Apoptotic-like Events Research Articles

The type-1 ribosome-inactivating protein OsRIP1 triggers caspase-independent apoptotic-like death in HeLa cells

Ribosome-inactivating proteins (RIPs) are capable of removing a specific adenine from 28S ribosomal RNA, thus inhibiting protein biosynthesis in an irreversible manner. In this study, recombinant OsRIP1, a type 1 RIP from rice (Oryza sativa L.), was investigated for its anti-proliferative properties. Human cervical cancer HeLa cells were incubated in the presence of OsRIP1 for 24–72 h. OsRIP1 treatment yielded an anti-proliferation response of the HeLa cells and resulted in apoptotic-like blebbing of the plasma membrane without causing DNA fragmentation. OsRIP1 labeled with FITC accumulated at the cell surface. Pull-down assays identified ASPP1 (Apoptosis-Stimulating Protein of p53 1) and IFITM3 (interferon-induced transmembrane protein 3) as potential interaction partners for OsRIP1. Transcript levels for several critical genes related to different signaling pathways were quantified by RT-qPCR. OsRIP1 provoked HeLa cells to undergo caspase-independent cell death, associated with a significant transcriptional upregulation of the apoptotic gene PUMA, interferon regulatory factor 1 (IRF1) and the autophagy-related marker LC3. No changes in caspase activities were observed. Together, these data suggest that apoptotic-like events were involved in OsRIP1-driven caspase-independent cell death that might trigger the IRF1 signaling pathway and LC3-mediated autophagy.

Albendazole induces oxidative stress and DNA damage in the parasitic protozoan Giardia duodenalis.

The control of Giardia duodenalis infections is carried out mainly by drugs, among these albendazole (ABZ) is commonly used. Although the cytotoxic effect of ABZ usually involves binding to β-tubulin, it has been suggested that oxidative stress may also play a role in its parasiticidal mechanism. In this work the effect of ABZ in Giardia clones that are susceptible or resistant to different concentrations (1.35, 8, and 250 μM) of this drug was analyzed. Reactive oxygen species (ROS) were induced by ABZ in susceptible clones and this was associated with a decrease in growth that was alleviated by cysteine supplementation. Remarkably, ABZ-resistant clones exhibited partial cross-resistance to H2O2, whereas a Giardia H2O2-resistant strain can grow in the presence of ABZ. Lipid oxidation and protein carbonylation in ABZ-treated parasites did not show significant differences as compared to untreated parasites; however, ABZ induced the formation of 8OHdG adducts and DNA degradation, indicating nucleic acid oxidative damage. This was supported by observations of histone H2AX phosphorylation in ABZ-susceptible trophozoites treated with 250 μM ABZ. Flow cytometry analysis showed that ABZ partially arrested cell cycle in drug-susceptible clones at G2/M phase at the expense of cells in G1 phase. Also, ABZ treatment resulted in phosphatidylserine exposure on the parasite surface, an event related to apoptosis. All together these data suggest that ROS induced by ABZ affect Giardia genetic material through oxidative stress mechanisms and subsequent induction of apoptotic-like events.

66 BUFFALO-BULL SEMEN-FERTILITY EVALUATION IN RELATION TO MOTILITY AND INTEGRITY OF ACROSOME, PLASMA MEMBRANE, AND SPERM DNA

The breeding-soundness examination is conducted to identify and select bulls with an acceptable reproductive efficiency. In buffalo, there is meagre information regarding fertility index in relation to sperm attributes so that a future breeding bull could be selected at the age of maturity. So, to predict the fertility of buffalo breeding bulls, the present study was conducted to assess the motility parameters and integrity of acrosome, plasma membrane, and DNA of cryopreserved semen of high- and low-fertile bulls. The fertility of bulls was classified on the basis of conception rates, where buffalo bulls with conception rate <35% were considered as low-fertility bulls and those with conception rate >55% were considered as high-fertility bulls. A computer-assisted semen analyser was used for motility and viability studies, whereas integrity of acrosome, plasma membrane, and DNA were assessed by Pisum sativum agglutinin–fluorescein isothiocyanate, Annexin-V/PI, and TUNEL assay kit, respectively, under the florescence microscope. At least 200 spermatozoa were evaluated from each group, and results were analysed by ANOVA. The level of significance was observed at P < 0.05. The mean (±SE) values for various motility parameters of sperm for high-fertile bulls were total motility (56.8 ± 3.2%), average path velocity (87.22 ± 1.6 µm s–1), straight linear velocity (68.93 ± 1.9 µm s–1), and curvilinear velocity (156.52 ± 4.3 µm s–1). These values were significantly higher than those of low-fertile bulls (43.8 ± 1.7%, 79.02 ± 2.4 µm s–1, 63.42 ± 1.2 µm s–1, and 142.37 ± 2.8 µm s–1, respectively). The amplitude of lateral head displacement (6.8 ± 0.07 v. 6.5 ± 0.1 µm), beat cross frequency (33.9 ± 0.4 v. 33.43 ± 0.5 Hz), straightness (79.2 ± 0.7 v. 78.7 ± 0.6%), linearity (45.5 ± 0.4 v. 45.5 ± 0.7%), and viability (71.2 ± 0.8 v. 68.9 ± 0.8%) did not differ in both groups. The average percentage of intactness of sperm acrosome of high-fertile bulls was significantly higher (81.82 ± 0.87%) than that of low-fertility bulls (76.86 ± 0.87%). Furthermore, to assess the functionality of plasma membrane of sperm, we analyzed different stages of apoptotic-like events. The percentage of apoptotic sperm differed significantly between high-fertility (15.59 ± 0.75%) and low-fertility (25.94 ± 0.5%) bulls. The percentages of early necrotic, necrotic, and viable sperm did not differ in 2 groups. The DNA integrity in high-fertility (90.24 ± 0.94%) and low-fertility (88.37 ± 0.91%) bulls was not significantly different. In conclusion, the various parameters such as average path velocity, curvilinear velocity, straight linear velocity, and total motility; acrosomal integrity; and percentage of apoptotic sperm are useful for evaluating the semen quality of a bull to reduce the risk of using poor-fertility bulls in an AI program.

Activation of Caspases-8 and -9 Occurs In Platelets From Children with Immune, but Not Chemotherapy-Related, Thrombocytopenia.

AbstractAbstract 3700Apoptotic-like processes in platelets are similar to those observed during apoptosis in the cytoplasm of nucleated cells: activation of caspase-8, caspase-9, and caspase-3, loss of mitochondrial inner membrane potential, and externalization of phosphatidylserine (PS) (Leytin et al, Br J Haematol 2006; Lopez et al, J Thromb Haemost 2009). We recently showed that platelets in pediatric primary immune thrombocytopenia (ITP) have activated caspase-3 (aCASP3) and externalized PS, both of which were reduced after IVIg administration (Speer et al, Blood 2008;112: 3417). To gain a more complete understanding of the apoptosis that occurs in ITP platelets, in the present study, we investigated whether caspase-8 and caspase-9 are also activated in platelets from children with ITP, and examined whether the increase in platelet count in response to IVIg is associated with a decrease in activated caspase-8 (aCASP8) and -9 (aCASP9) in platelets, as was observed for aCASP3. In addition, we measured the mitochondrial membrane potential in platelets before and after IVIg therapy. Children with primary ITP were enrolled in this prospective study. Severity of bleeding symptoms was assessed according to a pediatric bleeding score for ITP at the time of diagnosis. Blood samples were obtained at the time of diagnosis and after IVIg therapy for measurement of platelet count and for flow cytometric analyses of platelet apoptotic-like events. In citrated platelet-rich plasma, platelets were identified as CD42 positive events; aCASP8 and aCASP9 were measured as % platelets with bound FITC-fluorescent-labeled inhibitors of activated caspases; and mitochondrial membrane potential was measured as mean fluorescence intensity of the membrane potential sensitive fluorescent tetramethylrhodamine ethyl ester (TMRE). All patients (median age 5.4 yrs, n = 8) presented with typical symptoms of acute ITP with a bleeding score of 2 – 3 and had platelet counts < 20×109/L. Results from ITP patients were compared with 2 control groups, healthy children (platelet counts: 266–348 × 109/L, median age 6.8 yrs, n = 7) and children with thrombocytopenia as a result of chemotherapy for malignancies (cTP) (platelet counts: 3–51 × 109/L, median age 10.2 yrs, n = 7). ITP patients had significantly higher proportions of platelets with aCASP8 (17.5±5.1%) and aCASP9 (16.9±5.8%) compared with both healthy children (aCASP8 1.0±0.3%; aCASP9 1.1±0.3%) and children with cTP, (aCASP8 2.2±0.4%; aCASP9 1.9±0.4%) (p<0.01-0.05). In contrast, a loss of mitochondrial membrane potential was not observed in platelets from ITP patients at baseline, in healthy controls, or cTP. All ITP patients were treated with a maximum of 3 doses of IVIg (0.4 – 0.8 g/kg/dose) and showed a rise in platelet counts to > 20 × 109/L and amelioration of bleeding symptoms by 24 – 72 hours after IVIg administration. Concomitantly, the fractions of platelets with aCASP8 and aCASP9, decreased towards control values (ITP patients after IVIg: aCASP8 7.8±5.3%; aCASP9 6.9±2.1; p=0.5 for both compared to controls). Again no change in mitochondrial potential was observed after IVIg. In summary, we have demonstrated enhancement of the platelet apoptotic-like processes of aCASP8 and aCASP9 specifically in pediatric primary ITP, which were not observed in cTP. However, the platelet mitochondrial membrane potential was unchanged in ITP (before and after IVIg) and did not differ compared cTP and healthy children. Consistent with primary ITP, the patients' platelet counts were low and increased with IVIg administration. In parallel, IVIg led to a decrease of aCASP8 and aCASP9 in the patients' platelets. Together with our previously reported results (Speer et al, Blood 2008;112: 3417), we show that apoptotic events in platelets such as activation of caspases-8, -9, and -3 and PS exposure are increased specifically in ITP but not in cTP, and are decreased after IVIg treatment. As we detected no loss of the mitochondrial membrane potential in platelets from ITP patients, it may be that apoptotic processes in these platelets are not activated by mitochondrial signaling, but rather via an extrinsic signaling cascade including caspase-8, leading to the activation of caspase-3 and caspase-9. However, the complete signaling pathway leading to caspase-8 activation in platelets of pediatric ITP remains to be elucidated. Disclosures:No relevant conflicts of interest to declare.

Phosphatidylserine exposure and other apoptotic‐like events in Bernard‐Soulier syndrome platelets

In the Bernard-Soulier syndrome (BSS), the giant platelets are said to have increased phosphatidylserine (PS) surface exposure in the resting state and shortened survival in the circulation. When normal platelets are activated, they undergo many biochemical and morphological changes, some of which are apoptotic. Herein, we investigated apoptotic-like events in BSS platelets upon activation, specifically, PS exposure, microparticle (MP) formation, cell shrinkage, and loss of mitochondrial inner membrane potential (DeltaPsi(m)). Platelets from two unrelated BSS patients were examined in whole blood; agonists used were collagen, thrombin, PAR1- or PAR4-activating peptides (APs), or combinations of collagen with thrombin, and the PAR-APs. Flow cytometry was used to measure PS exposure (annexin A5 binding), platelet-derived MPs (forward scatter; events <0.75 microm size), and DeltaPsi(m) (TMRM fluorescence). PS exposure was increased on resting and activated BSS platelets, and this was independent of the platelet size. MP formation by BSS platelets was generally enhanced. Cell shrinkage occurred on activation to form smaller, PS-exposing platelets in BSS and controls. A proportion of PS-exposing BSS and control platelets exhibited DeltaPsi(m) loss, but unlike controls, there was also loss of DeltaPsi(m) in the BSS platelets not exposing PS. Thus, BSS platelets undergo apoptotic-like events upon activation, with PS exposure and MP formation being enhanced. These events may play a role in the shortened survival in BSS, as well as affecting thrombin generation.

Investigating caspases and other markers of apoptosis in ITP

Apoptosis is the main mechanism that regulates cell lifespan and the elimination of damaged or infected nucleatedcells. Although platelets are enucleate cells, they express atleast part of the apoptotic machinery known from nucleatedcells. In addition, they have been observed to undergoapoptotic-like events. This has been demonstrated by theactivation and translocation of Bcl-2 family members,cytochrome c release, activation of caspase-9 and ofcaspase-3 [1–10]. These observations indicate that plateletscontain the essential components needed to undergoprogrammed cell death executed in the cytoplasm. Recent-ly, in a murine model of ITP, it has been shown thatapoptotic-like processes can be induced by anti-plateletantibodies: injection of anti-GPIIb antibodies causedapoptotic-like events as well as profound thrombocytopenia[7, 11–14].In this murine ITP model, IVIg treatment wasable to decrease the apoptotic-like events and to improvethe low platelet count [7, 11–13].As no such study has been performed in paediatric ITPso far, we started to investigate whether in children withprimary ITP, an enhanced expression of markers ofapoptotic processes can be found. Furthermore, we areinterested to see if such markers correlate to platelet countand most important to clinical symptoms and if theydecrease after IVIg treatment.So far, we have shown in a limited number of patientsthat caspase-3 is activated in 40% of the platelets in patientswith primary ITP and decreases to 10% after IVIg treatment([15], manuscript in preparation). In parallel to caspase-3activation also bleeding signs decreased after IVIg therapy.In ongoing studies, we investigate caspase-3, caspase-9,caspase-8, PS-exposure and mitochondrial membrane po-tential by flow cytometry and caspases by Western blot. Wefurther will develop an immuno-fluorescence microscopyprotocol for the detection and quantification of activatedcaspases in bone marrow smears. Investigating and com-paring levels of activated caspases and other signs ofapoptosis in platelets and bone marrow samples isimportant to understand the initiation of apoptotic pro-cesses in platelets, whether apoptosis is already triggered inbone marrow or only in platelets in ITP patients. Further-more, it is important to understand whether apoptosismight contribute to the destruction of platelets in ITPpatients.To increase patient numbers, we invite other clinicians orlaboratories treating respectively investigating ITP tocollaborate with us.

Calpain inhibition by calpeptin does not prevent APLT activity reduction in PS-exposing platelets, but calpeptin has independent pro-apoptotic effects

Exposure of procoagulant phosphatidylserine (PS) on the surface of activated platelets is not readily reversible and this may propagate thrombosis. Persistence of PS exposure may be attributed, at least in part, to a continued reduction of the activity of aminophospholipid translocase (APLT), that transports PS from the outer to the inner membrane leaflet. We investigated whether calpain is involved in the inhibition of APLT activity. In flow cytometric investigations, using the inhibitors calpeptin or E64d at a concentration that blocks calpain activation, we found that calpain is not responsible for the reduction in APLT activity that results in persistence of PS exposure. Unexpectedly, we found that the inhibitors had additional effects independent of blocking calpain. Incubation of resting platelets with calpeptin resulted in a subpopulation of platelets with increased intracellular Ca(2+) and persistent PS exposure. The inhibitors also increased the proportion of platelets with persistent PS exposure in suspensions stimulated with thrombin and/or collagen or the Ca(2+)-ionophore A23187 under conditions in which calpain was not activated or in which its activation was completely blocked; P-selectin expression on thrombin and/or collagen-stimulated platelets was inhibited. Furthermore, in stimulated platelets, calpeptin increased the proportion of the PS-exposing platelets expressing a second apoptotic hallmark, collapsed mitochondrial inner membrane potential (DeltaPsi(m)). These additional effects of calpeptin on platelet regulation of intracellular Ca(2+) levels and apoptotic-like events should be taken into account when it is used as an inhibitor of calpain.

BH4 peptide derived from Bcl‐xL and Bax‐inhibitor peptide suppresses apoptotic mitochondrial changes in heat stressed bovine oocytes

Mitochondria play an important role in the integration and transmission of cell death signals mediated by the Bcl-2 family proteins. Experiments were conducted to determine whether the anti-apoptotic peptides BH4 domain of Bcl-xL (TAT-BH4) and Bax inhibitor peptide (BIP) suppresses heat stress (HS) injury in oocytes by reduction of apoptotic-like events. Cumulus-oocyte complexes (COCs) were matured at 39 degrees C (control) or 41 degrees C (HS) for 21 hr then placed in maturation medium containing 0 or 100 microM BIP in water and 0 or 1 microM TAT-BH4 in dimethyl sulfoxide (DMSO), or a combination of both peptides (BIP + BH4). Peptide effects on embryo development, DNA fragmentation, mitochondrial membrane potential (Delta(Psi)m), and mitochondrial DNA (mtDNA) copy number were measured. All groups were fertilized and cultured in vitro at 39 degrees C for 8 days. Compared to control, HS-treated oocytes induced a decrease in embryo development (P < 0.05), increase in proportion of TUNEL-positive chromatin in oocytes and blastocysts (P < 0.05), and loss of oocyte Delta(Psi)m (P < 0.001). In the presence of BIP or BIP + BH4, development of HS-treated oocytes into blastocysts was increased (P < 0.05). Conversely, COCs matured with TAT-BH4 at 41 degrees C showed reduced embryonic development (P < 0.05). Exposure of HS-treated to each or both peptides resulted in a reduction of TUNEL frequency in oocytes and blastocysts cells derived from these oocytes (P < 0.05). The loss of Delta(Psi)m in HS-treated oocytes was not restored by exposure to BIP + BH4 and there was no effect in mtDNA copy number. In conclusion, the present results show that HS-induced apoptosis in bovine oocytes involves Bax and BH4 domain-dependent pathways.

Apoptotic-Like Events in Bernard-Soulier Syndrome (BSS) Platelets.

Although anucleate, platelets are recognized to undergo apoptotic-like events, with some characteristics of activated platelets resembling those of apoptotic nucleated cells, e.g. membrane blebbing (microparticle (MP) formation), exposure of phosphatidylserine (PS). There are reports in the older literature that platelets from BSS patients expose more PS and have enhanced senescence. In the present study, using flow cytometry, we investigated apoptotic-like events, specifically PS exposure, MP formation, cell shrinkage and loss of mitochondrial inner membrane potential (ΔΨm) in platelets from 2 BSS patients (BSS-Ps), compared with platelets from a patient with another inherited macrothrombocytopenia - an MYH9-related disorder (MYH9-RD-P) - and from normal controls. Investigations were done using whole blood to avoid platelet manipulation, especially of the large platelets. Citrated blood was mixed with GPRP and Ca2+, and the agonists collagen (C; 20 μg/mL), thrombin (T; 1 U/mL), SFLLRN (PAR1 activating peptide (AP); 50 μM), AYPGKF (PAR4 AP; 250 μM), or combinations of C with T or the PAR APs. Platelets and MPs were identified as CD41-positive events; platelet size was expressed as median forward scatter (mFSC); MPs were defined as events <0.8 μm; PS exposure was measured as the % platelets with bound fluorescently-labeled annexin A5 and the mean fluorescence intensity (MFI) of bound annexin A5; and loss of ΔΨm was determined by decreased fluorescence of the potential-sensitive probe TMRM concurrently with PS exposure. Resting BSS and MYH9-RD platelets were larger than controls (mFSC of BSS-Ps: 136, 163; MYH9-RD-P: 126; controls: 50±2). Slightly more resting BSS platelets exposed PS than controls, as did MYH9-RD platelets (Table). Activation with C significantly increased the % platelets exposing PS, which was synergistically increased by T or the PAR1 and/or PAR4 APs; more BSS and MYH9-RD platelets exposed PS than did corresponding activated controls (Table).% PSExposureAgonist(s)BSS-P1BSS-P2MYH9-RD-Pcontrolsnone (resting platelets)3.03.53.01.3±0.4C27.029.937.97.0±1.5C+T11.335.364.513.5±1.7C+PAR1 AP20.143.649.516.4±3.1C+PAR4 AP31.647.849.718.4±3.7C+PAR1 AP+PAR4 AP33.449.554.820.6±3.4Interestingly, the platelets in the PS-exposing subpopulation were small (mFSC of BSS-Ps: 27, 30; MYH9-RD: 32; controls 18±1) and the annexin A5 MFI of this subpopulation of BSS and MYH9-RD platelets was .5-fold higher than that of controls irrespective of the platelet agonist(s). Unstimulated blood samples from patients and controls had 1–2% MPs; adding combinations of agonists led to the highest MP production, with control platelets producing 5–6% MPs, and BSS and MYH9-RD platelets, a 1.5–2.5-fold greater amount. Another apoptosis hallmark, cell shrinkage, was observed upon platelet activation in the non-PS-exposing population; in response to the various agonists, the size of this platelet population in patients and controls correlated inversely with the amount of PSexposing platelets formed (P<0.0.001) and the amount of MPs formed (P<0.0.001). In nucleated cells, dissipation of ΔΨm precedes PS exposure. However, activated BSS, MYH9-RD and control platelets exposed PS with or without a dissipated ΔΨm; ~50% of activated PS-exposing platelets did not exhibit loss of ΔΨm. While only 5–10% of activated non-PS-exposing control or MYH9-RD platelets exhibited ΔΨm loss, a substantial proportion, ~50%, of activated non-PS-exposing BSS platelets had a dissipated ΔΨm; in a rabbit model, we have previously found non-PS-exposing platelets with a collapsed ΔΨm to be associated with platelet senescence. In summary, we show that: PAR1 and PAR4 mediate the synergistic effect of T on C-stimulated PS expression on BSS, MYH9-RD and control platelets; the apoptotic-like event of cell shrinkage occurs in BSS platelets as well as MYH9-RD and control platelets; other such events, PS exposure and MP formation, are enhanced in BSS platelets compared with controls likely due to the large size of BSS platelets since they are also observed in MYH9-RD platelets; activation to expose PS can occur independently of ΔΨm dissipation in BSS, MYH9-RD and control platelets; and the loss of ΔΨm in non-PS-exposing platelets, an apoptotic-like event that is associated with platelet senescence and one that occurs only to a limited extent in MYH9-RD and control platelets, is a previously unrecognized characteristic of BSS platelets.

Apoptosis in Platelets from Pediatric Patients with Acute Immune Thrombocytopenic Purpura (ITP) Is Ameliorated by IV Ig.

Interaction of auto-antibodies with platelet surface glycoproteins (GP) has been implicated in the accelerated clearance of platelets in ITP. It was recently shown, using a murine model, that the ITP induced by injection of anti-GPIIb is associated with apoptotic-like processes in platelets. These apoptotic-like processes in platelets were similar to those observed during apoptosis in nucleated cells: activation of caspase-3 (aCASP3), loss of mitochondrial inner transmembrane potential and externalisation of phosphatidylserine (PS). Administration of IVIg, that ameliorates the ITP in this model, also inhibited aCASP3 and PS externalization (Leytin et al., 2006, Br J Haematol ; 133:78; Song et al., 2003, Blood ; 101:3708). Whether these observations in mice are applicable to patients with ITP had not been investigated. Thus, the aim of our study was to investigate whether platelets from paediatric patients with acute ITP show an enhancement of apoptotic-like processes. Further, we examined whether the increase in platelet count in response to IVIg in these patients is accompanied by an inhibition of apoptotic-like processes in platelets. Children with clinical and laboratory diagnosis of acute ITP were enrolled in this prospective study. Severity of bleeding symptoms was assessed according to a paediatric bleeding score for ITP at the time of diagnosis and after IVIg therapy. Blood samples were obtained at these time points for measurement of platelet count and for flow cytometric analyses of platelet apoptotic-like events. Specifically, platelet aCASP3 and PS externalisation were measured in citrated platelet-rich plasma. Platelets were identified as CD42 positive events; aCASP3 was measured as % platelets with bound FLICA-FITC fluorescence; and PS exposure as % platelets with bound annexin A5-FITC fluorescence. The fraction of young, reticulated platelets (RP) was analysed using thiazole orange. All patients (median age 7.5, n = 12) presented with typical symptoms of acute ITP with a bleeding score of 2 – 3. 10 patients had platelet counts below 10'000/micro l, and 2 had platelets counts over 10'000/micro l. ITP patients had increased levels of platelets with aCASP3 (n = 11) and PS exposure (n = 9) compared with healthy control children (see Figure) and increased reticulated platelets (15.6 ± 2.7 %, n = 10; controls: 1.2 ± 0.8, n = 11). The 10 patients with platelet counts below 10,000/micro l were treated with a maximum of 3 doses of IVIg (0.4 – 0.8 g/kg/dose). All patients showed a rise in platelet counts to above 20'000/micro l and amelioration of bleeding symptoms 24 – 72 hours after IVIg administration. Concomitantly, platelet aCASP3 and PS exposure decreased (see Figure), as did RP, to 3.8 ± 1.5 % (n = 9). One patient with an initial platelet count of 18,000/micro l was not treated. This patient showed 5% positive aCASP3 platelets, 3.5 % platelets with PS exposure and 3.5% RP, similar to controls; one month later, the platelet count increased spontaneously to 60'000/micro l with 10% aCASP positive platelets, 4% platelets with PS exposure and 3 % RP. One patient was lost to follow-up. In summary, we have demonstrated enhancement of the platelet apoptotic-like processes of aCASP3 and PS externalisation in paediatric acute ITP, similar to the reported murine model. Consistent with acute ITP, platelet counts were low and levels of RP were high; after administration of IVIg, platelet counts increased whereas levels of RP decreased. IVIg administration also led to a decrease of aCASP3 and PS exposure in the patients' platelets. Thus, amelioration of acute ITP in children by IVIg is accompanied by inhibition of enhanced apoptosis-like events in platelets. [Display omitted]

Bax activators potentiate coated-platelet formation.

Activation of platelets with collagen plus thrombin produces a subset of cells known as coated-platelets. Coated-platelets retain several alpha-granule proteins on their surface, express phosphatidylserine (PS), lose mitochondrial potential and release microparticles. A number of these characteristics are also observed in apoptotic cells, and this similarity leads to the hypothesis that mechanisms controlling initiation of apoptosis might also affect generation of coated-platelets. In this report, we demonstrate that BH3 mimetics, molecules that facilitate apoptosis by releasing pro-apoptotic Bax from its antiapoptotic partner Bcl-2, are able to promote coated-platelet formation as monitored by several different markers of these cells. Specifically, gossypol and methoxy-antimycin (MAM) promote fibrinogen retention, mitochondrial depolarization, and PS exposure upon activation with thrombin plus convulxin, a ligand of the glycoprotein VI collagen receptor. Gossypol also potentiates microparticle release by convulxin plus thrombin activated platelets although MAM does not. In addition, Bax activators together with thrombin generate coated-platelets, effectively bypassing the requirement for convulxin. These findings further support a close relationship between apoptotic-like events and the production of coated-platelets.

Apoptotic-like mitochondrial events associated to phosphatidylserine exposure in blood platelets induced by local anaesthetics.

Phosphatidylserine exposure in platelets is required for normal haemostasis and is also a hallmark of apoptosis. It results from activation of a phospholipid scramblase, which has been shown to be differently stimulated by Ca(2+)-influx and during apoptosis, thus suggesting that mitochondria may be involved in phosphatidylserine exposure in platelets. It is also well known that local anaesthetics can expose phosphatidylserine in platelets and affect the mitochondrial metabolism in other cells. Thus, the present study was undertaken to evaluate the specific involvement of mitochondria in phosphatidylserine exposure in platelets. For this purpose, we stimulated phosphatidylserine exposure by local anaesthetics and avoided any external Ca(2+)-influx by performing all experiments in the absence of added Ca(2+). We report that phosphatidylserine exposure, induced by the lipophilic local anaesthetics dibucaine and tetracaine, was accompanied by depolarization of the mitochondrial membrane, cytochrome c release, calpain-processing of caspases 9 and 3 to active enzymes, as well as a prolonged increase in both cytosolic and mitochondrial Ca(2+) concentrations. In contrast, in the absence of extracellular Ca(2+), the Ca(2+)-ionophore A23187 induced a smaller transient increase in both cytosolic and mitochondrial Ca(2+) concentrations, but did not induce any other phenomena, nor phosphatidylserine exposure. However, phosphatidylserine exposure and depolarization induced by dibucaine still occurred in spite of inhibition of intracellular Ca(2+) elevation. Thus we conclude that phosphatidylserine exposure in platelets is associated with mitochondrial apoptotic-like events. Therefore, we propose that mitochondria engagement in an apoptotic pathway in platelets could lead to PS exposure without the participation of Ca(2+).

Role of the long slender to short stumpy transition in the life cycle of the african trypanosomes

It is shown using mouse models that the African trypanosomes exert a significant drain upon their host's carbohydrate (energy) resources; and that the higher the parasitemia, the greater the energy demand. It is, therefore, hypothesized that the long slender (LS) to short stumpy (SS) transition evolved, in part, to help control the parasitemia and to increase host survival time. It is also suggested that the SS population is heterogeneous. One part of the population is tsetse infective, while a second older SS population is undergoing apoptotic-like events, which leads to their cell death and their stimulation of the host's immune response. This immune stimulation by the old dying SS forms would eliminate the major LS and SS variant antigen population, and produce the chronic relapsing infection. It is concluded that the SS stages during the apoptosis-like process are acting altruistically. They give their lives to insure the long-term survival of the host, and to insure renewed growth of the minor LS variants and new infective SS forms. This process is predicted to increase the probability for the successful transmission of the trypanosomes to a new host.

TPA induces apoptosis in MPC-11 mouse plasmacytoma cells grown in serum-free medium

Apoptotic-like events could be rapidly induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in cells of the mouse plasmacytoma cell line MPC-11 grown in serum-free medium. Indicators for apoptosis were morphological changes visualized by light and electron microscopy, such as chromatin condensation and the formation of cellular buds and fragments, as well as biochemical indices like the appearance of the so-called ‘DNA ladder’. Additionally, in these cells which are usually devoid of significant amounts of cytoplasmic intermediate filament (cIF) proteins, synthesis and accumulation of the cIF protein vimentin was rapidly induced by TPA treatment and almost all cells became vimentin-positive. Later on, substantial amounts of vimentin and lamin B degradation products appeared, and an increasing fraction of cells displayed low or even undetectable quantities of intact vimentin. This subpopulation was characterized via microscopy to be in the late stages of apoptosis. We suggest that in MPC-11 cells undergoing apoptosis in response to TPA treatment vimentin as well as lamin B are degraded, leading to a rearrangement and eventual loss of their respective filament networks.

Biochemical Identification of Apoptosis (Programmed Cell Death) in Granulosa Cells: Evidence for a Potential Mechanism Underlying Follicular Atresia*

In the present study, we examined the possibility that granulosa cell death during ovarian follicular atresia occurs by apoptosis (programmed cell death). To investigate this possibility, atresia was induced in immature female rats by injecting 15 IU PMSG. Controls received either vehicle or no treatment. PMSG-treated animals were killed on days 1-5 post-injection while controls were killed on days 1 or 5. The onset of atresia was assessed histologically by light microscopic inspection of 5 microns tissue sections and functionally by quantification of serum progesterone and estrogen levels. Apoptosis is characterized by the cleavage of genomic DNA into oligonucleosomal length fragments by a Ca2+/Mg(2+)-dependent endogenous endonuclease. Such fragments form a distinctive ladder pattern when separated electrophoretically. Accordingly, the occurrence of apoptosis in granulosa cells was assessed by examining the pattern of fragmented DNA in cell lysates after agarose gel electrophoresis. Gels were stained with ethidium bromide and DNA visualized by UV transillumination. The earliest morphological signs of atresia were detected 4 days after PMSG injection as evidenced by degeneration and detachment of granulosa cells from the basal lamina. Serum estrogen increased from basal to levels 7-fold over controls by day 3 after PMSG treatment, falling to control values by day 4 and thereafter. In contrast, progesterone remained basal for the first 3 days, rising to levels 3-fold and 8-fold above controls 4 and 5 days after PMSG treatment, respectively. Such shifts in the ratio of estrogen to progesterone production are known to be characteristic of follicular atresia. Finally, electrophoretic analysis of low mol wt DNA in granulosa cell lysates revealed a definitive ladder pattern of oligonucleosomal length DNA fragments (characteristic of apoptosis) on days 4 and 5 after PMSG injection. This pattern was not detectable on days 1 and 2 after treatment. Lysates obtained 3 days after PMSG treatment showed a faint apoptotic-like pattern of DNA fragments; a result consistent with other systems in which DNA cleavage begins before any morphological signs of death. Interestingly, a ladder pattern of DNA fragments was present in control lysates suggesting that granulosa cell death under normal (vs. induced) conditions of atresia in immature rats occurs by apoptosis. These data demonstrate an intimate association between apoptotic-like events and dying granulosa cells and thus support the possibility that apoptosis is involved in the induction of follicular atresia.