Effect of tannic acid on the Ehrlich ascites tumor cells.

Tannic acid was used to fix and stain Ehrlich ascites tumor cells in suspension. An increase in surface microvilli and cytoplasmic blebs in the tumor cells was observed. The mechanism of tannic acid induced surface morphological changes in tumor cells and the formation of a precipitate of protein-tannic acid-heavy metal complex are discussed.     

Effect of tannic acid on the Ehrlich ascites tumor cells

Summary Tannic acid was used to fix and stain Ehrlich ascites tumor cells in suspension. An increase in surface microvilli and cytoplasmic blebs in the tumor cells was observed. The mechanism of tannic acid induced surface morphological changes in tumor cells and the formation of a precipitate of protein-tannic acid-heavy metal complex are discussed.Acknowledgment. The author is grateful to Professor F. P. Lisowski for his advice and helpful criticism and to Mr Y. S. Tong for his expert technical assistance. This project was supported by a grant from the Hong Kong University Research Grants Committee.     

Regulation of caldesmon activity by Cdc2 kinase plays an important role in maintaining membrane cortex integrity during cell division

To study the mitosis-specific phosphorylation of caldesmon (CaD), we generated a mutant of the C-terminal fragment (amino acids 244–538) of human fibroblast CaD (CaD39-6F), as well as a mutant of the full-length CaD (CaD-6F), in which all six potential phosphorylation sites for Cdc2 kinase were abolished. The mitotic CaD39-6F-overexpressing cells required more time to progress from anaphase start to 50% cytokinesis, exhibited larger size, and abnormally formed numerous small blebs. In contrast, overexpression of the wild-type C-terminal fragment of CaD (CaD39) did not result in abnormal bleb formation, but led to larger size and prolonged the time requirement between anaphase start and 50% cytokinesis. Similar abnormal blebs were also observed in the CaD-6F-overexpressing cells. CaD-6F-overexpressing cells did not show larger size but required more time to progress from anaphase start to 50% cytokinesis. These results suggest that mitosis-specific phosphorylation of CaD plays a role in inhibiting bleb formation and that the N-terminal fragment of CaD is required for cell size determination. Received 4 September 2002; received after revision 25 November 2002; accepted 4 December 2002     

Tyrosine phosphorylation of cytoplasmic proteins in proliferating, differentiating, apoptotic HL-60 cells and blood neutrophils

Two-dimensional electrophoretic analysis was used to assess quantitative and qualitative changes in the expression and tyrosine phosphorylation of cytoplasmic proteins of proliferating, differentiating HL-60 cells and mature human blood neutrophils. The total tyrosine phosphorylation level of cytoplasmic proteins appeared approximately constant during the pre-commitment period, i.e., 6-24 h after induction of differentiation by 700 nM all-trans retinoic acid. At the time of granulocytic phenotype formation (48-120 h), the total level of tyrosine phosphorylation of cytoplasmic proteins increased significantly. Tyrosine phosphorylation of cytoplasmic proteins in matured blood neutrophils was significantly lower than that of cytoplasmic proteins of HL-60 cells differentiated for 96 h with retinoic acid. Immunoblotting with anti-Erk2 and anti-phosphotyrosine monoclonal IgG2bk antibodies showed that Erk2 was expressed and tyrosine-phosphorylated at different levels in HL-60 proliferating cells and in cells at all stages of differentiation. Our data showed that tyrosine phosphorylation of cytoplasmic proteins in differentiating HL-60 cells changes dramatically during the period of phenotype formation and is accompanied by increasing activity of Erk2. An increasing number of apoptotic cells appeared in the differentiating HL-60 cell population during the granulocyte maturation stage (48-120 h of differentiation). The appearance at this time of differentiation of a new set of tyrosine-phosphorylated cytoplasmic proteins (also distinctive for apoptotic HL-60 cells mediated by etoposide) together with an increasing number of apoptotic cells in the differentiating population strongly suggests that these proteins are associated with the apoptotic process.     

Heparin effects on cultured mammalian cells.

Plasmacytoma cells exposed to heparin exhibited zeiotic blebs in the G1 phase, S phase, and early G2 phase. Zeiosis was not seen in mitotic cells. This heparin effect was reversible. Also fibroblasts were sensitive to heparin. After trypsinization of fibroblasts, heparin produced large non-reversible zeiotic blebs in the cells, except in those in mitosis. The primary target for heparin is apparently to be sought among components of the cellular periphery.     

Scanning electron microscopy of antibody-dependent cell-mediated cytotoxicity of Friend leukemia virus-infected spleen cells

Summary Friend leukemia virus (FLV) infected splenocytes treated with rabbit anti-FLV serum and subsequently incubated with splenic lymphocytes from non-immune Balb/c mice were examined by scanning electron microscopy. Villous-covered lymphocytes adherred to the tumor cells and induced surface blebs, numerous membrane pores and eventual tumor cell lysis.     

Scanning electron microscopy of antibody-dependent cell-mediated cytotoxicity of Friend leukemia virus-infected spleen cells.

Friend leukemia virus (FLV) infected splenocytes treated with rabbit anti-FLV serum and subsequently incubated with splenic lymphocytes from non-immune Balb/c mice were examined by scanning electron microscopy. Villous-covered lymphocytes adherred to the tumor cells and induced surface blebs, numerous membrane pores and eventual tumor cell lysis.     

Ultrastructural aspects of the cell fusion induced by Visna virus on sheep choroid plexus cell in culture]

Sheep choroid plexus cells infected with low multiplicities of infection of Visna Virus were stellate and had long and thin processes containing filaments and forming cytoplasmic bridges between adjacent cells. Enlargement of the bridges resulted in the formation of multinucleated cells. Some glycoproteins were clustered on filaments outside the cell. The cytoplasmic changes showed : an intensive protein synthesis; numerous mitochondria closely associated with filaments and some lysosomes and numerous vesicules near the plasma membrane.     

Heat-shock protein 60 translocates to the surface of apoptotic cells and differentiated megakaryocytes and stimulates phagocytosis

Heat-shock protein 60 (Hsp60) is a highly conserved stress protein which has chaperone functions in prokaryotes and mammalian cells. Hsp60 is associated with the mitochondria and the plasma membrane through phosphorylation by protein kinase A, and is incorporated into lipid membranes as a protein-folding chaperone. Its diverse intracellular chaperone functions include the secretion of proteins where it maintains the conformation of precursors and facilitates their translocation through the plasma membrane. We report here that Hsp60 is concentrated in apoptotic membrane blebs and translocates to the surface of cells undergoing apoptosis. Hsp60 is also enriched in platelets derived from terminally differentiated megakaryocytes and expressed at the surface of senescent platelets. Furthermore, the exposure of monocytic U937 cells to Hsp60 enhanced their phagocytic activity. Our results suggests that externalized Hsp60 in apoptotic cells and senescent platelets influences events subsequent to apoptosis, such as the clearance of apoptotic cells by phagocytes.     

Surface morphological study of ehrlich ascites tumor cells exposed to microware irradiation and heat

Summary Microwave irradiation of EAT cells caused an increase in length and number of surface microvilli. The tumor cells tend to form large aggregates by means of extensive interdigitation of surface microvilli. On the other hand, heat hyperthermia caused a decrease of surface microvilli but an increase of surface blebs. Hence the surface morphology of EAT cells after in vitro exposure to microwave irradiation differs markedly from that after heat hyperthermia.Part of the results was presented a the Fifth meeting of the European Co-operative Hyperthermia Group, Essen, July 1983.     

Role of the pituitary in cyproheptadine-induced pancreatic beta-cell toxicity

Summary Hypophysectomized rats given cyproheptadine (40 mg/kg) for 10 days exhibited a loss of pancreatic immunoreactive insulin and ultrastructural changes in the cytoplasm of beta-cells. Sham-operated animals given cyproheptadine showed identical changes in pancreatic beta-cells except that cytoplasmic involvement progressed to the formation of large vacuoles. The pituitary is not directly involved with the cyproheptadine-induced depletion of pancreatic insulin but plays a role in the formation of large cytoplasmic vacuoles.Acknowledgments. This work was supported by U. S. Public Health Service, grant GM 12675.     

Histochemical localization of carbonic anhydrase in Malpighian tubules ofCulex pipiens

Summary Carbonic anhydrase (CA) activity has been localized histochemically by Hansson's method in Malpighian tubules ofCulex pipiens. The enzyme has been observed on membranes of the cytoplasmic inclusions of Malpighian cells; no CA activity has been found in other cytoplasmic structures. The possible meaning of the localization of the enzyme is discussed.     

Modulation of SMN nuclear foci and cytoplasmic localization by its C-terminus

The survival of motor neuron ( SMN1) gene product, SMN, is detected both in the cytoplasm and in nuclear gems and cajal bodies. We show here that SMN exon 6 is essential both for formation of its nuclear foci and for its cytoplasmic localization. However, exon 7 inhibits the formation of SMN nuclear foci but promotes SMN cytoplasmic localization. More interestingly, we find that a random C-terminal tag of five or more amino acids downstream of exon 6 is sufficient to inhibit the occurrence of multiple nuclear foci and to promote cytoplasmic localization of SMNDelta7, the primary product of the SMN2 gene. Moreover, SMNDelta7 proteins that bear spinal muscular atrophy mutations in exon 6 either showed defects in nuclear foci formation or enhanced cytoplasmic localization. We conclude that exon 6 and exon 7 synergistically regulate SMN distribution that may require specific exon 6 motifs but is independent of specific sequences in exon 7.     

Chemically mediated cell-to-cell contractile activation in isolated frog atrial cardiac cells

Summary A dying single frog atrial cardiac cell liberates an unknown substance which diffuses away from the dying cell and activates contractile activity in other isolated intact single cardiac cells within the vicinity of the dying cell.This investigation was supported by US Public Health Service, National Institutes of Health Grant HL 18943 and a Kansas Heart Association Grant-In-Aid.We wish to thank Dr Alan Chapman of the Department of Anatomy for doing the scanning electron microscopy of the cardiac cells.     

SEM observation of cultured,mitogen-stimulated leukocytes. Cells obtained from patients with acute myeloblastic and chronic lymphocytic leukemias

Summary SEM observation of acute myeloblastic leukemia cells, incubated for 20 h with the mitogens pokeweed and phytohaemagglutinin, showed these to have elongated structures that were either smooth or partially covered by thumblike figures. By contrast, the chronic lymphocytic leukemia cells possessed more compact shapes and some were covered with blebs of varying sizes.Supported by grant No. A3624 from the Natural Sciences and Engineering Research Council of Canada.We would like to acknowledge the help of Mr Jean-Luc Verville and Mrs Diane Keating from this Department. Our thanks also go to Dr J.F. Perdue, Lady Davis Institute for Medical Research of the Jewish General Hospital, Montreal, Canada, for the very generous use of his equipment and also to Mr Pierre Daigneault from M. E. C. A. Laboratories.     

The EM immunocytochemical demonstration of lysozyme in macrophage giant cells in sarcoidosis.

The giant cells (multinucleate macrophages) of human sarcoidosis have been shown by the unlabelled antibody immunoperoxidase technique at electron microscope level to contain lysozyme within cytoplasmic granules.     

S-nitrosoglutathione reductase activity of human and yeast glutathione-dependent formaldehyde dehydrogenase and its nuclear and cytoplasmic localisation

S-nitrosoglutathione (GSNO) formation represents a mechanism for storage and transport of nitric oxide. Analysis of human liver and Saccharomyces cerevisiae extracts has revealed the presence of only one enzyme able to significantly reduce GSNO, identified as glutathione-dependent formaldehyde dehydrogenase (FALDH). GSNO is the best substrate known for the human and yeast enzymes (kcat/Km = 444,400 and 350,000 mM(-1) min(-1), respectively). Although NADH is the preferred cofactor, some activity with NADPH (Km = 460 microM) can be predicted in vivo. The subcellular localization demonstrates a cytosolic and nuclear distribution of FALDH in living yeast cells. This agrees with previous results in rat, and suggests a role in the regulation of GSNO levels in the cytoplasmic and nuclear compartments of the eukaryotic cell.     

Aryl hydrocarbon hydroxylase activity in type II alveolar lung cells

Summary Type II alveolar lung cells metabolize polycyclic aromatic hydrocarbons as indicated by measurements of aryl hydrocarbon hydroxylase activity and binding of tritium labeled benzo(a)pyrene to nuclear and cytoplasmic components.     

Regulation of the type III InsP3 receptor and its role in beta cell function

The type III inositol 1,4,5-trisphosphate receptor (InsP3R) is an important intracellular calcium (Ca2+) release channel in the pancreatic beta cell. Pancreatic beta cells secrete insulin following a characteristic change in membrane potential that leads to an increase in cytoplasmic Ca2+. Both extracellular Ca2+ and Ca2+ mobilized from InsP3-sensitive stores contribute to this increase. RIN-m5F cells, an insulin-secreting beta cell line, preferentially express the type III InsP3R. These cells have been useful in determining the regulatory properties of the type III InsP3R and the role of this isoform in an intact cell. The type III InsP3R is ideal for signal initiation because high cytoplasmic Ca2+ does not inhibit its activity. Altered insulin secretion, the result of changes in Ca2+ handling by the beta cell, has significant clinical consequences.