API-1 (pyrido[2,3-d]pyrimidines) is a novel small-molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of cellular FLICE-inhibitory protein (c-FLIP) levels and TRAIL-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of death receptor 4 (DR4) or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1 but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Because other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity. 2012 AACR
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API-1 is a novel small molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation, and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of c-FLIP levels and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of DR4 or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis, but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1, but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Since other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity. PMID:22345097
Generalized systemic reactions to stinging hymenoptera venom constitute a potentially fatal condition in venom-allergic individuals. Hence, the identification and characterization of all allergens is imperative for improvement of diagnosis and design of effective immunotherapeutic approaches. Our aim was the immunochemical characterization of the carbohydrate-rich protein Api m 10, an Apis mellifera venom component and putative allergen, with focus on the relevance of glycosylation. Furthermore, the presence of Api m 10 in honeybee venom (HBV) and licensed venom immunotherapy preparations was addressed. Api m 10 was produced as soluble, aglycosylated protein in Escherichia coli and as differentially glycosylated protein providing a varying degree of fucosylation in insect cells. IgE reactivity and basophil activation of allergic patients were analyzed. For detection of Api m 10 in different venom preparations, a monoclonal human IgE antibody was generated. Both, the aglycosylated and the glycosylated variant of Api m 10 devoid of cross-reactive carbohydrate determinants (CCD), exhibited IgE reactivity with approximately 50% of HBV-sensitized patients. A corresponding reactivity could be documented for the activation of basophils. Although the detection of the native protein in crude HBV suggested content comparable to other relevant allergens, three therapeutical HBV extracts lacked detectable amounts of this component. Api m 10 is a genuine allergen of A. mellifera venom with IgE sensitizing potential in a significant fraction of allergic patients independent of CCD reactivity. Thus, Api m 10 could become a key element for component-resolved diagnostic tests and improved immunotherapeutic approaches in hymenoptera venom allergy. 2011 John Wiley & Sons A/S.
A model to predict fatigue crack growth of API pipeline steels in high pressure gaseous hydrogen has been developed and is presented elsewhere. The model currently has several parameters that must be calibrated for each pipeline steel of interest. This work provides a sensitivity analysis of the model parameters in order to provide (a) insight to the underlying mathematical and mechanistic aspects of the model, and (b) guidance for model calibration of other API steels.
In highly social bees, queen mandibular pheromone (QMP) is vital for colony life. Both Apis cerana (Ac) and Apis mellifera (Am) share an evolutionarily conserved set of QMP compounds: (E)-9-oxodec-2-enoic acid (9-ODA), (E)-9-hydroxydec-2-enoic acid (9-HDA), (E)-10-hydroxy-dec-2-enoic acid (10-HDA), 10-hydroxy-decanoic acid (10-HDAA), and methyl p-hydroxybenzoate (HOB) found at similar levels. However, evidence suggests there may be species-specific sensitivity differences to QMP compounds because Ac workers have higher levels of ovarian activation than Am workers. Using electroantennograms, we found species-specific sensitivity differences for a blend of the major QMP compounds and three individual compounds (9-HDA, 10-HDAA, and 10-HDA). As predicted, Am was more sensitive than Ac in all cases (1.3- to 2.7- fold higher responses). There were also species differences in worker retinue attraction to three compounds (9-HDA, HOB, and 10-HDA). In all significantly different cases, Am workers were 4.5- to 6.2-fold more strongly attracted than Ac workers were. Thus, Ac workers responded less strongly to QMP than Ac workers, and 9-HDA and 10-HDA consistently elicited stronger antennal and retinue formation responses.
In highly social bees, queen mandibular pheromone (QMP) is vital for colony life. Both Apis cerana (Ac) and Apis mellifera (Am) share an evolutionarily conserved set of QMP compounds: (E)-9-oxodec-2-enoic acid (9-ODA), (E)-9-hydroxydec-2-enoic acid (9-HDA), (E)-10-hydroxy-dec-2-enoic acid (10-HDA), 10-hydroxy-decanoic acid (10-HDAA), and methyl p-hydroxybenzoate (HOB) found at similar levels. However, evidence suggests there may be species-specific sensitivity differences to QMP compounds because Ac workers have higher levels of ovarian activation than Am workers. Using electroantennograms, we found species-specific sensitivity differences for a blend of the major QMP compounds and three individual compounds (9-HDA, 10-HDAA, and 10-HDA). As predicted, Am was more sensitive than Ac in all cases (1.3- to 2.7- fold higher responses). There were also species differences in worker retinue attraction to three compounds (9-HDA, HOB, and 10-HDA). In all significantly different cases, Am workers were 4.5- to 6.2-fold more strongly attracted than Ac workers were. Thus, Ac workers responded less strongly to QMP than Am workers, and 9-HDA and 10-HDA consistently elicited stronger antennal and retinue formation responses [corrected].
Apoptosis of host cells profoundly influences virus propagation and dissemination, events that are integral to influenza A virus (IAV) pathogenesis. The trigger for activation of apoptosis is regulated by an intricate interplay between cellular and viral proteins, with a strong bearing on IAV replication. Though the knowledge of viral proteins and mechanisms employed by IAV to induce apoptosis has advanced considerably of late, we know relatively little about the repertoire of host factors targeted by viral proteins. Thus, identification of cellular proteins that are hijacked by the virus will help us not only to understand the molecular underpinnings of IAV-induced apoptosis, but also to design future antiviral therapies. Here we show that the nucleoprotein (NP) of IAV directly interacts with and suppresses the expression of API5, a host antiapoptotic protein that antagonizes E2F1-dependent apoptosis. siRNA-mediated depletion of API5, in NP-overexpressed as well as IAV-infected cells, leads to upregulation of apoptotic protease activating factor 1 (APAF1), a downstream modulator of E2F1-mediated apoptosis, and cleavage of caspases 9 and 3, although a reciprocal pattern of these events was observed on ectopic overexpression of API5. In concordance with these observations, annexin V and 7AAD staining assays exhibit downregulation of early and late apoptosis in IAV-infected or NP-transfected cells on overexpression of API5. Most significantly, while overexpression of API5 decreases viral titers, cellular NP protein as well as mRNA levels in IAV-infected A549 cells, silencing of API5 expression causes a steep rise in the same parameters. From the data reported in this manuscript, we propose a proapoptotic role for NP in IAV pathogenesis, whereby it suppresses expression of antiapoptotic factor API5, thus potentiating the E2F1-dependent apoptotic pathway and ensuring viral replication.
Molecular dynamics of pure nifedipine and its solid dispersions with modified carbohydrates as well as the crystallization kinetics of active pharmaceutical ingredient (API) above and below the glass transition temperature were studied in detail by means of broadband dielectric spectroscopy (BDS), differential scanning calorimetry (DSC), and X-ray diffraction method. It was found that the activation barrier of crystallization increases in molecular dispersions composed of acetylated disaccharides, whereas it slightly decreases in those consisting of modified monocarbohydrates for the experiments carried out above the glass transition temperature. As shown by molecular dynamics simulations it can be related to the strength, character, and structure of intermolecular interactions between API and saccharides, which vary dependently on the excipient. Long-term physical stability studies showed that, in solid dispersions consisting of acetylated maltose and acetylated sucrose, the crystallization of nifedipine is dramatically slowed down, although it is still observable for a low concentration of excipients. With increasing content of modified carbohydrates, the crystallization of API becomes completely suppressed. This is most likely due to additional barriers relating to the intermolecular interactions and diffusion of nifedipine that must be overcome to trigger the crystallization process. 2ff7e9595c
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