcinoma cell line (AGS cells).METHODS: By using XTT-based cytotoxicityassay, flow cytometry using the TUNEL method, and quantitation of DNA fragmentation, both cell death and DNA fragmentation were detected in AGS cells treated with capsaicin. By using Western blotting methods, capsaicin reduced the expression of Bcl-2, the antiapoptotic protein, in AGS cells in a concentration-dependent manner.RESULTS: After incubation of AGS cells with capsaicin for 24 h, cell viability decreased significantly in a dose-dependent manner. After incubation of AGS cells with capsaicin for 24 h, apoptotic bodies also significantly increased, and were again correlated with the dose of capsaicin. When the concentration of capsaicin was 1 mmol/L, the amount of DNA fragments also increased. Similar results werealso in the lower traces.CONCLUSION: These results suggest that capsaicininduced cell death might be via a Bcl-2 sensitive apoptotic pathway. Therefore, capsaicin might induce protection from gastric cancer.     

Bone marrow sinus cell packing: a determinant of cell release.

Ultrastructural studies of erythropoietin effects on the bone marrow of control and hypertransfused (65 hct) mice revealed a decrease in adventitial cell cover of the sinus apertures in erythropoietin-treated animals. A more striking finding, however, was the marked inhibition of erythropoietin-induced reticulocytosis by hypertransfusion itself. Hypertransfusion of the erythropoietin-treated animals appeared to decrease the reticulocyte response by inhibiting reticulocyte response by marrow cords in addition to inhibiting erythroid proliferation. This inhibition of reticulocyte response was associated with clustering of reticulocytes around the marrow sinuses which were packed with red cells. Acute lowering of the hematocrit of erythropoietin-treated, hypertransfused animals to normal at the time of maximal reticulocyte response in control animals resulted in more than a twofold increase in reticulocytosis with 2 hr. It is suggested that (1) elevated levels of erythropoietin are associated with a diminution of the normal marrow-peripheral blood barrier, thereby contributing to the premature release of marrow elements and (2) the hematocrit is an important determinant of cell release from the marrow into the peripheral circulation.     

Analysis of co-factor function in a glucocorticoid-resistant small cell carcinoma cell line

Human small cell lung carcinoma (SCLC) tumours exhibit neuroendocrine differentiation, secreting hormones such as ACTH and related peptides. While glucocorticoids inhibit ACTH secretion from the pituitary, this does not occur in SCLC tumours and SCLC cell lines. Failure of glucocorticoids to suppress ACTH peptides is accompanied by a global lack of glucocorticoid action in a number of SCLC cell lines. In the human SCLC cell line, COR L103, activation of a human tyrosine aminotransferase (TAT3)-luciferase reporter gene is resistant to glucocorticoids despite similar glucocorticoid receptor (GR) expression to the glucocorticoid-sensitive A549 human lung cancer cell line; moreover, the GR is free of deleterious mutations. Over-expression of a wild-type GR restores glucocorticoid regulation of TAT3-luciferase, and this is enhanced when the activation function (AF)-2 domain is deleted but much reduced when the AF-1 domain is deleted. This suggests aberrant AF-2 activation domain function. We identified defective steroid receptor co-activator 1 (SRC1) recruitment to the GR AF-2 in COR L103 cells, although SRC1 was successfully recruited to the steroid X receptor with rifampicin, suggesting a defect in the GR. Analysis of other GR C-terminal co-factors identified increased expression of nuclear co-repressor (NCoR) in COR L103 cells. To determine the impact of this, NCoR was over-expressed in A549 cells, where it reduced GR transactivation by 55%. In summary, glucocorticoid resistance is associated with altered SRC protein recruitment and increased expression of NCoR in these SCLC cells, suggesting that glucocorticoid sensitivity may be modified by subtle changes in co-factor recruitment.     

The chiaroscuro stem cell: a unified stem cell theory

Hematopoiesis has been considered hierarchical in nature, but recent data suggest that the system is not hierarchical and is, in fact, quite functionally plastic. Existing data indicate that engraftment and progenitor phenotypes vary inversely with cell cycle transit and that gene expression also varies widely. These observations suggest that there is no progenitor/stem cell hierarchy, but rather a reversible continuum. This may, in turn, be dependent on shifting chromatin and gene expression with cell cycle transit. If the phenotype of these primitive marrow cells changes from engraftable stem cell to progenitor and back to engraftable stem cell with cycle transit, then this suggests that the identity of the engraftable stem cell may be partially masked in nonsynchronized marrow cell populations. A general model indicates a marrow cell that can continually change its surface receptor expression and thus responds to external stimuli differently at different points in the cell cycle.     

Establishment and characterization of a new Epstein-Barr virus transformed cell line from a human B cell lymphoma

Summary We have established a new cell line from a patient with centrocytic B cell lymphoma. Highly purified peripheral blood B cells from patient DUL (WBC counts 158,000/µl) were infected in vitro with Epstein-Barr virus (EBV), and CD 20⁺ B cells were cloned into 96 well culture plates with the aid of a cell sorter autoclone device. As shown by GTG-banding and Southern blot analysis, outgrowing EBV-positive clones had the same chromosomal abnormalities and identical monoclonal IgH gene rearrangement as the original EBV-genome-negative leukemic B cell clone. Surface marker analysis with a panel of monoclonal antibodies revealed identical patterns on EBV-negative and -positive clones, with the exception of PCA 1 (reactive with plasma cells) which was negative on freshly explanted leukemic B cells but positive on EBV-converted clones.List of abbreviations E sheep erythrocytes - EBV Epstein-Barr virus - FCC-NHL follicular centrocytic non-Hodgkin's Lymphoma - FCS fetal calf serum - LCL lymphoblastoid cell line(s) - mab monoclonal antibody - MNC mononuclear cells - NHL non-Hodgkin's lymphoma This work was supported by the Deutsche Forschungsgemeinschaft (SFB 322/B 2)This work forms part of the Ph. D. thesis of O. Janssen     

Characterisation of a new cell line (CJ18) from a patient with 'hairy' cell leukaemia

A cell line (CJ18) has been established from the peripheral blood of a patient with hairy cell leukaemia (HCL) in a leukaemic phase. They grow slowly in large clumps with a doubling time of 3-4 d. 8% show positivity for surface immunoglobulin G and a small percentage (5%) are positive for intracytoplasmic immunoglobulin. They are B1,Ia1 positive and CALL, TdT and OKM1 negative. Although they are Epstein Barr Virus Nuclear Antigen (EBNA) positive they have several features not found in other EBNA positive B lymphoblastoid cell lines which suggest they may be derived from the patient's leukaemic hairy cells. They are strongly positive for tartrate resistant acid phosphatase, esterase positive, contain numerous lysosomes and are able to phagocytose sheep red blood cells after exposure to tetradecanoyl-12, 13-phorbol acetate (TPA). Following exposure to retinoic acid and TPA they adhere to plastic with numerous slender processes, a feature seen in HCL cells.     

Akt as a mediator of cell death

Protein kinase B/Akt possesses prosurvival and antiapoptotic activities and is involved in growth factor-mediated neuronal protection. In this study we establish Akt deactivation as a causal mediator of cell death. Akt deactivation occurs in multiple models of cell death including N-methyl-d-aspartate excitotoxicity, vascular stroke, and nitric oxide (NO)- and hydrogen peroxide (H2O2)-elicited death of HeLa, PC12, and Jurkat T cells. Akt deactivation characterizes both caspase-dependent and -independent cell death. Conditions rescuing cell death, such as treatment with poly(ADP-ribose) polymerase or NO synthase inhibitors and preconditioning with sublethal concentrations of N-methyl-d-aspartate, restore Akt activity. Infection of neurons with adenovirus expressing constitutively active Akt prevents excitotoxicity, whereas phosphatidylinositol 3-kinase inhibitors or infection with dominant negative Akt induce death of untreated neuronal cells.