scholarly journals Bax- and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe.

1997 ◽  
Vol 8 (2) ◽  
pp. 325-339 ◽  
Author(s):  
J M Jürgensmeier ◽  
S Krajewski ◽  
R C Armstrong ◽  
G M Wilson ◽  
T Oltersdorf ◽  
...  
Keyword(s):  
Cell Death ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Mammalian Cells ◽  
Animal Cell ◽  
Chromatin Condensation ◽  
Interleukin 1 ◽  
Microscopic Analysis ◽  
Animal Cells ◽  
Electron Microscopic

The effects of the expression of the human Bcl-2 family proteins Bax, Bak, Bcl-2, and Bcl-XL were examined in the fission yeast Schizosaccharomyces pombe and compared with Bax-induced cell death in mammalian cells. Expression of the proapoptotic proteins Bax and Bak conferred a lethal phenotype in this yeast, which was strongly suppressed by coexpression of the anti-apoptotic protein Bcl-XL. Bcl-2 also partially abrogated Bax-mediated cytotoxicity in S. pombe, whereas a mutant of Bcl-2 (Gly145Ala) that fails to heterodimerize with Bax or block apoptosis in mammalian cells was inactive. However, other features distinguished Bax- and Bak-induced death in S. pombe from animal cell apoptosis. Electron microscopic analysis of S. pombe cells dying in response to Bax or Bak expression demonstrated massive cytosolic vacuolization and multifocal nuclear chromatin condensation, thus distinguishing this form of cell death from the classical morphological features of apoptosis seen in animal cells. Unlike Bax-induced apoptosis in 293 cells that led to the induction of interleukin-1 beta-converting enzyme (ICE)/CED-3-like protease activity, Bax- and Bak-induced cell death in S. pombe was accompanied neither by internucleosomal DNA fragmentation nor by activation of proteases with specificities similar to the ICE/CED-3 family. In addition, the baculovirus protease inhibitor p35, which is a potent inhibitor of ICE/CED-3 family proteases and a blocker of apoptosis in animal cells, failed to prevent cell death induction by Bax or Bak in fission yeast, whereas p35 inhibited Bax-induced cell death in mammalian cells. Taken together, these findings suggest that Bcl-2 family proteins may retain an evolutionarily conserved ability to regulate cell survival and death but also indicate differences in the downstream events that are activated by overexpression of Bax or Bak in divergent cell types.

Involvement of ICE family proteases in apoptosis induced by reoxygenation of hypoxic hepatocytes

1996 ◽  
Vol 271 (6) ◽  
pp. G949-G958 ◽  
Author(s):  
S. Shimizu ◽  
Y. Eguchi ◽  
W. Kamiike ◽  
Y. Akao ◽  
H. Kosaka ◽  
...  
Keyword(s):  
Cell Death ◽  
Chromatin Condensation ◽  
Interleukin 1 ◽  
Membrane Integrity ◽  
Morphological Characteristics ◽  
Electrophoretic Analysis ◽  
Dna Fragments ◽  
Electron Microscopic ◽  
Triggering Factor ◽  
Induced Apoptosis

Cell death due to reoxygenation after hypoxia was characterized in primary cultured hepatocytes. Fluorescence and electron microscopic analyses of reoxygenated hepatocytes revealed morphological characteristics of apoptosis, including chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Few necrotic hepatocytes, defined by loss of plasma membrane integrity, mitochondrial swelling, and formation of large vacuoles, were observed. Activation of interleukin-1 beta-converting enzyme (ICE)-like and CPP32/Yama-like proteases, which are known to drive apoptosis, was observed during reoxygenation, and addition of their respective inhibitors inhibited the induction of apoptosis, indicating the involvement of ICE family proteases in apoptosis by reoxygenation. Production of oxygen radicals was enhanced by reoxygenation of hypoxic cells, and reoxygenation-induced apoptosis was inhibited by oxygen radical scavengers, suggesting a role for reactive oxygen species as a triggering factor in cell death. Electrophoretic analysis revealed the presence of 50-kb DNA fragments but not oligonucleosomal DNA fragments in reoxygenation-induced apoptotic hepatocytes.

Overexpression Phenotypes of Plk1 and Ndrg2 in Schizosaccharomyces Pombe: Fission Yeast System for Mammalian Gene Study

2005 ◽  
Vol 277-279 ◽  
pp. 1-6 ◽  
Author(s):  
Young Joo Jang ◽  
Young Sook Kil ◽  
Jee Hee Ahn ◽  
Jae Hoon Ji ◽  
Jong Seok Lim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Rna Splicing ◽  
Mammalian Cells ◽  
Protein Modification ◽  
Genetic System ◽  
Functional Study ◽  
Mammalian Genes

The fission yeast, Schizosaccharomyces pombe is a single-celled free-living fungus that shares many features with cells of more complicated eukaryotes. Many of the genes required for the cell-cycle control, proteolysis, protein modification, and RNA splicing are highly conserved with those of higher eukaryotes. Moreover, fission yeast has the merit of genetics and its genetic system is already well characterized. As such, the current study evaluated the use of a fission yeast system as a tool for the functional study of mammalian genes and attempted to set up an assay system for novel genes. Since the phenotypes of a deletion mutant and the overexpression of a gene are generally analyzed for a functional study of specific genes in yeast, the present study used overexpression phenotypes to study the functions of mammalian genes. Therefore, based on using a thiamine-repressive promoter, two mammalian genes were expressed in fission yeast, and their overexpressed phenotypes compared with those in mammalian cells. The phenotypes resulting from overexpression were analyzed using a FACS, which analyzes the DNA contents, and a microscope. One of the selected genes was the mammalian Polo-like kinase 1 (Plk1), which is activated and plays a role in the mitotic phase of the cell division cycle. The overexpression of various constructs of Plk1 in the HeLa cells caused cell cycle defects, suggesting that the ectopic Plk1s blocked the endogenous Plk1 in the cells. As expected, when the constructs were overexpressed in the fission yeast system, the cells were arrested in mitosis and defected at the end of mitosis. As such, this data suggests that the Plk1-overexpressed phenotypes were similar in the mammalian cells and the fission yeast, thereby enabling the mammalian Plk1 functions to be approximated in the fission yeast. The other selected gene was the N-Myc downstream-regulated gene 2 (ndrg2), which is upregulated during cell differentiation, yet still not well characterized. When the ndrg2 gene was overexpressed in the fission yeast, the cells contained multi-septa. The septa were positioned well, yet their number increased per cell. Therefore, this gene was speculated to block cell division in the last stage of the cell cycle, making the phenotype potentially useful for explaining cell growth and differentiation in mammalian cells. Accordingly, fission yeast is demonstrated to be an appropriate species for the functional study of mammalian genes.

scholarly journals Analysis of Antigenicity and Topology of E2 Glycoprotein Present on Recombinant Hepatitis C Virus-Like Particles

2002 ◽  
Vol 76 (15) ◽  
pp. 7672-7682 ◽  
Author(s):  
Reginald F. Clayton ◽  
Ania Owsianka ◽  
Jim Aitken ◽  
Susan Graham ◽  
David Bhella ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mammalian Cells ◽  
Function Analysis ◽  
Recombinant Baculovirus ◽  
Microscopic Analysis ◽  
Surface Topology ◽  
Viral Glycoprotein ◽  
Electron Microscopic ◽  
Virus Like Particles

ABSTRACT Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2660) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2660 with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2660 but not VLPs or FL E1E2 complex. The antigenic differences between E2660, FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.

Acute phase response in exercise. III. Neutrophil and IL-1 beta accumulation in skeletal muscle

1993 ◽  
Vol 265 (1) ◽  
pp. R166-R172 ◽  
Author(s):  
R. A. Fielding ◽  
T. J. Manfredi ◽  
W. Ding ◽  
M. A. Fiatarone ◽  
W. J. Evans ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cross Sections ◽  
Immunohistochemical Staining ◽  
Rating Scale ◽  
Vastus Lateralis ◽  
Interleukin 1 ◽  
Microscopic Analysis ◽  
Neutrophil Infiltration ◽  
Neutrophil Accumulation ◽  
Electron Microscopic

Nine untrained men (22-29 yr) performed 45 min of downhill running (16% incline, 70% of maximum heart rate). Needle biopsies of the vastus lateralis were performed before, 45 min after, and 5 days after exercise. Immunohistochemical staining of muscle cross sections revealed a 135% increase in muscle interleukin-1 beta (IL-1 beta) immediately after and a 250% increase (P < 0.03) 5 days after exercise. Using a rating scale (0-3) for the presence of neutrophils, light microscopic examination showed a significant accumulation of neutrophils in muscle biopsies taken 45 min after and 5 days after exercise [before: 0.5 +/- 0.2, 45 min after: 1.5 +/- 0.3 (P < 0.01), and 5 days after: 1.2 +/- 0.2 (P < 0.04)]. In addition, electron microscopic analysis showed an increase in the percentage of damaged Z-bands relative to total Z-bands [before: 4.8 +/- 3.5%, 45 min after: 32.5 +/- 8.6% (P < 0.05), and 5 days after: 14.1 +/- 3.2%]. Neutrophil accumulation was positively correlated to intracellular Z-band damage (rho = 0.66, P < 0.001). Immunohistochemical staining for IL-1 beta was related to neutrophil accumulation in muscle (rho = 0.38, P < 0.06) and to plasma creatine kinase levels (rho = 0.416, P < 0.04). These data indicate that after eccentric exercise ultrastructural damage to skeletal muscle is associated with neutrophil infiltration and muscle IL-1 beta accumulation.

scholarly journals Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death.

1996 ◽  
Vol 133 (5) ◽  
pp. 1041-1051 ◽  
Author(s):  
M D Jacobsen ◽  
M Weil ◽  
M C Raff
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Apoptotic Cell ◽  
Interleukin 1 ◽  
Cell Types ◽  
Cysteine Proteases ◽  
A Cell ◽  
Bona Fide

In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS-induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.

scholarly journals Baculovirus Infection of Nondividing Mammalian Cells: Mechanisms of Entry and Nuclear Transport of Capsids

2001 ◽  
Vol 75 (2) ◽  
pp. 961-970 ◽  
Author(s):  
Nico-Dirk van Loo ◽  
Elisabetta Fortunati ◽  
Erich Ehlert ◽  
Martijn Rabelink ◽  
Frank Grosveld ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Nuclear Transport ◽  
Microscopic Analysis ◽  
Cell Types ◽  
Nuclear Pore ◽  
Fusion Event ◽  
Electron Microscopic ◽  
Cytoplasmic Transport ◽  
Baculovirus Infection

ABSTRACT We have studied the infection pathway of Autographa californica multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian cell types can be infected efficiently. In contrast to previous suggestions, our data show that the asialoglycoprotein receptor is not required for efficient infection. We demonstrate for the first time that this baculovirus can infect nondividing mammalian cells, which implies that the baculovirus is able to transport its genome across the nuclear membrane of mammalian cells. Our data further show that the virus enters via endocytosis, followed by an acid-induced fusion event, which releases the nucleocapsid into the cytoplasm. Cytochalasin D strongly reduces the infection efficiency but not the delivery of nucleocapsids to the cytoplasm, suggesting involvement of actin filaments in cytoplasmic transport of the capsids. Electron microscopic analysis shows the cigar-shaped nucleocapsids located at nuclear pores of nondividing cells. Under these conditions, we observed the viral genome, major capsid protein, and electron-dense capsids inside the nucleus. This suggests that the nucleocapsid is transported through the nuclear pore. This mode of transport seems different from viruses with large spherical capsids, such as herpes simplex virus and adenovirus, which are disassembled before nuclear transport of the genome. The implications for the application of baculovirus or its capsid proteins in gene therapy are discussed.

scholarly journals Plasmodesmata-like intercellular connections by plant remorin in animal cells

2019 ◽  
Author(s):  
Zhuang Wei ◽  
Shutang Tan ◽  
Tao Liu ◽  
Yuan Wu ◽  
Ji-Gang Lei ◽  
...  
Keyword(s):  
Intercellular Communication ◽  
Mammalian Cells ◽  
Communication Systems ◽  
Biochemical Characterization ◽  
Super Resolution ◽  
Microscopic Analysis ◽  
Filamentous Structure ◽  
Animal Cells ◽  
Specific Distribution ◽  
Intercellular Connections

SummaryPlasmodesmata (PD) are crucial structures for intercellular communication in multicellular plants with remorins being their crucial plant-specific structural and functional constituents. The PD biogenesis is an intriguing but poorly understood process. By expressing an Arabidopsis remorin protein in mammalian cells, we have reconstituted a PD-like filamentous structure, termed remorin filament (RF), connecting neighboring cells physically and physiologically. Notably, RFs are capable of transporting macromolecules intercellularly, in a way similar to plant PD. With further super-resolution microscopic analysis and biochemical characterization, we found that RFs are also composed of actin filaments, forming the core skeleton structure, aligned with the remorin protein. This unique heterologous filamentous structure might explain the molecular mechanism for remorin function as well as PD construction. Furthermore, remorin protein exhibits a specific distribution manner in the plasma membrane in mammalian cells, representing a lipid nanodomain, depending on its lipid modification status. Our studies not only provide crucial insights into the mechanism of PD biogenesis, but also uncovers unsuspected fundamental mechanistic and evolutionary links between intercellular communication systems of plants and animals.SignificanceRemorin is rising as a crucial lipid microdomain marker, as well as an essential component of plasmodesmata in plants. However, the biological role of remorin in plants is elusive. With a heterologous system, we found that remorin expression is able to form intercellular filamentous structure, namely remorin filament (RF), connecting neighboring mammalian cells functionally. By employing multiple approaches, we tested the functionality of RFs, as well as investigated their structures. RFs highly resemble plant plasmodesmata, in many ways, such as its morphology, molecular constitutes, and its ability to transport macromolecules intercellularly. Our study provides novel insights into the biogenesis of plasmodesmata and uncovers fundamental evolutionary links in molecular construction of intercellular connections in both plants and animals.

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