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  • Articles  (204)
  • Apoptosis  (163)
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  • Springer  (204)
  • 1995-1999  (204)
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  • Articles  (204)
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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Cellular and molecular life sciences 52 (1996), S. 942-949 
    ISSN: 1420-9071
    Keywords: Apoptosis ; transglutaminase ; signalling ; gene expression ; promoter elements ; retinoic acids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Clarification of the molecular details of forms of natural cell death, including apoptosis, has become one of the most challenging issues of contemporary biomedical sciences. One of the effector elements of various cell death pathways is the covalent cross-linking of cellular proteins by transglutaminases. This review will discuss the accumulating data related to the induction and regulation of these enzymes, particularly of tissue type transglutaminase, in the molecular program of cell death. A wide range of signalling pathways can lead to the parallel induction of apoptosis and transglutaminase, providing a handle for better understanding the exact molecular interactions responsible for the mechanism of regulated cell death.
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  • 2
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    Springer
    Cellular and molecular life sciences 52 (1996), S. 950-956 
    ISSN: 1420-9071
    Keywords: Apoptosis ; integrins ; lectins ; macrophage ; phagocytosis ; scavenger receptors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Effective removal of dying cells is crucial to a variety of processes in health and disease. Cells undergoing apoptosis are recognized and ingested intact by phagocytes, which are not stimulated to release inflammatory mediators. The alternative uncontrolled form of cell death, necrosis, is associated with release of cell contents with the potential to cause tissue damage and inflammation. Four distinct molecular mechanisms have been identified to date which mediate recognition by phagocytes of mammalian cells undergoing apoptosis, but further mechanisms remain to be discovered. The capacity for phagocyte removal of cells undergoing apoptosis may be closely regulated, for example by local cytokines.
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  • 3
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    Springer
    Cellular and molecular life sciences 52 (1996), S. 979-994 
    ISSN: 1420-9071
    Keywords: Apoptosis ; protein tyrosine kinase ; protein kinase C ; cell cycle ; PI3-kinase ; DNA-PK
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The transmission of signals from the plasma membrane to the nucleus involves a number of different pathways all of which have in common protein modification. The modification is primarily in the form of phosphorylation which leads to the activation of a series of protein kinases. It is now evident that these pathways are common to stimuli that lead to mitogenic and apoptotic responses. Even the same stimuli under different physiological conditions can cause either cell proliferation or apoptosis. Activation of specific protein kinases can in some circumstances protect against cell death, while in others it protects the cell against apoptosis. Some of the pathways involved lead to activation of transcription factors and the subsequent induction of genes involved in the process of cell death or proliferation. In other cases, such as for the tumour suppressor gene product p53, activation may be initiated both at the level of gene expression or through pre-existing proteins. Yet in others, while the initial steps in the pathway are ill-defined, it is clear that downstream activation of a series of cysteine proteases is instrumental in pushing the cell towards apoptosis. In this report we review the involvement of protein kinases at several different levels in the control of cell behavior.
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  • 4
    ISSN: 1420-9071
    Keywords: Apoptosis ; Ca2+/Mg2+-dependent endonuclease ; cumulus cell ; granulosa cell ; pig
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The porcine antral follicles, 3–6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, high activity of neutral Ca2+/Mg2+-dependent endonuclease and DNA ladder formation, estimated by electrophoresis, were noted in granulosa cells but not in cumulus cells. Extremely low activity of the endonuclease and no DNA ladder formation were observed in both types of cells obtained from healthy follicles. Moreover, apoptotic cells were observed histochemically among granulosa cells only. A good correlation (r=0.987) between the endonuclease activity of granulosa cells and the progesterone/estradiol ratio of follicular fluid in each follicle was found. These results suggest that apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pigs.
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  • 5
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    Cellular and molecular life sciences 52 (1996), S. 968-978 
    ISSN: 1420-9071
    Keywords: Apoptosis ; proteases ; ICE-like proteases ; protein substrates
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The interleukin-1 β-converting enzyme (ICE)-like family proteases have recently been identified as key enzymes in apoptotic cell death. Among these proteases one can identify specific activities which may be involved in cytokine production or in resident protein cleavage. Several factors influence the constitutive apoptotic mechanism and may provide insight into the role of protease(s) in apoptosis. Although it appears that ICE family members play a most important role in promoting apoptotic cell death, evidence has been advanced that other proteases are also involved in sequential or parallel steps of apoptosis. Activation of a particular protease can lead to processing molecules either of the same or different proteases, leading to an activation of a protease cascade. Here we attempt to summarize the current thinking concerning these proteases and their involvement in apoptosis.
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  • 6
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    Springer
    Cellular and molecular life sciences 52 (1996), S. 933-941 
    ISSN: 1420-9071
    Keywords: Apoptosis ; necrosis ; macromolecular degradation ; oncogenes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The process of programmed cell death, or apoptosis, has become one of the most intensively studied topics in biological sciences in the last two decades. Apoptosis as a common and universal mechanism of cell death, distinguishable from necrosis, is now a widely accepted concept after the landmark paper by Kerr, Wyllie and Currie in the early seventies [1]. Different components of the death machinery in eukaryotes are discussed in this issue.
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  • 7
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    Cellular and molecular life sciences 52 (1996), S. 957-962 
    ISSN: 1420-9071
    Keywords: Apoptosis ; cyclophilin ; DNA degradation ; NUC18 ; endonuclease
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Recent research has focused on identifying the biochemical events associated with the apoptotic process. These include specific degradation of the chromatin which was described by Wyllie in 1980 [1], with the report of the appearance of discretely sized DNA fragments from apoptotic rat thymocytes. The fragments corresponded in size to strands of DNA that were cleaved at internucleosomal regions and create a ‘ladder patterns’ when electrophoresed on an agarose gel. Because of its near universality, internucleosomal DNA degradation is considered a diagnostic hallmark of cells undergoing apoptosis. It is of great interest to identify the enzymes involved, and some of the candidates will be discussed.
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  • 8
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    Cellular and molecular life sciences 52 (1996), S. 995-1000 
    ISSN: 1420-9071
    Keywords: Apoptosis ; dNTP metabolism ; thymidine kinase ; interleukin-3 ; blc-2 ; hemopoietic cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Fidelity in DNA synthesis and repair is largely dependent on a balanced supply of deoxynucleotide triphosphate (dNTP) pools. Results from different groups have shown that alterations in dNTP supply result in DNA fragmentation and cell death with characteristics of apoptosis. We have recently shown that in apoptosis driven by deprivation of interleukin-3 (IL-3) in a murine hemopoietic cell line, there is a rapid imbalance in the availability of dNTP that precedes DNA fragmentation. In these cells, dNTP pool balance is closely coupled to the function of the salvage pathway of dNTP synthesis. Apoptosis, induced by treatment of these cells with drugs that inhibit the de novo dNTP synthesis, is prevented when dNTP precursors are supplied through the salvage pathway. IL-3 regulates thymidine kinase activity, suggesting that alterations in dNTP metabolism after IL-3 deprivation could be a relevant event in the commitment of hemopoietic cells to apoptosis.
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  • 9
    ISSN: 1572-9729
    Keywords: anaerobic metabolism ; fermentation ; methanogenesis ; nuclear magnetic resonance ; propionate ; syntrophic degradation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Energy, Environment Protection, Nuclear Power Engineering , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Propionate is an important intermediate in the anaerobic degradation of complex organic matter to methane and carbon dioxide. The metabolism of propionate-forming and propionate-degrading bacteria is reviewed here. Propionate is formed during fermentation of polysaccharides, proteins and fats. The study of the fate of 13C-labelled compounds by nuclear magnetic resonance (NMR) spectroscopy has contributed together with other techniques to the present knowledge of the metabolic routes which lead to propionate formation from these substrates. Since propionate oxidation under methanogenic conditions is thermodynamically difficult, propionate often accumulates when the rates of its formation and degradation are unbalanced. Bacteria which are able to degrade propionate to the methanogenic substrates acetate and hydrogen can only perform this reaction when the methanogens consume acetate and hydrogen efficiently. As a consequence, propionate can only be degraded by obligatory syntrophic consortia of microorganisms. NMR techniques were used to study the degradation of propionate by defined and less defined cultures of these syntrophic consortia. Different types of side-reactions were reported, like the reductive carboxylation to butyrate and the reductive acetylation to higher fatty acids.
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  • 10
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    Journal of biomedical science 5 (1998), S. 401-414 
    ISSN: 1423-0127
    Keywords: Oxidative stress ; Reactive oxygen species ; Excitotoxicity ; Hydroxyl radical ; Peroxynitrite ; Antioxidants ; Polyphenols ; Neurodegenerative disorders ; Apoptosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Oxidative insults, whether over-excitation, excessive release of glutamate or ATP caused by stroke, ischemia or inflammation, exposure to ionizing radiation, heavy-metal ions or oxidized lipoproteins may initiate various signaling cascades leading to apoptotic cell death and neurodegenerative disorders. Among the various reactive oxygen species (ROS) generated in the living organism, hydroxyl and peroxynitrite are the most potent and can damage proteins, lipids and nucleic acids. It appears that some natural antioxidants (tocopherol, ascorbic acid and glutathione) and defense enzyme systems (superoxide dismutase, catalase and glutathione peroxidase) may provide some protection against oxidative damage. Recent findings indicate several polyphenols and antioxidant drugs (probucol, seligilline) are effective in protecting the cells from ROS attack. Further development of these antioxidant molecules may be of value in preventing the development of neurodegenerative diseases.
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  • 11
    ISSN: 1423-0127
    Keywords: TNFα ; Fas ; FasL ; Apoptosis ; Cytokine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The CD3+/TCRαβ+ T-cell-mediated hepatic inflammation induced byPropionibacterium acnes could be divided into an acute and a chronic phase. The acute phase occurred within 72 h after injection and displayed hepatic apoptosis. Anti-TNFα antibody inhibited both theP. acnes-induced hepatic apoptosis and lymphocyte infiltration seen in this phase, indicating the involvement of this cytokine. Thereafter, a chronic phase was manifested from days 7 to 14 after injection. It was characterized as granulomatous inflammation admixed with apoptosis of infiltrating lymphocytes and some hepatocytes. Immunohistochemical staining showed that the infiltrating lymphocytes displayed TNFα, TNF type I receptor and a variety of cytokines including IL-1β, IL-4, IL-6, IL-10, IFNγ or IL-12. Interestingly, in naive mice, the arteries in the liver constitutively expressed IFNγ. Its expression appeared to be substantially increased at 48 h, decreased at 72 h, and increased again on day 14 afterP. acnes injection. Furthermore, Fas or FasL was only detected on the lymphocytes within the granuloma. We conclude thatP. acnes can induce a TNFα-mediated acute hepatic apoptosis which subsequently progress to a T-cell-mediated granulomatous hepatitis with increased expression of multiple cytokines and Fas/FasL.
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  • 12
    ISSN: 1423-0127
    Keywords: Thyroid hormone receptor ; Xenopus laevis ; Metamorphosis ; Apoptosis ; Cell proliferation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The thyroid hormone (T3)-dependent amphibian metamorphosis involves degeneration of larval tissues through programmed cell death (apoptosis) and concurrent proliferation and differentiation of adult cell types. As the mediators of the causative effects of T3 on metamorphosis, both thyroid hormone receptor (TR) α and β genes have been found to be expressed in different tissues during this process. In particular, theXenopus TRβ genes have been shown to be regulated by T3 at the transcriptional level and their expression correlates with organ-specific metamorphosis. We demonstrate here by in situ hybridization that theXenopus TRβ genes are regulated in a cell-type specific manner that correlates with tissue transformation. In particular, they are found to be expressed in the larval intestinal epithelial cells prior to their apoptotic degeneration and in the proliferating cells of the adult epithelium, connective tissue, and muscles. However, they are repressed again upon the differentiation of these adult cells. These results implicate that TRβ participates both in inducing apoptosis and stimulating cell proliferation during development.
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  • 13
    ISSN: 1423-0127
    Keywords: Apoptosis ; Cell cycle ; 3-Deazaadenosine analogs ; L1210 lymphocytic leukemia cells ; c-myc ; NF-kappa B
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A new class of potent apogens (apoptosis-inducing agents) has been identified, consisting of 3-deazaadenosine (DZA), 3-deaza-(±)aristeromycin (DZAri) and 1-β-D-arabinofuranosyl-1H-imidazo[4,5-c]pyridine (ara-3-deazaadenine; DZAra-A). They are inhibitors ofS-adenosylhomocysteine hydrolase and indirect inhibitors of methylation. Furthermore, they have also been found to form 3-deaza-nucleotide analogs. The DZA analogs, DZA, DZAri, and DZAra-A, induced DNA fragmentation in a dose- and time-dependent manner, reaching a maximum at 250 µM after 72 h. Cycloheximide at 0.5 µg/ml completely blocked the DNA fragmentation induced by 250 µM of each of the analogs. Interestingly, exogenous 100 µM L-homocysteine thiolactone abrogated the DNA fragmentation caused by DZAri and DZAra-A, but not by DZA. Flow cytometric analysis showed that DZA arrested the cells in the G2/M phase, whereas the S phase was arrested by DZAri. Correlated with the effect of DZA was a rapid decrease in the expression of c-myc, whereasnur77 and GAPDH were unaffected. In comparison, there was an elevated expression of IFN-γ mRNA without apparent change inbax, p53 or GAPDH mRNA after 24 h. After treatment with DZA, there was an elevated expression of NF-κB DNA binding activity, which became more pronounced at 24 h. Simultaneously, there was an apparent disappearance of AP-1 activity. Thus, DZA most likely inhibited the RNA synthesis of c-myc, a reduction of which could trigger a cascade of gene transcription leading to apoptosis in L1210 cells.
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  • 14
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    Journal of biomedical science 6 (1999), S. 433-438 
    ISSN: 1423-0127
    Keywords: AIDS dementia complex ; Antioxidants ; Apoptosis ; Cerebral atrophy ; gp120 ; HIV-1 protease ; Human neuroblastoma cell ; Neuroprotection ; Protease inhibitor (KNI-272)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A significant number of adult male patients with acquired immunodeficiency syndrome develop cerebral atrophy and progressive brain disorders such as dementia complex and neuropsychiatric problems. Upon entering the brain via activated macrophages or microglias, the human immunodeficiency type 1 virus (HIV-1) may produce cytotoxic factors such as HIV-1 envelope protein (gp 120) and protease. Owing to significant proteolysis of nonviral proteins, the protease derived from HIV-1 may be detrimental to brain cells and neurons. Our results revealed that HIV-1 protease, at nanomolar concentrations, was as potent as gp 120 in causing neurotoxicity in human neuroblastoma neurotypic SH-SY5Y cells. As shown by the Oncor ApopTag staining procedure, HIV-1 protease significantly increased the number of apoptotic cells over the serum-free controls. Moreover, HIV-1 protease-induced neurotoxicity was blocked by a selective protease inhibitor, kynostatin (KNI-272). Antioxidants such as 17β-estradiol, melatonin, andS-nitrosoglutathione also prevented protease-induced neurotoxicity. These findings indicate that oxidative proteolysis may mediate HIV-1 protease-induced apoptosis and the degeneration of neurons and other brain cells. Centrally active protease inhibitors and antioxidants may play an important role in preventing cerebral atrophy and associated dementia complex caused by HIV-1.
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  • 15
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    Development genes and evolution 204 (1995), S. 418 
    ISSN: 1432-041X
    Keywords: Lepidopteran insect ; Butterfly wing ; Programmed cell death ; Apoptosis ; Wing morphogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The outline of the adult wing of lepidopteran insects (butterflies and moths) emerges as a result of disappearance of a group of cells at the periphery of the pupal wing. Histological observation of the pupal wing of Pieris rapae showed that, just after apolysis of the wing epithelium from the pupal cuticle, there occurs a rapid and localized decrease of the number of cells at the periphery of the wing. This decrease occurs through cell death, which lasts 1–1.5 days at 20°C. Dying cells lose contact with the neighbouring cells and show condensation of chromatin and cytoplasm. They then appear to be phagocytosed by neighbouring epithelial cells or discharged through the basal surface of the epithelium into the lumen within the wing and taken up by phagocytes. Fragmentation of DNA in the nuclei was detected in the dead cells or their debris. These results indicate that programmed cell death in the lepidopteran wing proceeds through a mechanism closely similar to that of apoptosis in the vertebrate.
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  • 16
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    Development genes and evolution 208 (1998), S. 37-45 
    ISSN: 1432-041X
    Keywords: Key words wingless ; Wnt ; Drosophila ; Brain development ; Apoptosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  We have studied the role of the wingless gene in embryonic brain development of Drosophila. wingless is expressed in a large domain in the anlage of the protocerebrum and also transiently in smaller domains in the anlagen of the deutocerebrum and tritocerebrum. Elimination of the wingless gene in null mutants has dramatic effects on the developing protocerebrum; although initially generated, approximately one half of the protocerebrum is deleted in wingless null mutants by apoptotic cell death at late embryonic stages. Using temperature sensitive mutants, a rescue of the mutant phenotype can be achieved by stage-specific expression of functional wingless protein during embryonic stages 9–10. This time period correlates with that of neuroblast specification but preceeds the generation and subsequent loss of protocerebral neurons. Ectopic wingless over-expression in gain-of-function mutants results in dramatically oversized CNS. We conclude that wingless is required for the development of the anterior protocerebral brain region in Drosophila. We propose that an important role of wingless in this part of the developing brain is the determination of neural cell fate.
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  • 17
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    Journal of biomedical science 4 (1997), S. 269-278 
    ISSN: 1423-0127
    Keywords: c-Myc ; Oncogene ; Transcription ; Cancer ; Metabolism ; Apoptosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Themyc gene was initially discovered as a prototypical retrovirally transduced oncogene. Over the decades, abundant evidence has emerged to support a causal role for the activated cellular gene, c-myc, in animal and human tumors. The gene encodes an oncogenic helix-loop-helix leucine zipper transcription factor that acts as a heterodimer with its partner protein, Max, to activate genes regulating the cell cycle machinery as well as critical metabolic enzymes. The additional ability of c-Myc to repress transcription of differentiation-related genes suggest that c-Myc is a central and key molecular integrator of cell proliferation, differentiation and metabolism.
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  • 18
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    Journal of biomedical science 5 (1998), S. 185-191 
    ISSN: 1423-0127
    Keywords: TGF-β ; Bcl-2 ; Apoptosis ; Antioxidative enzyme ; Reactive oxygen species
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Transforming growth factor-β (TGF-β) has been shown to induce apoptosis on normal hepatocytes and hepatoma cells both in vitro and in vivo. However, how the TGF-β induces apoptosis is still not clear. We examined the expression of anti-apoptosis proteins and sensitivity to TGF-β in three well differentiated human hepatoma cell lines. Two TGF-β sensitive cell lines Hep3B and HuH7 totally lacked Bcl-2. In contrast, the TGF-β resistant HepG2 cells expressed a substantial amount of Bcl-2. All three cell lines expressed equal amounts of Bcl-XL, Bcl-XS and Bax. Overexpression of Bcl-2 in Hep3B and HuH7 cells protected them from TGF-β-induced apoptosis. TGF-β treatment increased intracellular peroxide production and suppressed the expression of glutathione-S-transferase in the Hep3B cells, and these effects were partially suppressed by the overexpression of Bcl-2. These results suggest that Bcl-2 may protect cell from TGF-β-F-induced apoptosis by interfering TGF-β generated signals leading to induce reactive oxygen species production.
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  • 19
    ISSN: 1423-0127
    Keywords: Retroviral gene transfer ; Apoptosis ; Antisense ; Growth factor receptors ; C-Abl
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The c-abl proto-oncogene is ubiquitously expressed during mammalian development. Activated forms of c-Abl proteins are oncogenic and have been shown to suppress apoptosis. The biological role of normal c-Abl protein is unknown. In this study, we have introduced c-abl antisense sequences into various hemopoietic cells by retroviral gene transfer. Introduction and expression of the antisense sequence effectively reduced the amount of c-Abl protein in a number of transduced hemopoietic cells, that consequently underwent apoptosis. When factor-dependent cell lines were examined, we observed that the addition of sufficient amounts of growth factors could suppress apoptosis in myeloid but not in lymphoid lines. The ability of myeloid cells to be rescued by growth factors correlated with upregulation of mRNA level of IL-3 receptor subunits. Our data suggest that c-Abl provides an anti-apoptotic signal during mammalian cell growth, and that myeloid and lymphoid cells are different in their resistance to apoptosis.
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  • 20
    ISSN: 1423-0127
    Keywords: Autoimmunity ; Apoptosis ; IL-2 ; FasL ; Tolerance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Studies of several gene knockout mice suggest an interesting association of a moderate T cell response with systemic autoimmune diseases. In addition, CD95 ligand (FasL) expression in some strains of these mice is impaired. Because FasL is critically involved in regulating peripheral tolerance, there may be a link between autoimmune diseases and a moderate T cell response that cannot activate the FasL gene. Here, we propose that there are two thresholds of T cell activation. When moderately stimulated, T cells can be activated to the low (1st) threshold, which permits the induction of CD40L, IL-2, IL-4, and other components that help the immune response. The high (2nd) activation threshold can only be achieved by a strong and concurrent stimulation through TCR and IL-2R. Once the high threshold is reached, FasL is produced to induce apoptosis of the activated T and B cells. In the absence of the FasL-mediated downregulation, the activated B cells become efficient antigen-presenting cells for self-antigens and excellent responders for T cell help. Such an exacerbating condition, induced by recurrent and moderate activation, favors the development of autoreactive T cells and autoantibody production. Evidence supporting this hypothesis and some predictions that can be tested are described.
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  • 21
    ISSN: 1423-0127
    Keywords: Glioma ; Apoptosis ; Vandate ; Akt ; PKB
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The dual signal hypothesis of apoptosis holds that a common signal can activate both apoptotic and proliferative pathways. The fate of a cell is dependent on which of these two pathways predominates. In the MAPK family of kinases, ERK and JNK have been proposed to mediate apoptosis whereas the PI3K-stimulated kinase, Akt/PKB, has been shown to inhibit apoptosis. The object of this study was to determine the role of these kinases in a glioma model of apoptosis. We have previously shown that K252a induces apoptosis and inhibits kinase activity. In this study we confirm these results and shown that the protein tyrosine phosphatase inhibitor sodium vanadate activates ERK, JNK and Akt/PKB, but does not stimulate proliferation. Vanadate did protect T98G cells from K252a-induced apoptosis, an effect that was abolished by addition of the PI3K inhibitor wortmannin. This suggests that PI3K and Akt/PKB may be responsible for mediating vanadate's protective effect on glioma cells. We conclude that the intracellular balance between protein phosphorylation pathways is a critical determinant of both cell proliferation and cell death.
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  • 22
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    Development genes and evolution 208 (1998), S. 295-303 
    ISSN: 1432-041X
    Keywords: Key words Euprymna scolopes ; Vibrio fischeri ; Apoptosis ; Symbiosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Associations with pathogenic bacteria have recently been shown to initiate apoptotic programs in the cells of their animal hosts, where host cell death is hypothesized to be a response of the immune system, either initiated as a mechanism of host defense or bacterial offense. In this study, we present evidence that bacterial initiation of apoptosis is neither restricted to pathogenesis nor to the initation of an immune response. In the cooperative association between the sepiolid squid Euprymna scolopes and the luminous bacterium Vibrio fischeri, the bacteria induce a dramatic morphogenesis of the host tissues during the first few days of interaction between these partners. The most striking change is the bacteria-triggered loss of an extensive superficial epithelium that potentiates the infection process. Our analyses of these tissues revealed that the bacteria induce apoptosis in the cells that comprise this epithelium within hours of the interaction with bacteria. Ultrastructural analysis revealed that after 24 h the integrity of the epithelium had been lost, i.e., the basement membrane had degenerated and the majority of the cells exhibited signs of apoptosis, most notably chromatin condensation. Analysis of these tissues with probes that reveal intracellular acidification showed that the cells first undergo an initial acidification beginning about 6–8 h after exposure to V. fischeri. As determined by end-labeling of DNA fragments, extensive endonuclease activity was detected at approximately 16–20 h post-infection. These data provide evidence that cooperative bacteria can participate in the remodeling of host tissues through the induction of host apoptotic programs.
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  • 23
    ISSN: 1432-041X
    Keywords: Key words Direct development ; Frog development ; Body wall ; Morphogenetic movement ; Apoptosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Eleutherodactylus coqui develops directly from a large 3.5-mm egg to a froglet, without an intervening tadpole stage. We have examined the development of the body wall, a structure whose behavior has been altered in this derived development. In an event that is unusual for amphibian embryos, the yolk mass is secondarily surrounded by the body wall, which originates near the embryo’s trunk. The epidermis of the body wall is marked by melanophores, and the rectus abdominis, which will form the ventral musculature, is near its leading edge. As the body wall expands, the epidermis, melanophores, and rectus abdominis all move from the dorsal side to close over the yolk at the ventral midline. The original ectoderm over the yolk undergoes apoptosis, as it is replaced by body wall epidermis. Intact muscles are not required for ventral closure of the body wall, despite their normal presence near the advancing edge. Comparative examination of embryos of Xenopus laevis and Rana pipiens suggests that ventral closure does not occur in species with tadpoles. The expansion of dorsal tissues over the yolk, as illustrated by E. coqui, may have been important in the origin of amniote embryos.
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  • 24
    ISSN: 1432-0983
    Keywords: Key words Fission yeast ; Caspases ; Bcl-2 ; Apoptosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two pro-apoptotic proteases, caspase-1 and caspase-3, have been expressed as full-length proteins in the fission yeast Schizosaccharomyces pombe. Both proteins autoprocess to generate the corresponding active enzyme and both are lethal to the yeast cell. Lethality is due to catalytic activity since the expression of the inactive mutant forms of both caspases does not result in an obvious phenotype. Caspase-expressing yeast can be rescued by co-expression of the baculovirus protein p35, a known inhibitor of the caspase family. Co-expression of Bcl-2, another anti-apoptotic protein, does not prevent the cell death induced by either caspase. However, Bcl-2 is itself cleaved by both caspase-1 and caspase-3 at two adjacent recognition sites, YEWD31′A and DAGD34′V respectively, immediately downstream from the N-terminal BH4 domain, a region of Bcl-2 which is essential for its anti-apoptotic activity; similar cleavage of Bcl-2 by caspases has been demonstrated in mammalian cells. Hence, key elements of the apoptotic pathway can be reliably reconstituted in fission yeast, opening the way to exploit yeast in order to study the control of apoptosis. Furthermore, the activity of caspase-3, although not caspase-1, can be demonstrated in vitro using chromogenic substrates. This offers the possibility of using caspase-producing strains of yeast to screen for chemical inhibitors either in vivo or in vitro.
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  • 25
    ISSN: 1432-072X
    Keywords: Key wordsClostridium acetobutylicum ; Lactate ; utilization ; Acetate utilization ; Acetone-butanol ; fermentation ; Lactate dehydrogenase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Clostridium acetobutylicum strain P262 utilized lactate at a rapid rate [600 nmol min–1 (mg protein)–1], but lactate could not serve as the sole energy source. When acetate was provided as a co-substrate, the growth rate was 0.05 h–1. Butyrate, carbon dioxide and hydrogen were the end products of lactate and acetate utilization, and the stoichiometry was 1 lactate + 0.4 acetate → 0.7 butyrate + 0.6 H2 + 1 CO2. Lactate-grown cells had twofold lower hydrogenase than glucose-grown cells, and the lactate-grown cells used acetate as an alternative electron acceptor. The cells had a poor affinity for lactate (Ks = 1.1 mM), and there was no evidence for active transport. Lactate utilization was catabolyzed by an inducible NAD-independent lactate dehydrogenase (iLDH) that had a pH optimum of 7.5. The iLDH was fivefold more active with d-lactate than l-lactate, and the K m for d-lactate was 3.2 mM. Lactate-grown cells had little butyraldehyde dehydrogenase activity, and this defect did not allow the conversion of lactate to butanol.
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  • 26
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    Journal of industrial microbiology and biotechnology 15 (1995), S. 414-417 
    ISSN: 1476-5535
    Keywords: fermentation ; scale up ; secondary metabolite ; physostigmine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A reliable and scalable fermentation process was developed for production of the acetylcholine esterase inhibitor physostigmine employingStreptomyces griseofuscus NRRL 5324. Initial fermentation in small-scale bioreactors reached physostigmine levels of approximately 60 mg L−1 after 139 h. Optimization of both process operating parameters and production medium composition rapidly yielded a seven-fold increase in physostigmine titer. The scaled up process routinely produced physostigmine titers of approximately 400 mg L−1 during a fermentation cycle of 180 h, and supported the rapid production of large amounts of physostigmine. A physostigmine production of 500 mg L−1 representing an eight-fold improvement over the original performance, was achieved using a glucose/ammonium fed-batch process.
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  • 27
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    Journal of industrial microbiology and biotechnology 16 (1996), S. 102-109 
    ISSN: 1476-5535
    Keywords: Millet ; Pennisetum typhoides ; liquefaction ; saccharification ; baker's yeast ; Saccharomyces cerevisiae ; fermentation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A fermentation medium based on millet (Pennisetum typhoides) flour hydrolysate and a four-phase feeding strategy for fed-batch production of baker's yeast,Saccharomyces cerevisiae, are presented. Millet flour was prepared by dry-milling and sieving of whole grain. A 25% (w/v) flour mash was liquefied with a thermostable 1,4-α-d-glucanohydrolase (EC 3.2.1.1) in the presence of 100 ppm Ca2+, at 80°C, pH 6.1–6.3, for 1 h. The liquefied mash was saccharified with 1,4-α-d-glucan glucohydrolase (EC 3.2.1.3) at 55°C, pH 5.5, for 2 h. An average of 75% of the flour was hydrolysed and about 82% of the hydrolysate was glucose. The feeding profile, which was based on a model with desired specific growth rate range of 0.18–0.23 h−1, biomass yield coefficient of 0.5 g g−1 and feed substrate concentration of 200 g L−1, was implemented manually using the millet flour hydrolysate in test experiments and glucose feed in control experiments. The fermentation off-gas was analyzed on-line by mass spectrometry for the calculation of carbon dioxide production rate, oxygen up-take rate and the respiratory quotient. Off-line determination of biomass, ethanol and glucose were done, respectively, by dry weight, gas chromatography and spectrophotometry. Cell mass concentrations of 49.9–51.9 g L−1 were achieved in all experiments within 27 h of which the last 15 h were in the fedbatch mode. The average biomass yields for the millet flour and glucose media were 0.48 and 0.49 g g−1, respectively. No significant differences were observed between the dough-leavening activities of the products of the test and the control media and a commercial preparation of instant active dry yeast. Millet flour hydrolysate was established to be a satisfactory low cost replacement for glucose in the production of baking quality yeast.
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  • 28
    ISSN: 1476-5535
    Keywords: fermentation ; fermentation monitoring ; fermentation control ; fermentation software ; Escherichia coli
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The advent of inexpensive computers and associated control and data acquisition software makes possible the development of sophisticated, configurable, integrated monitoring and control systems for small-scale laboratory and pilot-scale fermentors at low cost. We describe here the implementation of such a system, the interfacing of off-line instruments to enhance real time data analysis, low level process control and several substrate feeding protocols.
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  • 29
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    Bioscience reports 17 (1997), S. 67-76 
    ISSN: 1573-4935
    Keywords: Apoptosis ; necrosis ; mitochondria ; megachannel ; permeability transition ; programmed cell death ; poteases
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Mitochondrial permeability transition (PT) involves the formation of proteaceous, regulated pores, probably by apposition of inner and outer mitochondrial membrane proteins which cooperate to form the mitochondrial megachannel (=mitochondrial PT pore). PT has important metabolic consequences, namely the collapse of the mitochondrial transmembrane potential, uncoupling of the respiratory chain, hyperproduction of superoxide anions, disruption of mitochondrial biogenesis, outflow of matrix calcium and glutathione, and release of soluble intermembrane proteins. Recent evidence suggests that PT is a critical, rate limiting event of apoptosis (programmed cell death): (i) induction of PT suffices to cause apoptosis; (ii) one of the immediate consequences of PT, disruption of the mitochondrial transmembrane potential (ΔΨm), is a constant feature of early apoptosis; (iii) prevention of PT impedes the ΔΨm collapse as well as all other features of apoptosis at the levels of the cytoplasma, the nucleus, and the plasma membrane; (iv) PT is modulated by members of the apoptosis-regulatory bcl-2 gene family. Recent data suggest that the acquisition of the apoptotic phenotype, including characteristic changes in nuclear morphology and biochemistry (chromatin condensation and DNA fragmentation), depends on the action of apoptogenic proteins released from the mitochondrial intermembrane space.
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  • 30
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    Bioscience reports 17 (1997), S. 237-250 
    ISSN: 1573-4935
    Keywords: Apoptosis ; DNA fragmentation ; mitochondrial DNA ; oxidative damage
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract The molecular genetics and bioenergetics of oxidative damage, fragmentation, and fragility of mitochondrial DNA in cellular apoptosis is reviewed in connection with the “redox mechanism of ageing”.
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  • 31
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    Plant molecular biology 28 (1995), S. 739-750 
    ISSN: 1573-5028
    Keywords: alcohol dehydrogenase ; fermentation ; gene expression ; pollen ; pyruvate decarboxylase ; respiration ; tobacco
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We characterized the genes coding for the two dedicated enzymes of ethanolic fermentation, alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC), and show that they are functional in pollen. Two PDC-encoding genes were isolated, which displayed reciprocal regulation: PDC1 was anaerobically induced in leaves, whereas PDC2 mRNA was absent in leaves, but constitutively present in pollen. A flux through the ethanolic fermentation pathway could be measured in pollen under all tested environmental and developmental conditions. Surprisingly, the major factor influencing the rate of ethanol production was not oxygen availability, but the composition of the incubation medium. Under optimal conditions for pollen tube growth, approximately two-thirds of the carbon consumed was fermented, and ethanol accumulated into the surrounding medium to a concentration exceeding 100 mM.
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  • 32
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    Theoretical and applied genetics 96 (1998), S. 1022-1026 
    ISSN: 1432-2242
    Keywords: Key words Chromosome elongation ; Deletion ; Mitosis ; Disturbed development ; Fertility ; Apoptosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Chromosomes elongated beyond a critical size by balanced rearrangements reduce the viability and fertility of field bean individuals. The severity of symptoms, ranging from growth retardation to early death of seedlings, increases with the length of the longest chromosome arm. This is paralleled by the incomplete separation of sister chromatids during nuclear division, resulting in chromatin connections between daughter nuclei which become disrupted by cell-wall formation and yield chromatid deletions detectable as micronuclei. By means of the TUNEL assay we show that, compared to the wild-type, a 〉6-fold higher number of meristematic cells of karyotypes with chromosome arms surpassing the limit of tolerance reveal apoptotic nuclei and are prone to die. Thus, terminal chromatid deletions apparently trigger unscheduled apoptosis. Extensive cell death in meristems is eventually responsible for reduced growth, disturbed development and reduced seed set. Differentiated root tissues and microspores did not reveal apoptotic nuclei.
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  • 33
    ISSN: 1432-0878
    Keywords: Key words Adenosine ; Nucleosides ; Neurotoxicity ; Embryogenesis ; Apoptosis ; Chick
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Previous work has shown that nucleosides produce apoptosis in sympathetic ganglion (SG) cells in vitro. The present study examined the effects of nucleosides on the development of the chick embryo in vivo with special attention to the SG and the optic tectum of the central nervous system. In the presence of an adenosine deaminase inhibitor, adenosine and 2’-deoxyadenosine (2’-dAdo) produced different toxicity patterns: both adenosine and 2’-dAdo were toxic to E3 embryos, but only 2’-dAdo was toxic at later stages (E6 1/2, E11). Dosage experiments on E6 1/2 embryos showed that adenosine was less toxic than 2’-dAdo and that 2’-dAdo in sublethal doses was teratogenic. We also examined the effects of 2’-dAdo on embryonic chicken SG and optic tectum in vivo to determine whether sublethal doses of 2’-dAdo produced cell death in these centers on E6 1/2 and 10. In the E6 1/2 SG, 2’-dAdo produced significant neuron loss (83%) and a decrease in SG volume (65%); however, at E10, there was only minor cell loss (7%) and no significant change in SG volume. In the optic tectum at E6 1/2, cell loss was confined mainly to the tectal ventricular zone, but there was little sign of cell loss in this organ at E10. Since cell production is vigorous in the SG and optic tectum at E6 1/2 but relatively low at E10, 2’-dAdo appears to work by stopping cell proliferation. The ineffectiveness of 2’-dAdo at E10 may result from the lethality of 2’-dAdo to the embryo at low concentrations (30 µM) in vivo, well below the apoptosis-inducing concentrations employed in vitro (100–300 µM). These data extend previous findings showing that purine and pyrimidine metabolism plays an important role in development.
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  • 34
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    Cell & tissue research 279 (1995), S. 379-383 
    ISSN: 1432-0878
    Keywords: NADPH-diaphorase ; Myenteric plexus-Ileum ; Proximal colon ; Age ; Programmed cell survival and cell death ; Apoptosis ; Rat (Sprague Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The effect of age on the distribution of NADPH-diaphorase-containing neurones was investigated in the myenteric plexus of ileum and proximal colon of embryonic day-19 rats, as well as in rats at postnatal day 4, 6 months and 26 months. The mean percentage of NADPH-diaphorase-stained neurones per ganglion was established using protein gene product 9.5 (protein found in most if not all neurones)-immunostained neurones as 100%. The results revealed that there was a significant relative increase in NADPH-diaphorase-positive neurones with increasing age in the myenteric plexus of proximal colon with nearly all protein gene product 9.5-immunoreactive neurones staining for NADPH-diaphorase in 26-month-old rats. This was in marked contrast with the ileum, where no significant relative increase in NADPH-diaphorase-positive neurones was seen in aged rats. The implications of these findings in relation to programmed cell survival and cell death are discussed.
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  • 35
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    Cell & tissue research 282 (1995), S. 527-529 
    ISSN: 1432-0878
    Keywords: Key words: Gonadotropin-releasing hormone receptor ; Endometrium ; Apoptosis ; Mouse
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Circumstantial evidence from a previous study indicated that antibodies generated against a synthetic N-terminal extracellular domain mouse pituitary gonadotropin-releasing hormone (GnRH) receptor peptide acted directly on the murine uterus affecting endometrial regression. Affinity-purified polyclonal sheep antibodies were used to assess tissue-specificity of antibody reactions in diestrous mice. Antibody binding was localized by immunofluorescence staining to anterior pituitary gland and endometrium. Ovary, brain, liver, kidneys, heart, lungs, spleen, gastrointestinal tract, adrenal glands, thymus, thyroid gland, muscle, and adipose were unreactive. Fragmented deoxyribonucleic acid, a marker of programmed cell death/apoptosis, was detected by digoxigenin labeling-immunoperoxidase in endometrial (but not pituitary) glands of animals injected with antipeptide antibodies or native ligand. It appears that luteal phase endometrium of mice expresses a GnRH receptor moiety that is coupled to a cell death (endonuclease) transduction pathway.
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  • 36
    ISSN: 1432-0878
    Keywords: Key words: Parkinson’s disease ; Dopamine neurons ; Transfection ; Conditioned medium ; GDNF ; BDNF ; Apoptosis ; Bonnet monkey ; Macaca radiata ; Rat (Sprague Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Fibroblasts derived from the cerebral cortex of an aged Bonnet monkey (Macaca radiata) were utilized to express recombinant cDNAs encoding rat glial-cell-line-derived neurotrophic factor (GDNF) and human prepro brain-derived neurotrophic factor (BDNF) by lipofection. The cells showed stable expression and secretion of biologically active proteins. Conditioned medium from fibroblasts expressing BDNF or GDNF increased the number of surviving mesencephalic tyrosine-hydroxylase-immunoreactive neurons after 7 days in culture. The trophic effects of BDNF and GDNF were examined at two different plating densities of embryonic mesencephalic cells. At 50 000 cells/cm2 plating density, treatment of the mesencephalic cultures with BDNF-conditioned medium increased the number of tyrosine-hydroxylase-immunoreactive neurons by about 40% compared with vector-transfected control. At the same plating density, GDNF-conditioned medium increased the number of surviving tyrosine-hydroxylase-immunoreactive neurons above the vector-transfected control by 30%. When the tissue was plated at a higher density, viz., 75 000 cells/cm2, the number of tyrosine-hydroxylase-immunoreactive neurons increased by 41% with BDNF-conditioned medium, and by 56% with GDNF-conditioned medium above vector-transfected controls. Conditioned medium from cells secreting GDNF was also found to reduce the number of apoptotic tyrosine-hydroxylase-immunoreactive cells by 50%.
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  • 37
    ISSN: 1432-0878
    Keywords: Key words. Neuroendocrine system ; Lung ; Neuroepithelial bodies ; Apoptosis ; Thymectomy ; Golden hamster
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Effects of postnatal age and neonatal thymectomy on the numbers and characteristics of pulmonary neuroepithelial bodies (NEB) were investigated in 14-day- compared with 2.5-month-old hamsters. Left lung sections were stained for the marker PGP 9.5 and used for light-microscopic quantification, while the right lungs were processed for an electron-microscopic survey of the NEB ultrastructural features. For the first time, it is clearly demonstrated that, depending on the sampling method, the number of NEB may rise or fall with age; when considering the entire lung volume, the actual number of NEB doubles, whereas when studying a constant surface area, their number apparently decreases. Also, the proportion of alveolar NEB as well as luminal contact increase on normal development. In neonates, in contrast to older animals, apoptosis is clearly present in NEB, and approximately 10% of the NEB are associated with inflammatory cells. In some cases, the dead cells have properties of both apoptosis, disintegration and cytoplasmic degeneration. The presence of intracorpuscular neutrophilic granulocytes correlates with cellular death and innervation of the NEB. Thymectomy causes only minor effects on the pulmonary neuroendocrine system. It is argued that development of the NEB and of their innervation continue during the postnatal period.
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  • 38
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    Cell & tissue research 292 (1998), S. 435-445 
    ISSN: 1432-0878
    Keywords: Key words p53 ; Apoptosis ; Chemotherapy ; Cell cycle ; Differentiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Loss of wild-type p53 activity is thought to be a major predictor of failure to respond to radiotherapy and chemotherapy in various human cancers. This assumption is largely based on some cell-death studies in p53-knockout mice and on correlations of p53 status assessed by immunochemistry or single-strand conformational polymorphism (SSCP) analysis, and responses to therapy in human cancers in vivo. In principle, p53 may enhance chemosensitivity by promoting apoptosis via transcription-independent mechanisms as well as transcriptional activation of proapoptotic genes such as bax and transcriptional repression of antiapoptotic genes such as bcl-2. Drug-induced suicide mediated by the CD95/CD95 ligand system may also involve a p53-controlled pathway. Yet, p53 may decrease chemosensitivity by promoting p21-mediated and p21-independent growth arrest, DNA repair, and differentiation, and by enhancing the transcription of antiapoptotic genes such as bcl-x. Cell-culture work indicates that the effects of altering the p53 status on chemosensitivity depend very much on the cellular context. Disruption of p53 function in otherwise normal, nonneoplastic cells may enhance rather than decrease chemosensitivity. However, targeted p53 gene disruption in some cell types obtained from p53-knockout mice results in enhanced rather than decreased sensitivity, e.g., to irradiation. Transformed cells that have retained wild-type p53 function tend to acquire chemoresistance when p53 function is disabled, with few exceptions. Thus, preexisting molecular alterations or consecutive accumulation of molecular alterations after loss of p53 rather than the loss of wild-type p53 activity per se may confer chemoresistence to tumor cells. Moreover, p53 accumulation resulting from the increased half-life of mutant p53 proteins can act as a gain-of-function mutation, presumably as a consequence of multiple protein-protein interactions. Finally, significant tumor cell-type- and drug-specific patterns of modulation of chemosensitivity by p53 are beginning to emerge. Transfer of wild-type p53 genes into tumor cells commonly induces growth arrest but may render these cells relatively more resistant to most chemotherapeutic drugs. Therefore, careful experimental in vitro and in vivo studies are required before chemotherapy-supported p53 gene therapy for human cancer is introduced into clinical practice.
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  • 39
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    Cell & tissue research 286 (1996), S. 55-62 
    ISSN: 1432-0878
    Keywords: Key words: Taste bud ; Denervation ; Apoptosis ; Cell death ; TUNEL method ; Glossopharyngeal nerve ; Circumvallate papilla ; Mouse (dd)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Apoptotic cells in the taste buds of mouse circumvallate papillae after the sectioning of bilateral glossopharyngeal nerves were examined by the method of DNA nick-end labeling (TUNEL), together with standard electron microscopy. The taste buds decreased in number and size 3–11 days after denervation and disappeared at 11 days. The TUNEL method revealed only a few positively stained nuclei in normal taste buds but, in those of mice 1–5 days after denervation, the number of positive nuclei had increased to 3–5 times that of taste buds from normal mice. Electron-microscopic observation after denervation demonstrated taste bud cells containing condensed and fragmentary nuclei in a cytoplasm with increased density. The results show that taste bud cells under normal conditions die by apoptosis at the end of their life span, and that gustatory nerve sectioning causes apoptosis of taste bud cells with taste buds decreasing in number and ultimately disappearing.
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  • 40
    ISSN: 1432-0878
    Keywords: Key words: Retinoids ; Vaginal epithelium ; Differentiation ; Keratin ; Apoptosis ; Estradiol ; Progesterone ; Rat (Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. In rodents, the vaginal epithelium shows cyclic changes with an alternating pattern of keratinization under estrogen control and mucification under progesterone control. Retinoids are powerful regulators of cell differentiation, an excess of retinoids suppressing the keratinizing differentiation of keratinocytes. Here, we have examined the vaginal epithelium during the estrous cycle and compare the effects of retinoids on both types of hormonally induced differentiation, i.e. keratinization and mucification. All-trans retinoic acid was administred either by daily injections during the estrous cycle or by a single injection before the estrogen rise; these two protocols gave similar results. Retinoic acid suppressed estrogen-induced vaginal keratinization and cytokeratin K10 expression (a biochemical marker of terminal differentiation). Progesterone-induced mucification was not impaired; however, retinoic acid impeded mucous cell desquamation, suggesting an effect of retinoic acid on cell adhesiveness. Retinoic acid induced the appearance of apoptotic-like cells, as revealed by immunocytochemical staining of DNA fragmentation.
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  • 41
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    Cell & tissue research 279 (1995), S. 379-383 
    ISSN: 1432-0878
    Keywords: Key words: NADPH-diaphorase ; Myenteric plexus ; Ileum ; Proximal colon ; Age ; Programmed cell survival and cell death ; Apoptosis ; Rat (Sprague Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The effect of age on the distribution of NADPH-diaphorase-containing neurones was investigated in the myenteric plexus of ileum and proximal colon of embryonic day-19 rats, as well as in rats at postnatal day 4, 6 months and 26 months. The mean percentage of NADPH-diaphorase-stained neurones per ganglion was established using protein gene product 9.5(protein found in most if not all neurones)-immunostained neu- rones as 100%. The results revealed that there was a significant relative increase in NADPH-diaphorase-positive neurones with increasing age in the myenteric plexus of proximal colon with nearly all protein gene product 9.5-immunoreactive neurones staining for NADPH-diaphorase in 26-month-old rats. This was in marked contrast with the ileum, where no significant relative increase in NADPH-diaphorase-positive neurones was seen in aged rats. The implications of these findings in relation to programmed cell survival and cell death are discussed.
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  • 42
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    Cell & tissue research 282 (1995), S. 527-529 
    ISSN: 1432-0878
    Keywords: Gonadotropin-releasing hormone receptor ; Endometrium ; Apoptosis ; Mouse
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Circumstantial evidence from a previous study indicated that antibodies generated against a synthetic N-terminal extracellular domain mouse pituitary gonadotropin-releasing hormone (GnRH) receptor peptide acted directly on the murine uterus affecting endometrial regression. Affinity-purified polyclonal sheep antibodies were used to assess tissue-specificity of antibody reactions in diestrous mice. Antibody binding was localized by immunofluorescence staining to anterior pituitary gland and endometrium. Ovary, brain, liver, kidneys, heart, lungs, spleen, gastrointestinal tract, adrenal glands, thymus, thyroid gland, muscle, and adipose were unreactive. Fragmented deoxyribonucleic acid, a marker of programmed cell death/apoptosis, was detected by digoxigenin labeling-immunoperoxidase in endometrial (but not pituitary) glands of animals injected with antipeptide antibodies or native ligand. It appears that luteal phase endometrium of mice expresses a GnRH receptor moiety that is coupled to a cell death (endonuclease) transduction pathway.
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  • 43
    ISSN: 1432-0878
    Keywords: Key words: Thymus ; Thymic epithelial cells ; Cell culture ; Organ culture ; Apoptosis ; Mouse (C57BL/6J)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Apoptosis of murine thymocytes was examined either in intact fetal thymus lobes or in thymus cell suspensions, both cultured alone or in the presence of either a cortical (TEC 1.4) or a medullary (TEC 2.3) thymic epithelial cell line. Both TECs induced a pronounced increase of apoptosis in 24-h cultivated single thymus cell suspensions but not in spleen or bone marrow cell cultures. Co-culture of thymocytes with murine fibroblasts did not enhance apoptosis of the thymus cells. A similar enhancement of thymocyte apoptosis was observed with dialysed culture supernatants derived from both TEC lines, the active component(s) having a molecular weight of 〉30 kDa. In contrast, the cortical TEC 1.4 had a pronounced apoptosis inducing effect on intact fetal thymus lobes cultivated for six days, whereas the medullary TEC 2.3 had only a marginal influence. TEC 1.4 also induced a significant alteration in the ratio of CD4+CD8+ to CD4-CD8- cells. It is concluded that both the cortical and medullary epithelial cell lines are able to induce thymocyte apoptosis but that a large proportion of the cells within the intact thymus stroma is refractory to the respective signal(s) of the medullary epithelial cell line.
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  • 44
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    Cell & tissue research 281 (1995), S. 413-419 
    ISSN: 1432-0878
    Keywords: Apoptosis ; Mammary gland ; Lactation ; Involution ; DNA laddering ; Mouse
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mammary involution after cessation of milk removal is associated with extensive loss of secretory epithelial cells. Ultrastructural changes and the appearance of oligonucleosomal DNA laddering in ethidium bromide-stained gels indicates that cell loss during involution occurs by apoptosis. In this study, a technique for nick end-labelling of genomic DNA with radiolabelled deoxynucleotide has been used to monitor the induction of programmed cell death in mice after litter removal at peak lactation. This technique proved more sensitive than conventional ethidium bromide staining, and results suggested that apoptosis was induced rapidly by milk stasis, before extensive tissue re-modelling had begun. Oligonucleosomal DNA laddering on agarose gels was detected within 24 h of milk stasis, and increased progressively for at least 4 days. Nick-end labelling also detected laddering before litter removal, suggesting that programmed cell death is a normal feature of the lactating tissue. The DNA end-labelling technique was also adapted for in situ visualisation of apoptotic cells in tissue sections. By this criterion, apoptotic cells were identified in both the secretory epithelium lining the alveoli of the gland and, increasingly with prolonged milk stasis, amongst those sloughed into the alveolar lumen. The results demonstrate the utility of these techniques for study of mammary cell death and suggest that, whilst apoptosis is rapidly induced by milk stasis, it is also a normal physiological event in the lactating mammary gland.
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  • 45
    ISSN: 1432-0878
    Keywords: Key words: Adrenal cortex ; Apoptosis ; DNA fragmentation ; DNA 3′-end labeling ; Adrenocorticotropic hormone ; Hypophysectomy ; Rat (Sprague Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Adrenocortical cell apoptosis was studied by using an established in vivo model, the hypophysectomized rat, and an in vitro model, viz., rat adrenal glands in short-term organ culture. In vivo, apoptosis (biochemical autoradiographic analysis of internucleosomal DNA cleavage) was weak and not apparent until 12–24 h after hypophysectomy. In situ histochemical localization of 3′-end DNA strand breaks revealed that apoptosis in vivo occurred nearly exclusively in subpopulations of zona reticularis cells. Adrenocorticotropic hormone (ACTH) maintenance completely blocked these indices of apoptosis. By contrast, apoptosis (DNA fragmentation) in cultured rat adrenal glands without ACTH was extensive and relatively rapid, being apparent after 1 h and increasing with the duration of incubation. ACTH attenuated (by 44%) but did not completely block apoptosis in vitro. Thus, ACTH appears to be the sole pituitary hormone that forestalls apoptosis of terminally differentiated adrenocortical (zona reticularis) cells. However, the discrepancy between in vitro and in vivo models in terms of the magnitude and rate of DNA fragmentation suggests that, in vivo, other factors finely regulate the magnitude of adrenocortical apoptotic cell death.
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  • 46
    ISSN: 1432-0878
    Keywords: Key words Mammary gland ; Lactation ; Secretion ; Proliferation ; Apoptosis ; Milk ; African elephant ; Loxodonta africana (Proboscidea)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The lactating mammary gland of the African elephant (Loxodonta africana) has been studied with a panel of morphological techniques focusing on (1) the functional changes during the secretory process, (2) proliferative process [by application of proliferating cell nuclear antigen (PCNA) immunohistochemistry] and apoptotic phenomena [by use of the TUNEL technique] in the individual lobules, and (3) components of milk and milk-fat-globule membrane. In the lactating gland, the lobules are variably differentiated; within a lobule, however, the alveoli are usually similarly differentiated. The morphology of their alveoli suggests a classification of the lobules into types 1–3. Lobules of type 1 are composed of immature tubular alveoli with mitotic figures and numerous PCNA-positive nuclei; advanced type 1 alveoli contain abundant glycogen and specific secretory granules. Lobules of type 2 are further subdivided. In type 2a lobules, the epithelial cells of the alveoli form tall apical protrusions, which in part are occupied by small lipid droplets and which are pinched off in an apocrine fashion. The number of lysosomes varies considerably. Type 2b is the most common type, with striking basal membrane foldings, abundant rough endoplasmic reticulum cisterns, large Golgi apparatus, numerous mitochondria, lipid droplets, and protein vesicles with 30- to 90-nm-wide casein micelles. The lipid droplets are pinched off with minimal amounts of cytoplasm. Type 2c is composed of alveoli with a cuboidal epithelium and few signs of secretory activity. Increasing expression of peanut-agglutinin-binding sites parallels the maturation and differentiation of the glandular cells. Type 3 lobules are marked by numerous TUNEL-positive nuclei and large lipid droplets and are apparently degenerating structures. Cytokeratin (CK) 14 is usually present in the myoepithelial cells; CK 19 and CK 7 mark ductal and immature alveolar epithelia. Milk protein content varies between 2.6% and 6.3%, and casein micelles range from 35 to 90 nm in diameter. The diameter of intra-alveolar milk fat globules ranges from 5 to 25 µm and the membranes bear a filamentous surface coat composed of membrane-anchored mucins; gel-electrophoretic analysis of these mucins from different individuals demonstrates the presence of mucin MUC 1, which is expressed with considerable genetic heterogeneity.
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  • 47
    ISSN: 1617-4623
    Keywords: Key words Proteasome ; Apoptosis ; sug-1 ; Drosophila melanogaster ; Nervous system
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The proteasome plays essential roles in a variety of cellular processes, including degradation of the bulk of cellular proteins, degradation of short-lived proteins such as cell cycle regulators, generation of antigenic peptides, and mediating programmed cell death. One of the best characterized subunits of the 26S proteasome is encoded by the yeast gene SUG1. We report here the cloning and characterization of the Drosophila homolog of this gene, Pros45. At the protein level, Pros45 is highly conserved with respect to its homologs in a variety of taxa: it shows 74% identity to yeast Sug1; 86% to mouse m56/mSug1/FZA-B; 87% to human Trip1; and 97% to moth 18-56. Using a genomic clone as a probe for in situ hyridization to polytene chromesomes, we demonstrated that Pros45 maps to 19F, near the base of the X chromosome. Use of a pros45 cDNA clone as a probe revealed a second site of hybridization at 99CD. Pros45 mRNA is found in the unfertilized egg and in all cells of the early embryo. By the end of embryogenesis, Pros45 is expressed predominantly in the central nervous system. Targeted expression of Pros45 in a variety of different cells using the Gal4 UAS P-element system failed to generate an overt phenotype. This study provides the foundation for further examination of the role of the 26S proteasome in homeostasis and development in Drosophila.
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  • 48
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    Protoplasma 196 (1997), S. 197-211 
    ISSN: 1615-6102
    Keywords: Apoptosis ; Programmed cell death ; Tracheary element ; Xylogenesis ; Zinnia elegans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We used various microscopic and labeling techniques to examine events occurring during the programmed cell death (PCD) of plant tracheary elements (TEs) developing in vitro. TEs differentiating in vitro synthesize a secondary cell wall which is complex in composition and pattern at approximately 72 h after hormone manipulation. The timing of PCD events was established relative to this developmental marker. Cytoplasmic streaming continues throughout secondary wall synthesis, which takes 6 h to complete in a typical cell. Vital dye staining and ultrastructural analysis show that the vacuole and plasma membrane are intact during secondary cell wall synthesis, but the cytoplasm becomes less dense in appearance, most likely through the action of confined hydrolysis by small vacuoles which are seen throughout the cell at this time. The final, preeminent step of TE PCD is a rapid collapse of the vacuole occurring after completion of secondary cell wall synthesis. Vacuole collapse is an irreversible commitment to death which results in the immediate cessation of cytoplasmic streaming and leads to the complete degradation of cellular contents, which is probably accomplished by release of hydrolytic enzymes sequestered in the vacuole. This event represents a novel form of PCD. The degradation of nuclear DNA is detectable by TUNEL, an in situ labeling method, and appears to occur near or after vacuole collapse. Our observations indicate that the process of cellular degradation that produces the hollow TE cell corpse is an active and cell-autonomous process which is distinguishable morphologically and kinetically from necrosis. Although TE PCD does not resemble apoptosis morphologically, we describe the production of spherical protoplast fragments by cultured cells that resemble apoptotic bodies but which are not involved in TE PCD. We also present evidence that, unlike the hypersensitive response (HR), TE PCD does not involve an oxidative burst. While this evidence does not exclude a role for reactive oxygen intermediates in TE PCD, it does suggest TE PCD is mechanistically distinct from cell death during the HR.
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  • 49
    ISSN: 1615-6102
    Keywords: Actin ; Actin-binding proteins ; Apoptosis ; F9 embryonal carcinoma cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary During the execution phase of apoptosis, the cell undergoes a set of morphological changes which reveal the activation of a complex machinery leading the cell to its disruption into small, spherical, membrane-bounded fragments called apoptotic bodies. In the present study, we have focused on the implications of the micro-filament network in the early stages of the active phase of apoptosis. By using confocal microscopy, we have analysed the location of the actin microfilaments and two actin-binding proteins, α-actinin and myosin, in F9 embryonal carcinoma cells undergoing apoptosis during the stages previous to their fragmentation. Our results show that these proteins locate in the centre of the disrupting cell and form a three-dimensional structure which suggests the existence of a fully functional contractile system involved in the fragmentation of the cell and the formation of apoptotic bodies.
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  • 50
    ISSN: 1615-6102
    Keywords: Antioxidants ; Apoptosis ; Glutathione ; N-Acetylcysteine ; Oxidation-reduction ; Thiols
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In previous studies, oxidants such as hydrogen peroxide (H2O2) or hydroperoxy fatty acids were shown to induce apoptosis in the CEM human T cell line as demonstrated by the cleavage of cellular DNA into a ∼ 180-base pair “ladder”. Oxidant-induced DNA fragmentation was detectable within 3 h and inhibitable by various antioxidants. In the present study, apoptosis is shown to also be induced by the addition of low doses (0.1–3 mM) of N-acetyl-L-cysteine (NAC), reduced glutathione (GSH) or cysteine. By contrast, higher concentrations (≥10 mM) of the same thiols displayed a paradoxical lack of toxicity. Thiol-induced apoptosis was completely prevented by the addition of BAPTA-AM, an intracellular calcium chelator, or by simultaneous treatment with 5 mM pyruvate which forms a thiazolidine complex with sulfhydryl compounds. Catalase or glutathione peroxidase, but not Superoxide dismutase, protected the cells from thiol-induced apoptosis demonstrating a role for H2O2. The ability of thiol compounds to either evoke or prevent oxidative stress implies a unique role for these agents in the control of apoptosis in lymphoid cells.
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  • 51
    ISSN: 1615-6102
    Keywords: Antitumor sulfonylurea ; Apoptosis ; pH control ; Growth ; Hela cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The antitumor sulfonylureas appear to inhibit both mitochondrial activity in susceptible human colon lines and to inhibit the oxidation of NADH by isolated plasma membrane vesicles from HeLa cells. The results reported here describe the morphological appearance of HeLa cells treated with the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984). The cells remain viable for several days although the rate of increase in cell number is slowed especially at high concentrations of the drug. Cells become smaller with normal nuclei or maintain a normal size but contain multiple or enlarged nuclei. The morphological observations suggest that the drug may somehow interfere with the ability of the cells to enlarge following cytokinesis. Between 72 and 96 h, the cells begin to die. Cell death is accompanied by a condensed and fragmented appearance of the nuclear DNA as revealed by fluorescence microscopy with 4′,6-diamidino-2-phenylindole suggestive of apoptosis. Early transients in loss of pH control (4 min after sulfonylurea addition) and an increase in cytoplasmic calcium (4 h after sulfonylurea addition) were observed but were small and perhaps secondary to the mechanism responsible for the failure of the cells to grow and ensuing cell death.
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  • 52
    ISSN: 1573-0778
    Keywords: amino acids ; endothelium ; fermentation ; glutamine consumption ; microcarriers ; spinner cultivation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Primary bovine aortic endothelial cells were cultivated in serum supplemented medium without any additional growth factors. The anchorage dependent cells were propagated on Dormacell® microcarriers with covalently bound dimeric DEAE-groups at the surface of the dextrane beads. Cultivations were performed in 200 ml spinner cultures containing 1 g l−1 to 3 g l−1 of microcarriers. Out of five types of Dormacell® microcarriers with different ion exchange capacities ranging from 0.30 up to 0.65 meq g−1, corresponding to nitrogen contents from 1.2% to 2.9%, respectively, optimal attachment and growth of endothelial cells were obtained with beads of highest nitrogen content (2.9%). Cells were seeded withca. 5 viable cells per microcarrier being sufficient to achieve fully confluent microcarriers after 4 to 5 days. Glucose concentrations decreased from 21 mM to uppermost half of the original concentrations. 4 mM glutamine was rapidly consumed and virtually exhausted after the cells reached confluency. Lactate concentrations raised to a maximum of 7 mM in spinner cultures, but was found to be reutilized in the stationary phase after glutamine limitation occurred. Serine was found to be the second most prominent amino acid being almost exhausted at confluency whereas alanine was produced in noteworthy amounts. Considerable decrease was determined for threonine, lysine and arginine; low consumption rates were observed for leucine, phenylalanine and methionine. All other amino acids did not alter significantly throughout cultivation. These data support that bovine aortic endothelial cells are capable to utilize glucose and glutamine as well as lactic acid (after glutamine exhaustion) as energy and/or carbon source. Finally, batch cultures in a 2 liter membrane stirred bioreactor with bubble-free aeration were performed to produce large quantities of endothelial cells using microcarrier concentrations of 3 g l−1.
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  • 53
    ISSN: 1573-0778
    Keywords: Apoptosis ; programmed cell death ; CHO cells ; batch culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Chinese hamster ovary cells grown under conditions which are optimal for the production of a genetically engineered protein in batch culture, lose significant viability shortly after entering the stationary phase. This cell death was investigated morphologically and was found to be almost exclusively via apoptosi. Furthermore, cells were analyzed by flow cytometry using a fluorescent DNA end-labeling assay to label apoptotic cells, in conjunction with cell cycle analysis using propidium iodide. Apoptotic cells could be detected by this method, and by the radioactive end-labeling of extracted DNA, on all days of culture from day 1 to day 7; however, the degree of apoptotic cell death increased dramatically when the cells entered the stationary phase, rising to 50–60% of the total cell number at the termination of the culture. Flow cytometric analysis showed that the majority of cells underwent apoptosis whilst in G1/G0 and formed an apoptotic population with high DNA FITC end-labeling and hypodiploid propidium iodide binding. Additionally, the ability or inability to secrete specific protein products did not appear to interfere with the development of the apoptotic population with time.
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  • 54
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    Apoptosis 1 (1996), S. 75-80 
    ISSN: 1573-675X
    Keywords: Apoptosis ; calpain ; protein kinase C ; serine protease inhibitor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The effects of a synthetic serine protease inhibitor, FOY-305, and its derivatives, ONO-3403 and FO-349, on the proliferation of mouse NIH3T3 cells were investigated. At concentrations between 10 and 100 μg/ml, three protease inhibitors induced a moderate suppression of cell growth. However, only ONO-3403 showed severe cytotoxicity at concentrations higher than 200 μg/ml. Results of TUNEL staining and DNA fragmentation analysis indicated that ONO-3403 induced apoptosis at the high concentrations. Biochemical analysis has shown that ONO-3403 directly enhanced the amidolytic activity of purified μ-calpain at a concentration higher than 100 μg/ml while FOY-305 and FO-349 showed less effects. When the cell extract was incubated in the presence of ONO-3403, specific degradation of a few proteins including protein kinase C was observed. Similar degradation was also observed by addition of μ-calpain to the extract. These results imply that ONO-3403 is a specific stimulator of calpain. It seems reasonable to conclude that increase in calpain activity results in apoptosis.
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  • 55
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    Apoptosis 1 (1996), S. 153-160 
    ISSN: 1573-675X
    Keywords: Apoptosis ; cyclobutane pyrimidine dimers ; photoreactivation ; poly(ADP-ribose) polymerase ; UV-damage
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Rat kangaroo(Potorous tridactylus) cells efficiently repair 254 nm ultraviolet light (UV) induced cyclobutane pyrlmidine dimers (CPDs) through photoreactivation, leading to an enhancement of survival when cells are exposed to photoreactivation light (PRL) immediately after UV-irradiation. This work presents evidence that at least part of the UV-irradiated cells die through apoptosis, as demonstrated by DNA fragmentation and chromatin condensation. The induction of this kind of cell death can be reversed through photoreactivation immediately after irradiation, indicating that CPDs are essential signals for the initiation of apoptosis by UV-irradiation. Exposure to PRL 24 h after UV-irradiation does not reverse the induction of apoptosis, implying that the cells are committed to die at this time after irradiation. Inhibition of DNA synthesis during this period of time following UV-irradiation, and before exposure to PRL, does not avoid apoptosis. Since similar results were obtained in Go confluent and G1/S synchronized cells, the signals for the UV-induced apoptosis do not seem to be related to a specific phase of cell cycle. Nevertheless, by adding 3-aminobenzamide (3AB—an inhibitor of poly(ADP-ribose) polymerase) in the cell medium after UV-irradiation, apoptosis endpoints were partially reversed if cells are exposed to PRL 24 h later. This result strongly indicates that poly(ADP-ribose) is an intermediary signal for UV-induced apoptosis in mammalian cells.
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  • 56
    ISSN: 1573-675X
    Keywords: Apoptosis ; Bax ; Bcl-2 ; CDC 2 ; cyclin B1 ; paclitaxel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Paclitaxel is a potential cancer chemotherapeutic agent for ovary, breast, and head and neck cancers; its effects on nasopharyngeal carcinoma (NPC) have not been reported previously. This study investigated the cytotoxic mechanism of paclitaxel in two NPC cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with pacli-taxel showed convoluted nuclei, condensed chromatin and decreased cellular and nuclear volume, and also exhibited genomic DNA degradation into multiple oligonucleosomal fragments, suggesting that pacli-taxel induced apoptosis in these cells. The effects of paclitaxel on apoptosis-related proteins including Bcl-2, Bax and CDC 2 were also detected. Although the levels of Bcl-2 and Bax were not changed in NPC cells following treatment with 5 nM-1 μM of paclitaxel, phosphorylation of Bcl-2 was significantly observed in the cells treated with 1 μM of paclitaxel for 12 hours. In addition, cyclin B1-associated CDC 2 kinase was highly activated in the NPC cells exposed to paclitaxel even at low (5 nM) concentration, and this result is associated with the finding that low concentration of paclitaxel is able to induce apoptosis in NPC cells.
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  • 57
    ISSN: 1573-675X
    Keywords: Apoptosis ; bcr-abl ; cisplatin ; chronic myeloid ; leukaemia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Chronic myeloid leukaemia (CML) cell lines expressing the bcr-abl fusion gene are resistant to drug-induced apoptosis. Using a human CML cell line (EM2), we investigated the effects of cisplatinum (DDP), Doxorubicin and Tallimustine on the level of p210, the product of the hybrid bcr-abl gene, and on the induction of apoptosis. DDP exposure of this cell line resulted in a decrease of p210 levels with a concomitant activation of apoptosis. At all the concentrations tested, neither Doxorubicin nor Tallimustine were able to induce DNA fragmentation nor to reduce the levels of the fusion protein p210. The reduction in the p210 levels induced by DDP were also observed at mRNA level as observed with RT-PCR, suggesting that, at least in part, the decrease in p210 levels was the result of a reduction in the transcription of the bcr-abl fusion gene. The DDP-induced DNA fragmentation and decrease in p210 levels, were observed in EM2 cells but not in another human CML cell line (K562) which overexpresses the fusion gene. In K562 cells the levels of bcr-abl, although decreased, remained well detectable after DDP treatment. Data indicate that it may be possible to investigate compounds able to contrast the resistance to DNA-damage induced apoptosis of CML cell lines.
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  • 58
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    Apoptosis 2 (1997), S. 19-24 
    ISSN: 1573-675X
    Keywords: Apoptosis ; bcl-2 gene family ; hormones ; mammary gland involution ; proteinase ; tissue remodelling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Apoptosis is a process of programmed cell death. Mammary gland involution is a tissue remodelling process. Mammary epithelial cell apoptosis is an integral component of tissue remodelling but it is only one element. Equally important are the factors which degrade basement membrane and extracellular matrix. Both operations are required for completion of mammary gland involution. The primary apoptotic process occurs first and is temporally distinct from the second stage of involution typified by lobular-alveolar collapse. Local factors related to milk accumulation trigger the first stage, but loss of systemic hormonal stimulation governs the second stage. Changes in the expression patterns of cell cycle control genes and bcl-2 family member genes are found in the first stage. Proteinase gene activation dominates the second stage. These findings support a two stage model of mammary gland involution. Both mammary epithelial cell apoptosis and mammary gland remodelling advance through a process which includes both loss of survival factors and gain of death factors. This review focuses on signalling pathways and genetic controls which are activated and repressed during mammary gland involution.
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  • 59
    ISSN: 1573-675X
    Keywords: Apoptosis ; bone marrow ; cord blood ; CD34+CD4+ cells ; fas antigen ; HIV-1 infection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Haematologic abnormalities accompany the majority of HIV-1 infections. At present it is unclear whether this is due directly to HIV infection of hematopoietic progenitor cells, or whether this results from an indirect mechanism secondary to HIV infection. Here we provide evidence for an indirect mechanism, whereby hematopoietic progenitor cells undergo HIV gp120-induced apoptosis (programmed cell death) even in the absence of HIV infection. Freshly isolated, purified human hematopoietic progenitor CD34+ cells, derived from both umbilical cord blood and bone marrow, co-expressed the CD4 marker at low density on their surface. Although these CD34+CD4+ cells theoretically should be capable of productive infection by HIV, we found that HIV-IIIB could not establish productive infection in these cells. Nonetheless, gp120 from IIIB could bind the cells. Thus, binding of gp120 did not correlate with infectivity. Furthermore, binding of gp120 was a specific event, leading to apoptosis upon crosslinking with anti-gp120 through a fas-dependent mechanism. If apoptosis is also observed in vivo even in uninfected hematopoietic cells, this could contribute significantly to the impairment in hematopoietic cell number and function. Our data suggest a novel indirect mechanism for depletion of CD34+ and CD34+-derived cells even in the absence of productive viral infection of these cells.
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  • 60
    ISSN: 1573-675X
    Keywords: Apoptosis ; CD4 ; HIV ; PMA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The progressive loss of CD4 T lymphocytes is one of the hallmarks of HIV infection. The reverse correlation observed in vivo, between plasmatic HIV levels and CD4 T lymphocyte counts, supports the concept that direct HIV-mediated cell death contributes to this depletion. Previously, we and others have demonstrated, in vitro, that interactions between membrane-expressed HIV-envelope glycoprotein complexes and CD4 ecto-molecules are critical to cell killing which occurs mainly by apoptosis. Here, by the use of a co-culture model, in which chronically HIV-1 infected cells trigger apoptosis in uninfected CD4+ target cells, we have investigated the role of different CD4 domains in HIV envelope-mediated apoptosis. Target cells were A201 lymphoblastoid cell lines expressing wild-type CD4 or mutant forms of CD4. We show that the cytoplasmic domain of CD4 was not required for apoptosis induction. In contrast, the HIV permissive cell line expressing a CD4/CD8 chimeric molecule which contains only the first 171 amino acids of CD4, appeared to be resistant to HIV-induced apoptosis; thus suggesting that the D3-D4 CD4 module plays somewhat a regulatory role. Pre-treatment of wild-type CD4 expressing target cells by the phorbol ester PMA which leads to down-regulation of CD4, completely abolished apoptosis. Interestingly, in cells expressing CD4 devoid of its cytoplasmic domain, PMA blocked partially cell death without affecting, as expected, the CD4 expression. Taken together, these results demonstrate that although CD4 expression is essential for HIV envelope induced apoptosis, the apoptotic signal could be delivered in the absence of its cytoplasmic domain. Consistent with this, we suggest that other membrane associated molecule(s) are recruited for the signalling to initiate apoptosis.
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  • 61
    ISSN: 1573-675X
    Keywords: Apoptosis ; ERK ; JNK ; p53 ; Rb
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The molecular events associated with apoptosis induced by two distinct triggers (1) serum withdrawal and (2) etoposide treatment were investigated in the human lung carcinoma cell line A549. Although both serum withdrawal and etoposide treatment resulted in internucleosomal DNA fragmentation, the morphologic features were distinct. Serum deprived apoptotic cells appeared small, round and refractile, with little evidence of nuclear fragmentation; etoposide-induced apoptotic cells appeared enlarged and flattened and displayed prominent nuclear fragmentation. p53 and p21/waf1 protein levels were elevated in etoposide-treated cells, but not in cells subjected to serum with-drawal. Apoptosis induced by both treatments was accompanied by a significant reduction in Rb protein levels. However, etoposide treatment led to hypo-phosphorylation of Rb, while serum withdrawal did not alter the Rb phosphorylation pattern. Serum withdrawal-induced apoptosis was correlated with activation of JNK and suppression of ERK activities, while both JNK and ERK activities were slightly elevated during etoposid- induced apoptosis. Together, these results support the hypothesis that apoptosis induced by serum withdrawal and etoposide treatment occurs through different pathways and involves distinct mediators.
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  • 62
    ISSN: 1573-675X
    Keywords: Apoptosis ; electroporation ; HL-60
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Cell death was induced by electroporation in HL60 cells, a human promyeloid leukaemia strain, in order to determine by both morphological and biochemical criteria whether necrotic or apoptotic processes occurred. Cells sampled at several times after electroporation were analyzed for the assessment of the following end-points: (i) chromosomal DNA fragmentation; (ii) cell viability; (iii) mono- and oligonucleosomes in the cytoplasmic fraction; (iv) apoptotic index; and (v) morphology of treated cells. The results indicate that about 50% of the cells killed by electroporation die through necrosis, while the remaining 50% of the cells undergo apoptosis. Chromosome damage was also studied by cytogenetic analysis at metaphase. The possibility of killing tumour cells by electroporation, as a variant of electrotherapy, constitutes, in our opinion, a promising procedure in cancer therapy, avoiding the undesirable side effects normally derived from treatment with cytotoxic drugs.
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  • 63
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    Apoptosis 2 (1997), S. 359-367 
    ISSN: 1573-675X
    Keywords: Apoptosis ; cadmium ; metallothionein ; superoxide ; zinc-finger
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Cell death resulting from cadmium (Cd) intoxication has been confirmed to occur through apoptosis by morphological and biochemical studies. However it is still not clear whether Cd itself or metallothionein (MT) induced by Cd is the major factor responsible for the apoptosis. Although apoptosis is inducible by exposure of cells to various stimuli, the common pathway involved is generally accepted to be activation of endonucleases that induce internucleosomal cleavage of DNA, resulting in the ‘ladder’ formation observed upon agarose gel electrophoresis and the chromatin condensation seen by electron microscopy. Cd does not seem to activate the endonuclease in vitro. However, Cd itself can be associated with apoptosis through indirect oxidative stress by inhibition of antioxidant enzymes and possible interaction with zinc finger protein. In addition to the direct effect of Cd, MT appears to play dual roles in apoptosis induction: one as a Cd carrier by which Cd accumulates in the nucleus, and the other as an inhibitor of zinc finger proteins, which include transcriptional factors related to apoptosis such as the product of the apoptosis resistance gene A20. In this review, we demonstrated that the mode of cell death following Cd exposure is associated with intracellular movement of Cd and MT. A possible mechanism for Cd-induced apoptosis is also discussed.
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  • 64
    ISSN: 1573-675X
    Keywords: Apoptosis ; experimental cancer therapy ; L1210 leukaemia ; proteasome ; proteasome inhibitor ; TNF
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Of a number of factors involved in apoptosis, protease activity may play a crucial role. We show that N-benzyloxycarbonyl-Ile-Glu( O-t-butyl)-Ala-leucinal (PSI), a selective inhibitor of the chymotrypsin-like activity of the proteasome, induces massive apoptosis in murine leukaemia L1210 cells. At 50 nM concentration, PSI induces a block of cytokinesis, while higher concentrations (500 nM) cause S phase block and massive apoptosis. Z-Leu-leucinal, a specific calpain inhibitor, did not induce apoptosis. In contrast to previous reports, TNF-α did not enhance apoptosis when combined with PSI. Our results suggest that proteasome inhibitors may be considered as potential anti-neoplastic agents.
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  • 65
    ISSN: 1573-675X
    Keywords: Apoptosis ; endothelial ; p53
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Transfection of the wild-type p53 gene into an immortalized human endothelial cell line (ECV-304) by recombinant adenoviral delivery resulted in high level expression of the wild-type p53 protein and induction of apoptosis. Increases in the number of apoptotic cells were observed within 12 h after infection of ECV-304 cells with recombinant p53 adenovirus, as deter-mined by the appearance of internucleosomal DNA fragmentation ladders and by TUNEL and electron microscopic analyses. Control cells infected with a β-galactosidase recombinant adenovirus exhibited little or no increase in apoptosis over uninfected cells. The expression of Waf-1 and Bax gene products were in-creased substantially in apoptotic ECV-304 cells as determined by Northern blot, reverse transcription-PCR and immunoblotting analyses. Lesser increases in the expression of the PCNA gene were detected in ECV-304 cells undergoing apoptosis. Both control and apoptotic ECV-304 cells did not express detectable levels of Bcl-2 mRNA or protein in Northern blotting and immunoblotting analyses, respectively. The data suggest a role for the Bax gene product in p53-mediated apoptosis of endothelial cells.
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  • 66
    ISSN: 1573-675X
    Keywords: Apoptosis ; IAP ; NAIP ; TNF
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Multicellular organisms have evolved elaborate signal transduction pathways for maintaining homeostasis through the control of cell proliferation and death. The recent surge of interest in the regulation of programmed cell death has led to the rapid identification of many proteins involved in controlling and executing apoptosis. The inhibitors of apoptosis proteins (IAPs) constitute a family of highly conserved death suppressing proteins that were first identified in baculoviruses, and that has recently expanded to include at least two homologues in Drosophila melanogaster and four in rodents and humans. In this article we review the current state of IAP research. Two of the IAPs, HIAP-1 and HIAP-2, have been placed within the TNFα induced cell death pathway which involves two receptors for TNFα and multiple, overlapping signal transduction proteins. A third, X-linked gene termed XIAP, is ubiquitously expressed and appears to have a broad range of suppressor activity to a variety of apoptotic triggers. The fourth member, NAIP, has been identified as the protein product of a candidate gene for the inherited neuromuscular disorder, spinal muscular atrophy (SMA). The neuroprotective activity of NAIP in an in vivo model of cerebral ischemia has also been demonstrated.
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  • 67
    ISSN: 1573-675X
    Keywords: Apoptosis ; cancer therapy ; drug resistance ; LMB-7
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Immunotoxins are presently being evaluated as novel agents for cancer therapy. The direct mechanism by which immunotoxins kill cancer cells is inhibition of protein synthesis, but cytotoxicity due to induction of apoptosis has also been observed with these agents. Some cancers that express high levels of BCL-2 are relatively resistant to apoptosis inducing agents. It is therefore important to determine to what degree the toxicity of ricin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas exotoxin derived immunotoxins towards cancer cells can be attributed to inhibition of protein synthesis, and to what degree to subsequent induction of apoptosis. We compared the sensitivity of MCF-7 breast cancer cells that were stably transfected with a BCL-2 expression plasmid and thus protected against apoptosis and of MCF-7 cells transfected with a control plasmid towards ricin, diphtheria and Pseudomonas toxin, a Pseudomonas toxin-derived immunotoxin (LMB-7) and tumour necrosis factor α (TNF). We found that BCL-2 mediated inhibition of apoptosis renders the cells almost completely resistant (1000-fold) to tumour necrosis factor, but the same cells were only 3–10 fold more resistant to cytotoxicity induced by immunotoxin LMB-7 as well as Pseudo-monas exotoxin, diphtheria toxin and ricin. We next studied several leukaemia cell lines with variable levels of BCL-2 expression and found them quite sensitive to a Pseudomonas exotoxin containing immunotoxin independent of the level of BCL-2. Our data indicate that although BCL-2 overexpression can have a modest effect on sensitivity to an immunotoxin, cell lines derived from patients are still very sensitive to immunotoxins.
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  • 68
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    Keywords: Apoptosis ; differentiation ; OK-432 ; oral squamous cell carcinoma ; radiotherapy ; tegafur
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    Notes: Abstract Twenty patients with oral squamous cell carcinoma having mainly stage II or III lesions without distant metastasis, were treated with tegafur and streptococcal agent, OK-432, in combination with radiotherapy. As a consequence, 16 cases among the treated 20 cases showed complete remission by this therapy alone. Especially, we have found that the squamous cell carcinoma arising in non-keratinizing oral epithelium rather than in keratinizing oral epithelium has better response to this therapy. Among the 16 cases with complete remission (CR) by the current therapy, 10 cases were histopathologically diagnosed as well-differentiated squamous cell carcinoma and six cases as moderately differentiated squamous cell carcinoma. When we examined immunohistochemically the expres-sion of various antigens such as proliferating cell nuclear antigen (PCNA), p53 and LeY or the presence of DNA fragmentation by nick-end labelling in the biopsy materials taken at the first visit to our clinic from 20 patients treated with the current therapy, the CR group showed a significantly increased LeY expres-sion level ( p〈 0.05) and DNA fragmentation rate ( p〈 0.05) as compared with the partial response (PR, n= 3) + no change (NC, n= 1) group. On the other hand, the CR group with respect to PCNA expression level was significantly decreased as compared with the PR + NC group ( p〈 0.05). From these findings, it can be considered that the therapy for oral squamous cell carcinoma by UFT and OK-432 in combination with radiotherapy is very effective, which may be associated with differentiation or apoptosis in oral squamous carcinoma cells. In addition, we present the clinical findings and results of immunohistochemical staining for the biopsy materials obtained from four CR cases treated with the current therapeutic method.
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  • 69
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    Keywords: Apoptosis ; caspases ; CHO-K1 ; CPP32 ; ICE family proteases ; staurosporine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Apoptosis is a distinct form of programmed cell death that plays an important role in many biological processes.Although the phenotypes of apoptotic cells are well documented, little is known of the central mechanismleading to programmed cell death. Over the past few years, a number of ICE/CED-3 family proteases(also termed caspases) have been discovered and implicated as the common effectors of apoptosis. Inthis report, we demonstrate that induction of apoptosis in CHO-K1 cells by staurosporine, a broad spectruminhibitor of protein kinases, results in an increase in DEVD-dependent protease activity. These events werefollowed by nuclear DNA fragmentation and cell death. Inhibition of the DEVD-cleaving activity by a synthetictetrapeptide inhibitor DEVD-CHO, blocked staurosporine-induced downstream apoptotic phenotypes, suchas morphological characteristics and DNA fragmentation. These results suggest that staurosporine-inducedapoptosis in CHO-K1 cells is mediated through the CPP32/caspase-3-like cysteine proteases.
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  • 70
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    Apoptosis 3 (1998), S. 75-82 
    ISSN: 1573-675X
    Keywords: Apoptosis ; BCL-2 ; BCL-X ; cancer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Apoptosis is a genetically controlled cell death process that is required for normal development and tissue homeostasis. Suppression of apoptosis can confer a growth advantage to cells and contribute to cancer; many cancers are relatively resistant to apoptosis, including that induced by radiation or chemotherapeutics. Mutations which inactivate pro-apoptotic or activate anti-apoptotic proteins in cancer cells are therefore likely to be responsible for some of these differences. BCL-2 family proteins are key regulators of apoptosis and there is evidence supporting a role for mutation of BCL-2 family proteins in cancer. This includes well established events such as activation of BCL-2 via translocations in follicular lymphoma, as well as more recent observations implicating activation of Bcl-XL expression and frameshift and missense mutations of BAX and BCL-2 in cancer.
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  • 71
    ISSN: 1573-675X
    Keywords: Apoptosis ; DNA fragmentation ; DNase γ ; endonuclease
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We report here on the nucleotide sequence of the cDNA encoding human DNase γ, which is a candidate for an apoptotic endonuclease. The cDNA clone isolated from a human spleen cDNA library is composed of a 918 bp open reading frame encoding a 305 amino acid precursor protein for DNase γ. Northern blot analysis reveals that the expression of a single transcript of 1.5 kb DNase γ mRNA is detected in the spleen and liver. The chromosomal localization of DNase γ gene is mapped to chromosome 3 at region p21.1-p14.2 by fluorescence in situ hybridization (FISH). Characterization of thioredoxin-DNase γ fusion protein (Trx-hDNase γ) shows that the recombinant protein has a Ca2+/Mg2+- or Mn2+-dependent endonuclease activity that cleaves chromatin DNA to nucleosomal units. The optimum pH is around 7.2. Zn2+ and aurintricarboxylic acid (ATA) inhibits the activity in dose-dependent manners. These properties are identical to those of purified DNase γ.
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  • 72
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    Apoptosis 3 (1998), S. 97-103 
    ISSN: 1573-675X
    Keywords: Apoptosis ; DNA fragmentation ; DNase γ ; neurogenesis ; PC12 cells ; programmed cell death
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract DNase γ, which cleaves chromosomal DNA into nucleosomal units (DNA ladder formation), has been suggested to be the critical component of apoptotic machinery. Using rat pheochromocytoma PC12 cells, which are differentiated to sympathetic neurons by nerve growth factor (NGF), we investigated whether DNase γ-like enzyme is present in neuronal cells and is involved in neuronal cell death. The nuclear auto-digestion assay for DNase catalyzing internucleosomal DNA cleavage revealed that nuclei from neuronal differentiated PC12 cells contain acidic and neutral endonucleases, while nuclei from undifferentiated PC12 cells have only acidic endonuclease. The DNA ladder formation observed in isolated nuclei from neuronal differentiated PC12 cells at neutral pH requires both Ca2+ and Mg2+, and is sensitive to Zn2+. The molecular mass of the neutral endonuclease present in neuronal differentiated PC12 cell nuclei is 32000 as determined by activity gel analysis (zymography). The properties of the neuronal endonuclease present in neuronal differentiated PC12 cell nuclei were similar to those of purified DNase γ from rat thymocytes and splenocytes. Interestingly, in neuronal differentiated PC12 cells, internucleosomal DNA fragmentation is observed following NGF deprivation, whereas undifferentiated PC12 cells fail to exhibit DNA ladder formation during cell death by serum starvation. These results suggest that the DNase γ-like endonuclease present in neuronal differentiated PC12 cell nuclei is involved in internucleosomal DNA fragmentation during apoptosis, induced by NGF deprivation.
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  • 73
    ISSN: 1573-675X
    Keywords: Apoptosis ; bcl-2 ; cisplatin resistance ; p53
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Since apoptosis is the primary mode of cell death induced by cisplatin, the role of apoptosis and apoptosis-related gene products in cisplatin resistance was investigated in four human cisplatin-resistant cell lines of different tumour type. A common feature of the resistant sublines was a reduced susceptibility to drug-induced apoptosis compared to parental sensitive lines. Loss of wild-type p53 function was not a general event associated with the development of drug resistance. An increased bcl-2 expression was found in resistant cells characterized by mutant p53 (A431/Pt and IGROV-1/Pt), whereas in osteosarcoma (U2-OS/Pt) and in ovarian carcinoma (A2780/CP) cells with wild-type p53, bcl-2 levels were markedly reduced. U2-OS/Pt cells had a 16-fold increase in the level of Bcl-xL protein. Stable transfection of U2-OS cells with bcl-xL cDNA conferred a low level of drug resistance to cisplatin, suggesting that overexpression of this gene contributes to the ci splatin-resistant phenotype of this osteosarcoma cell system. In conclusion, these observations suggest a variable contribution of apoptosis-related genes to cisplatin resistance depending on the biological background of the cell system and presumably reflecting different pathways of apoptosis.
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  • 74
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    Apoptosis 3 (1998), S. 1-7 
    ISSN: 1573-675X
    Keywords: Apoptosis ; cell cycle ; differentiation ; p27Kip1 ; proteasome
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A characteristic feature of apoptotic cell death is the activation of a cascade of cytoplasmic proteases that results in the cleavage of a limited number of target proteins. A central role in these proteolytic events has been assigned to members of the capase family. However, the use of low molecular weight proteasomal inhibitors has also demonstrated that protein degradation or processing by the ubiquitin-proteasome system of the cell has a decisive impact on cell survival and death as well, depending on the cell type and/or the proliferative status of the cells studied. Treatment of proliferating cells with proteasome inhibitors leads to cell death, potentially involving an internal signalling conflict between accumulating levels of the cdk inhibitor p27Kip1 and c-myc. In contrast, in terminally differentiated cells the same compounds have the opposite effect of blocking apoptosis, possibly by preventing proteasome-mediated degradation of a capase inhibitor. In this review the role of proteasome-mediated proteolysis in the dying cell is discussed and apparently conflicting results are integrated into a working hypothesis which functionally locates the proteasome upstream of capase3-like enzymes.
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  • 75
    ISSN: 1573-675X
    Keywords: Apoptosis ; Bax ; Bcl-x ; corpus luteum ; GnRH; ; rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We have demonstrated that continuous administration of a GnRH-agonist (GnRH-Ag) in the rat during early pregnancy suppressed plasma progesterone levels within 8 h after the commencement of treatment. The purpose of the present study is to evaluate the hypothesis that in vivo GnRH-Ag treatment induces apoptotic cell death during early pregnancy. Rats were treated individually on day 8 of pregnancy with 5 μg/day of GnRH-Ag via an osmotic minipump. Sham-controlled rats received no treatment. Rats were killed at 4, 8 or 24 h after the treatment. At autopsy, blood samples were obtained and ovaries were removed. The corpora lutea (CL) from one ovary were removed for DNA laddering and RNA studies. As early as 8 h after the commencement of treatment, GnRH-Ag suppressed serum progesterone levels as expected, and increased the degree of DNA degradation in the CL that was time-dependent. At 24 h after the treatment, GnRH-Ag increased the Bax, a cell death gene, expression in the CL. Collectively, these data suggest that GnRH-Ag treatment induces apoptosis during early pregnancy in the rat.
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  • 76
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    Keywords: Apoptosis ; Bax ; Bcl-2 ; HC11 ; mammary gland ; prolactin
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    Topics: Biology , Medicine
    Notes: Abstract The effect of prolactin on apoptosis and the expression of bcl-2 and bax in HC11 mouse mammary epithelial cells were investigated. Flow cytometric analysis of Bcl-2 level (FITC-conjugated monoclonal anti-Bcl-2 antibody and FITC-conjugated monoclonal anti-IgG1 antibody as a negative control), number of apoptotic cells and cell cycle phases (DNA stained with DAPI) was performed. Bax transcript was measured using the RT-PCR method with GAPDH serving as a reference gene. Administration of prolactin (5lg/ml) in the presence of insulin stimulated differentiation of mammary epithelial cells, which manifested in stopping cells at G0/G1 phase, cell swelling and increase of cell number with enhanced protein content. Moreover, prolactin highly significantly reduced the extent of apoptosis of HC11 cells during 48 h of incubation. Nevertheless, the apoptotic cell number rose with increased time length of cell culture, probably due to the resulting high cell density and EGF withdrawal from t he incubation medium. The antiapoptotic effect of prolactin was associated with up-regulation of bcl-2 expression, shown as an increase in cell numbers expressing this protooncogene and elevated Bcl-2 content in these cells. A negative relationship (r=−0.87, p≤0.001) between the number of apoptotic cells and those expressing bcl-2 was also found. Prolactin administration lowered Bax transcript by 68.8% and 70.7% after 3 and 6h, respectively. In conclusion, the results presented indicate that stimulation of bcl-2 expression with simultaneous suppression of bax may be key events in the mechanism of antiapoptotic action of prolactin in HC11 mammary epithelial cells.
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  • 77
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    Apoptosis 3 (1998), S. 317-334 
    ISSN: 1573-675X
    Keywords: Apoptosis ; cell death ; ceramide ; caspase ; Bcl-2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Knowledge about the molecular regulators of apoptosis is rapidly expanding. Cell death signals emanating from death receptors or internal cell injury detectors launch a number of signaling pathways which converge on several key families of proteins including specialized proteases and endonucleases which play a critical role in the execution of the death order. In this review, we summarize recent discoveries relating to the signaling pathways involved, the death receptors, the caspase family of apoptotic proteases, Bcl-2 family members, the sphingolipid ceramide, and the tumor suppressor p53. In particular, we focus on the role played by ceramide as a coordinator of the stress response and as a candidate biostat in the detection of cell injury.
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  • 78
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    Keywords: Apoptosis ; cell adhesion ; fibronectin fragment ; matrix metalloproteinase ; mesangial cell.
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract We previously showed that in passive Heymann nephritis (PHN) rats, a large quantity of fibronectin (FN) fragments containing the central cell-binding (CCB) domain and adjacent domains are generated in the kidney and excreted into urine (Nishizawa et al., Biol Pharm Bull 1998; 21: 429–433). To ascertain whether the FN fragments could affect the progression of PHN, we investigated the effect of a 150 K FN fragment containing the CCB and carboxyl-terminal heparin-binding (Hep 2) domains on cultured rat mesangial cells. When rat mesangial cells cultured on FN-coated plates were exposed to the 150 K FN fragment, some mesangial cells detached from the FN substrate and then underwent apoptosis as judged by nuclear and DNA fragmentations. The 150 K FN fragment competitively inhibited the mesangial cell adhesion to the FN substrate in a dose-dependent manner. Furthermore, gelatinzymography of the conditioned medium of mesangial cells showed that the 150 K FN fragment induced and/or poteintiated the extracellular matrix (ECM)-degrading proteinases including matrix metalloproteinases (MMPs) of mesangial cells. These results indicate that the 150 K FN fragment may elicit mesangial cell apoptosis by disrupting the mesangial cell adhesion through two distinct ways: the inhibition of mesangial cell adhesion and the ECM-degradation by the 150 K FN fragment-induced MMPs. Thus, FN fragments containing the CCB and adjacent domains generated in the kidneys of PHN rats may be involved in the evolution of the renal injury.
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  • 79
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    Keywords: Apoptosis ; non-small cell lung carcinoma ; p53 ; phosphorylation.
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract The natural metabolic byproduct of estradiol, 2-methoxyestradiol (2-MeOE2), induces apoptosis in human lung cancer cells by a p53-dependent mechanism. The expression of wild-type p53 isoforms was investigated in H1299 non-small cell lung carcinoma cells induced into apoptosis by 2-MeOE2. H1299 cells lack endogenous p53 and undergo predominantly a G1 arrest when infected with a recombinant wild-type p53 adenovirus. However, when H1299 cells transfected with p53 were treated with 2-MeOE2, they underwent rapid and extensive apoptosis. H1299 cells expressing mutant his273 p53 were unaffected by 2-MeOE2, indicating a dependence of 2-MeOE2-mediated apoptosis on the presence of a functional p53. Analysis of wild-type p53 phosphoisoforms in H1299 cells by two-dimensional gel electrophoresis revealed that 2-MeOE2 induced a unique group of acidic p53 isoforms. Although most of the wild-type p53 in untreated H1299 cells migrated as at least five diffuse species with isoelectric points from pH 5.5–6.3, as many as nine additional forms migrating toward the acidic region with pI values from 4.4–5.3 were detected in 2-MeOE2-treated apoptotic cells. Two other agents known to induce apoptosis, vinblastine and actinomycin D, induced a similar pattern of acidic p53 species as that observed for 2-MeOE2. The results indicated that the induction of apoptosis in H1299 cells by 2-MeOE2 is dependent on the upregulation of specific p53 isoforms. Identification of the specific p53 phosphoisoforms induced by MeOE2 will be an important step in understanding the regulation and function of p53 in apoptosis.
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  • 80
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    Apoptosis 4 (1999), S. 255-261 
    ISSN: 1573-675X
    Keywords: Apoptosis ; cell cycle ; G2/M phase ; Fas ; Jurkat cell.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The relationship between the cell cycle and Fas-mediated apoptosis was investigated using Jurkat cells. Analysis of the inducibility of apoptosis by anti-Fas antibody during the cell cycle synchronized by the thymidine double-block method, showed that apoptosis was induced in only 50% of the G2/M phase cells, while most of cells in the other phases underwent apoptosis. These observations indicate that G2/M phase cells are more resistant to Fas-mediated apoptosis than cells in other phases. Furthermore, a detailed analysis of G2/M phase found that only 20–30% of the cells underwent apoptosis 12 h after the removal of the second thymidine block (pre-G2/M phase). This suggests that Fas-mediated apoptosis is potently suppressed during the pre-G2/M phase. A possible explanation for the observation that cells in the pre-G2/M phase are less sensitive to anti-Fas antibody is lower expression level of Fas. To test this possibility, Fas expression levels on the cell surface during the cell cycle were examined. The content of Fas on the cell surface, however, did not change appreciably during the cell cycle. Thus, the suppression of apoptosis in the pre-G2/M phase is determined downstream after the receipt of the apoptotic signal through Fas.
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  • 81
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    Keywords: Apoptosis ; B/T blood lymphocytes ; γ-glutamyltransferase ; glutathione ; glutathione S transferase ; membrane permeability ; type I (IDDM) ; type II (NIDDM) diabetes.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We have compared the concentrations of intracellular glutathione (GSH), glutathione-dependent antioxidative enzymes, the cell death rate and immunophenotype profile of peripheral blood mononuclear cells (PBMC) from healthy donors and from patients with insulin-dependent type I (IDDM) or non insulin-dependent type II (NIDDM) diabetes mellitus. The IDDM and NIDDM patients had above-normal absolute lymphocyte counts, whereas the percentages of CD3, CD4 and CD8 T lymphocytes were significantly reduced. In contrast, the absolute number and percentage of B lymphocytes was higher in diabetic patients than in healthy donors. The low intracellular reduced glutathione (GSH) and the unbalanced profile of key enzymes involved in GSH metabolism, gamma glutamyltransferase (γ-GT) and glutathione-S-transferase (GST), account for the increased oxidative status of PBMC from diabetic patients. The plasma membranes of PBMC from diabetic patients were less permeable to propidium iodide than those of PBMC from healthy donors, indicating that the apoptotic cell death rate was lower in the cells from diabetic patients. These differences are potentially useful markers of pathogenic metabolic changes which occur during clinical diabetes and if they are confirmed could be used to identify the onset of diabetes.
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  • 82
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    Keywords: Apoptosis ; fluorescence microscopy ; seminoma ; TdT.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Apoptosis morphology (DNA condensation) and internucleosomal DNA cleavage (TdT assay) were measured simultaneously on double fluorescence labeled testis tumor sections, employing conventional immunofluores cence microscopy. Six different apoptosis indices (AI) were determined based either solely on morphological or biochemical criteria, or on a combination of both processes. Measurements were performed in metastasized and non-metastasized seminoma, and in histological regions located distantly and associated with the tumor. Preliminary results on 19 histologies revealed that up to 66% of apoptotic cells were not detected, depending on the method used for apoptosis detection. Besides, no changes of solely morphologically defined AI was found in the different histological regions. By contrast, significant changes (p 〈 0.0004) in the different histological regions were detected when measuring AIs, e.g., defined by DNA fragmentation occuring without DNA condensation in apoptotic cells. Those changes were not detected in metastasized seminoma. These data, for the first time allow a comparison of two widely used approaches for apoptosis detection. Furthermore, the results reveal differences in apoptotic processes in tissue associated with non-metastasized seminoma detectable by a modified evaluated TdT assay but not by morphological changes, although this TdT method fails to show the total amount of apoptotic cells.
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  • 83
    ISSN: 1573-675X
    Keywords: Apoptosis ; bcl-2 ; Ca2+ ; flow cytometry ; hybridoma ; K+ ; Mg2+ ; necrosis.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We have addressed the possibility that Ca2+, Mg2+ and K+ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death. By removing Ca2+, Mg2+ or K+ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability. The differences were explained in terms of a possible reduction in their respective intracellular levels. From several lines of evidence, the deprivation of K+ ions was the most detrimental to cellular growth and viability and induced significant levels of early apoptotic cells. Another effect of this deprivation was to weaken the plasma membranes without causing membrane breakdown; exposure to high agitation rates confirmed fragility of the cell membranes. Removal of Mg2+ caused a reduction in the levels of early apoptotic cells and predisposed cells to high levels of primary necrotic death. The lower levels of apoptotic cells failed to demonstrate the classic nuclear morphology associated with apoptosis, while retaining other apoptotic features. These results highlighted the importance of utilizing several assays for the determination of apoptosis. The absence of Ca2+ appeared to be the mildest insult, but its deprivation did accelerate a significant decline in culture by increasing apoptotic death. Hybridoma cells overexpressing the apoptotic suppresser gene bcl-2 were protected from the predominantly necrosis inducing effects of Mg2+ ion deprivation and apoptosis inducing effects of Ca2+ ion deprivation. However, apoptosis was not as effectively suppressed in bcl-2 cells responding to incubation in K+ free medium. The inclusion of bcl-2 activity in the mechanisms of Ca2+ Mg2+ or K+ deprivation induced cell death emphasizes a close relationship between ionic dissipation and the apoptotic process.
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  • 84
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    Apoptosis 4 (1999), S. 365-372 
    ISSN: 1573-675X
    Keywords: Apoptosis ; chemoprevention ; colorectal cancer ; cyclooxygenase ; NSAIDs.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Epidemiological, clinical and animal studies indicate non-steroidal anti-inflammatory drugs (NSAIDs) to be chemopreventive for colorectal cancer. The best established target for NSAIDs are the two isoforms of cyclooxygenase (COX), a key enzyme in the biosynthesis of prostaglandins. Recent investigations using human colorectal tumor cell lines have focused on the cellular and molecular mechanisms potentially underlying the chemopreventive effect of NSAIDs. These studies have used ‘traditional’ NSAIDs and their metabolites which either do not inhibit COX, are non-selective for the COX isoforms or selectively inhibit COX-1 over COX-2, and recently developed NSAIDs that are highly selective for COX-2. In vitro, apoptosis is the dominant anti-proliferative effect of each of these classes of NSAID and sensitivity to NSAID-induced apoptosis increases with the malignant potential of the tumor cells. Limited in vivo evidence backs up these findings. Cell cycle arrest also contributes to the in vitro growth inhibitory effect of traditional NSAIDs. The induction of apoptosis by NSAIDs may result from the inhibition of the COX isoforms but other as yet undefined paths to NSAID-induced apoptosis clearly exist. A member of each class of NSAID is under trial as a chemopreventive agent for colorectal cancer.
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  • 85
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    Keywords: Apoptosis ; carcinogenesis ; colon cancer ; cyclooxygenase ; nonsteroidal anti-inflammatory drugs.
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract NSAIDs are potent chemopreventive agents for colon cancer. Although their mechanism of action is unknown, it probably relates to their modulation of colon epithelial cell kinetics, i.e. apoptosis and/or cell proliferation. NSAIDs are pleiotropic in their biochemical activities; their best known effect is inhibition of prostaglandin H synthase (PHS), the enzyme catalyzing the biosynthesis of prostaglandins. Current data appear to lead to two conflicting conclusions. One body of data indicates that PHS is important in induction of apoptosis and colon carcinogenesis and that its inhibition by NSAIDs is required for induction of apoptosis and their overall chemopreventive effect. Another set of data indicates that NSAIDs may induce apoptosis and prevent colon cancer without inhibiting the activity of PHS. Both sides of this argument are presented and discussed. This apparent contradiction may be resolved if one accepts that both mechanisms are correct but that they act on different steps of this multistep process.
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  • 86
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    Apoptosis 1 (1996), S. 40-48 
    ISSN: 1573-675X
    Keywords: Apoptosis ; endonuclease ; intracellular pH ; ion channels ; review
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Programmed cell death is a stereotyped and highly conserved process leading to deletion of unwanted cells. The process may be initiated in response to physiologic signals (Fas ligation, removal of extra-cellular matrix or growth factors), or pathologic events (DNA damage, hypoxia/reperfusion injury, viral infection). Some of the signal transduction pathways between a specific stimulus and the commitment to apoptosis are being worked out, although these may not represent general pathways for all triggers of apoptosis. In addition, the cell is able to integrate a variety of signals, some favoring apoptosis and some favoring survival, and to make a life-or-death decision. This has been termed the ‘judgment phase’, whereas once the cell is irreversibly committed to apoptosis, the ‘execution phase’ is initiated. The biochemical features of the ‘execution phase’ are still unclear; DNA cleavage probably represents one of the final events of the execution phase, but what about the multitude of proteases that participate in the process, phosphatidylserine externalization, transglutaminase activation, and, of course, the subject of this discussion, cytoplasmic acidification? Where do these events fit into the process, and what are the relationships of one to the other? This review will address the following points: (1) Is cytoplasmic acidification a universal feature of apoptosis, and is it essential? The reported cases examined to date will be evaluated. (2) How is cytoplasmic acidification accomplished? Is acidification sufficient for apoptosis to occur? (3) What are the consequences of acidification, particularly with respect to other biochemical features of apoptosis? (4) Finally, I would like to advance the hypothesis that acidification may represent the cellular mechanism for integration of multiple signals; cytoplasmic acidification could represent the point of no return on the road to apoptosis.
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    Apoptosis 1 (1996), S. 147-152 
    ISSN: 1573-675X
    Keywords: Apoptosis ; DNA fragmentation ; DNase γ ; programmed cell death ; thymocytes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The physiological and pathological importance of cell death by apoptosis has recently been recognized. One of the hallmarks of apoptosis is the enzymatic cleavage of genomic DNA into nucleosomal oligomers. The identification of an endonuclease responsible for apoptosis might help to explain how this cell suicide is regulated and why DNA is cleaved. Here, we found that γ type of DNase was retained in apoptotic rat thymocyte nuclei. Homogeneously purified DNase γ (Mr = 33 kDa) from the apoptotic nuclei was revealed to be a Ca2+/Mg2+-dependent endonuclease and inhibited by Zn2+. This enzyme cleaved chromosomal DNA with 3′-hydroxyl (OH) and 5′-phosphoryl (P) ends. The cleavage ends and its divalent cation dependencies match those observed in apoptotic thymocytes induced by X-irradiation or glucocorticoid treatment, indicating that this endonuclease is a central component of the thymic apoptosis machinery.
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  • 88
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    Keywords: Apoptosis ; cell proliferation ; IL-2 ; Ras
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract Oncoproteins of the Ras family have been extensively studied because of their implication in human cancer. Their roles have been primarily assigned to the commandment of cell proliferation and suppression of apoptosis, which has also been demonstrated by the involvement of Ras activation in the signal transduction pathways triggered by most cytokine receptors. Nevertheless, the functions of Ras proteins have been extended in the last years by the findings showing that they can also act as promoters or enhancers of apoptosis in various systems and conditions. These considerations have raised the issue as to how the signals delivered by Ras are regulated and translated in terms of cellular responses, suggesting that signal complementation may direct the final fate of cells. As an example, the interleukin-2 receptor system may represent a useful model in which the meaning of Ras signals may be evaluated in terms of interactions with other simultaneous signalling events, since knowledge of the biochemical events triggered by the interaction of interleukin-2 with its cell surface receptor in lymphocytes has allowed the proposal of a complete signalling model arranged in three independent channels, one of which is mediated by Ras.
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  • 89
    ISSN: 1573-675X
    Keywords: Apoptosis ; CD4+ lymphocytes ; cell penetration ; HIV-1 ; mitochondrial dysfunction ; Vpr
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We have previously shown that expression of HIV-1 vpr in yeast results in cell growth arrest and structural defects, and identified a C-terminal domain of Vpr as being responsible for these effects in yeast.1 In this report we show that recombinant Vpr and C-terminal peptides of Vpr containing the conserved sequence HFRIGCRHSRIG caused permeabilization of CD4+ T lymphocytes, a dramatic reduction of mitochondrial membrane potential and finally cell death. Vpr and Vpr peptides containing the conserved sequence rapidly penetrated cells, co-localized with the DNA, and caused increased granularity and formation of dense apoptotic bodies. The above results suggest that Vpr treated cells undergo apoptosis and this was confirmed by demonstration of DNA fragmentation by the highly sensitive TUNEL assay. Our results, together with the demonstration of extracellular Vpr in HIV infected individuals,2,3 suggest the possibility that extracellular Vpr could contribute to the apoptotic death and depletion of bystander cells in lymphoid tissues4,5 during HIV infection.
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  • 90
    ISSN: 1573-675X
    Keywords: Apoptosis ; α-fodrin ; calpain ; thymocytes ; x-irradiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The α-subunit (240 kDa) of fodrin was found to be digested selectively to a 120 kDa fragment during apoptosis of rat thymocytes in vivo and in vitro. This fragment was detected by an antibody (Ab) against full length α-fodrin, but not by the anti-N-terminal sequence (GMMPR) of the μ-calpain-generated 150 kDa fragment Ab or the anti-PEST sequence of α-fodrin Ab. On the other hand, basal levels of the 150 kDa fragment were constantly recognized by these three antibodies during apoptosis. The production of the 120 kDa fragment during apoptosis was not affected by the addition of calpain inhibitors such as Ac-LLLnal and E-64d, despite inhibition of the generation of the 150 kDa fragment. When x-irradiated thymocytes were incubated in the presence of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), both production of the 120 kDa fragment and apoptosis were suppressed. Purified μ- and m-calpain did not catalyze the formation of the 120 kDa fragment from purified α-fodrin in vitro. These results suggest that a protease different from calpains is involved in the major process of α-fodrin proteolysis to a 120 kDa fragment during thymic apoptosis.
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  • 91
    Electronic Resource
    Electronic Resource
    Springer
    Apoptosis 2 (1997), S. 77-83 
    ISSN: 1573-675X
    Keywords: Apoptosis ; bcl-2 ; CPP32 ; thiol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Reduced thiols (e.g., cysteine) are important in the maintenance of lymphocyte cell viability and growth. L1210 monocytic leukaemia cells were known to have a limited ability to uptake cystine, and they require cysteine for cell growth. L1210 cells underwent apoptosis when cultured without thiol-bearing and dithiol-cleaving compounds, adding thiols suppressed the apoptosis and promoted cell growth. A specific inhibitor of interleukin-1 β-converting enzyme (ICE)-like and CPP32-like proteases could suppress L1210 cell apoptosis induced by thiol deprivation. The cell lysates of apoptotic L1210 cells exhibited protease activity that could cleave DEVD-AMC, but not YVAD-AMC, and so CPP32-like proteases, but not ICE-like proteases, were activated and participated in apoptosis. The addition of thiols could suppress CPP32-like protease activation. Although the cell death-suppressor bcl-2-family proteins (bcl-2 and bcl-XL) were recently found to suppress the activation of CPP32-like proteases, the expression levels of death-suppressor bcl-2-family proteins did not change when thiols were added. These results suggest that reduced thiols maintain L1210 cell survival by inhibiting the activation of CPP32-like proteases without changing the anti-apoptotic bcl-2-family protein expression.
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  • 92
    ISSN: 1573-675X
    Keywords: Apoptosis ; cytokeratin ; drug resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Cytokeratins are one group of intermediate filament proteins responsible for the integrity of cell structure, and have been recently reported to play a role in conferring a drug resistance phenotype. MAb Cx-99 is a monoclonal antibody exhibiting the specificity toward its corresponding antigen which was recently identified as the cytokeratin-19 protein. In the present study, we found that the level of cytokeratin-19 in cervical cancer cells could be decreased by incubation of cancer cells with MAb Cx-99. The reduction of cytokeratin-19 level had a killing effect on cervical carcinoma SIHA and HeLa S3 cell lines. The DNA ladder pattern, convoluted nuclei and blebbing morphology were observed with these cells after exposure to MAb Cx-99 for 72 h, suggesting that the cytotoxic mechanism of reduced cytokeratin-19 was mediated by induction of apoptosis. Moreover, the MAb Cx-99 treatment could increase the cytotoxicities of cancer chemotherapeutic agents such as cisplatin and vinblastine to both cervical carcinoma cell lines. The LD80 values were at least 15-fold reduced when cancer cells were treated with cisplatin or vinblastine in the presence of MAb Cx-99. These results suggest that the functional role of cytokeratin-19 was associated with the apoptosis prevention and drug resistance of cervical cancer cells.
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  • 93
    ISSN: 1573-675X
    Keywords: Apoptosis ; CPP32 ; ICE ; ICE inhibitors ; ICE-related protease
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract IL-1β converting enzyme (ICE) and ICE-related proteases (IRPs) have been suggested to play a central role in apoptosis. We report the use of peptidic ICE inhibitors to reassess the role of this enzyme in the apoptosis induced by Fas or TNFα receptor ligation in Jurkat cells, U937 cells or monocytes. Our results show that inhibition of IL-1β processing can be dissociated from inhibition of apoptosis. Indeed, two out of three com-pounds active on ICE are not inhibitory for apoptosis. This shows that ICE is not required for progression in the apoptotic pathway, but that one or several IRPs are necessary. In addition, Western blot analysis of cell lysates shows that both ICE and CPP32 precursors disappear rapidly after apoptosis induction, while ICH-1L precursor remains intact. Concomitant appearance of cleavage products can be visualized for CPP32, but not for ICE, suggesting that the former is proteolytically activated. In addition, this precursor cleavage can be blocked by an ICE inhibitor active on apoptosis. Altogether, our data support the hypothesis that one or several IRPs are necessary for apoptosis and are responsible for ICE and CPP32 cleavage during this process.
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  • 94
    ISSN: 1573-675X
    Keywords: Apoptosis ; bcl-2 ; dopamine ; Parkinson's disease ; PC-12 ; proto-oncogene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract It is currently believed that excessive oxidant stress induced by metabolism of dopamine (DA), plays a major role in the pathogenesis of the selective nigrostriatal neuronal loss in Parkinson's disease. We recently showed that the neurotransmitter DA, in physiological concentrations, is capable of initiating apoptosis in cultured, post-mitotic sympathetic neurons. Bcl-2 is a proto-oncogene that blocks apoptosis. We now report that Bcl-2 is a powerful inhibitor of DA toxicity in PC-12 pheochromocytoma cells. We induced stable expression of Bcl-2 in PC-12 cells by transfection with recombinant pCMV5 expression vector, containing mouse bcl-2 (coding-sequence) cDNA. Cells expressing Bcl-2 manifested marked resistance to otherwise lethal (300 uM) in vitroconcentrations of DA. This protective effect was reflected in the trypan-blue test of cell survival, 3 H-thymidine incorporation and inhibition of the characteristic apoptotic morphologic alterations in scanning electron microscopic studies. Bcl-2 and associated control systems of apoptosis may have an important physiological role in restraining the apop-tosis-triggering potential of DA in nigrostriatal neurons. This novel field of research may yield insights into the pathogenesis of Parkinson's disease and lead to development of novel therapeutic approaches.
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  • 95
    ISSN: 1573-675X
    Keywords: Apoptosis ; beta cells ; cytokines ; Islets of Langerhans ; nitric oxide ; peroxynitrite ; superoxide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We have shown that nitric oxide treatment for 30–90 min causes inhibition of insulin secretion, DNA damage and disturbs sub-cellular organization in rat and human islets of Langerhans and HIT-T15 cells. Here rat islets and beta-cell lines were treated with various free radical generating systems S-nitrosoglutathione (nitric oxide), xanthine oxidase plus hypoxanthine (reactive oxygen species), 3-morpholinosydnonimine (nitric oxide, super-oxide, peroxynitrite, hydrogen peroxide) and peroxynitrite and their effects over 4 h to 3 days compared with those of the cytokine combination interleukin-1β, tumour necrosis factor-α and interferon-γ. End points examined were de novo protein synthesis, cellular reducing capacity, morphological changes and apoptosis by acridine orange cytochemistry, DNA gel electrophoresis and electron microscopy. Treatment (24–72 h) with nitric oxide, superoxide, peroxynitrite or combined cytokines differentially decreased redox function and inhibited protein synthesis in rat islets of Langerhans and in insulin-containing cell lines; cytokine effects were arginine and nitric oxide dependent. Peroxynitrite gave rare apoptosis in HIT-T15 cells and superoxide gave none in any cell type, but caused the most beta cell-specific damage in islets. S-nitroso-glutathione was the most effective agent at causing DNA laddering or chromatin margination characteristic of apoptotic cell death in insulin-containing cells. Cytokine-induced apoptosis was observed specifically in islet beta cells, combined cytokine effects on islet function and death most resembled those of the mixed radical donor SIN-1.
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  • 96
    ISSN: 1573-675X
    Keywords: Apoptosis ; enzyme immunoassay ; gel electrophoresis ; growth factor ; immunohistochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Many isolated adult cardiocytes do not survive beyond the early days of culture, but why they die has not been defined. We examined the possibility of apoptosis as the mechanism of death in cultured atrial and ventricular rat cardiocytes. Calcium-tolerant cardiocytes isolated by enzymatic dissociation were cultured with a medium containing FBS. Nucleosomal DNA fragmentation was detected by electrophoresis of DNA extracted from the cardiocytes, by immunohistochemical in situ DNA nick-end labelling of single cells, and by enzyme immunoassay for in vitro quantification in cytoplasmic fraction. Electrophoresis on the 5th to 14th day of culture revealed the ladder appearance characteristic of internucleosomal DNA cleavage in apoptosis with a consistent single peak of increased cytoplasmic DNA fragments. After the 14th day, the cytoplasmic DNA fragments decreased, and the ladder appearance could no longer be detected by electrophoresis. Cardiocytes positive with nick-end labelling were seen by the 5th day, and then increased in number over the remaining days. These results indicate that many isolated cardiocytes die spontaneously by apoptosis within the first 2 weeks of culture, suggesting a possible signal dependence for survival of adult cardiocytes. In addition to chemical signal depletion in culture, other possible explanations for this apoptosis include the absence of an electric signal during culture, lack of contractile activity, and initial loss of intercellular connections.
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  • 97
    Electronic Resource
    Electronic Resource
    Springer
    Apoptosis 2 (1997), S. 265-282 
    ISSN: 1573-675X
    Keywords: Apoptosis ; cell cycle ; clonogenicity ; p53 ; radiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Ionizing radiation can be an effective inducer of apoptosis and studies of many aspects of the pathways and mechanisms involved in this apoptosis induction have been published. This review stresses two aspects: the relationship between apoptosis and loss of clonogenic ability in irradiated cells and the time course for the appearance of apoptosis after radiation exposure. Although it was initially assumed that apoptosis occurred relatively quickly (within hours) after irradiation, evidence is presented and discussed here showing that apoptosis can occur at long times after irradiation (out to 20 days) in some cell types. This late, or delayed, apoptosis occurs after the cells have divided once or several times. The impact of delayed apoptosis on loss of clonogenicity after irradiation remains unclear. It seems likely that in some cell types, e.g., fibroblasts, the occurrence of late apoptosis is minimal and may have little impact on long term cell survival of the population, but in at least one instance, with a cell line of hematopoietic origin, it appears that late apoptosis can account for all the loss of clonogenicity in irradiated cells. The role of p53 in radiation-induced apoptosis is also discussed, with data presented showing that both p53-dependent and independent pathways for radiation-induced apoptosis exist, depending on the cell type.
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  • 98
    ISSN: 1573-675X
    Keywords: Apoptosis ; cerebral ischemia ; electron microscopy ; necrosis ; TUNEL staining
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Electron microscopy and terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end-labelling (TUNEL) were used to illustrate the different stages and subcellular alterations of cell degeneration that occur in the striatum of the rat after transient focal ischemia. Degenerating neurons exhibited different morphological types: apoptosis Type 1 (aggregation of dense masses of chromatin beneath the 'intact' nuclear membrane) and Type 2 (high cytoplasmic vacuolization), and necrosis. These profiles were localized in different part of the striatum. Type 1 was found in the head of the caudate putamen, Type 2 in the middle part of the striatum and necrosis in the striatal core. These ultrastructural results demonstrated that apoptosis occurs in neurons following focal ischemia in the striatal penumbra. In contrast, necrosis can be observed in the ischemic core, the region maximally affected by the ischemia. Finally, the presence of astrocytes throughout both the penumbra and ischemic core displaying numerous cytoplasmic vacuoles suggested an activation of glial cells.
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  • 99
    ISSN: 1573-675X
    Keywords: Apoptosis ; high-molecular-weight DNA ; oligonucleosomal DNA ; UV-B
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We investigated changes typical for apoptosis in various cell lines after UV-B irradiation. Using established methods for detection of apoptosis we demonstrate changes of cellular morphology, phosphatidylserine (PS) exposure, ollgonucleosomal DNA fragmentation and generation of hypochrome nuclei. To isolated high-molecular-weight (hmwt) DNA fragments we engaged a new method avoiding pulse field gel electrophoresis. Most UV-B irradiated cell lines showed oligonucleosomal DNA fragmentation, hypochrome nuclei, morphological changes, annexin-V binding and positive TUNEL reaction. However, no oligonucleosomal DNA fragmentation could be detected in Raji and HaCaT cells. Whereas HaCaT cells displayed all other changes typical for apoptosis, Raji cells were TUNEL negative, formed low amounts of hmwt DNA and showed an 'atypically' low hypochrome shift. Nevertheless, UV-B irradiated Raji cells excluded propidium iodide (PI), bound annexin-V and stopped proliferation. This suggests that Raji cells underwent growth arrest with exposure of PS being the only feature of apoptosis. However, in the presence of phagocytes expressing the phosphatidylserine receptor these cells would share the removal pathway with apoptotic cells. Since UV-B induced programmed cell death differs in dependence of cells under investigation, the failure to detect oligonucleosomal DNA fragmentation or chromatin condensation is not suitable to exclude programmed (apoptotic?) cell death.
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  • 100
    ISSN: 1573-675X
    Keywords: Apoptosis ; cervical carcinoma ; cytokeratin-19 ; tumour marker
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Cytokeratin-19 is an intermediate filament protein associated with the integrity of cell structure, and its elevated expression has been reported to correlate with the disease progression of oesophagus and lung cancers. In this study, we examined the level of cytokeratin-19 in five cervical cancer cell lines by immunobinding and Western blotting analyses. Compared with two control cell lines, FS-4 (foreskin cell line) and G9T (glioma cell line), all five cervical carcinoma cell lines (Caski, CC7T, ME180, HeLa and SIHA) showed higher cytokeratin-19 expression. By double-staining flow cytometry, expression of cytokeratin-19 in cervical cancer cells was suggested to be in a cell cycle-independent manner. Furthermore, we could specifically localize the SIHA cell-derived tumours in nude mice by injecting with cytokeratin-19-recognized radiolabelled MAb Cx-99 antibody, suggesting the possibility of using cytokeratin-19 as a marker of cervical carcinoma. A clinical investigation was therefore performed on 19 patients (11 patients with cervical carcinoma and eight patients with benign neoplasia). In the 11 patients having cervical carcinoma, all eight patients with advanced stages and one out of three patients with early stage diseases showed higher cytokeratin-19 protein contents than the other 10 patients with benign neoplasia. This suggested that elevation of cytokeratin-19 level was associated with cervical cancer staging. In addition, we have studied the biological significance of elevated cytokeratin-19 level in malignant cervical cancer. The apoptotic rate of cervical carcinoma cells in response to cisplatin was increased if their cellular cytokeratin-19 level was reduced by specific antibody MAb Cx-99. These results indicated that elevation of cytokeratin-19 expression could associate with the apoptotic resistance and malignant progression of cervical carcinoma.
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