Caspase-1 Is Not Involved in CD95/Fas-Induced Apoptosis in Jurkat T Cells

It is now well established that the caspases, a family of cysteine proteases, play a key role in apoptosis. Although overexpressing each of the caspases in cells triggered apoptosis, the precise role and contribution of individual caspases are still unclear. Caspase-1, the first caspase discovered, was initially implicated in mammalian apoptosis because of its similarity to the gene productced-3.Using whole cells as well as anin vitrosystem to study apoptosis, the role of caspase-1 in Fas-mediated apoptosis in Jurkat T cells was examined in greater detail. Using various peptide-based caspase inhibitors, our results showed thatN-acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone efficiently blocked Fas-mediated apoptosis in Jurkat T cells, whereasN-acetyl-Tyr-Val-Ala-Asp aldehyde, which is more specific for caspase-1, had little effect. Cell lysates derived from anti-Fas-stimulated cells, which readily induced apoptotic nuclei morphology and DNA fragmentation in isolated thymocyte nuclei, had no caspase-1 activity using proIL-1β as a substrate. Time-course studies showed no caspase-1 activity during the activation of apoptosis in Jurkat cells by agonistic Fas antibodies. Furthermore, no pro-caspase-1 protein nor activated form of the protein was detected in normal or apoptotic Jurkat cells. In contrast, both caspase-2 and caspase-3 were readily detected as proenzymes in control cells and their activated forms were detected in apoptotic cells. Incubation of recombinant active caspase-1 with control cell lysates did not activate the apoptotic cascade as shown by the lack of detectable apoptotic nuclei promoting activity using isolated nuclei as substrate. However, under similar conditions proIL-1β was readily processed into the mature cytokine, indicating that the recombinant caspase-1 remained active in the presence of control cell lysates. Taken together our results demonstrate that caspase-1 is not required for the induction of apoptosis in Jurkat T cells mediated by the Fas antigen.     

The Expression of Fn14 via Mechanical Stress-activated JNK Contributes to Apoptosis Induction in Osteoblasts

Bone mass is maintained by the balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. It is well known that adequate mechanical stress is essential for the maintenance of bone mass, whereas excess mechanical stress induces bone resorption. However, it has not been clarified how osteoblasts respond to different magnitudes of mechanical stress. Here we report that large-magnitude (12%) cyclic stretch induced Ca²⁺ influx, which activated reactive oxygen species generation in MC3T3-E1 osteoblasts. Reactive oxygen species then activated the ASK1-JNK/p38 pathways. The activated JNK led to transiently enhanced expression of FGF-inducible 14 (Fn14, a member of the TNF receptor superfamily) gene. Cells with enhanced expression of Fn14 subsequently acquired sensitivity to the ligand of Fn14, TNF-related weak inducer of apoptosis, and underwent apoptosis. On the other hand, the ASK1-p38 pathway induced expression of the monocyte chemoattractant protein 3 (MCP-3) gene, which promoted chemotaxis of preosteoclasts. In contrast, the ERK pathway was activated by small-magnitude stretching (1%) and induced expression of two osteogenic genes, collagen Ia (Col1a) and osteopontin (OPN). Moreover, activated JNK suppressed Col1a and OPN induction in large-magnitude mechanical stretch-loaded cells. The enhanced expression of Fn14 and MCP-3 by 12% stretch and the enhanced expression of Col1a and OPN by 1% stretch were also observed in mouse primary osteoblasts. These results suggest that differences in the response of osteoblasts to varying magnitudes of mechanical stress play a key role in switching the mode of bone metabolism between formation and resorption.     

Gliadin Peptides Induce Tissue Transglutaminase Activation and ER-Stress through Ca2+ Mobilization in Caco-2 Cells

Background

Celiac disease (CD) is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG) seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca²⁺ homeostasis and tTG activity.

Methods/Principal Findings

We studied Ca²⁺ homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31–43 and 57–68 rapidly mobilized Ca²⁺ from intracellular stores. Specifically, peptide 31–43 mobilized Ca²⁺ from the endoplasmic reticulum (ER) and mitochondria, whereas peptide 57–68 mobilized Ca²⁺ only from mitochondria. We also found that gliadin peptide-induced Ca²⁺ mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31–43, but not peptide 57–68, induces an increase of tTG expression. Finally, we monitored the expression of glucose-regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31–43, but not of peptide 57–68, induces the expression of both genes.

Conclusions

By inducing Ca²⁺ mobilization from the ER, peptide 31–43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31–43 and 57–68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin–tTG crosslinking in enterocytes and specialized antigen-presenting cells.     

Intracellular Ca2+ regulation in CD3 stimulated Jurkat T cells involves H+ fluxes.

Triggering the CD3/TCR complex of T lymphocytes induces a rapid rise in cytosolic free calcium followed by a slowly declining plateau. The level of this plateau depends on external pH, the more alkalinized media leading to higher values. Neither a pH-dependent binding of mAb, nor a perturbation of internal pH can account for this effect. In a sodium-free medium, or in the presence of dimethylamiloride Ca2+, elevation is accompanied by an acidification of the cells; both of them depend, to the same extent, on external calcium concentration. TPA inhibits CD3-, but not ionomycin-induced Ca2+ and H+ raises, indicating that it acts more probably on Ca2+ influx, rather than on its efflux. These results suggest that intracellular calcium could be regulated by a Ca2+/H+ ATPase which drives H+ in and Ca2+ out. In the presence of external Na+, H+ should return to the medium by the Na+/H+ exchanger.     

Suppression by Thimerosal of Ex-Vivo CD4+ T Cell Response to Influenza Vaccine and Induction of Apoptosis in Primary Memory T Cells

Thimerosal is a preservative used widely in vaccine formulations to prevent bacterial and fungal contamination in multidose vials of vaccine. Thimerosal was included in the multidose non-adjuvanted pandemic 2009 H1N1 vaccine Panenza. In the context of the analysis of the ex-vivo T cell responses directed against influenza vaccine, we discovered the in vitro toxicity Panenza, due to its content in thimerosal. Because thimerosal may skew the immune response to vaccines, we investigated in detail the ex-vivo effects of thimerosal on the fate and functions of T cells in response to TCR ligation. We report that ex-vivo exposure of quiescent or TCR-activated primary human T cells to thimerosal induced a dose-dependent apoptotic cell death associated with depolarization of mitochondrial membrane, generation of reactive oxygen species, cytochrome c release from the mitochondria and caspase-3 activation. Moreover, exposure to non-toxic concentrations of thimerosal induced cell cycle arrest in G0/G1 phase of TCR-activated T cells, and inhibition of the release of proinflammatory cytokines such as IFN gamma, IL-1 beta, TNF alpha, IL-2, as well as the chemokine MCP1. No shift towards Th2 or Th17 cells was detected. Overall these results underline the proapoptotic effect of thimerosal on primary human lymphocytes at concentrations 100 times less to those contained in the multidose vaccine, and they reveal the inhibitory effect of this preservative on T-cell proliferation and functions at nanomolar concentrations.     

Tripeptidyl Peptidase II Regulates Sperm Function by Modulating Intracellular Ca2+ Stores via the Ryanodine Receptor

Recent studies have identified Ca²⁺ stores in sperm cells; however, it is not clear whether these Ca²⁺ stores are functional and how they are mobilized. Here, in vitro and in vivo, we determined that tripeptidyl peptidase II antagonists strongly activated the cAMP/PKA signaling pathway that drives sperm capacitation-associated protein tyrosine phosphorylation. We demonstrated that in the absence of Ca²⁺, TPIII antagonists elevated the intracellular Ca²⁺ levels in sperm, resulting in a marked improvement in sperm movement, capacitation, acrosome reaction, and the in vitro fertilizing ability. This antagonist-induced release of intracellular Ca²⁺ could be blocked by the inhibitors of ryanodine receptors (RyRs) which are the main intracellular Ca²⁺ channels responsible for releasing stored Ca²⁺. Consistent with these results, indirect immunofluorescence assay using anti-RyR antibodies further validated the presence of RyR₃ in the acrosomal region of mature sperm. Thus, TPPII can regulate sperm maturation by modulating intracellular Ca²⁺ stores via the type 3 RyR.     

Cadmium Impairs Autophagy Leading to Apoptosis by Ca2+-Dependent Activation of JNK Signaling Pathway in Neuronal Cells

Neurochemical Research - Autophagy, a process for self-degradation of intracellular components and dysfunctional organelles, is closely related with neurodegenerative diseases. It has been shown...     

Induction of SENP1 in Endothelial Cells Contributes to Hypoxia-driven VEGF Expression and Angiogenesis

SENP1 (SUMO-specific protease 1) has been shown to be essential for the stability and activity of hypoxia-inducible factor 1 (HIF-1α) under hypoxia conditions. However, it is unknown how SENP1 activation and hypoxia signaling are coordinated in the cellular response to hypoxia. Here, we report the essential role of SENP1 in endothelial cells as a positive regulator of hypoxia-driven VEGF production and angiogenesis. SENP1 expression is increased in endothelial cells following exposure to hypoxia. Silencing of HIF-1α blocks SENP1 expression in cell response to hypoxia. Mutation of the hypoxia response element (HRE) on the Senp1 promoter abolishes its transactivation in response to hypoxia. Moreover, silencing of SENP1 expression decreases VEGF production and abrogates the angiogenic functions of endothelial cell. We also find that the elongated endothelial cells in embryonic brain section and vascular endothelial cells in embryonic renal glomeruli in Senp1^−/− mice are markedly reduced than those in wild-type. Thus, these results show that hypoxia implies a positive feedback loop mediated by SENP1. This feedback loop is important in VEGF production, which is essential for angiogenesis in endothelial cells.     

Systems Modeling of Ca2+ Homeostasis and Mobilization in Platelets Mediated by IP3 and Store-Operated Ca2+ Entry

Resting platelets maintain a stable level of low cytoplasmic calcium ([Ca²⁺]_cyt) and high dense tubular system calcium ([Ca²⁺]_dts). During thrombosis, activators cause a transient rise in inositol trisphosphate (IP₃) to trigger calcium mobilization from stores and elevation of [Ca²⁺]_cyt. Another major source of [Ca²⁺]_cyt elevation is store-operated calcium entry (SOCE) through plasmalemmal calcium channels that open in response to store depletion as [Ca²⁺]_dts drops. A 34-species systems model employed kinetics describing IP₃-receptor, DTS-plasmalemma puncta formation, SOCE via assembly of STIM1 and Orai1, and the plasmalemma and sarco/endoplasmic reticulum Ca²⁺-ATPases. Four constraints were imposed: calcium homeostasis before activation; stable in zero extracellular calcium; IP₃-activatable; and functional SOCE. Using a Monte Carlo method to sample three unknown parameters and nine initial concentrations in a 12-dimensional space near measured or expected values, we found that model configurations that were responsive to stimuli and demonstrated significant SOCE required high inner membrane electric potential (>−70 mV) and low resting IP₃ concentrations. The absence of puncta in resting cells was required to prevent spontaneous store depletion in calcium-free media. Ten-fold increases in IP₃ caused saturated calcium mobilization. This systems model represents a critical step in being able to predict platelets’ phenotypes during hemostasis or thrombosis.     

Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation

Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly studied as regulators of the immune response and potential therapeutic agents for autoimmune disorders (1). To date, 15 galectins have been identified and classified according with the structural organization of their distinctive monomeric or dimeric carbohydrate recognition domain for β-galactosides (2, 3). Galectins are secreted by unconventional mechanisms and once outside the cells bind to and cross-link multiple glycoconjugates both at the cell surface and at the extracellular matrix, modulating processes as diverse as cell adhesion, migration, proliferation, differentiation, and apoptosis (4–10). Several galectins have been involved in T cell homeostasis because of their capability to kill thymocytes, activated T cells, and T cell lines (11–16). Pro-apoptotic galectins might contribute to shape the T cell repertoire in the thymus by negative selection, restrict the immune response by eliminating activated T cells at the periphery (1), and help cancer cells to escape the immune system by eliminating cancer-infiltrating T cells (17). They have also a promising therapeutic potential to eliminate abnormally activated T cells and inflammatory cells (1). Studies on the mostly explored galectins, Gal-1, -3, and -9 (14, 15, 18–20), as well as in Gal-2 (13), suggest immunosuppressive complementary roles inducing different pathways to apoptosis. Galectin-8 (Gal-8)⁴ is one of the most widely expressed galectins in human tissues (21, 22) and cancerous cells (23, 24). Depending on the cell context and mode of presentation, either as soluble stimulus or extracellular matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis (6, 7, 9, 10, 22, 25). Its role has been mostly studied in relation to tumor malignancy (23, 24). However, there is some evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or inflammatory disorders. For instance, the intrathymic expression and pro-apoptotic effect of Gal-8 upon CD4^highCD8^high thymocytes suggest a role for Gal-8 in shaping the T cell repertoire (16). Gal-8 could also modulate the inflammatory function of neutrophils (26), Moreover Gal-8-blocking agents have been detected in chronic autoimmune disorders (10, 27, 28). In rheumatoid arthritis, Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid cells, but can be counteracted by a specific rheumatoid version of CD44 (CD44vRA) (27). In systemic lupus erythematosus (SLE), a prototypic autoimmune disease, we recently described function-blocking autoantibodies against Gal-8 (10, 28). Thus it is important to define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with specific integrins, such as α1β1, α3β1, and α5β1 but not α4β1, and as a matrix protein promotes cell adhesion and asymmetric spreading through activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) (10). These early effects occur within 5–30 min. However, ERK1/2 signaling supports long term processes such as T cell survival or death, depending on the moment of the immune response. During T cell activation, ERK1/2 contributes to enhance the expression of interleukin-2 (IL-2) required for T cell clonal expansion (29). It also supports T cell survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by other previously activated T cells (30, 31). Later on, ERK1/2 is required for activation-induced cell death, which controls the extension of the immune response by eliminating recently activated and restimulated T cells (32, 33). In activation-induced cell death, ERK1/2 signaling contributes to enhance the expression of FasL and its receptor Fas/CD95 (32, 33), which constitute a preponderant pro-apoptotic system in T cells (34). Here, we ask whether Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling (35) deserves special attention. cAMP/PKA signaling plays an immunosuppressive role in T cells (36) and is altered in SLE (37). Phosphodiesterases (PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA during T cell activation (38, 39). PKA has been described to control the activity of ERK1/2 either positively or negatively in different cells and processes (35). A little explored integration among ERK1/2 and PKA occurs via phosphatidic acid (PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA plays roles in signaling interacting with a variety of targeting proteins that bear PA-binding domains (40). In this way PA recruits Raf-1 to the plasma membrane (41). It is also converted by phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG), which among other functions, recruits and activates the GTPase Ras (42). Both Ras and Raf-1 are upstream elements of the ERK1/2 activation pathway (43). In addition, PA binds to and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP levels and PKA down-regulation (44). The regulation and role of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell homeostasis, because it is also unknown whether galectins stimulate the PLD/PA pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our results for the first time show that a galectin increases the PA levels, down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE activity, and induces an ERK1/2-dependent expression of the pro-apoptotic factor FasL. The enhanced PDE activity induced by Gal-8 is required for the activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces apoptosis in human peripheral blood mononuclear cells (PBMC), especially after activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other galectins the property of killing activated T cells contributing to the T cell homeostasis. The pathway involves a particularly integrated signaling context, engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its susceptibility to inhibition by anti-Gal-8 autoantibodies.     

Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells

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Chymotryptic-type protease inhibitors block the increase in Ca2+ and Il-2 production in activated Jurkat T cells

Several chymotryptic-type protease inhibitors were found to inhibit both anti-CD3 mAb- and PHA-induced rise in Ca2+ and IL-2 production in Jurkat T cells. The magnitude of inhibition was a function of the effectors used to stimulate Ca2+ entry and depended on the concentration of the inhibitors. Neither tryptic-type protease inhibitors nor an elastase substrate prevented anti-CD3 mAb- or PHA-induced Ca2+ rise in Jurkat cells. The inhibitory effect of N-alpha-p-tosyl-L-phenylalanine chloromethyl-ketone on anti-CD3 mAb- and PHA-induced rise in Ca2+ resulted from a rapid increase in Ca2+ efflux. The inhibitors which were effective on Ca2+ mobilization also inhibited IL-2 production initiated by an anti-CD3 mAb in the presence of 12-O-tetradecanoylphorbol-13-acetate, and to a lesser extent by PHA or the calcium ionophore A23187. No inhibition of IL-2 production was observed when tryptic-type protease inhibitors or the elastase inhibitor were used. In addition, membrane preparations from Jurkat cells were found to hydrolyze the chymotryptic substrate Suc-Ala-Ala-Phe-paranitroaniline, an effect markedly inhibited by N-alpha-p-tosyl-L-phenylalanine chloromethylketone. Moreover, this inhibitor protected one potential endogenous substrate (Mr 38 kDa) from proteolysis. Taken together, these observations show that chymotryptic-type protease inhibitors block the responses generated by the binding of anti-CD3 mAb to Jurkat cells, and suggest that a chymotryptic-like membrane protease contributes to T cell activation.     

Protein kinase C inhibits the transplasma membrane influx of Ca2+ triggered by 4-aminopyridine in Jurkat T lymphocytes

4-aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).     

Induction of Apoptosis in Human Leukemia Cells by the CDK1 Inhibitor

We have examined the effects of the CDK1 inhibitor CGP74514A on cell cycle- and apoptosis-related events in human leukemia cells. An 18-hr exposure to 5 mM CGP74514A induced mitochondrial damage (i.e., loss of Dym) and apoptosis in multiple human leukemia cell lines (e.g., U937, HL-60, KG-1, CCRF-CEM, Raji, and THP; range 30-95%). In U937 cells, CGP74514A- induced apoptosis (5 mM) became apparent within 4 hr and approached 100% by 24 hr. The pan- caspase inhibitor Boc-fmk and the caspase-8 inhibitor IETD-fmk opposed CGP74514A-induced caspase-9 activation and PARP degradation, but not cytochrome c or Smac/DIABLO release. CGP74514A-mediated apoptosis was substantially blocked by ectopic expression of full-length Bcl- 2, a loop-deleted mutant Bcl-2, and Bcl-xL. CGP74514A treatment (5 mM; 18 hr) resulted in increased p21CIP1 expression, p27KIP1 degradation, diminished E2F1 expression, and dephosphorylation of p34cdc2. It also induced early (i.e., within 2 hr) inhibition of CDK1 activity and dephosphorylation of pRb, followed by pRb degradation, but did not block pRb phosphorylation at CDK2- and CDK4- specific sites. These findings indicate that the selective CDK1 inhibitor, CGP74514A, induces complex changes in cell cycle-related proteins in human leukemia cells accompanied by extensive mitochondrial damage, caspase activation, and apoptosis.

Key Words:

Leukemia, CDK1 Inhibitor, Apoptosis, CGP74514A