EPILEPSIA, Issue 6 2003
Hilmi Uysal
Summary: Purpose: Mesial temporal sclerosis (MTS) is characterized by neuronal loss in the hippocampus. Studies on experimental models and patients with intractable epilepsy suggest that apoptosis may be involved in neuronal death induced by recurrent seizures. Methods: We searched evidence for apoptotic cell death in temporal lobes resected from drug-resistant epilepsy patients with MTS by using the terminal deoxynucleotidyl transferase (TdT) and digoxigenin-11-dUTP (TUNEL) method and immunohistochemistry for Bcl-2, Bax, and caspase-cleaved actin fragment, fractin. The temporal lobe specimens were obtained from 15 patients (six women and nine men; mean age, 29 ± 8 years). Results: Unlike that in normal adult brain, we observed Bcl-2 immunoreactivity in some of the remaining neurons dispersed throughout the hippocampus proper as well as in most of the reactive astroglia. Bax immunopositivity was increased in almost all neurons. Fractin immunostaining, an indicator of caspase activity, was detected in ,10% of these neurons. Des pite increased Bax expression and activation of caspases, we could not find evidence for DNA fragmentation by TUNEL staining. We also could not detect typical apoptotic changes in nuclear morphology by Hoechst-33258 or hematoxylin counterstaining. Conclusions: These data suggest that either apoptosis is not involved in cell loss in MTS, or a very slow rate of cell demise may have precluded detecting TUNEL-positive neurons dying through apoptosis. Increased Bax expression and activation of caspases support the latter possibility. [source]

Apoptotic and behavioral sequelae of mild brain trauma in mice

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2007
David Tweedie
Abstract Mild traumatic brain injury (mTBI) is a not uncommon event in adolescents and young adults. Although it does not result in clear morphological brain defects, it is associated with long-term cognitive, emotional, and behavioral problems. Herein, we characterized the biochemical and behavioral changes associated with experimental mTBI in mice that may act as either targets or surrogate markers for interventional therapy. Specifically, mTBI was induced by 30-g and 50-g weight drop, and at 8 and 72 hr thereafter markers of cellular apoptosis,caspase-3, Bax, apoptosis-inducing factor (AIF), and cytochrome-c (Cyt-c),were quantified by Western blot analysis in hippocampus ipsilateral to the impact. Levels of amyloid-, precursor protein (APP) were also measured, and specific behavioral tests,passive avoidance, open field, and forced swimming (Porsolt) paradigms,were undertaken to assess learning, emotionality, and emotional memory. In the absence of hemorrhage or infarcts, as assessed by triphenyltetrazolium chloride staining, procaspase-3 and Bax levels were markedly altered following mTBI at both times. No cleaved caspase-3 was detected, and levels of AIF and Cyt-c, but not APP, were significantly changed at 72 hr. Mice subjected to mTBI were indistinguishable from controls by neurological examination at 1 and 24 hr, and by passive avoidance/open field at 72 hr, but could be differentiated in the forced swimming paradigm. In general, this model mimics the diffuse effects of mTBI on brain function associated with the human condition and highlights specific apoptotic proteins and a behavioral paradigm as potential markers for prospective interventional strategies. © 2007 Wiley-Liss, Inc. [source]

Apoptotic Changes in the Epithelium Germinativum of the Cat (Felis catus s. domestica, L. 1758) at Different Ages and Breeding Seasons

REPRODUCTION IN DOMESTIC ANIMALS, Issue 4 2008
MJ SiemieniuchArticle first published online: 25 FEB 200
Contents Apoptosis (programmed cell death) could be considered as a physiological process that takes part in a healthy organism, which helps to maintain organism homeostasis. The visible deterioration of semen quality and the number of germ cells is accompanied by a seasonal decrease of the reproductive activity in some species. This post-effect cascade is caused by apoptosis, which is the primary mechanism responsible for the elimination of germ cells during spermatogenesis. The aim of our study was to assess apoptotic changes in the epithelium germinativum in cat testes at different ages. One hundred and two pairs of testes were obtained from domestic cats aged between 4 months and 10 years. The paraffin-embedded tissue sections were labelled using the Oncogene and Calbiochem Research Products DNA Fragmentation Detection Kit (Cat# QIA21; Darmstadt, Germany), which allows the recognition of apoptotic nuclei in tissue sections with Fragment End Labelling (FragELTM) of DNA. The activity of apoptotic processes in cat testes collected from the spring-summer period compared with the autumn-winter season revealed that, 59.42% and 51.51%, respectively, males testes were characterized by insignificant changes. The obtained data revealed a distinctive apoptotic changes in the young animal testes before spermatogenesis onset. An intensification of programmed death cells in the epithelium germinativum in the elder cats (between 3,6 and 6,10 years) was not observed. Apoptotic changes slightly intensified in cats aged between 12 and 36 months. [source]

Cell Death Mechanisms Following Traumatic Brain Injury

BRAIN PATHOLOGY, Issue 2 2004
Ramesh Raghupathi PhD
Neuronal and glial cell death and traumatic axonal injury contribute to the overall pathology of traumatic brain injury (TBI) in both humans and animals. In both head-injured humans and following experimental brain injury, dying neural cells exhibit either an apoptotic or a necrotic morphology. Apoptotic and necrotic neurons have been identified within contusions in the acute post-traumatic period, and in regions remote from the site of impact in the days and weeks after trauma, while degenerating oligodendrocytes and astrocytes have been observed within injured white matter tracts. We review and compare the regional and temporal patterns of apoptotic and necrotic cell death following TBI and the possible mechanisms underlying trauma-induced cell death. While excitatory amino acids, increases in intracellular calcium and free radicals can all cause cells to undergo apoptosis, in vitro studies have determined that neural cells can undergo apoptosis via many other pathways. It is generally accepted that a shift in the balance between pro- and anti-apoptotic protein factors towards the expression of proteins that promote death may be one mechanism underlying apoptotic cell death. The effect of TBI on cellular expression of survival promoting-proteins such as Bcl-2, Bcl-xL, and extracellular signal-regulated kinases, and death-inducing proteins such as Bax, c-Jun N-terminal kinase, tumor-suppressor gene, p53, and the calpain and caspase families of proteases are reviewed. In light of pharmacologic strategies that have been devised to reduce the extent of apoptotic cell death in animal models of TBI, our review also considers whether apoptosis may serve a protective role in the injured brain. Together, these observations suggest that cell death mechanisms may be representative of a continuum between apoptotic and necrotic pathways. [source]

Apoptotic and Anti-Apoptotic Synaptic Signaling Mechanisms

BRAIN PATHOLOGY, Issue 2 2000
Mark P. Mattson
Although several prominent morphological features of apoptosis are evident in the cell body (e.g., cell shrinkage, membrane blebbing, and nuclear DNA condensation and fragmentation) the biochemical and molecular cascades that constitute the cell death machinery can be engaged in synaptic terminals and neurites. Initiating events such as oxyradical production and calcium influx, and effector processes such as Par-4 production, mitochondrial alterations and caspase activation, can be induced in synapses and neurites. Several prominent signal transduction pathways in synaptic terminals play important roles in either promoting or preventing neuronal death in physiological and pathological conditions. For example, activation of glutamate receptors in postsynaptic spines can induce neuronal apoptosis, whereas local activation of neurotrophic factor receptors in presynaptic terminals can prevent neuronal death. Factors capable of inducing nuclear chromatin condensation and fragmentation can be produced locally in synaptic terminals and neurites, and may propogate to the cell body. Recent findings suggest that, beyond their roles in inducing or preventing cell death, apoptotic and anti-apoptotic cascades play roles in synaptic plasticity (structural remodelling and long-term functional changes). For example, caspase activation results in proteolysis of glutamate receptor (AMPA) subunits, which results in altered neuronal responsivity to glutamate. Activation of neurotrophic factor receptors in synaptic terminals can result in local changes in energy metabolism and calcium homeostasis, and can induce long-term changes in synaptic transmission. The emerging data therefore suggest that synapses can be considered as autonomous compartments in which both pro- and anti-apoptotic signaling pathways are activated resulting in structural and functional changes in neuronal circuits. A better understanding of such synaptic signaling mechanisms may reveal novel approaches for preventing and treating an array of neurodegenerative conditions that are initiated by perturbed synaptic homeostasis. [source]

Cytologic features and frequency of plasmacytoid dendritic cells in the lymph nodes of patients with histiocytic necrotizing lymphadenitis (Kikuchi-Fujimoto disease)

DIAGNOSTIC CYTOPATHOLOGY, Issue 7 2010
I.A.C., Koji Kishimoto C.T., Ph.D.
Abstract Histiocytic necrotizing lymphadenitis (HNL), also known as Kikuchi-Fujimoto disease, is a benign and self-limiting disease. It is histologically characterized by nodal lesions that show the infiltration of histiocytes, lymphoid cells, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs), along with either apoptotic or karyorrhexic nuclear debris. pDCs have been proposed to be lymphoid early-committed immature DCs which are positive for CD123, CD303, CD68, and HLA-DR but negative for fascin, a mature DC marker, as well as CD13 and CD33,which are mDC markers. In the present study, we analyzed the cytomorphologic features and frequency of pDCs in the lymph nodes of HNL patients. Because the cytologic apprearance of pDCs with Papanicolau staining was quite similar to that of large lymphocytes, immunocytochemistry against CD123 was necessary for the distinction of pDCs. Counting the number of CD123-positive pDCs in the HNL lymph nodes revealed that pDCs more frequently infiltrated the lymph nodes in the setting of HNL than in either reactive lymphadenitis or T and B cell lymphoma. In addition, interestingly, the numberof pDCs did not depend on the age of the HNL lesion, thus suggesting that pDCs are excellent indicators for the cytologic diagnosis of HNL. Diagn. Cytopathol. 2010. © 2009 Wiley-Liss, Inc. [source]

Production of Taxol fromPhyllosticta spinarum, an endophytic fungus ofCupressus sp.

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2008
R. Senthil Kumaran
Abstract Taxol production during the cultivation on a modified liquid and potato dextrose broth medium was indicated for the first time to occur in Phyllosticta spinarum, an endophytic fungus isolated from the needles of Cupressus sp. The presence of taxol in the fungal culture filtrate was confirmed by chromatographic and spectroscopic methods of analysis. The amount of taxol produced by this fungus was quantified by high performance liquid chromatography. The maximum amount of taxol production was obtained in this fungus when grown on M1D medium (235,,g/L) followed by PDB medium (125,,g/L). The results indicate that P.,spinarum is an excellent candidate for taxol production. The production rate was 4.7,×,103 -fold higher than that found in the culture broth of an earlier reported fungus, Taxomyces andreanae. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of human cancer cells tested in an apoptotic assay. [source]

Microcystin-LR modulates selected immune parameters and induces necrosis/apoptosis of carp leucocytes,

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2010
Anna Rymuszka
Abstract Microcystins (MCs) are potent hepatotoxins acting by the inhibition of protein phosphatase 1 and 2A, and may promote liver tumors. Moreover, studies also suggest they are nephrotoxic. The aim of the present study was to assess possible in vitro effects of microcystin-LR (which contains the amino acids leucine and arginine, the most widely studied and distributed variant of all microcystins) on the selected immune functions of the cells isolated from the head kidney of carp. In the experiments, pure microcystin-LR (MC-LR), was used at concentrations of 0.01, 0.1, 0.5, and 1,µg/ml RPMI-1640 medium. Leucocytes (lymphocytes and phagocytes) were isolated by centrifugation on a density gradient. Lymphocyte proliferation, intracellular production of reactive oxygen species by phagocytes, and the presence of apoptotic and/or necrotic cells were assessed. The respiratory burst activity of phagocytic cells was increased at the lowest toxin concentration used in the study, but it was decreased at higher concentrations. Using a sensitive luminescent immunoassay, MC-LR was observed to have no influence on the T-cell proliferation but decreased the proliferation of B lymphocytes. Moreover, it was noted that MC-LR induced necrosis to a higher degree than apoptosis in fish leucocytes. The results of the present study suggest the modulatory potency of microcystin-LR on fish leucocytes. Environ. Toxicol. Chem. 2010;29:569,574. © 2009 SETAC [source]

Distinct caspase pathways mediate necrosis and apoptosis in subpopulations of hippocampal neurons after status epilepticus

EPILEPSIA, Issue 2010
Maria-Leonor Lopez-Meraz
Summary Status epilepticus in the immature brain induces neuronal injury in the hippocampal formation, but the mode and mechanism of death are poorly understood. Our laboratory has recently investigated the role of caspase-3, -8, and -9 in neuronal injury, using a lithium,pilocarpine model of status epilepticus in 2-week-old rat pups. Our results showed that dying neurons in the dentate gyrus and CA1-subiculum area do not share the same mechanism of death. In CA1-subiculum, caspase-8 upregulation preceded caspase-3 activation in morphologically necrotic neurons. The pan-caspase inhibitor Q-VD-OPH reduced CA1 damage, showing that caspases contribute to status epilepticus,induced necrosis. In the dentate gyrus, dying neurons were caspase-9 and -3 immunoreactive and morphologically apoptotic. It is not clear why the same seizures cause different types of cell death in neurons that are connected in series along the same hippocampal circuit, but the apoptotic dentate neurons express doublecortin, and do not express calbindin-D28k, suggesting that their immaturity may be a factor in producing an apoptotic mode of death. [source]

Histologic and morphologic effects of valproic acid and oxcarbazepine on rat uterine and ovarian cells

EPILEPSIA, Issue 1 2010
Ali Cansu
Summary Purpose:, To determine the histologic and morphologic effects of valproic acid (VPA) and oxcarbazepine (OXC) on rat uterine and ovarian cells. Methods:, Fifty-six female prepubertal Wistar rats (21,24 days old and weighing between 47.5 and 58.1 g) were divided equally into four groups, which were given drinking water (controls), 300 mg/kg/day of VPA, 100 mg/kg/day of OXC or VPA + OXC via gavage, for 90 days. Ovaries and uteri of rats on proestrous and diestrous phases of estrous cycle were extirpated and placed in a fixation solution. The tissue specimens were assessed with apoptosis (TUNEL) staining protocols, eosinophil counting, and electron microscopic techniques. Results:, In uteri, apoptosis in stroma, mitochondrial swelling, and cristolysis were observed in the VPA group, and OXC led to negative effects on epithelial cell and intracellular edema. In ovaries, both drugs increased apoptosis and intracytoplasmic edema. Organelle structure disruption was also observed in the OXC group. More conspicuous degenerative modifications were determined in the VPA + OXC group. In uteri, the number of TUNEL-positive luminal epithelial cells was 7.20 ± 1.32 in controls, and significantly increased to 29.60 ± 1.58, 34.20 ± 2.53, and 54.80 ± 2.04 in VPA, OXC, and VPA + OXC groups, respectively (p < 0.001). The highest number of TUNEL-positive glandular epithelium cells was observed in the VPA + OXC group; however, the number of TUNEL-positive stroma cells was highest in the VPA group. The highest number of eosinophils in stroma was in the VPA group. Conclusion:, VPA and OXC trigger apoptotic and degenerative effects on rat uterine and ovarian cells. VPA also prevents implantation of embryo to the uterus and causes abortion via endometrial eosinophil infiltration. [source]

Resveratrol modulates apoptosis and oxidation in human blood mononuclear cells

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 9 2003
G. A. Losa
Abstract Background, We examined the effect of resveratrol (RS), a nonflavonoid polyphenolic phytoalexin found in grapes and red wine, and RS coincubated with the oxidant 2-deoxy-D-ribose (dR), on apoptosis and on the oxidative metabolic status of normal human peripheral blood mononuclear cells (PBMNCs) isolated ex vivo from healthy donors. Material and methods, Apoptosis was measured by changes of membrane permeability to propidium iodide (PI), plasma membrane exposure of phosphatidylserine (PS) and intracellular caspase activity. Oxidative status was assessed by recording the intracellular glutathione concentration (GSH), the activities of the enzymes y -glutamyltransferase (y- GT) and glutathione-S-transferase (GST), and intracellular lipid peroxidation (MDA). Results, Neither apoptotic nor oxidative parameters were affected by culturing PBMNCs in medium containing RS up to 20 µM for 5 days, while the frequency of cells with intermediate permeability to PI (17% ± 5) increased at 50 µM of RS. Thus resveratrol was slightly toxic, but there was little apoptosis in these cells. Peripheral blood mononuclear cells were also grown first in medium plus RS for 24 h and then for 96 h in medium containing RS plus 10 mM of dR, an oxidant sugar that is apoptogenic for human lymphocytes. The apoptotic changes triggered by dR were counteracted by the phytoalexin in a dose-dependent manner, but RS activity was absent at the lowest concentration (5 µM) and significantly reduced at the highest concentration used (50 µM). In PBMNCs coincubated with 20 µM of RS and 10 mM of dR the antioxidant effect of RS manifested with a significant reduction of caspases-3, -8, y- GT, GST activities and MDA content. Conclusions, Peripheral blood mononuclear cells acquire antioxidant capacity when treated with RS. Grape resveratrol may make a useful dietary supplement for minimizing oxidative injury in immune-perturbed states and human chronic degenerative diseases. [source]

The antiapoptotic effects of blood constituents in patients with chronic lymphocytic leukemia

EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2003
Yonit Bomstein
Abstract: Objective: Clonal B-lymphocytes of chronic lymphocytic leukemia (B-CLL) are characterized by decreased sensitivity to programmed cell death and, therefore, they accumulate in vivo. However, these malignant cells die rapidly in vitro. In the current study we concentrated on the contribution of autologous serum (AS) and lymphocyte subsets to the survival of the malignant cells in vitro. Methods: Mononuclear cells from the peripheral blood of 26 CLL patients and 24 controls were incubated overnight in the presence or absence of AS and heat-inactivated AS (HI-AS) or fetal calf serum (FCS). Also, isolated B cells were incubated at different concentrations in the presence of AS and/or isolated T cells. The level of apoptosis of CD19+ cells was measured by flow cytometry. Results: Spontaneous apoptosis of unfractionated B-CLL cells incubated with AS, FCS or without serum was significantly lower than the rate of B-cell death in the control group, in similar culture conditions. AS had an antiapoptotic effect on unfractionated B-CLL cells when compared with FCS. The rate of apoptosis of B-CLL cells was directly associated with stage. HI of AS had a variable effect, which was related to the stage of the disease. High concentrations of B cells and the addition of autologous T cells reduced the rate of apoptosis when incubated without serum. The antiapoptotic effect of T cells was most prominent in progressive stages. Conclusions: B-CLL cells exhibit decreased spontaneous apoptosis, which is partially prevented by humoral (AS) and cellular (T cells and B-CLL cells) factors. The equilibrium between apoptotic and antiapoptotic factors changes with disease progression. [source]

Systemic autoimmune disease induced by dendritic cells that have captured necrotic but not apoptotic cells in susceptible mouse strains

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2005
Liang Ma
Abstract Systemic lupus erythematosus (SLE) is an autoimmune disorder of a largely unknown etiology. Anti-double-stranded (ds) DNA antibodies are a classic hallmark of the disease, although the mechanism underlying their induction remains unclear. We demonstrate here that, in both lupus-prone and normal mouse strains, strong anti-dsDNA antibody responses can be induced by dendritic cells (DC) that have ingested syngeneic necrotic (DC/nec), but not apoptotic (DC/apo), cells. Clinical manifestations of lupus were evident, however, only in susceptible mouse strains, which correlate with the ability of DC/nec to release IFN-, and to induce the pathogenic IgG2a anti-dsDNA antibodies. Injection of DC/nec not only accelerated disease progression in the MRL/MpJ- lpr/lpr lupus-prone mice but also induced a lupus-like disease in the MRL/MpJ-+/+ wild-type control strain. Immune complex deposition was readily detectable in the kidneys, and the mice developed proteinuria. Strikingly, female MRL/MpJ-+/+ mice that had received DC/nec, but not DC/apo, developed a ,butterfly' facial lesion resembling a cardinal feature of human SLE. Our study therefore demonstrates that DC/nec inducing a Th1 type of responses, which are otherwise tightly regulated in a normal immune system, may play a pivotal role in SLE pathogenesis. [source]

Cross-presentation of a human tumor antigen delivered to dendritic cells by HSV VP22-mediated protein translocation

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2004
Arvind Chhabra
Abstract Dendritic cells (DC) capture antigens from apoptotic and/or necrotic tumor cells and cross-present them to T,cells, and various ways of delivering tumor antigens to DC in vitro and in vivo are being pursued. Since fusions of antigenic proteins with the HSV integument protein VP22 are capable of intercellular trafficking, this approach has been exploited for delivery of antigens to antigen-presenting cells. Adenoviral vectors were used to express the tumor-associated-but-self-antigen MART-1 fused to HSV VP22 in MART-1-negative A375 melanoma cells and in DC. When expressed in A375 cells and allowed to spread to DC across a transwell barrier, the VP22-MART-1 fusion protein localized to both early and late endosomal structures of the DC. The DC loaded with the VP22-MART-1 fusion by intercellular trafficking efficiently presented the MART-127,35 epitope to MART-127,35 -specific CTL. Furthermore, transloaded DC were capable of expanding the population of MART-127,35 -specific CTL. Thus, a tumor antigen acquired by intercellular trafficking can be cross-presented by DC. This experimental approach should therefore be useful not only for studying the mechanism of cross-presentation but also for vaccine development. [source]

N -methyl- d -aspartate-triggered neuronal death in organotypic hippocampal cultures is endocytic, autophagic and mediated by the c-Jun N-terminal kinase pathway

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2003
Tiziana Borsello
Abstract Acute excitotoxic neuronal death was studied in rat organotypic hippocampal slices exposed to 100 µmN -methyl- d -aspartate. Fulgurant death of pyramidal neurons occurred in the CA1 and CA3 regions and was already detectable within 2 h of the N-methyl- d -aspartate administration. Morphologically, the neuronal death was neither apoptotic nor necrotic but had the hallmarks of autophagic neuronal death, as shown by acid phosphatase histochemistry in both CA1 and CA3 and by electron microscopy in CA1. The dying neurons also manifested strong endocytosis of horseradish peroxidase or microperoxidase, occurring probably by a fluid phase mechanism, and followed, surprisingly, by nuclear entry. In addition to these autophagic and endocytic characteristics, there were indications that the c-Jun N-terminal kinase pathway was activated. Its target c-Jun was selectively phosphorylated in CA1, CA3 and the dentate gyrus and c-Fos, the transcription of which is under the positive control of c-Jun N-terminal kinase target Elk1, was selectively up-regulated in CA1 and CA3. All these effects, the neuronal death itself and the associated autophagy and endocytosis, were totally prevented by a cell-permeable inhibitor of the interaction between c-Jun N-terminal kinase and certain of its targets. These results show that pyramidal neurons undergoing excitotoxic death in this situation are autophagic and endocytic and that both the cell death and the associated autophagy and endocytosis are under the control of the c-Jun N-terminal kinase pathway. [source]

Transforming growth factor-,1 expression is up-regulated in maturation-stage enamel organ and may induce ameloblast apoptosis

EUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2 2009
Masahiro Tsuchiya
Transforming growth factor-,1 (TGF-,1) regulates a variety of cellular responses that are dependent on the developmental stage and on the origins of the cell or the tissue. In mature tissues, and especially in tissues of epithelial origin, TGF-,1 is generally considered to be a growth inhibitor that may also promote apoptosis. The ameloblast cells of the enamel organ epithelium are adjacent to and responsible for the developing enamel layer on unerupted teeth. Once the enamel layer reaches its full thickness, the tall columnar secretory-stage ameloblasts shorten, and a portion of these maturation-stage ameloblasts become apoptotic. Here we investigate whether TGF-,1 plays a role in apoptosis of the maturation-stage ameloblasts. We demonstrate in vitro that ameloblast lineage cells are highly susceptible to TGF-,1-mediated growth arrest and are prone to TGF-,1-mediated cell death/apoptosis. We also demonstrate in vivo that TGF-,1 is expressed in the maturation-stage enamel organ at significantly higher levels than in the earlier secretory-stage enamel organ. This increased expression of TGF-,1 correlates with an increase in expression of the enamel organ immediate-early stress-response gene and with a decrease in the anti-apoptotic Bcl2 : Bax expression ratio. We conclude that TGF-,1 may play an important role in ameloblast apoptosis during the maturation stage of enamel development. [source]

Effect of dopamine on rat diaphragm apoptosis and muscle performance

EXPERIMENTAL PHYSIOLOGY, Issue 4 2006
Janet D. Pierce
The purpose of this study was to determine whether dopamine (DA) decreases diaphragm apoptosis and attenuates the decline in diaphragmatic contractile performance associated with repetitive isometric contraction using an in vitro diaphragm preparation. Strenuous diaphragm contractions produce free radicals and muscle apoptosis. Dopamine is a free radical scavenger and, at higher concentrations, increases muscle contractility by simulating ,2 -adrenoreceptors. A total of 47 male Sprague,Dawley rats weighing 330,450 g were used in a prospective, randomized, controlled in vitro study. Following animal anaesthetization, diaphragms were excised, and muscle strips prepared and placed in a temperature-controlled isolated tissue bath containing Krebs,Ringer solution (KR) or KR plus 100 ,m DA. The solutions were equilibrated with oxygen (O2) at 10, 21 or 95% and 5% carbon dioxide, with the balance being nitrogen. Diaphragm isometric twitch and subtetanic contractions were measured intermittently over 65 min. The diaphragms were then removed and, using a nuclear differential dye uptake method, the percentages of normal, apoptotic and necrotic nuclei were determined using fluorescent microscopy. There were significantly fewer apoptotic nuclei in the DA group diaphragms than in the KR-only group diaphragms in 10 and 21% O2 following either twitch or subtetanic contractions. Dopamine at 100 ,m produced only modest increases in muscle performance in both 10 and 21% O2. The attenuation of apoptosis by DA was markedly greater than the effect of DA on muscle performance. Dopamine decreased diaphragmatic apoptosis, perhaps by preventing the activation of intricate apoptotic pathways, stimulating antiapoptotic mechanisms and/or scavenging free radicals. [source]

Chlamydia pneumoniae infections prevent the programmed cell death on THP-1 cell line

FEMS MICROBIOLOGY LETTERS, Issue 1 2002
C.Romano Carratelli
Abstract Chlamydia pneumoniae is an obligate intracellular bacterium which frequently causes airway infection in humans and has been implicated in chronic inflammatory disease and atherosclerosis. Here we show that infection with C. pneumoniae protects THP-1 cells against the apoptosis which spontaneously occurs in macrophages in the absence of an activation signal. Analysis by flow cytometry at different post-infection times revealed that 50±7% of THP-1 cells were apoptotic at 48 h after onset of the experiments, whereas C. pneumoniae -infected cultures (multiplicity of infection, MOI = 30) displayed only 18±4% of cells in apoptosis. At MOI = 20 and MOI = 10 the cells susceptible to apoptosis at 48 h were 28±5% and 35±6% respectively. Moreover, the results show that heat-inactivated bacteria do not give significant protection against apoptosis, even at higher MOI (MOI = 30), while UV-treated Chlamydia did provide a degree of protection against apoptosis. These data suggest that the anti-apoptotic effect of C. pneumoniae requires a heat-labile component released during infection, and that the effect is not lipopolysaccharide-dependent. [source]

Human remyelination promoting antibody inhibits apoptotic signaling and differentiation through Lyn kinase in primary rat oligodendrocytes

GLIA, Issue 15 2010
J. Watzlawik
Abstract Purpose: Human remyelination promoting IgM mAbs target oligodendrocytes (OLs) and function in animal models of multiple sclerosis (MS). However, their mechanism of action is unknown. This study seeks to identify the cellular mechanism of action of a recombinant human IgM on OL survival. Methods: Binding of rHIgM22 to the surface of rat OLs was studied by co-localization with various markers. RHIgM22-mediated effects on apoptotic signaling in OLs, differentiation markers, and signaling molecules were detected by Western blotting and immunoprecipitation. Results: RHIgM22 co-localized with integrin ,3 but not other integrin ,-chains in OLs. Downstream of integrin ,3 we identified Src family kinase (SFK) Lyn as a key player of rHIgM22-mediated actions in OLs. Lyn immunoprecipitated in a complex together with integrin ,v,3 and PDGF,R. Lyn expression was 9-fold up-regulated and Lyn activation was 3-fold higher inrHIgM22-treated OL cultures compared with controls. RHIgM22 inhibited apoptotic signaling by greater than 10-fold reduction of caspase-3 and capsase-9 cleavage and reduced by 4-fold expression of differentiation markers MBP and MOG in OLs. SFK inhibitors PP2 and SU6656 inhibited Lyn activity and restored caspase-cleavage in OLs. A human IgM that did not promote remyelination and medium wereused as controls. Conclusions: rHIgM22 prevented apoptotic signaling andinhibited OL differentiation by Lyn implying thatIgM-mediated remyelination is due toprotection of OPC and OLs rather than promotion of OPC differentiation. © 2010 Wiley-Liss, Inc. [source]

The therapeutic potential of the proteasome in leukaemia,

HEMATOLOGICAL ONCOLOGY, Issue 2 2008
Scott Marshall McCloskey
Abstract Many cellular processes converge on the proteasome, and its key regulatory role is increasingly being recognized. Proteasome inhibition allows the manipulation of many cellular pathways including apoptotic and cell cycle mechanisms. The proteasome inhibitor bortezomib has enhanced responses in newly diagnosed patients with myeloma and provides a new line of therapy in relapsed and refractory patients. Malignant cells are more sensitive to proteasome inhibition than normal haematopoietic cells. Proteasome inhibition enhances many conventional therapies and its role in leukaemia is promising. Copyright © 2008 John Wiley & Sons, Ltd. [source]

Adiponectin protects LPS-induced liver injury through modulation of TNF-, in KK-Ay obese mice

HEPATOLOGY, Issue 1 2004
Takayuki Masaki
Adiponectin, an adipocytokine, has been identified in adipose tissue, and its receptors are widely distributed in many tissues, including the liver. The present study was performed to clarify the role of adiponectin in lipopolysaccharide (LPS)-induced liver injury using KK-Ay obese mice. We analyzed the effects of adiponectin pretreatment on liver injury induced by D -galactosamine/LPS (GalN/LPS) in KK-Ay obese mice. GalN/LPS treatment induced significant increases in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the blood, apoptotic and necrotic changes in hepatocytes, and/or showed a high degree of lethality. The GalN/LPS-induced liver injury was more pronounced in KK-Ay obese mice than in lean controls. Pretreatment with adiponectin ameliorated the GalN/LPS-induced elevation of serum AST and ALT levels and the apoptotic and necrotic changes in hepatocytes, resulting in a reduction in lethality. In addition, pretreatment with adiponectin attenuated the GalN/LPS-induced increases in serum and hepatic tumor necrosis factor , (TNF-,) levels and increased peroxisome proliferator-activated receptor (PPAR) , messenger RNA expression in the liver. Furthermore, abdominal macrophages from KK-Ay obese mice pretreated with adiponectin in vitro exhibited decreased LPS-induced TNF-, production compared with controls. Finally, adiponectin pretreatment also ameliorated TNF-,-induced liver injury. In conclusion, these findings suggest that adiponectin prevents LPS-induced hepatic injury by inhibiting the synthesis and/or release of TNF-, of KK-Ay obese mice. (HEPATOLOGY 2004;40:177,184.) [source]

,-Arrestin 2 regulates toll-like receptor 4-mediated apoptotic signalling through glycogen synthase kinase-3,

IMMUNOLOGY, Issue 4 2010
Hui Li
Summary Toll-like receptor 4 (TLR4), a key member of the TLR family, has been well characterized by its function in the induction of inflammatory products of innate immunity. However, the involvement of TLR4 in a variety of apoptotic events by an unknown mechanism has been the focus of great interest. Our investigation found that TLR4 promoted apoptotic signalling by affecting the glycogen synthase kinase-3, (GSK-3,) pathway in a serum-deprivation-induced apoptotic paradigm. Serum deprivation induces GSK-3, activation in a pathway that leads to subsequent cell apoptosis. Intriguingly, this apoptotic cascade is amplified in presence of TLR4 but greatly attenuated by ,-arrestin 2, another critical molecule implicated in TLR4-mediated immune responses. Our data suggest that the association of ,-arrestin 2 with GSK-3, contributes to the stabilization of phospho-GSK-3,, an inactive form of GSK-3,. It becomes a critical determinant for the attenuation of TLR4-initiated apoptosis by ,-arrestin 2. Taken together, we demonstrate that the TLR4 possesses the capability of accelerating GSK-3, activation thereby deteriorating serum-deprivation-induced apoptosis; ,-arrestin 2 represents an inhibitory effect on the TLR4-mediated apoptotic cascade, through controlling the homeostasis of activation and inactivation of GSK-3,. [source]

Mechanism of antigen presentation after hypertonic loading of soluble antigens

IMMUNOLOGY, Issue 4 2002
Georg A. Enders
Summary Hypertonic loading of proteins into cells has been used to introduce soluble proteins into the major histocompatibility complex class I pathway of antigen presentation followed by cytotoxic T-lymphocyte (CTL) induction. The precise mechanism for this pathway is not completely understood. The antigen is either processed and presented by/on the same cell or by professional antigen-presenting cells (APC) after taking up the antigen from damaged or apoptotic cells. After loading labelled ovalbumin (OVA), it could be co-precipitated with the proteasome complex, supporting the role of this pathway for antigen processing. The processing speed however, appeared to be slow since intact OVA could be detected inside the cells even after 18 hr. This corresponded well with the processing of OVA by isolated proteasomes. On the other hand, enough peptides for recognition of target cells by CTLs were generated in this reaction. One reason for the low level of processing might be that hypertonic loading may damage the cells and inhibit direct processing. In fact, at least 50% of the cells became positive for Annexin V binding after hypertonic loading which indicates severe membrane alterations usually associated with the progress of apoptosis. Annexin V binds to phosphatidylserine residues which also serve as ligand for CD36 expressed on monocytes and some immature dendritic cells. This may direct the phagocytic pathway to hypertonically loaded cells and thus enable professional APCs to present OVA-peptides. Therefore, in addition to the direct processing of OVA, CTLs can be primed by professional APC after uptake of apoptotic, OVA-loaded cells. [source]

Linear relationship between Wnt activity levels and apoptosis in colorectal carcinoma cells exposed to butyrate

INTERNATIONAL JOURNAL OF CANCER, Issue 4 2004
Darina L. Lazarova
Abstract We have reported that butyrate, a fatty acid produced by dietary fiber that induces cell cycle arrest, differentiation and/or apoptosis in colorectal carcinoma (CRC) cells in vitro, modulates Wnt activity in 2 CRC cell lines (Bordonaro et al., Int. J. Cancer, 2002; 97:42,51). Our study determines how changes in the levels of Wnt activity induced by butyrate relate to the effects of butyrate on apoptosis, cell cycle arrest and differentiation of CRC cells. In 10 human CRC cell lines a direct relationship was shown between apoptosis and butyrate-induced increase in Wnt activity, as well as between suppressed clonal growth and increased Wnt activity. No correlation existed between butyrate-induced increase in Wnt activity and differentiation. The direct relationship between apoptosis and Wnt activity was supported by analyses of DLD-1 and HCT-116 cells expressing a dominant negative form of Tcf4, and therefore, with repressed Wnt activity, as well as by measuring the ratio of apoptotic to live cells in flow cytometry-sorted cell fractions with high and low Wnt activity. Novel flow cytometric methodology was utilized to show that butyrate differentially increases the number of cells with Wnt activity in different CRC cell lines. Thus, CRC cell lines in which butyrate upregulated Wnt activity to relatively high levels were most susceptible to the apoptotic effects of butyrate, whereas cell lines in which butyrate modestly modulated Wnt activity were less affected. © 2004 Wiley-Liss, Inc. [source]

Morphological features of Murray Valley encephalitis virus infection in the central nervous system of swiss mice

INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 1 2000
Vance Matthews
We have examined the histological and ultrastructural features of CNS infection with Murray Valley encephalitis (MVE) virus in mice inoculated with a virulent parental strain (BH3479). Light microscopic examination revealed neuronal necrosis in the olfactory bulb and hippocampus of MVE-infected brains by 5 days post-infection (pi). Electron microscopy of these regions showed endoplasmic reticulum membrane proliferation, and tubular and spherical structures in the cisternae of the endoplasmic reticulum, Golgi complex and nuclear envelope. At seven to eight days pi, infected neurones exhibited chromatin condensation and extrusion, nuclear fragmentation, loss of segments of the nuclear envelope, reduced surface contact with adjacent cells and loss of cytoplasmic organelles. This cell injury was particularly noticeable in the proximal CA3 and distal CA1 regions of the hippocampus. The inflammatory cell profile consisted of macrophages, lymphocytes and especially neutrophils, and many of these inflammatory cells were apoptotic. High mortality rates in the BH3479-infected population of mice correlated with the intense polymorphonuclear and mononuclear leucocyte inflammatory infiltrate in the CNS. [source]

Radiation-induced lysosomal iron reactivity: Implications for radioprotective therapy

IUBMB LIFE, Issue 7 2006
H. Lennart Persson
Abstract A novel mechanism of radiosensitization involves radiation-enhanced autophagy of damaged mitochondria and various metalloproteins, by which iron accumulates within lysosomes. Hydrogen peroxide, formed by the radiolytic cleavage of water, generates in the presence of lysosomal redox-active iron extremely reactive hydroxyl radicals by Fenton-type chemistry. Subsequent peroxidative damage of lysosomal membranes initiates release of harmful content from ruptured lysosomes that triggers a cascade of events eventuating in DNA damage and apoptotic or necrotic cell death. This article reviews the role of lysosomal destabilization in radiation-induced cell damage and death. The potential effects of iron chelation therapy targeted to the lysosomes for protection of normal tissues against unwanted effects by radiation is also discussed. iubmb Life, 58: 395-401, 2006 [source]

Yeast Programmed Cell Death: An Intricate Puzzle

IUBMB LIFE, Issue 3 2005
P. Ludovico
Abstract Yeasts as eukaryotic microorganisms with simple, well known and tractable genetics, have long been powerful model systems for studying complex biological phenomena such as the cell cycle or vesicle fusion. Until recently, yeast has been assumed as a cellular 'clean room' to study the interactions and the mechanisms of action of mammalian apoptotic regulators. However, the finding of an endogenous programmed cell death (PCD) process in yeast with an apoptotic phenotype has turned yeast into an 'unclean' but even more powerful model for apoptosis research. Yeast cells appear to possess an endogenous apoptotic machinery including its own regulators and pathway(s). Such machinery may not exactly recapitulate that of mammalian systems but it represents a simple and valuable model which will assist in the future understanding of the complex connections between apoptotic and non-apoptotic mammalian PCD pathways. Following this line of thought and in order to validate and make the most of this promising cell death model, researchers must undoubtedly address the following issues: what are the crucial yeast PCD regulators? How do they play together? What are the cell death pathways shared by yeast and mammalian PCD? Solving these questions is currently the most pressing challenge for yeast cell death researchers.IUBMB Life, 57: 129-135, 2005 [source]

Cell proliferation and death in the brain of active and hibernating frogs

JOURNAL OF ANATOMY, Issue 2 2009
Silvia Cerri
Abstract ,Binomial' cell proliferation and cell death have been studied in only a few non-mammalian vertebrates, such as fish. We thought it of interest to map cell proliferation/apoptosis in the brain of the frog (Rana esculenta L.) as this animal species undergoes, during the annual cycle, physiological events that could be associated with central nervous system damage. Therefore, we compared the active period and the deep underground hibernation of the frog. Using western blot analysis for proliferating cell nuclear antigen (PCNA), we revealed a positive 36 kDa band in all samples and found higher optical density values in the hibernating frogs than in active frogs. In both active and hibernating frogs, we found regional differences in PCNA-immunoreactive cells and terminal transferase dUTP nick-end labelling apoptotic cells in the ventricular zones and parenchyma areas of the main encephalon subdivisions. During the active period of the frogs, the highest concentration of PCNA-immunoreactive cells was found in the ventricle dorsal zone of the cerebral hemispheres but only some of the cells were apoptotic. By contrast, the tectal and cerebellar ventricular zones had a small or medium amount of PCNA-immunoreactive cells, respectively, and a higher number of apoptotic cells. During hibernation, an increased PCNA-immunoreactive cell number was observed in both the brain ventricles and parenchyma compared with active frogs. This increase was primarily evident in the lateral ventricles, a region known to be a proliferation ,hot spot'. Although differences existed among the brain areas, a general increase of apoptotic cell death was found in hibernating frogs, with the highest number of apoptotic cells being detected in the parenchyma of the cerebral hemispheres and optic tectum. In particular, the increased number of apoptotic cells in the hibernating frogs compared with active frogs in the parenchyma of these brain areas occurred when cell proliferation was higher in the corresponding ventricular zones. We suggest that the high number of dying cells found in the parenchymal regions of hibernating frogs might provide the stimulus for the ventricular zones to proliferate. Hibernating frogs could utilize an increased cell proliferation in the brain areas as a neuroprotective strategy to face cell death and the onset of neurological damages. Therefore, the hibernator promises to be a valuable model for studying the mechanisms naturally carried out by the central nervous system in order to adapt itself or survive adverse conditions. [source]

An ultrastructural study of cell death in the CA1 pyramidal field of the hippocapmus in rats submitted to transient global ischemia followed by reperfusion

JOURNAL OF ANATOMY, Issue 5 2007
Aline De Souza Pagnussat
Abstract In the course of ischemia and reperfusion a disruption of release and uptake of excitatory neurotransmitters occurs. This excitotoxicity triggers delayed cell death, a process closely related to mitochondrial physiology and one that shows both apoptotic and necrotic features. The aim of the present study was to use electron microscopy to characterize the cell death of pyramidal cells from the CA1 field of the hippocampus after 10 min of transient global ischemia followed by short reperfusion periods. For this study 25 adult male Wistar rats were used, divided into six groups: 10 min of ischemia, 3, 6, 12 and 24 h of reperfusion and an untouched group. Transient forebrain ischemia was produced using the 4-vessel occlusion method. The pyramidal cells of the CA1 field from rat hippocampus submitted to ischemia exhibited intracellular alterations consistent with a process of degeneration, with varied intensities according to the reperfusion period and bearing both apoptotic and necrotic features. Gradual neuronal and glial modifications allowed for the classification of the degenerative process into three stages: initial, intermediate and final were found. With 3 and 6 h of reperfusion, slight and moderate morphological alterations were seen, such as organelle and cytoplasm edema. Within 12 h of reperfusion, there was an apparent recovery and more ,intact' cells could be identified, while 24 h after the event neuronal damage was more severe and cells with disrupted membranes and cell debris were identified. Necrotic-like neurons were found together with some apoptotic bodies with 24 h of reperfusion. Present results support the view that cell death in the CA1 field of rat hippocampus submitted to 10 min of global transient ischemia and early reperfusion times includes both apoptotic and necrotic features, a process referred to as parapoptosis. [source]

Exposure to mixtures of endosulfan and zineb induces apoptotic and necrotic cell death in SH-SY5Y neuroblastoma cells, in vitro

JOURNAL OF APPLIED TOXICOLOGY, Issue 5 2007
Zhenquan Jia
Abstract A number of epidemiological studies have demonstrated a strong association between the incidence of Parkinson's disease and pesticide exposure. Earlier it was demonstrated that exposure to the pesticides endosulfan and zineb, alone and in combination, caused neurodegeneration in vivo. It was hypothesized that these pesticides cause neurotoxicity, in part, by enhancing apoptotic cell death. SH-SY5Y human neuroblastoma cells, which retain a catecholaminergic phenotype, were exposed to endosulfan, zineb or a combination of these chemicals, in vitro. For mixture studies, concentrations of pesticides (100 µm each) were chosen based on LC25 (lethal concentration) that would result in minimum cell death. Exposure to a mixture of pesticides exhibited significantly (P , 0.05) higher toxicity than each one alone. Both pesticides were found to cause apoptotic cell death that was concentration (50,400 µm) dependent. A flow cytometric (7-aminoactinomycin D) assay was used to distinguish live, early apoptotic and late apoptotic/necrotic populations. Exposure to mixtures of the pesticides enhanced both early apoptosis and late apoptosis/necrosis compared with either chemical alone. Visual evaluation using a DNA ladder assay and a fluorescence Annexin V/PI assay confirmed the contribution of both apoptotic and necrotic processes. These findings suggest that the cytotoxicity of endosulfan and zineb, both individually and in mixtures, is associated with the occurrence of early and late apoptotic/necrotic processes in SH-SY5Y human neuroblastoma cells and support the contention that pesticide-induced neuronal cell death leading to neurodegenerative disease may, at least in part, be associated with early and late apoptosis of dopaminergic neurons. Copyright © 2007 John Wiley & Sons, Ltd. [source]