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1
Casasampere, Mireia, Núria Bielsa, Daniel Riba, Laura Bassas, Ruijuan Xu, Cungui Mao, Gemma Fabriàs, José-Luis Abad, Antonio Delgado, and Josefina Casas. "New fluorogenic probes for neutral and alkaline ceramidases." Journal of Lipid Research 60, no. 6 (March 29, 2019): 1174–81. http://dx.doi.org/10.1194/jlr.d092759.
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New fluorogenic ceramidase substrates derived from the N-acyl modification of our previously reported probes (RBM14) are reported. While none of the new probes were superior to the known RBM14C12 as acid ceramidase substrates, the corresponding nervonic acid amide (RBM14C24:1) is an efficient and selective substrate for the recombinant human neutral ceramidase, both in cell lysates and in intact cells. A second generation of substrates, incorporating the natural 2-(N-acylamino)-1,3-diol-4-ene framework (compounds RBM15) is also reported. Among them, the corresponding fatty acyl amides with an unsaturated N-acyl chain can be used as substrates to determine alkaline ceramidase (ACER)1 and ACER2 activities. In particular, compound RBM15C18:1 has emerged as the best fluorogenic probe reported so far to measure ACER1 and ACER2 activities in a 96-well plate format.
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Petrera, Agnese, Beat Amstutz, Magda Gioia, Janine Hähnlein, Antonio Baici, Petra Selchow, Davide M. Ferraris, et al. "Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates." Biological Chemistry 393, no. 7 (July 1, 2012): 631–40. http://dx.doi.org/10.1515/hsz-2012-0106.
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Abstract Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identified as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fluorogenic substrate at moderately acidic pH values (i.e., 6.3), which corresponds to those reported for the Mtb phagosome where this enzyme should exert its pathological activity. Substrate specificity of Zmp1 was investigated by screening a peptide library. Several sequences derived from biologically relevant proteins were identified as possible substrates, including the neuropeptides bradykinin, neurotensin, and neuropeptide FF. Further, subsequences of other small bioactive peptides were found among most frequently cleaved sites, e.g., apelin-13 and substance P. We determined the specific cleavage site within neuropeptides by mass spectrometry, observing that hydrophobic amino acids, mainly phenylalanine and isoleucine, are overrepresented at position P1′. In addition, the enzymatic mechanism of Zmp1 toward these neuropeptides has been characterized, displaying some differences with respect to the synthetic fluorogenic substrate and indicating that the enzyme adapts its enzymatic action to different substrates.
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STEPANIAK, L. "Comparison of Different Peptidase Substrates for Evaluation of Microbial Quality of Aerobically Stored Meats." Journal of Food Protection 63, no. 10 (October 1, 2000): 1447–49. http://dx.doi.org/10.4315/0362-028x-63.10.1447.
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Different aminopeptidase and endopeptidase substrates were assessed for the detection of enzymatic activity of microorganisms collected from the surface of aerobically cold-stored pork and beef. The most sensitive substrates were fluorogenic Ala-7-amino-4-methylcoumarin (Ala-AMC) or Leu-AMC and colorogenic Ala-p-nitroanilide (Ala-pNA). Activity on natural oligopeptides, e.g., bradykinin or αs1 casein fragment 1 to 23, was very low. The correlation coefficient (r) between log surface counts of 66 meat samples and log fluorescence or absorbance after incubation of surface microbial cells for 2 h with Ala-AMC, Leu-AMC, and Ala-pNA was 0.89, 0.83, and 0.82, respectively. A distinct yellow color was obtained with Ala-pNA when the surface count was ∼106 CFU/cm2. Although correlation and sensitivity was better, no clear advantage is obtained with the use of the fluorogenic Ala-AMC or Leu-AMC instead of Ala-pNA, a substrate proposed by Alvarado et al. (J. Food Sci. 57:1330, 1992) for rapidly assessing the microbial quality of refrigerated meat. The correlation coefficient (r) between time of cold storage and surface count was 0.69.
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Knappe, Sabine, Susanne Till, Gabriele Gerstenbauer, Friedrich Scheiflinger, and Michael Dockal. "The Application of Thrombin Generation Test Is Compromised By Antithrombin III Deficiency." Blood 126, no. 23 (December 3, 2015): 4663. http://dx.doi.org/10.1182/blood.v126.23.4663.4663.
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Abstract Background: Antithrombin III (ATIII) is a stoichiometric inhibitor of factor Xa and thrombin and a major physiological regulator of coagulation. Reduction of ATIII levels, for example in patients with congenital ATIII deficiency, causes a rise in active thrombin which is associated with risk of venous and arterial thrombosis (1, 2). The calibrated automated thrombogram (CAT) assay is widely used to measure thrombin generation in human plasma. Therewith, the fluorogenic substrate Z-G-G-R-AMC is cleaved by the thrombin emerging over time in the plasma sample. Thus, an increase in fluorescence signal reflects the amount of thrombin provided that fluorogenic substrate is in excess over thrombin. To account for substrate consumption, plasma color, and 'inner filter' effect, the fluorescence signal is compared to alpha-2-macroglobulin thrombin complex, the thrombin calibrator. In this complex, thrombin is protected from its physiological inhibitors, but retains its ability to cleave small substrates (3). Aim: We assessed the technical feasibility of measuring the effect of ATIII reduction on thrombin generation in normal and FVIII-inhibited human plasma in the tissue factor (TF) triggered CAT assay. Methods: CAT was performed in ATIII deficient plasma with 1 pM of TF to trigger thrombin generation. The plasma was supplemented with 0.125 - 2.5 µM ATIII to achieve ATIII plasma levels of 5 - 100% of normal. The ATIII titration was also done in ATIII deficient plasma inhibited with a goat polyclonal anti-human FVIII antibody to simulate hemophilia A conditions. Raw data analysis focused on the velocity of fluorescence signal increase, which is usually automatically converted to nM of thrombin by the Thrombinoscope software. Results: Reduced ATIII levels resulted in an apparent concentration dependent increase in thrombin generation in both normal and FVIII inhibited human plasma. For example, a reduction from 2.5 to 1.25 µM ATIII resulted in an apparent increase of 45 - 55% in thrombin peak and ETP values. Most importantly, analysis of the raw data showed that the fluorescence signal reached a plateau after 30 min (normal plasma) and 50 min (FVIII-inhibited plasma) in samples with <1.25 µM ATIII. The plateau (at ~1700 FU) is due to complete depletion of fluorescent substrate. In comparison, samples with ³1.25 µM ATIII did not reach a plateau and show substrate conversion until the end of the assay. In summary, a plasma level of at least 1.25 µM ATIII (50% of normal) was required to obtain thrombin generation profiles which were not compromised by fluorogenic substrate depletion. Below 1.25 µM ATIII, it is not possible to distinguish between inhibition of thrombin and excessive consumption of fluorescence substrate, and therefore, data may be misinterpreted. Conclusion: Thrombin generation at low ATIII plasma levels is difficult to interpret as the physiological inhibition of the generated thrombin is reduced. Consequently, the thrombin substrate is consumed, resulting in substrate depletion. Therefore, we conclude that the CAT assay is technically not suitable to assess the effect of ATIII levels < 1.25 µM ATIII corresponding to 50% of normal plasma. Disclosures Scheiflinger: Baxalta Innovations GmbH: Employment.
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Hodges, Heather L., Robert A. Brown, John A. Crooks, Douglas B. Weibel, and Laura L. Kiessling. "Imaging mycobacterial growth and division with a fluorogenic probe." Proceedings of the National Academy of Sciences 115, no. 20 (April 27, 2018): 5271–76. http://dx.doi.org/10.1073/pnas.1720996115.
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Control and manipulation of bacterial populations requires an understanding of the factors that govern growth, division, and antibiotic action. Fluorescent and chemically reactive small molecule probes of cell envelope components can visualize these processes and advance our knowledge of cell envelope biosynthesis (e.g., peptidoglycan production). Still, fundamental gaps remain in our understanding of the spatial and temporal dynamics of cell envelope assembly. Previously described reporters require steps that limit their use to static imaging. Probes that can be used for real-time imaging would advance our understanding of cell envelope construction. To this end, we synthesized a fluorogenic probe that enables continuous live cell imaging in mycobacteria and related genera. This probe reports on the mycolyltransferases that assemble the mycolic acid membrane. This peptidoglycan-anchored bilayer-like assembly functions to protect these cells from antibiotics and host defenses. Our probe, quencher-trehalose-fluorophore (QTF), is an analog of the natural mycolyltransferase substrate. Mycolyltransferases process QTF by diverting their normal transesterification activity to hydrolysis, a process that unleashes fluorescence. QTF enables high contrast continuous imaging and the visualization of mycolyltransferase activity in cells. QTF revealed that mycolyltransferase activity is augmented before cell division and localized to the septa and cell poles, especially at the old pole. This observed localization suggests that mycolyltransferases are components of extracellular cell envelope assemblies, in analogy to the intracellular divisomes and polar elongation complexes. We anticipate QTF can be exploited to detect and monitor mycobacteria in physiologically relevant environments.
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Baskova, I. P., O. V. Kharitonova, and L. L. Zavalova. "Lysozyme activity of the salivary gland secretion of the medicinal leech h. verbana, h. medicinalis and h. orientalis." Biomeditsinskaya Khimiya 57, no. 5 (2011): 511–18. http://dx.doi.org/10.18097/pbmc20115705511.
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Salivary gland secretions of three species of the medicinal leech differ in the level of lysozyme peptidoglycan-lysing activity. Using the synthetic fluorogenic substrate, 4-methyl-umbelliferyl tetra N-acetyl-β-chitotetraosid, the glycosidase activity (as one of peptidoglycan-lysing activities) of salivary gland secretion of three species of the medicinal leech was quantitatively evaluated in comparison with egg lysozyme. It is supposed, that lysozyme activity of the leech secretions is determined not only by 5 isoforms of destabilase-lysozyme, but by some other enzymes which can utilize this substrate. These may be lysozymes other than i- (invertebrate) lysozymes (such as destabilase-lysozyme, or related enzymes).
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KIPPER, Kalle, Priit VÄLJAMÄE, and Gunnar JOHANSSON. "Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as ‘burst’ kinetics on fluorescent polymeric model substrates." Biochemical Journal 385, no. 2 (January 7, 2005): 527–35. http://dx.doi.org/10.1042/bj20041144.
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Reaction conditions for the reducing-end-specific derivatization of cellulose substrates with the fluorogenic compound, anthranilic acid, have been established. Hydrolysis of fluorescence-labelled celluloses by cellobiohydrolase Cel7A from Trichoderma reesei was consistent with the active-site titration kinetics (burst kinetics), which allowed the quantification of the processivity of the enzyme. The processivity values of 88±10, 42±10 and 34±2.0 cellobiose units were found for Cel7A acting on labelled bacterial cellulose, bacterial microcrystalline cellulose and endoglucanase-pretreated bacterial cellulose respectively. The anthranilic acid derivatization also provides an alternative means for estimating the average degree of polymerization of cellulose and, furthermore, allows the quantitative monitoring of the production of reducing end groups on solid cellulose on hydrolysis by cellulases. Hydrolysis of bacterial cellulose by cellulases from T. reesei revealed that, by contrast with endoglucanase Cel5A, neither cellobiohydrolases Cel7A nor Cel6A produced detectable amounts of new reducing end groups on residual cellulose.
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Andrews, Wallace H., Clyde R. Wilson, and Paul L. Poelma. "Glucuronidase Assay in a Rapid MPN Determination for Recovery of Escherichia coli from Selected Foods." Journal of AOAC INTERNATIONAL 70, no. 1 (January 1, 1987): 31–34. http://dx.doi.org/10.1093/jaoac/70.1.31.
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Abstract Glucuronidase is present in most strains of Escherichia coli but absent in most other enteric microorganisms; therefore, an assay for this enzyme is useful for determining the presence of the organism. The substrate 4-methylumbelliferyl beta-D-glucuronide (MUG) is incorporated into either lauryl tryptose (LT) broth or EC medium; the inoculated tubes are then incubated under specified conditions and examined under longwave UV light for the presence of a fluorogenic glucuronidase end product. When compared with the 10-day most probable number (MPN) procedure of AOAC, the LT-MUG and the EC-MUG tests required 24 and 96 h, respectively, and gave comparable mean log MPN values for samples of crabmeat, sunflower kernels, and walnut pieces. However, false-positive and falsenegative reactions were observed with foods tested by both of these rapid methods. Overall, method sensitivity was not compromised by using the LT-MUG rather than the EC-MUG method. Incorporation of 25 αg MUG/mL into LT broth resulted in diminished fluorescence of positive reactions, whereas MUG concentrations of 50 and 100 αg/mL provided decisive fluorogenic reactions.
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Poelma, Paul L., Clyde R. Wilson, and Wallace H. Andrews. "Rapid Fluorogenic Enumeration of Escherichia coli in Selected, Naturally Contaminated High Moisture Foods." Journal of AOAC INTERNATIONAL 70, no. 6 (November 1, 1987): 991–93. http://dx.doi.org/10.1093/jaoac/70.6.991.
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Abstract An assay for the enzyme glucuronidase was used to determine the presence of Escherichia coli in selected, naturally contaminated high moisture foods. Raw pork sausage, ground turkey, and ground beef were inoculated into tubes containing the substrate 4-methylumbelliferyl beta-D-glucuronide (MUG) in lauryl tryptose (LT) medium. After incubation at 35°C for 24 h, the inoculated LT-MUG tubes were examined under longwave ultraviolet light for the presence of a fluorogenic glucuronidase end product. A fluorescing tube indicated the presumptive presence of E. coli. The 10 day most probable number method of the AOAC and the LT-MUG procedure gave comparable recoveries of E. coli.
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Moberg, Lloyd J., Mary K. Wagner, and Lisa A. Kellen. "Fluorogenic Assay for Rapid Detection of Escherichia coli in Chilled and Frozen Foods: Collaborative Study." Journal of AOAC INTERNATIONAL 71, no. 3 (May 1, 1988): 589–602. http://dx.doi.org/10.1093/jaoac/71.3.589.
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Abstract A collaborative study was conducted to compare a proposed LSTMUG method with the AOAC official method for Escherichia coli detection. E. coli produces an enzyme, β-glucuronidase, which cleaves the substrate, 4-methyl-umbelliferyl-β-D-glucuronide (MUG), to yield a fluorescent end product. Incorporation of the MUG substrate into lauryl tryptose broth (LST) enables a rapid quantitative method for screening E. coli, which is detected by fluorescence of the medium under longwave UV light. In this collaborative study, 5 food samples, 2 frozen (entree sauce/gravy and dairy topping) and 3 chilled (hamburger, pork sausage, and cheese), were tested for E. coli detection by 17 collaborating laboratories. Results indicate that the LST-MUG method is equal to or better than the current AOAC method for detecting E. coli. The LST-MUG method has been adopted official first action
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Ethier, Sara J., Saulius Butenas, and Richard J. Jenny. "Fluorogenic Assays for Functional Factor VIIa and Tissue Factor." Blood 104, no. 11 (November 16, 2004): 1740. http://dx.doi.org/10.1182/blood.v104.11.1740.1740.
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Abstract Blood coagulation is initiated when cryptic tissue factor (TF) becomes exposed on the surface of vascular cells where it can bind circulating factor VIIa (fVIIa). The resulting fVIIa/TF enzyme complex catalyzes the activation of certain blood zymogens that propagate the coagulation event. As a key enzyme for the initiation of coagulation, circulating fVIIa levels have been viewed as an indicator of hemostatic potential and may also be an indicator for the risk of developing cardiovascular disease. The formation of the fVIIa/TF complex is also the basis of specific coagulation assays. Natural or synthetic TF is employed in the prothrombin time (PT assay) where it is used to initiate coagulation in vitro. Therefore, fVIIa and TF play important roles both in vivo and in vitro. Despite such important roles, there have been no simple methods described for measuring the active forms of either protein. This report describes the initial development of an assay for fVIIa and TF that employs the use of an Aminonaphthalenesulfonamide-based (ANSN) fluorogenic substrate. The ANSN substrate is hydrolyzed by fVIIa in a TF-dependent manner, and by titrating fVIIa into a system containing excess TF, the assay becomes sensitive to the fVIIa concentration. Inversely, by titrating TF into a system containing excess fVIIa, the assay becomes sensitive to the TF concentration. This approach has led to the development of both a rapid kinetic and an end-point assay method for each analyte providing detection ranges down to 156 and 1.56 pM respectively. The utility of these assays is displayed by the following applications. Thromboplastin reagents are generally qualified against a W.H.O. standard and are assigned a numerical value known as the International Sensitivity Index (ISI). Theoretically the ISI value should be affected by the quantity and quality of TF in each preparation. An inverse relationship between ISI value and the amount of functional TF would support this theory. To prove this relationship the TF assay was employed to examine two thromboplastin reagents with ISI values of 1.84 and 1.01. The concentration of functional TF measured by this assay was 10.8 nM and 14.6 nM respectively. Because most commercial thromboplastin reagents are prepared by adding TF on a mass basis (as opposed to a functional basis), target ISI values are often missed, leading to an excessive number of failed lots. The TF functional assay provides a method for assigning a specific activity to each TF preparation. By adding TF on a functional basis, lot-to-lot variability would be minimized and target ISI values would be easily achieved. To substantiate this claim, we have determined the functional activity of seven different lots of purified recombinant human TF. With each lot analyzed at a fixed concentration, we observed a broad range of specific activity (range: 8,250 U/sec/mg -14,500 U/sec/mg). These data support the need for a functional TF assay to be incorporated in the manufacturing process of thromboplastin reagents. Lastly, the utility of the fVIIa assay was demonstrated in an experiment using factor VII-deficient plasma samples that were spiked with known quantities of fVIIa. Plasma samples spiked with fVIIa in the range of 0.5-2nM returned mean assay values within 71–100% of the expected values. These data demonstrate the feasibility of using the fVIIa assay to monitor fVIIa levels in hemophilia patients receiving recombinant fVIIa therapy. Further studies will address the circulating concentrations of fVIIa and TF in “normal” and disease state plasma samples.
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Al Dieri, Raed, Erik De Smedt, Suzette Béguin, and H. Hemker. "Thrombin generation, a function test of the haemostaticthrombotic system." Thrombosis and Haemostasis 96, no. 11 (2006): 553–61. http://dx.doi.org/10.1160/th06-07-0408.
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SummaryBy the use of a fluorogenic thrombin substrate and continuous calibration of each individual sample, it is now possible to obtain a thrombin generation (TG) curve (or thrombogram) in plasma, with or without platelets, in an easy routine procedure at high throughput and with an acceptable experimental error (< 5%). Evidence is growing that the parameters of the thrombogram, and notably the area under the curve (endogenous thrombin potential, ETP), are useful in assessing bleeding-or thrombotic risk and its modification by antithrombotic-or haemostatic treatment. Available data strongly suggest that conditions (congenital, acquired, drug-induced) that increase TG all cause a thrombotic tendency and that conditions that decrease TG prevent thrombosis but, beyond a limit, cause bleeding. Diminution of TG is a common denominator of all antithrombotic treatment, including anti-platelet drugs. The thrombogram can also be used as a tool in the search for new antithrombotics and reflects the haemorrhagic or thrombotic side effects of other drugs (e.g. oral contraceptives).The thrombogram thus is a promising new approach to clinical management of bleeding and thrombotic disease as well as a tool in drug research and epidemiology. Our experience at this moment is insufficient, however, to already clearly define its limits.
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Hallaert, Delfine Y. H., Heike Schmidlin, Eric Eldering, and Marinus H. J. Van Oers. "GSI-1, a Putative Notch Inhibitor, Induces Apoptosis in B-CLL Cells Via Proteasomal Inhibition and Noxa Upregulation." Blood 110, no. 11 (November 16, 2007): 3113. http://dx.doi.org/10.1182/blood.v110.11.3113.3113.
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Abstract Mainly based on the observation that overexpression of CD23 in B-CLL cells is regulated by Notch2, deregulation of the Notch pathway has been suggested to contribute to the pathogenesis of B cell chronic lymphocytic leukemia (B-CLL). The aim of the present study was to assess a possible functional role of the Notch pathway in CLL. To this end we performed two kind of experiments. First we co-cultured primary CLL cells with either L cells or OP9 cells expressing the Notch ligands DeltaLike1 (DL1) and Jagged1 (Jag1). This did not affect cell survival in CLL. Next we evaluated the effect of the g-secretase inhibitors GSI-1 and GSI-9 (DAPT) which have been shown to block intracellular processing of all four Notch receptors. Here we encountered a surprising dichotomy: whereas DAPT was unable to induce apoptosis in CLL, it did inhibit lineage commitment of early thymic precursors by means of DL-1 triggering of the Notch1 receptor (Dontje et al, Blood 15 March 2006, Vol. 107, No. 6). In contrast we found that GSI-1 was a potent inducer of apoptosis in CLL, but did not affect Notch1 dependent lineage commitment, indicating that GSI-1-induced apoptosis in B-CLL is not due to inhibition of Notch signaling. Instead, we observed efficient GSI-1 mediated blocking of the proteasome, measured using a 20S proteasomal activity assay (Fig. 1). The blocking activity was equivalent to that observed with two well known proteasome inhibitors, Bortezomib and MG-132. In contrast DAPT had no effect on proteasome activity. Furthermore, GSI-1-induced apoptosis was associated with a transcription-independent accumulation of the BH3-only protein Noxa. The pivotal role of Noxa in GSI-1 mediated apoptosis was demonstrated via RNAi in a model system. Importantly, p53 functionality proved not to be required for GSI-1-induced apoptosis. In summary, we have shown that GSI-1 efficiently blocks the proteasome and that this induces p53-independent apoptosis in B-CLL. Therefore, GSI-1, or similar compounds that inhibit g-secretase activity, may be an effective treatment option in B-CLL. Figure 1: Proteasome activity upon GSI-1 treatment The enzymatic activity of the 20S proteasome was measured after adding Bortezomib (20 nM), MG-132 (0.5 μM), GSI-1 (5 μM), DAPT (5 μM), Roscovitine (25 μM) and Fludarabine (100 μM) to cytoplasmic extracts from freshly isolated B-CLL patients. After 15 minutes incubation, the fluorogenic proteasome substrate LVVY-AMC was added. Monitoring the increase in fluorescence over time allowed quantification of the enzymatic activity. Figure 1:. Proteasome activity upon GSI-1 treatment The enzymatic activity of the 20S proteasome was measured after adding Bortezomib (20 nM), MG-132 (0.5 μM), GSI-1 (5 μM), DAPT (5 μM), Roscovitine (25 μM) and Fludarabine (100 μM) to cytoplasmic extracts from freshly isolated B-CLL patients. After 15 minutes incubation, the fluorogenic proteasome substrate LVVY-AMC was added. Monitoring the increase in fluorescence over time allowed quantification of the enzymatic activity.
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Yang, Won Seok, Jin Ju Kim, Mee Jeong Lee, Eun Kyoung Lee, and Su-Kil Park. "Ectodomain Shedding of RAGE and TLR4 as a Negative Feedback Regulation in High-Mobility Group Box 1-Activated Aortic Endothelial Cells." Cellular Physiology and Biochemistry 51, no. 4 (2018): 1632–44. http://dx.doi.org/10.1159/000495651.
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Background/Aims: High-mobility group box 1 (HMGB1) elicits inflammatory responses through interactions with the receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4). We investigated how RAGE and TLR4 expressions are regulated after HMGB1 stimulation in cultured human aortic endothelial cells (HAECs). Methods: RAGE and TLR4 expressions were analyzed by Western blot analysis and immunofluorescence staining. A disintegrin and metalloprotease 17 (ADAM17) activity was measured using a fluorogenic substrate. Results: Upon treatment with HMGB1, both RAGE and TLR4 began to decrease in cell lysate and remained decreased up to 24 h. The decrease in cellular RAGE and TLR4 was accompanied by an increase of N-terminal fragment of RAGE and TLR4 in culture supernatant, indicating ectodomain shedding of the receptors. HMGB1 activated p38 mitogen-activated protein kinase (p38 MAPK) and ADAM17, while HMGB1-induced ADAM17 activation was inhibited by SB203580, a p38 MAPK inhibitor. HMGB1-induced ectodomain shedding of RAGE and TLR4 was prevented by siRNA depletion of ADAM17 as well as TAPI-2, an inhibitor of ADAM family, and SB203580. HMGB1 pretreatment abolished p38 MAPK activation in response to 2nd HMGB1 stimulation. In the cells depleted of ADAM17, HMGB1-induced p38 MAPK activation was prolonged. siRNA depletion of RAGE, but not TLR4, suppressed HMGB1-induced p38 MAPK activation. Conclusion: In response to HMGB1 stimulation, HAECs rapidly undergo ectodomain shedding of RAGE and TLR4, and thereby become insensitive to further HMGB1 stimulation. ADAM17, activated through RAGE-p38 MAPK pathway, is implicated in the ectodomain cleavage of the receptors.
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Tripodi, Armando. "Thrombin Generation Assay and Its Application in the Clinical Laboratory." Clinical Chemistry 62, no. 5 (May 1, 2016): 699–707. http://dx.doi.org/10.1373/clinchem.2015.248625.
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Abstract BACKGROUND A gap exists between in vivo and ex vivo coagulation when investigated by use of the coagulation tests prothrombin time (PT) and activated partial thromboplastin time (APTT). The thrombin generation assay (TGA) has been developed to fill this gap. CONTENT TGA evaluates thrombin generation (resulting from the action of the procoagulant driver) and decay (resulting from the action of the anticoagulant driver), thus assessing the balance between the two. Coagulation of the test plasma (platelet poor or platelet rich) is activated by small amounts of tissue factor and phospholipids, and the reaction of thrombin generation is continuously monitored by means of a thrombin-specific fluorogenic substrate. Among the parameters derived from the thrombin-generation curve, the most important is the endogenous thrombin potential, defined as the net amount of thrombin that test plasmas can generate on the basis of the relative strength of the pro- and anticoagulant drivers. TGA is therefore the candidate assay to investigate hypo- or hypercoagulability. SUMMARY From my analysis of the literature, I draw the following conclusions. There is strong evidence that TGA is helpful to elucidate coagulation mechanisms in various clinical conditions that until recently were poorly understood (chronic liver disease; diabetes; inflammatory bowel disease, myeloproliferative neoplasms, nonalcoholic fatty liver disease). TGA is a promising laboratory tool for investigating hemorrhagic coagulopathies and monitoring replacement therapy in hemophiliacs, predicting the risk of recurrent venous thromboembolism after a first event, and monitoring patients on parenteral or oral anticoagulants. These applications require clinical trials in which TGA results are combined with specific clinical end points.
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Miller, Timothy M., Krista L. Moulder, C. Michael Knudson, Douglas J. Creedon, Mohanish Deshmukh, Stanley J. Korsmeyer, and Eugene M. Johnson. "Bax Deletion Further Orders the Cell Death Pathway in Cerebellar Granule Cells and Suggests a Caspase-independent Pathway to Cell Death." Journal of Cell Biology 139, no. 1 (October 6, 1997): 205–17. http://dx.doi.org/10.1083/jcb.139.1.205.
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Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 μM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax −/− neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increased cleavage of DEVD–aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.
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Bansal, Vinod, Kristiyana Kaneva, Debra Hoppensteadt, Josephine Cunanan, and Jawed Fareed. "Variations in the Circulating Heparin Levels During Maintenance Hemodialysis in End Stage Renal Disease Patients." Blood 120, no. 21 (November 16, 2012): 3412. http://dx.doi.org/10.1182/blood.v120.21.3412.3412.
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Abstract Abstract 3412 Introduction: Unfractionated heparin has remained the anticoagulant of choice for hemodialysis patients. The anticoagulation facilitates blood flow through the dialysis circuit and assures the patency of the dialysis membrane. Wide variations in the heparinization responses have been observed in patients undergoing this procedure. The purpose of this investigation was to measure circulating heparin levels in patients prior to and after hemodialysis. Methods: This study included 119 ESRD patients undergoing maintenance hemodialysis after appropriate IRB approval and patient consent. For the 3–4 hour hemodialysis duration, a heparin loading dose of 1000 Units followed by two additional dosages of 500 Units/hour were administered. Citrated blood samples were collected prior to and immediately after the dialysis session. The blood samples were centrifuged for 15 minutes at 3000 g at 4°C and platelet poor plasma (PPP) was extracted. Citrated plasma was frozen at −70°C and analyzed utilizing such clot based methods as Activated Partial Thromboplastin Time (APTT), Heptest and Prothrombinase Induced Clotting Time (PiCT). Circulating Anti-Xa levels were measured using a chromogenic substrate method. Thromboplastin induced thrombin generation was also measured using a fluorogenic substrate method, Thrombin Generation Assay (TGA). The Antithrombin (AT) levels in each of these patients were also measured using a functional assay. The circulating levels of heparin were determined using a calibration curve constructed from the heparin used in the dialysis unit. Results were computed for the individual tests and heparin concentrations were obtained using the assay based calibration procedures. Results: In the clot based assays such as APTT and Heptest, no significant differences between pre and post plasma samples were noted. The circulating levels of heparin were from 0 to 1.08 U/ml with a mean of 0.07 ± 0.11 for the APTT and a range of 0 to 1.98 for the Heptest with a mean of 0.09 ± 0.26 U/ml. In the PiCT test the range was from 14.0 to 300 seconds for the pre dialysis samples with a mean of 32.0 ± 38.2, whereas for the post samples the range was from 15.2 to 110 with a mean of 29.6 ± 14.0. For the Anti-Xa levels the % inhibition for the two groups was similar. The circulating Anti-Xa levels in the pre dialysis samples ranged from 0 to 0.77 with a mean of 0.10 ± 0.14, whereas the post level ranged from 0 to 0.51 with a mean of 0.13 ± 0.11. For the thrombin generation, the % inhibition levels ranged from 0 to 100% pre dialysis with a mean of 34.2 ± 34% and ranged 0 to 100% post dialysis with a mean of 44.5 ± 34.4%. The Antithrombin levels ranged from 28 to 130% with a mean of 86.6 ± 19.5% in the pre dialysis samples. There was no significant difference between pre and post dialysis samples using APTT, Heptest and PiCT, whereas the Thrombin generation and Anti-Xa resulted in a statistically significant p value < 0.05 when comparing the two groups. Conclusion: Wide variations in circulating heparin levels are noted in maintenance hemodialysis patients at pre dialysis and post dialysis time periods. Some patients exhibit higher levels of heparin due to a vascular access flush. These results also suggest that the use of heparin in maintenance hemodialysis patients in repeated regimen results in a steady state hypocoagulation as evidenced by the inhibition of thrombin generation, circulating Anti-Xa level and the prolongation of various clotting times. Disclosures: No relevant conflicts of interest to declare.
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Supinski, Gerald S., and Leigh A. Callahan. "Caspase activation contributes to endotoxin-induced diaphragm weakness." Journal of Applied Physiology 100, no. 6 (June 2006): 1770–77. http://dx.doi.org/10.1152/japplphysiol.01288.2005.
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Infections produce significant respiratory muscle weakness, but the mechanisms by which inflammation reduces muscle force remain incompletely understood. Recent work suggests that caspase 3 releases actin and myosin from the contractile protein lattice, so we postulated that infections may reduce skeletal muscle force by activating caspase 3. The present experiments were designed to test this hypothesis by determining 1) diaphragm caspase 3 activation in the diaphragm after endotoxin and 2) the effect of a broad-spectrum caspase inhibitor, Z-Val-Ala-Asp(OCH3)-fluoromethylketone (zVAD-fmk), and a selective caspase 3 inhibitor, N-acetyl-Asp-Glu-Val-Asp-al (DEVD-CHO), on endotoxin-induced diaphragm weakness. Caspase 3 activation was assessed by measuring caspase protein levels and by measuring cleavage of a fluorogenic substrate. Diaphragm force was measured in response to electrical stimulation (1–150 Hz). Caspase-mediated spectrin degradation was assessed by Western blotting. Parameters were compared in mice given saline, endotoxin (12 mg/kg ip), endotoxin plus zVAD-fmk (3 mg/kg iv), zVAD-fmk alone, or endotoxin plus DEVD-CHO (3 mg/kg iv). Endotoxin increased diaphragm active caspase 3 protein ( P < 0.003), increased caspase 3 activity ( P < 0.002), increased diaphragm spectrin degradation ( P < 0.001), and reduced diaphragm force ( P < 0.001). Administration of zVAD-fmk or DEVD-CHO prevented endotoxin-induced weakness (e.g., force in response to 150-Hz stimulation was 23.8 ± 1.4, 12.1 ± 1.3, 23.5 ± 0.8, 22.7 ± 1.3, and 24.4 ± 0.8 N/cm2, respectively, for control, endotoxin, endotoxin plus zVAD-fmk, endotoxin plus DEVD-CHO, and zVAD-fmk alone treated groups, P < 0.001). Caspase inhibitors also prevented spectrin degradation. In conclusion, endotoxin administration elicits significant diaphragm caspase 3 activation and caspase-mediated diaphragmatic weakness.
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Fareed, Jawed, Nasir Sadeghi, Daniel Kahn, Josephine Cunanan, Kimberly Bartosiak, Job Harenberg, and Debra Hoppensteadt. "Tissue Factor Mediated Activation of Prothrombin Complex Concentrates (PCCs) Is Differently Inhibited by Dabigatran, Rivaroxaban and Apixaban. Potential Clinical Implications." Blood 120, no. 21 (November 16, 2012): 3410. http://dx.doi.org/10.1182/blood.v120.21.3410.3410.
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Abstract Abstract 3410 The newly developed oral anticoagulants represent specific antithrombin (dabigatran, Boehinger, Ingelheim) and antifactor Xa agents (rivaroxaban, Bayer Health Care/Jhonsen) and apixaban, Bristol Myers Squibb/Pfizer). Prothrombin Complex Concentrates (PCCs) such as profilnine® and beriplex® are reported to partially neutralize the anticoagulant effects of these agents. Since these PCCs are capable of generating factor Xa and thrombin, the newer anticoagulants may be neutralized differentially by the proteases generated by PCCs. Coagulation and thrombosis are activated substantially by tissue factor in vivo. The purpose of this study is to compare the inhibitory effects of dabigatran, rivaroxaban and apixaban in tissue factor mediated thrombin generation using profilnine, by utilizing various approaches to characterize activation products including thrombin. Materials and Methods Dabigatran, rivaroxaban, and apixaban were synthesized and/or commercially obtained. Profilnine (Grifols Biologicals Inc.) was also commercially obtained. One commercial lot of a recombinant thrombin preparation Recothrom® was obtained from ZymoGenetics Inc for the development of polyclonal antibodies. To generate specific antisera, individual groups of rabbits (n = 3–6) were challenged repeatedly with human recombinant thrombin, over a 9-month period. At the end of this time the antisera from each rabbit was collected and pooled. Immunglobulin (IgGs) were isolated using a protein G column (HiTrap Protein G HP – GE Helathcare Bio-Science Crop). Buffered profilnine (2.5 u/ml) was activated with routinely used tissue factor reagents by adding commercially available PT reagents such as thromboplastin C, neoplastinPlus, and simplastin at a 1:4 ratio and incubated for 30 minutes. The activation of profilnine was measured by using thrombin generation utilizing a fluorogenic substrate method (Technoclone) and the protease generation profile was evaluated using mass spectrometry method (SELDI), SDS-PAGE analysis and immunoblotting using a specific antithrombin (Recothrombin) antibody to profile the activation products. Similar studies were carried out in profilnine supplemented with graded amount of various oral anticoagulants in the concentration range of 0–2.5ug/ml. Results All tissue factors produce varying degrees of time dependent activation of profilnine as measured by consumption of prothrombin peak at 71 KDa and generation of thrombin peaks at 3l–37 KDa as observed in the SELDI. Varying amounts of prothrombin generation at 52 KDa was also evident. Distinct immunoblot for thrombin in western blotting analysis was consistent with SDS-PAGE and SELDI analysis showing the generation of thrombin. The anti-Xa agents blocked the generation of thrombin whereas dabigatran failed to produce this effect. This phenomenon was also observed in all three methods used to study generation of the thrombin when using other PCCs such as octaplex and thromboplex activated by various tissue factors. In the fluorometric thrombin generation assays both apixaban and rivaroxaban produced a relatively stronger inhibition of thrombin generation (IC50= 20–200ng/ml) wheras > 500ng/ml for dabigatran in various PCCs. Conclusion These results suggest that in contrast to dabigatran both rivaroxaban and apixaban produce a much stronger inhibition of tissue factor mediated generation of the thrombin in PCCs. Inhibition of the functional generation of thrombin was weaker with dabigatran in contrast to apixiban and rivaroxiban. The observed ex-vivo neutralization profile of these agents by PCCs may be due to the differential interactions with the protease generated during their activation. These differences along with the compositional variations in the PCCs should be taken into account while considering prothrombin complexes for the neutralization of new oral anticoagulants. Disclosures: No relevant conflicts of interest to declare.
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Spencer, Andrew, Simon Harrison, Francis Burrows, Paul Mainwaring, Timothy Price, Michael J. Millward, Peeter Padrik, et al. "Marizomib Overcomes Compensatory Hyperactivation of Trypsin-like and Caspase-like Subunits to Provide Pan-Proteasome Subunit Inhibition in Patients with Multiple Myeloma and Solid Tumors." Blood 126, no. 23 (December 3, 2015): 5375. http://dx.doi.org/10.1182/blood.v126.23.5375.5375.
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Abstract Background: Proteasome inhibitors (PIs) are a highly active drug class in multiple myeloma (MM), but development of resistance is commonly observed. Although all clinical-stage PIs are effective inhibitors of chymotrypsin-like (CT-L) proteasome subunit activity, a possible mechanism of resistance is compensatory hyperactivation of the caspase-like (C-L) and trypsin-like (T-L) subunits. Marizomib (MRZ) is a novel, irreversible, second-generation PI under development for the treatment of MM and malignant glioma. MRZ potently inhibits the 3 subunits of the 20S proteasome with a specificity and activity distinct from that of bortezomib and carfilzomib and their oral analogs ixazomib and oprozomib. Methods: In the clinical study NPI-0052-102, we evaluated the pharmacodynamic (PD) activity of MRZ against all three proteasome subunits in whole blood after single, or repeated administration, via two schedules in patients (pts) with previously treated advanced malignancies (solid tumors or hematologic). Pts received MRZ via Schedule A - weekly dosing for 3 doses in 4-week cycles by IV infusion over 1 to 10 min, or Schedule B - twice-weekly dosing for 4 doses in 3-week cycles by IV infusion over 10 min to 2 hr. Blood samples were collected on Days 1 and 15 (Schedule A) or 1 and 11 (Schedule B), pelleted by centrifugation and frozen within 48 hrs. Pellets were lysed and the activity of all 3 proteasomal subunits was assayed using specific fluorogenic substrates. Results: Partial or complete inhibition of all three proteasome subunits was achieved with both once-weekly and twice-weekly MRZ dosing. The rank order of sensitivity, CT-L > T-L > C-L, was as expected from the biochemical and cellular potencies of the drug. For CT-L activity, proteasome inhibition was dose dependent, with both the initial effect (on Cycle 1, Day 1 - C1D1) and the peak effect of proteasome inhibition increasing in a dose-dependent manner. CT-L inhibition was modest at the lowest dose levels (≤ 0.15 mg/m2), reaching moderate levels after repeat dosing. At intermediate dose levels of 0.3 to 0.55 mg/m2, CT-L inhibition was 31% to 75% on C1D1, rising to 70% to 93% after repeat dosing. At doses of 0.7 mg/m2 and above, inhibition of CT-L activity was usually complete within the first cycle. By contrast, C-L and T-L activities were unchanged or increased in the first cycle, suggesting compensatory hyperactivation in response to effective blockade of CT-L activity. Importantly, however, this response was overcome by repeat dosing with MRZ, and inhibition of T-L and C-L activity was noted across all dose levels. For C-L activity, treatment with MRZ at the recommended Phase 2 dose (RP2D) of 0.5 mg/m2 with twice-weekly dosing resulted in inhibition of up to 39% by Cycle 2 and was maintained, when tested, through Cycles 4 and 6. Treatment at the RP2D of 0.7 mg/m2 for once-weekly dosing resulted in C-L inhibition of 14% to 37% by the end of the first cycle, rising to 31% to 50% by the end of Cycle 2. Blockade of T-L activity was more robust after multiple cycles of MRZ therapy. Although inhibition of the T-L subunit was absent on C1D1 in patients receiving 0.5 to 0.55 mg/m2, inhibition of up to 80% was achieved by Cycle 2 and maintained for the duration of treatment. At the once-weekly RP2D of 0.7 mg/m2, T-L inhibition of 29% to 56% was achieved by the end of the first cycle, rising to 64% to 78% by the end of Cycle 2. Conclusions: The PD activity of MRZ against all three proteolytic subunits was assessed in patients with MM, solid tumors, and advanced lymphomas. At the twice-weekly and once-weekly RP2Ds, complete inhibition of CT-L activity was observed within 1-2 cycles of therapy, but accompanied by compensatory hyperactivation of the C-L and T-L subunits. This phenomenon in red cells suggests that it may be due to allosteric interactions within the catalytic core of the 20S proteasome rather than de novo synthesis of additional proteasomes. Ongoing MRZ therapy was able to overcome this adaptive response, resulting in robust pan-subunit proteasome inhibition within 2-6 cycles, most probably due to the cumulative effect of multiple exposures to MRZ. Due to their reversible binding mode (bortezomib, ixazomib) or monospecificity for the CT-L site (carfilzomib, oprozomib), other clinical PIs are predicted to lack this capability. This unique property of MRZ may explain in part its clinical activity in patients with MM resistant to both bortezomib and carfilzomib. Disclosures Off Label Use: marizomib treatment for multiple myeloma and solid tumours.. Harrison:AbbVie: Research Funding; Janssen: Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding. Burrows:Triphase Accelerator Corporation: Consultancy. Reich:Triphase Accelerator Corporation: Consultancy. Trikha:Triphase Accelerator Corporation: Employment.
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Bianchi, Giada, Laura Oliva, Paolo Cascio, Niccolo’ Pengo, Andrea Orsi, Francesca Fontana, Elena Pasqualetto, et al. "Proteasome Stress Causes Apoptotic Sensitivity of Multiple Myeloma Cells to Proteasome Inhibition." Blood 112, no. 11 (November 16, 2008): 247. http://dx.doi.org/10.1182/blood.v112.11.247.247.
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Abstract Proteasome inhibitors (PI) proved to be extremely effective against different types of cancer, particularly against Multiple Myeloma (MM), a frequent and still incurable plasma cell malignancy. Phase II clinical trials showed that more than 50% of MM patients fail to respond to bortezomib, the only PI currently approved for clinical use. However, the mechanisms of action and bases of individual susceptibility to PI remain largely unclear, with no reliable predictor of response identified so far. Recent evidences linking proteasome activity and Ig synthesis to susceptibility to PI suggest that the exquisite sensitivity of MM cells (MMC) to PI might be explained by an imbalance between the efficiency of the ubiquitin (Ub)-proteasome pathway and the demand for proteasome-mediated degradation. We set out to explore this hypothesis both in vitro and ex vivo. To achieve this aim, we employed human MM cell lines characterized by differential apoptotic sensitivity to PI (U266 and RPMI8226, fairly resistant cell lines, versus MM.1S, an extremely sensitive one) and primary, patient derived MMC. In MM cell lines, we found that high apoptotic sensitivity to PI is associated with lower expression of active proteasomes (as assessed by decreased expression of cleaved catalytic subunits and enzymatic assays with fluorogenic substrates in cell extracts), together with higher proteasomal workload (demonstrated by higher proteasome-dependent loss of TCA-insoluble radioactivity in pulse-chase assays). Indeed, MM.1S cells displayed 2–3 times lower proteasomal activity as compared to the more resistant U266 and RPMI8226 cells, both on a per cell basis and upon normalization by protein content. Together with the reduced proteasome capacity, MM.1S cells showed a consistently higher production of client proteins for the Ub-proteasome pathway. Such an increased load appears to be the consequence of a higher production of Rapidly Degraded Polypeptides (RDP). These are newly synthesized proteins which are quickly redirected to proteasome-mediated degradation. The imbalance between proteasomal load and capacity results in remarkable accumulation of poly-Ub proteins at the expense of free Ub (as established by both western blotting and immunofluorescence), unveiling basal proteasome stress in PI-sensitive MMC. In order to establish a causal link between proteasome stress and sensitivity to PI, we pharmacologically modulated either proteasome expression or workload and successfully altered PI-induced apoptosis. As predicted, increasing proteasome workload by means of ER stressors (e.g. tunicamycin, thapsigargin, brefeldin A) dramatically enhances susceptibility to PI, while a raise in proteasomal activity (achieved by exploiting the proteasome stress response, an adaptive mechanism by which mammalian cells induce proteasome biogenesis in response to either decreased proteasome function or increased proteasomal demand), confers marked resistance to PI-induced apoptosis. Having established cause-effect relationships between determinants of proteasome stress and vulnerability to PI in vitro, we then asked if our model could be used to predict responsiveness to PI in MM patients. In keeping with this hypothesis, intracellular immunostaining in primary, patient-derived MMC reveals that accumulation of poly-Ub proteins specifically hallmarks neoplastic plasma cells, indicating that the cancer compartment in MM patients suffers from proteasome stress. Moreover, poly-Ub levels positively correlate with Ig content, both intra- and inter-patient, suggesting a direct effect of Ig synthesis and/or retention on proteasome functional load. Finally, overall proteasome activity of primary MMC inversely correlates with the intrinsic apoptotic sensitivity to PI as assessed ex vivo, providing a rationale for the assessment of this parameter as a potential predictor of the in vivo response to bortezomib or other PI. Altogether, our data indicate that the balance between proteasome workload and degradative capacity represents a critical determinant of apoptotic sensitivity of MMC to PI, providing both a novel predictive tool of potential prognostic value and the framework for novel combination therapies aimed at exacerbating proteasome stress in MM.
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Brinkman, Herm Jan, Erica Sellink, Bas de Laat, and Koen Mertens. "Differential Anticoagulant Effects of Protein S on Vascular Cells and Platelets." Blood 112, no. 11 (November 16, 2008): 2026. http://dx.doi.org/10.1182/blood.v112.11.2026.2026.
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Abstract Background: Protein S is a vitamin K-dependent plasma protein and involved in down-regulation of the coagulation process. Protein S serves as a cofactor of activated protein C (APC) in the proteolytic inactivation of activated factor V and VIII. Protein S is also able to exert its anticoagulant activity independent of APC, e.g. by supporting the anticoagulant activity of tissue factor pathway inhibitor (TFPI). The anticoagulant properties of protein S have been thoroughly characterized by in vitro methods. However, fewer studies focus on protein S function on vascular cells. These studies are limited to model systems employing purified coagulation factors. The aim of this study was to investigate the role of protein S in plasma that is in contact with natural cell membranes, including endothelial cells, smooth muscle cells and platelets. Method: We employed thrombography to evaluate protein S function in 50 % v/v recalcified citrated plasma in the presence of washed platelets, cultured umbilical vein endothelial cells or cultured umbilical artery smooth muscle cells. Since we aimed at a comparison between different cellular membranes, micro-particle free plasma was used. As a reference, we also examined synthetic phospholipid membranes composed of phosphatidyl serine, phosphatidyl choline and phosphatidyl ethanolamine in a 2/6/2 molar ratio. Thrombin activity was measured employing the fluorogenic substrate z-Gly-Gly- Arg-AMC. Protein S activity was probed with CLB-PS13, an antibody directed against the protein S Gla-domain. The APC-independent activity of protein S was assayed in the presence of an inhibitory antibody against protein C. In studies employing phospholipids, thrombin generation was triggered with relipidated tissue factor (TF). Expression of TF on endothelial cells was induced during a 6-hour preincubation with PMA. Results: In the presence of CLB-PS13, the APC-independent activity of protein S became apparent as an increase in peak height in the thrombogram. Lag time, time to peak and area under the curve remained essentially unaffected. Peak height was increased two-fold when examining phospholipids at standard conditions (4 μM lipids and 1 pM TF). This increase in peak height by CLB-PS13 was concentration dependent and complete at 10 μg/ml IgG. Increasing the TF concentration from 1 to 5 pM resulted in loss of the APC-independent activity of protein S on these membranes. APC cofactor activity was assessed in the presence of APC. Addition of APC resulted in inhibition of the thrombin formation on phospholipids with an IC50 of 0.4 nM. CLB-PS13 completely abolished this decrease in thrombin generation up to 50 nM APC, irrespective whether 1 or 5 pM TF was present. Our results are compatible with the view that at high procoagulant stimuli the TFPI-cofactor activity of protein S is abolished. Furthermore, these results show that in plasma APC is completely dependent on protein S. Protein S activity on platelets was studied in the presence of 1 pM TF. As for synthetic lipid membranes and in the absence of APC, CLB-PS13 increased the peak height in the thrombogram. APC inhibited platelet mediated thrombin generation (IC50 = 19 nM), and this inhibition was completely abolished by CLB-PS13. These observations suggest that platelets support both APC-dependent and APC-independent anticoagulant activities of protein S. Thrombography on TF-expressing endothelial cells and smooth muscle cells revealed massive thrombin generation that could not be enhanced with CLB-PS13, indicating that protein S does not contribute to regulation of thrombin formation under these conditions. The APC-dependent activity of protein S became apparent as an abolished inhibition by APC in the presence of CLB-PS13. However, APC proved relatively inefficient in inhibiting thrombin generation on TF-expressing vascular cells (IC50 > 50 nM). Inhibition of TF restored the APC-independent protein S activity. Conclusion: Our results indicate that, in plasma, vascular cells and platelets are able to support the APC-dependent as well as the APC-independent anticoagulant activities of protein S. The APC-independent activity on vascular cells is abolished upon increasing TF expression, while the APC-dependent activity of protein S is limited by the relatively low anticoagulant activity of APC on these cell surfaces. We conclude that protein S activity on cells require relatively high levels of APC or low expression of TF.