Apoptosis induced by Semliki Forest virus is RNA replication dependent and mediated via Bak.

The RNA alphavirus Semliki Forest (SFV) triggers apoptosis in various mammalian cells, but it has remained controversial at what infection stage and by which signalling pathways host cells are killed. Both RNA synthesis-dependent and -independent initiation processes and mitochondrial as well as death receptor signalling pathways have been implicated. Here, we show that SFV-induced apoptosis is initiated at the level of RNA replication or thereafter. Moreover, by expressing antiapoptotic genes from recombinant SFV (replicons) and by using neutralizing reagents and gene-knockout cells, we provide clear evidence that SFV does not require CD95L-, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)- or tumor necrosis factor-mediated signalling but mitochondrial Bak to trigger cytochrome c release, the fall in the mitochondrial membrane potential, apoptotic protease-activating factor-1/caspase-9 apoptosome formation and caspase-3/-7 activation. Of seven BH3-only proteins tested, only Bid contributed to effective SFV-induced apoptosis. However, caspase-8 activation and Bid cleavage occurred downstream of Bax/Bak, indicating that truncated Bid formation serves to amplify rather than trigger SFV-induced apoptosis. Our data show that SFV sequentially activates a mitochondrial, Bak-mediated, caspase-8-dependent and Bid-mediated death signalling pathway that can be accurately dissected with gene-knockout cells and SFV replicons carrying antiapoptotic genes.

Granzyme B-induced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs.

Granzyme B (gzmB) of cytotoxic T lymphocytes (CTL) is essential for recovery from intracellular pathogens, but the molecular basis of its action is still unresolved. Here, we analyzed gzmB-mediated death pathways under physiological conditions using ex vivo virus-immune CTLs that express perf and gzmB, but not gzmA (gzmB(+)CTL). We show that gzmB(+)CTL abrogate target cell proliferation most likely by inducing cell death, independent of caspases and mitochondrial signaling. In addition, the data reveal that gzmB(+)CTL independently induce pro-apoptotic processes either via caspase-3/-7, leading to plasma membrane perturbance and ROS production or via Bid/Bak/Bax, resulting in cytochrome c release and that both pathways elicit loss of DeltaPsi(m). Our data provide evidence for a pleiotropic pro-apoptotic function of gzmB presumably to counteract evasion strategies of pathogens and to control tumors.

The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis.

Bcl-2 is a mitochondrial- and perinuclear-associated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2 alpha for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2 alpha. A truncation mutant of bcl-2 alpha lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-alpha-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-II) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.

Unsymmetrical diborane(4) derivatives by copper mediated B-B coupling.

A novel, versatile and modular route to unsymmetrical diborane(4) derivatives bearing either two different diol moieties or one diol and one diamine moiety is presented. Utilising the very basic approach of reacting a boron nucleophile with a boron electrophile to establish the B-B bond allows a simple variation of the two individual boron moieties. The copper(i) boryl complexes used as nucleophilic boron sources are readily accessible from commercially available symmetrical diborane(4) derivatives, whilst equally readily available boron halide derivatives are used as electrophiles. Seven previously inaccessible diborane(4) derivatives were obtained and fully characterised, including single crystal X-ray structure determinations, illustrating the broad scope of the method.

Bak but not Bax is essential for Bcl-xS-induced apoptosis.

Bcl-x(S), a proapoptotic member of the Bcl-2 protein family, is localized in the mitochondria and induces apoptosis in a caspase- and BH3-dependent manner by a mechanism involving cytochrome c release. The way in which Bcl-x(S) induces caspase activation and cytochrome c release, as well as the relationship between Bcl-x(S) and other proapoptotic members of the Bcl-2 family, is not known. Here we used embryonic fibroblasts derived from mice deficient in the multidomain proapoptotic members of the Bcl-2 family (Bax and Bak) and the apoptotic components of the apoptosome (Apaf-1 and caspase-9) to unravel the cascade of events by which Bcl-x(S) promotes apoptosis. Our results show that Bak but not Bax is essential for Bcl-x(S)-induced apoptosis. Bcl-x(S) induced activation of Bak, which in turn promoted apoptosis by apoptosome-dependent and -independent pathways. These findings provide the first evidence that a proapoptotic Bcl-2 family protein induces apoptosis exclusively via Bak.

[Results of hip arthroscopy in sports related groin pain]. / Ergebnisse der Hüftarthroskopie bei sportabhängigen Leistenschmerzen.

INTRODUCTION: The study analyses the intraoperative findings and the clinical results of hip arthroscopy in sports related groin pain. METHODS: Between june 1998 and october 2002 we performed hip arthroscopy in 30 athletes (12 female, 18 male) with a history of sports related groin pain. Their average age was 36 (20 to 68) years. All patients had a clinical follow-up- examination at an average of 20 (12 to 48) months postoperative. The result was rated using the Larson-Score. RESULTS: We found a lesion of the acetabular labrum and performed a partial resection at 17 patients (57 %) (synovitis: n = 28 [93 %] loose bodies: n = 6 [20 %] torn ligamentum teres: n = 4 [13 %], others: n = 2 [6 %]). 11 patients (37 %) had a cartilage degeneration grade II in the Outerbridge classification (III degrees : n = 9 [30 %], I degrees : n = 4 [13 %], 0 degrees : n = 6 [20 %]). Preoperative 14 patients (47 %) complained severe groin pain (moderate: n = 14 [47 %], slight: n = 2 [6 %]) against only 3 patients (10 %) with severe groin pain at the follow-up examination (moderate: n = 11 [37 %], slight: n = 16 [53 %]). Following hip arthroscopy 28 patients (94 %) returned to full sports activity. The Larson-Score was increased significantly (p < 0.05) rating 43 (10 to 64) points preoperative to 59 (28 to 80) points at the follow-up. DISCUSSION: We found that persistent sports related groin pain was frequently caused by an intraarticular hip disorder. Following hip arthroscopy pain could be reduced in most patients as a return to full sports activity.

Overxpression of Bcl-2 inhibits UVA-mediated immediate apoptosiinrat 6 fibroblasts: evidence for the involvement of Bcl-2 as an antioxidant.

We examined the effect of broad spectrum UVA (320-380 nm) and UVB (290-320 nm) radiation on the induction of apoptosis in the rat 6 fibroblast cell line (R6). UVA, but not UVB, induces apoptosis in this cell line. The morphological changes and DNA ladders associated with apoptosis occurred within the first 4 h after UVA irradiation, a phenomenon referred to as "immediate" apoptosis. From previous studies, it is known that Bcl-2 inhibits most types of apoptotic cell death. Overexpression of mouse Bcl-2 in the R6 fibroblasts inhibited the UVA-induced immediate apoptosis. The induction of the heme oxygenase 1 (HO-1) gene by UVA is a general response to oxidative stress. As a marker of oxidative stress, we monitored the effect of Bcl-2 overexpression on the level of HO-1 mRNA accumulation after UVA irradiation. The results showed that the overexpression of Bcl-2 in the R6 fibroblasts strongly reduces the level of HO-1 induction from 12.5- to 4.9-fold. We propose that Bcl-2 expression inhibits UVA-induced immediate apoptosis via an antioxidant pathway, suppressing either the generation or effects of specific UVA-mediated reactive oxygen species.

The clerodane diterpene casearin J induces apoptosis of T-ALL cells through SERCA inhibition, oxidative stress, and interference with Notch1 signaling.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that preferentially affects children and adolescents. Over 50% of human T-ALLs possess activating mutations of Notch1. The clerodane diterpene casearin J (CJ) is a natural product that inhibits the sarcoendoplasmatic reticulum calcium ATPase (SERCA) pump and induces cell death in leukemia cells, but the molecular mechanism of cytotoxicity remains poorly understood. Here we show that owing to SERCA pump inhibition, CJ induces depletion of the endoplasmic reticulum calcium pools, oxidative stress, and apoptosis via the intrinsic signaling pathway. Moreover, Notch1 signaling is reduced in T-ALL cells with auto-activating mutations in the HD-domain of Notch1, but not in cells that do not depend on Notch1 signaling. CJ also provoked a slight activation of NF-κB, and consistent with this notion a combined treatment of CJ and the NF-κB inhibitor parthenolide (Pt) led to a remarkable synergistic cell death in T-ALL cells. Altogether, our data support the concept that inhibition of the SERCA pump may be a novel strategy for the treatment of T-ALL with HD-domain-mutant Notch1 receptors and that additional treatment with the NF-κB inhibitor parthenolide may have further therapeutic benefits.

Apoptotic crosstalk between the endoplasmic reticulum and mitochondria controlled by Bcl-2.

Apoptosis involves mitochondrial steps such as the release of the apoptogenic factor cytochrome c which are effectively blocked by Bcl-2. Although Bcl-2 may have a direct action on the mitochondrial membrane, it also resides and functions on the endoplasmic reticulum (ER), and there is increasing evidence for a role of the ER in apoptosis regulation as well. Here we uncover a hitherto unrecognized, apoptotic crosstalk between the ER and mitochondria that is controlled by Bcl-2. After triggering massive ER dilation due to an inhibition of secretion, the drug brefeldin A (BFA) induces the release of cytochrome c from mitochondria in a caspase-8- and Bid-independent manner. This is followed by caspase-3 activation and DNA/nuclear fragmentation. Surprisingly, cytochrome c release by BFA is not only blocked by wild-type Bcl-2 but also by a Bcl-2 variant that is exclusively targeted to the ER (Bcl-2/cb5). Similar findings were obtained with tunicamycin, an agent interfering with N-linked glycosylations in the secretory system. Thus, apoptotic agents perturbing ER functions induce a novel crosstalk between the ER and mitochondria that can be interrupted by ER-based Bcl-2.

Expression of calmodulin and calmodulin binding proteins in rat fibroblasts stably transfected with protein kinase C and oncogenes.

Molecular mechanisms leading to elevated calmodulin (CaM) expression in cancer have not yet been discovered. We have quantitated the levels of transcripts derived from all three CaM genes in a variety of the same origin rat fibroblasts transformed with oncogenes in combination with gene for protein kinase C using Northern blot analysis with three CaM gene specific cDNA probes. Five species of CaM mRNA were detected in all these cells. Surprisingly many of the investigated cell lines exhibited a decreased content of all CaM mRNAs as compared to control cells with CaMI and CaMII transcripts showing the most pronounced alterations. In contrast, CaM protein levels were increased in all these cell lines as determined by a radioimmunoassay. These results suggest that oncogenic up-regulation of CaM synthesis takes place posttranscriptionally. Several CaM binding proteins were found at different concentrations in the studied cell lines depending on the oncogenes used for transformation. However, CaM overexpression does not seem to affect the overall levels of CaM binding proteins.

Apoptosis without caspases: an inefficient molecular guillotine?

Since the discovery that the cysteine protease CED-3 was essential for developmental death in the nematode C. elegans, the search has been on to identify homologous proteases governing mammalian apoptosis. Fourteen of these proteases, now called caspases, have been found to date, and studies with natural or chemical inhibitors, and more recently knock-out mice, confirmed the involvement of at least a subset of these proteases in various forms of mammalian apoptosis. However, there has been recent evidence that some apoptotic morphologies, such as cell shrinkage, membrane blebbing and nuclear condensation, are not blocked by caspase inhibitors and that the cells continue to die in a protracted and inefficient manner. This has led to the notion that caspases are not required for all aspects of apoptosis in mammals. Here we review the current knowledge about caspase-independent apoptosis, discuss the strengths and weaknesses of the reasoning that led to its proposition and provide insights into its possible regulation and physiological significance.

Failure of Bcl-2 family members to interact with Apaf-1 in normal and apoptotic cells.

CED-9 blocks programmed cell death (apoptosis) in the nematode C. elegans by binding to and neutralizing CED-4, an essential activator of the aspartate-directed cysteine protease (caspase) CED-3. In mammals, the CED-9 homologs Bcl-2 and Bcl-xL also block apoptosis by interfering with the activation of CED-3-like caspases. However, it is unknown whether this occurs by binding to the CED-4 homolog Apaf-1. Whilst two groups previously detected an interaction between Bcl-xL and Apaf-1 in immunoprecipitates,1,2 another group found no interaction between Apaf-1 and any of ten individual members of the Bcl-2 family using the same experimental approach.3 In this study, we aimed to resolve this discrepancy by monitoring the binding of Apaf-1 to three Bcl-2 family members within cells. Using immunofluorescence and Western blot analysis, we show that whilst Apaf-1 is a predominantly cytoplasmic protein, Bcl-2, Bcl-xL and Bax mostly reside on nuclear/ER and mitochondrial membranes. This pattern of localization is maintained when the proteins are co-expressed in both normal and apoptotic cells, suggesting that Bcl-2, Bcl-xL or Bax do not significantly sequester cytoplasmic Apaf-1 to intracellular membranes. In addition, we confirm that Apaf-1 does not interact with Bcl-2 and Bcl-xL in immunoprecipitates. Based on these data, we propose that Apaf-1 is not a direct, physiological target of Bcl-2, Bcl-xL or Bax.

Bcl-x(S) can form homodimers and heterodimers and its apoptotic activity requires localization of Bcl-x(S) to the mitochondria and its BH3 and loop domains.

Proteins of the Bcl-2 family regulate apoptosis, some antagonizing cell death and others, such as Bcl-x(S), promoting it. We previously showed that expression of Bcl-x(S) in PC12 cells is a useful system for studying the mechanism of Bcl-x(S)-induced apoptosis. To further investigate this apoptotic effect and its prevention by anti-apoptotic agents, we assessed the role of distinct Bcl-x(S) domains, via the study of their mutations, on the ability of Bcl-x(S) to induce apoptosis and to localize to the mitochondria, as well as the ability of these domains to counteract the effects of anti-apoptotic agents on Bcl-x(S). Deletion of the transmembrane domain (DeltaTM) prevented the localization of Bcl-x(S) DeltaTM to the mitochondria and the ability of this mutant to induce apoptosis. Deletion of the amino acids GD 94-95 from the BH3 domain, or deletion of the loop region, impaired the ability of these mutants to induce apoptosis but not their localization to the mitochondria. Deletion of the BH4 domain or destruction of the caspase cleavage site in the loop region (by replacing amino acid D61 with A61) did not affect either the localization of these mutants to the mitochondria or their ability to induce cell death. It thus appears that Bcl-x(S)-induced apoptosis in PC12 cells is mediated by localization of Bcl-x(S) to the mitochondria by a process that requires the transmembrane domain. Furthermore, once localized to the mitochondria Bcl-x(S) requires the BH3 domain, and to a lesser extent the loop domain, for its subsequent activity. The anti-apoptotic agents Bcl-2 and Bcl-x(L), the caspase inhibitor Z-VAD-FMK, and nerve growth factor (NGF) did not prevent Bcl-x(S) localization to the mitochondria, and did not require the BH4 or the loop domains of Bcl-x(S) for their survival effect. Bcl-x(S) is capable of forming homodimers with itself and heterodimers with Bcl-x(L) or Bcl-2. Accordingly co-expression of Bcl-x(S) DeltaTM with Bcl-x(S), Bcl-2, or Bcl-x(L) leads to a change in the subcellular distribution of Bcl-x(S) DeltaTM, from a diffuse distribution throughout the cell to a more defined distribution. Moreover co-immunoprecipitation experiments directly demonstrated that Bcl-x(S) can associate with GFP-Bcl-x(S), Bcl-x(L), or Bcl-2. These results suggest that such Bcl-x(S) interactions may be important for the mechanism of action of this protein.

Serine proteases mediate apoptosis-like cell death and phagocytosis under caspase-inhibiting conditions.

Effective execution of apoptosis requires the activation of caspases. However, in many cases, broad-range caspase inhibitors such as Z-VAD.fmk do not inhibit cell death because death signaling continues via basal caspase activities or caspase-independent processes. Although death mediators acting under caspase-inhibiting conditions have been identified, it remains unknown whether they trigger a physiologically relevant cell death that shows typical signs of apoptosis, including phosphatidylserine (PS) exposure and the removal of apoptotic cells by phagocytosis. Here we show that cells treated with ER stress drugs or deprived of IL-3 still show hallmarks of apoptosis such as cell shrinkage, membrane blebbing, mitochondrial release of cytochrome c, PS exposure and phagocytosis in the presence of Z-VAD.fmk. Cotreatment of the stressed cells with Z-VAD.fmk and the serine protease inhibitor Pefabloc (AEBSF) inhibited all these events, indicating that serine proteases mediated the apoptosis-like cell death and phagocytosis under these conditions. The serine proteases were found to act upstream of an increase in mitochondrial membrane permeability as opposed to the serine protease Omi/HtrA2 which is released from mitochondria at a later stage. Thus, despite caspase inhibition or basal caspase activities, cells can still be phagocytosed and killed in an apoptosis-like fashion by a serine protease-mediated mechanism that damages the mitochondrial membrane.

[Treatment of incomplete meniscal lesions in athletes]. / Die Therapie des inkompletten Meniskusrisses beim Sportler.

PURPOSE: Arthroscopic treatment of complete meniscal lesions is well established. Nevertheless there is discussion, how to treat incomplete meniscal tears, especially in younger and active patients. This study was designed to evaluate our standard-therapy without refixation of the meniscus. METHOD: Between 7/89 and 3/01 in 47 patients (48 knees, Ø age 29 years) an incomplete meniscal lesion following sports injury was found. The lesions were revitalized by "needling" or shaving. We performed no refixation. All patients had a postoperative flexion limit in an orthosis for 6 weeks. The follow-up examination was performed 6.5 (2 - 14) years postoperative. RESULTS: The avarage Lysholm-Score increased significantly from 55 points preoperative to 92 points at the follow-up examination. The Tegner-Score increased from 3.3 points preoperative to 6.2 points at the follow-up examination. The overall result was rated "exellent" and "good" by 83 % of the patients, "fair" by 15 % of the patients and "poor" by 2 % of the patients. Return to sports activity was possible at an avarage of 7 (3 - 12) months postoperative. CONCLUSIONS: In summary we found, that shaving and "needling" of an incomplete meniscal lesion in combination with partial synovectomi and standardized postoperative treatment leads to a high healing rate. A limited flexion for 6 weeks postoperativ in an orthosis at full weight bearing is recommended. In case of complete healing of the lesion the chondroprotective and joint stabilizing function of the meniscus, especially in young and active patients is obtained.

Selective B-B bond activation in an unsymmetrical diborane(4) by [(Me3P)4Rh-X] (X = Me, OtBu): a switch of mechanism?

The unsymmetrical diborane(4) pinB-B((RN)2(C6H4)) (R = Me, Bn) reacts with [(Me3P)4Rh-X] (X = Me, OtBu) giving predominantly either [(Me3P)4Rh-Bpin] or [(Me3P)3Rh-B((RN)2(C6H4))] depending on X. At low temperatures in the presence of excess PMe3 the unprecedented equatorial boryl complex [(Me3P)4Rh-B((MeN)2(C6H4))] is formed.

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Modulation of presynaptic calcium homeostasis by nitric oxide.

In the present work, we have adapted established microfluorimetric techniques based on the calcium indicator Fura-2, for the study of synaptosomal calcium homeostasis regulation in an immobilized synaptosomal preparation from the rat hippocampus. With this tool, we have addressed the actions of two proposed interneuronal messengers, nitric oxide (NO) and arachidonic acid (AA). NO donors (sodium nitroprusside, SNP and hydroxylamine, HX) and AA induced an increase in depolarization-induced calcium transients (both in magnitude and duration). However, resting calcium levels were not modified by NO, whereas AA application resulted in an steady increase in Ca. The effects of SNP were blocked when EGTA was present between depolarizations, suggesting that a minimum level of internal calcium load is required for NO effects. The effects of NO on Cai transients are persistent up to 90 min after drug application, and could be involved in some of the forms of synaptic plasticity where NO plays a role.

NMDA-induced increase in [Ca2+]i and 45Ca2+ uptake in acutely dissociated brain cells derived from adult rats.

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-D-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca2+]i measured with fluo3 and 45Ca2+ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 microM) and N-methyl-DL-aspartate (200 microM) increased [Ca2+]i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated 45Ca2+ uptake about 16-10% in the same regions. The increases in [Ca2+]i and 45Ca2+ uptake were inhibited by 40% in the presence of 1 mM MgCl2 and by 90-50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca2+]i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca2+]i increase in adult age.