Academic literature on the topic 'Inhibitor Activity'
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Journal articles on the topic "Inhibitor Activity"
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Sumanadasa, Subathdrage D. M., Christopher D. Goodman, Andrew J. Lucke, Tina Skinner-Adams, Ishani Sahama, Ashraful Haque, Tram Anh Do, Geoffrey I. McFadden, David P. Fairlie, and Katherine T. Andrews. "Antimalarial Activity of the Anticancer Histone Deacetylase Inhibitor SB939." Antimicrobial Agents and Chemotherapy 56, no. 7 (April 16, 2012): 3849–56. http://dx.doi.org/10.1128/aac.00030-12.
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ABSTRACTHistone deacetylase (HDAC) enzymes posttranslationally modify lysines on histone and nonhistone proteins and play crucial roles in epigenetic regulation and other important cellular processes. HDAC inhibitors (e.g., suberoylanilide hydroxamic acid [SAHA; also known as vorinostat]) are used clinically to treat some cancers and are under investigation for use against many other diseases. Development of new HDAC inhibitors for noncancer indications has the potential to be accelerated by piggybacking onto cancer studies, as several HDAC inhibitors have undergone or are undergoing clinical trials. One such compound, SB939, is a new orally active hydroxamate-based HDAC inhibitor with an improved pharmacokinetic profile compared to that of SAHA. In this study, thein vitroandin vivoantiplasmodial activities of SB939 were investigated. SB939 was found to be a potent inhibitor of the growth ofPlasmodium falciparumasexual-stage parasitesin vitro(50% inhibitory concentration [IC50], 100 to 200 nM), causing hyperacetylation of parasite histone and nonhistone proteins. In combination with the aspartic protease inhibitor lopinavir, SB939 displayed additive activity. SB939 also potently inhibited thein vitrogrowth of exoerythrocytic-stagePlasmodiumparasites in liver cells (IC50, ∼150 nM), suggesting that inhibitor targeting to multiple malaria parasite life cycle stages may be possible. In an experimentalin vivomurine model of cerebral malaria, orally administered SB939 significantly inhibitedP. bergheiANKA parasite growth, preventing development of cerebral malaria-like symptoms. These results identify SB939 as a potent new antimalarial HDAC inhibitor and underscore the potential of investigating next-generation anticancer HDAC inhibitors as prospective new drug leads for treatment of malaria.
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Böhm, Martina, Manuela Krause, Charis Von Auer, Wolfgang Miesbach, and Inge Scharrer. "The Frequency and the Significance of ADAMTS-13 Neutralising Inhibitors in 62 Patients with Non-Familial Thrombotic Thrombocytopenic Purpura." Blood 104, no. 11 (November 16, 2004): 3945. http://dx.doi.org/10.1182/blood.v104.11.3945.3945.
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Abstract Neutralising inhibitors against ADAMTS-13 are detected in 51–67% of patients with Thrombotic Thrombocytopenic Purpura (TTP). These ADAMTS-13 inhibitors have not been very well characterised and the diagnostic or the prognostic value of these inhibitors is not established. In the present study, we measured ADAMTS-13 activity and the corresponding inhibitor titer in 96 samples from 62 patients with TTP at various stages of their disease. All patients presented with non-familial TTP. For patients with severe ADAMTS-13 activity without detectable inhibitor heritable ADAMTS-13 deficiency was excluded by either normalisation of ADAMTS-13 activity in remission or by detecting normal ADAMTS-13 activity in first degree family members. ADAMTS-13 activity was quantified by measuring the residual ristocetin cofactor activity of the substrate. The inhibitor against ADAMTS-13 was detected by mixing patient plasma, either neat or diluted, with normal plasma. The inhibitor concentration neutralising 50% of ADAMTS-13 activity in a 1:1 dilution of patient plasma with normal plasma was defined as 1 U/ml. Inhibitors were considered non-detectable (<0.4 U/ml), if residual ADAMTS-13 activity in the mixture was higher than 75%. Samples with ADAMTS-13 activity >6.25% were heat-inactivated (30 min at 56°C) before testing for inhibitory activity. We found severe ADAMTS-13 deficiency in 89% (24/27) of the samples from patients with acute untreated TTP. 87% (21/24) of these samples were positive for inhibitory activity. The inhibitor titer ranged from 0.4 to 62 U/ml with a median of 1 U/ml. One patient with acute TTP demonstrated ADAMTS-13 activity of 34% despite an inhibitor titer of 0.6 U/ml. The sensitivity of a positive inhibitor test for the diagnosis of TTP was thus 82%. The inhibitor titer before initiation of therapy could not be correlated with the platelet count, the CRP-level or the response to PE-therapy, if patients with an index episode and patients with a relapse were analysed separately. Severe ADAMTS-13 activity was detected in 15/31 samples collected from patients during plasma exchange therapy. 93% (14/15) of these samples were positive for an inhibitor with a titer ranging between 0.6 and 47 U/ml (median: 3 U/ml). 12 of 37 patients tested in remission presented with severe ADAMTS-13 deficiency, 6 of them were positive for inhibitory activity (range: 1–52 U/ml; median: 5 U/ml). The inhibitor titer for patients, which were analysed during acute untreated TTP as well as in remission (n=5), was notable not related to the stage of disease. Five patients with positive inhibitory activity at admission demonstrated mild ADAMTS-13 deficiency in remission without detectable inhibitor. In contrast, we detected inhibitory activity of 0,6–0,8 U/ml in 3 samples from two patients with measurable ADAMTS-13 activity. These low titer inhibitors were only detectable, if samples were heat inactivated before performing the inhibitor assay. Our data demonstrate, that inhibitors against ADAMTS-13 are very heterogeneous. It is highly suspected, that some of these inhibitors can either completely or partly neutralise ADAMTS-13 function in vivo without being detectable in vitro. Inhibitors against ADAMTS-13 might, on the other side, not always completely inhibit ADAMTS-13 function, since they can occur in patients with high residual ADAMTS-13 activity. Inhibitor titers show a wide variation and the clinical significance of the inhibitor titer before, during and after therapy needs to be further investigated.
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Aghaali, Negar, Mohammad Ghadamyari, Vahid Hosseininaveh, and Nasir Saberi Riseh. "PROTEASE INHIBITOR FROM THE CRUDE EXTRACT OF PLANT SEEDS AFFECTS THE DIGESTIVE PROTEASES IN HYPHANTRIA CUNEA (LEP.: ARCTIIDAE)." Journal of Plant Protection Research 53, no. 4 (October 1, 2013): 338–46. http://dx.doi.org/10.2478/jppr-2013-0051.
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Abstract Proteases are one of the most important digestive enzymes in the midgut of Hyphantria cunea Drury. Proteases are responsible for protein digestion. In the present study, we evaluated the efficiency of some plant inhibitors on proteases in the gut of the H. cunea. Last instar larvae were collected from mulberry trees. The digestive system of the larvae was used as an enzyme source. The total proteolytic and trypsin activity were assessed by the hemoglobin and BApNA, respectively, as the substrate. The evaluation of the total proteolytic and trypsin activities in various pHs showed the highest relative activity at a pH of 11. Also, the inhibitory effect of inhibitors extracted from Alhagi maurorum Medik., Lathyrus sativus L., Vicia faba L., Prosopis farcta (Banks & Sol.) Eig., and Panicum miliaceum L. on the digestive protease of the fall webworm was measured. Protease inhibitors extracted from A. maurorum, P. farcta and P. miliaceum showed negligible inhibition but L. sativus was able to inhibit 34.72% and 100% of the total activity of proteolytic and trypsin, respectively. Also, the total proteolytic and trypsin activities were inhibited by the inhibitor from V. faba, at 22.27% and 100%, respectively. The zymogram pattern of trypsin with nitro-cellulose membranes showed 2 isoforms in the gut of H. cunea. The inhibitor from L. sativus completely inhibited both isoforms. Gel electrophoresis of proteolitytic activity revealed at least 6 isoforms the inhibitor extracted from L. sativus; completely inhibiting some of them. The inhibitor from L. sativus was purified by ammonium sulfate precipitation and gel-filtration. The molecular mass of the inhibitor was determined as 45 kDa. The highest inhibition of trypsin activity by the inhibitor from L. sativus occurred at a pH of 10. The stability of the inhibitor from L. sativus was evaluated at different pHs and temperatures. The results showed that the inhibitor from L. sativus was stable at a pH of 11.0, and showed 45% inhibition on trypsin activity at a pH of 11. Also, this inhibitor revealed stability up to 50°C.
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Jang, Seong, Bill Strickland, Lynda Finis, Jeffrey J. Koojiman, Janneke J. Melis, Guido J. Zaman, and Jan V. Tornout. "Abstract 4014: Comparative biochemical kinase activity analysis identifies rivoceranib as the most selective VEGFR-2 inhibitor compared with other TKIs with known activity against VEGFR-2." Cancer Research 83, no. 7_Supplement (April 4, 2023): 4014. http://dx.doi.org/10.1158/1538-7445.am2023-4014.
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Abstract Introduction: Vascular endothelial growth factor receptor 2 (VEGFR-2) is a key regulator of tumor angiogenesis that is highly expressed in several tumor types and is a known target for anti-cancer therapy. Yet the clinical use of VEGFR-2 inhibitors has been challenged by limited efficacy and various side effects, potentially due to the low selectivity of these TKIs for VEGFR-2. Thus, potent VEGFR-2 inhibitors with improved selectivity are needed. Rivoceranib is an oral tyrosine kinase inhibitor (TKI) that potently and selectively inhibits VEGFR-2. A comparison of the potency and selectivity of VEGFR-2 inhibitors can provide a rationale for selecting a specific TKI for anticancer therapy in the clinic. Methods: Binding of rivoceranib to VEGFR-2 was determined on a Biacore T200. The affinity constant (KD) was derived from the association and dissociation rate constants. Inhibitory potency of rivoceranib and 10 FDA-approved reference inhibitors on kinase enzyme activity was determined using mobility shift assays (MSA) or immobilized metal ion affinity particle (IMAP) assays. The half-maximum inhibitory activity (IC50) of the 11 inhibitors on VEGFR-2 was determined in 10-point dose-response curves. The selectivity of the inhibitors was determined on 270 wild-type kinases at a fixed concentration of each inhibitor. Rivoceranib was tested at 10- and 100-times IC50 (160 nmol/L and 1.6 µmol/L). Reference inhibitors were tested at 1 µmol/L (35 to 1056-times IC50). Results: Rivoceranib had a KD of 3 nmol/L on VEGFR-2. In enzyme activity assays, rivoceranib had intermediate potency compared with the 10 reference inhibitors, with a VEGFR-2 kinase inhibition IC50 value of 16 nmol/L. Analysis of the residual activity of the panel of 270 kinases in the presence of rivoceranib or the reference inhibitors showed wide variation in selectivity for VEGFR-2, with rivoceranib identified as the most selective inhibitor (activity of 16 additional kinases inhibited by >50% at 1.6 µmol/L). Tivozanib, the most potent VEGFR-2 inhibitor, displayed greater than 50% inhibitory activity against more than 70 additional kinases. Sunitinib was identified as the least selective inhibitor included in this study, inhibiting 125 additional kinases by >50%. Conclusion: Variations in selectivity among TKIs with similar anti-VEGFR-2 potency can help explain differences in their clinical toxicity profiles, which may be partially due to variant inhibitory effects against TKIs other than VEGFR-2. This comparative biochemical analysis highlights the potential for rivoceranib to address clinical limitations associated with the poor selectivity of currently available VEGFR-2 inhibitors. Rivoceranib is under ongoing investigation as monotherapy and in combination with chemotherapy in various tumor types. Citation Format: Seong Jang, Bill Strickland, Lynda Finis, Jeffrey J. Koojiman, Janneke J. Melis, Guido J. Zaman, Jan V. Tornout. Comparative biochemical kinase activity analysis identifies rivoceranib as the most selective VEGFR-2 inhibitor compared with other TKIs with known activity against VEGFR-2. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4014.
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Li, Qi, Hongyu Yang, Jun Mo, Yao Chen, Yue Wu, Chen Kang, Yuan Sun, and Haopeng Sun. "Identification by shape-based virtual screening and evaluation of new tyrosinase inhibitors." PeerJ 6 (January 26, 2018): e4206. http://dx.doi.org/10.7717/peerj.4206.
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Targeting tyrosinase is considered to be an effective way to control the production of melanin. Tyrosinase inhibitor is anticipated to provide new therapy to prevent skin pigmentation, melanoma and neurodegenerative diseases. Herein, we report our results in identifying new tyrosinase inhibitors. The shape-based virtual screening was performed to discover new tyrosinase inhibitors. Thirteen potential hits derived from virtual screening were tested by biological determinations. Compound 5186-0429 exhibited the most potent inhibitory activity. It dose-dependently inhibited the activity of tyrosinase, with the IC50 values 6.2 ± 2.0 µM and 10.3 ± 5.4 µM on tyrosine and L-Dopa formation, respectively. The kinetic study of 5186-0429 demonstrated that this compound acted as a competitive inhibitor. We believe the discoveries here could serve as a good starting point for further design of potent tyrosinase inhibitor.
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Sato, Shun, Kana Yamamoto, Moeno Ito, Katsutoshi Nishino, Takanao Otsuka, Kazuhiro Irie, and Masaya Nagao. "Enhancement of Inhibitory Activity by Combining Allosteric Inhibitors Putatively Binding to Different Allosteric Sites on Cathepsin K." Molecules 28, no. 10 (May 19, 2023): 4197. http://dx.doi.org/10.3390/molecules28104197.
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Background: Cathepsin K, which is involved in bone resorption, is a good target for treating osteoporosis, but no clinically approved medicine has been developed. Recently, allosteric inhibitors with high specificity and few side effects have been attracting attention for use in new medicines. Methods: Cathepsin K inhibitors were isolated from the methanol extract of Chamaecrista nomame (Leguminosae) using cathepsin K inhibition activity-assisted multi-step chromatography. Standard kinetic analysis was employed to examine the mechanism of cathepsin K inhibition when an isolated inhibitor and its derivative were used. The allosteric binding of these cathepsin K inhibitors was supported by a docking study using AutoDock vina. Combinations of allosteric cathepsin K inhibitors expected to bind to different allosteric sites were examined by means of cathepsin K inhibition assay. Results: Two types of cathepsin K inhibitors were identified in the methanol extract of Chamaecrista nomame. One type consisted of cassiaoccidentalin B and torachrysone 8-β-gentiobioside, and inhibited both cathepsin K and B with similar inhibitory potential, while the other type of inhibitor consisted of pheophytin a, and inhibited cathepsin K but not cathepsin B, suggesting that pheophytin a binds to an allosteric site of cathepsin K. Kinetic analysis of inhibitory activity suggested that pheophytin a and its derivative, pheophorbide b, bind allosterically to cathepsin K. This possibility was supported by a docking study on cathepsin K. The cathepsin K inhibitory activity of pheophytin a and pheophorbide b was enhanced by combining them with the allosteric inhibitors NSC 13345 and NSC94914, which bind to other allosteric sites on cathepsin K. Conclusions: Different allosteric inhibitors that bind to different sites in combination, as shown in this study, may be useful for designing new allosteric inhibitory drugs with high specificity and few side effects.
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Pintus, Francesca, Delia Spanò, Angela Corona, and Rosaria Medda. "Antityrosinase activity ofEuphorbia characiasextracts." PeerJ 3 (October 13, 2015): e1305. http://dx.doi.org/10.7717/peerj.1305.
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Tyrosinase is a well-known key enzyme in melanin biosynthesis and its inhibitors have become increasingly important because of their potential use as hypopigmenting agents. In the present study, the anti-melanogenic effect of aqueous and ethanolic extracts fromEuphorbia characiasleaves, stems, and flowers in cell-free and cellular systems was examined. All the extracts showed inhibitory effects against mushroom tyrosinase with leaf extracts exhibiting the lowest IC50values of 24 and 97 µg/mL for aqueous and ethanolic extracts respectively. Enzyme kinetic analysis indicated that leaf aqueous extract acts as a mixed type inhibitor, while ethanolic extract shows a competitive inhibition effect on mushroom tyrosinase using L-DOPA as substrate. In addition, the inhibitory effect of leaf extracts on tyrosinase activity and melanin production was examined in murine melanoma B16F10 cells. Cellular tyrosinase activity as well as levels of melanin synthesis are reduced in a dose-dependent manner by extracts in cells treated withα-melanocyte stimulating hormone (α-MSH). The effects are comparable, and sometimes even better, than that of kojic acid, a well known tyrosinase inhibitor used for reference. All these results suggest thatE. characiascould be a great source of the natural inhibitors from tyrosinase and has the potential to be used as a whitening agent in therapeutic fields.
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Hs, Ranjini, Padmanabha Udupa Eg, Shobha U. Kamath, Manjunath Setty, Basavaraj Hadapad, and Asha Kamath. "AN IN VITRO STUDY OF CINNAMOMUM ZEYLANICUM AS NATURAL INHIBITOR OF ANGIOTENSIN-CONVERTING ENZYME (ACE) ON SHEEP (OVIS ARIES) TISSUES." Asian Journal of Pharmaceutical and Clinical Research 9, no. 5 (September 1, 2016): 249. http://dx.doi.org/10.22159/ajpcr.2016.v9i5.13424.
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ABSTRACTObjective: The present study was aimed to find the angiotensin-converting enzyme (ACE) inhibitory activity using the methanolic extract ofCinnamomum zeylanicum (as a natural inhibitor) on sheep tissues as the enzyme source.Methods: Hippuryl-histidyl-leucine (HHL) as a substrate, tissue ACE activity was measured spectrophotometrically at 228 nm. For an incubationperiod of 30 minutes at 37°C, the linearity of ACE activity of kidney, lung, and testis enzyme was established. A known medicinal plant C. zeylanicumwas used as natural inhibitor of ACE. In this enzyme assay, inhibitory effect of methanolic extract of C. zeylanicum on kidney, lung and testicular ACEwas determined. ACE activity was confirmed by captopril, a standard inhibitor of ACE.Results: In the presence of a methanolic extract of C. zeylanicum (10:1), ACE activity was determined and this has inhibited ACE activity verysignificantly. C. zeylanicum leaves extract has reduced sheep kidney, lung, and testis ACE activity by 70.06%, 12.63%, and 20.23%, respectively.Conclusion: Significant inhibition was observed in the kidney ACE than in lung and testis ACE activity. This can propose that there may be a possiblerole in controlling blood pressure or reduction in cardiovascular diseases. Some plants with the great medicinal property may be considered aspromising sources of natural inhibitors of ACE for medicine and commercial uses. This comprehensive study may show numerous beneficial effects asa potential therapeutic agent for lowering blood pressure.Keywords: Angiotensin-converting enzyme, Natural angiotensin-converting enzyme inhibitor, Kinetic assay, Hippuryl-histidyl-leucine, Cinnamomumzeylanicum, Cardiovascular diseases.
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CHEN, Ching-Jiunn, Hui-Sheng HUANG, Yu-Tsong LEE, Chia-Yi YANG, and Wen-Chang CHANG. "Characterization and purification of a lipoxygenase inhibitor in human epidermoid carcinoma A431 cells." Biochemical Journal 327, no. 1 (October 1, 1997): 193–98. http://dx.doi.org/10.1042/bj3270193.
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A lipoxygenase inhibitor in the cytosolic fraction of human epidermoid carcinoma A431 cells was characterized and purified. The cytosolic inhibitor lost the inhibitory activity upon heating at 75 °C for 15 min or pretreating with 1 mg/ml trypsin at 37 °C for 60 min. Cytosol, after dialysis, lost the inhibitory activity but its inhibitory activity recovered when 1 mM GSH was added to the dialysate. The inhibitory activity of cytosol was also abolished by treatment either with 1 mM iodoacetate at 4 °C for 1 h or with 0.5 mM H2O2. The pI of the inhibitor was approx. 7.0. In addition to 12-lipoxygenase, the inhibitor inhibited the activities of 5-lipoxygenase and fatty acid cyclo-oxygenase in a cell-free system. The inhibitor was purified by a series of column chromatographies using CM Sephadex C-50, Sephadex G-100 SF and Mono P columns. A major 22 kDa protein was obtained that was distinct from selenium-dependent glutathione peroxidase.
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Miao, Yuxi, Guanzhu Chen, Xinping Xi, Chengbang Ma, Lei Wang, James F. Burrows, Jinao Duan, Mei Zhou, and Tianbao Chen. "Discovery and Rational Design of a Novel Bowman-Birk Related Protease Inhibitor." Biomolecules 9, no. 7 (July 14, 2019): 280. http://dx.doi.org/10.3390/biom9070280.
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Anuran amphibian skin secretions are a rich source of peptides, many of which represent novel protease inhibitors and can potentially act as a source for protease inhibitor drug discovery. In this study, a novel bioactive Bowman-Birk type inhibitory hexadecapeptide of the Ranacyclin family from the defensive skin secretion of the Fukien gold-striped pond frog, Pelophlax plancyi fukienesis, was successfully isolated and identified, named PPF-BBI. The primary structure of the biosynthetic precursor was deduced from a cDNA sequence cloned from a skin-derived cDNA library, which contains a consensus motif representative of the Bowman-Birk type inhibitor. The peptide was chemically synthesized and displayed a potent inhibitory activity against trypsin (Ki of 0.17 µM), as well as an inhibitory activity against tryptase (Ki of 30.73 µM). A number of analogues of this peptide were produced by rational design. An analogue, which substituted the lysine (K) at the predicted P1 position with phenylalanine (F), exhibited a potent chymotrypsin inhibitory activity (Ki of 0.851 µM). Alternatively, a more potent protease inhibitory activity, as well as antimicrobial activity, was observed when P16 was replaced by lysine, forming K16-PPF-BBI. The addition of the cell-penetrating peptide Tat with a trypsin inhibitory loop resulted in a peptide with a selective inhibitory activity toward trypsin, as well as a strong antifungal activity. This peptide also inhibited the growth of two lung cancer cells, H460 and H157, demonstrating that the targeted modifications of this peptide could effectively and efficiently alter its bioactivity.
Dissertations / Theses on the topic "Inhibitor Activity"
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Stott, Holly Rosannah. "MMP-12 activity during vascular remodelling." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/23609.
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Matrix metalloproteinases (MMPs) are required for tissue remodelling processes, including angiogenesis. MMP activity is generally proangiogenic but MMP-12 is suggested to be antiangiogenic and its precise role is still unclear. The work in this thesis describes the synthesis of an MMP-12 inhibitor and activity probe to address the hypothesis that MMP-12 inhibits angiogenesis. An inhibitor, synthesised in-house, selectively inhibited MMP-12 in in vitro recombinant enzyme assays. An activity probe, also synthesised in-house, was selective for MMP-12 in in vitro recombinant enzyme assays. The function of MMP-12 during angiogenesis was assessed using murine models of angiogenesis; the in vivo sponge implantation, and the ex vivo aortic ring assays. Angiogenesis and MMP activity were imaged in vivo in sponges in C57Bl6/J mice over 7 − 21 days (D) using commercial probes (MMPSense™ and AngioSense™). MMP-12 protein concentration and activity were higher in sponges during early angiogenesis (D 3 − 7) when gene expression of vascular endothelial growth factor (a proangiogenic marker) was also high. Gene expression for MMP-12 and platelet-derived growth factor receptor (a marker of vascular maturation) were both higher on D 21 as angiogenesis started to stabilise. The MMP-12 activity probe was unsuccessful in selectively detecting MMP-12 activity in sponge lysate mixtures from D 7 − 21. Administration of an MMP-12 inhibitor did not increase angiogenesis in the sponges in vivo. Additionally, sponges implanted in MMP-12-/- mice did not exhibit significant changes in angiogenesis or MMP activity when imaged in vivo using commercial probes (MMPSense™ and AngioSense™) on D 7. Supporting this, histological analysis of the sponges (removed on D 21) showed that deletion of MMP-12 also did not increase angiogenesis within the sponges.
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Eckelman, Brendan P. "The regulation of caspase activity by the inhibitor of apoptosis proteins." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC IP addresses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3288846.
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Thesis (Ph. D.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed June 2, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 188-214).
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Gora, Kasia G. (Kasia Gabriela). "SciP : a novel inhibitor of CtrA transcriptional activity in Caulobacter crescentus." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/68426.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, September 2011.Cataloged from PDF version of thesis. "August 8, 2011."
Includes bibliographical references.
Cells must sense changes in their environment and respond appropriately in order to survive. A common survival strategy is for cells to translate an environmental signal into the activity of a transcription factor they effects a change in gene expression. In this way, cells can express a gene just-in-time for its biological function. In addition, cells can coordinate the activity of many transcription factors to construct genetic regulatory networks that integrate many inputs to control complex cellular behaviors. In this thesis, I use the model system Caulobacter crescentus to examine the regulation of CtrA, the essential transcription factor at the core of the Caulobacter cell cycle regulatory network. CtrA regulates the activity of approximately 100 cell cycle genes many of which are critical for cell cycle progression. CtrA activity is regulated at the level of abundance, post-transcriptional modification, and here I show that CtrA is also regulated by a novel protein-protein interaction. I identify SciP, a GI specific inhibitor of CtrA transcriptional activity and show that SciP forms a complex with CtrA at CtrA dependent promoters. The SciP/CtrA interaction likely prevents CtrA from recruiting RNA polymerase thereby blocking the activation of transcription. In addition, I show that SciP is restricted to G1 by regulated proteolysis. I identify the Lon as the SciP protease and show that Lon is required for SciP proteolysis in vivo and that E. coli Lon degrades SciP in vitro. Finally I engineer a stable allele of sciP and show SciP proteolysis is critical for proper cell cycle progression.
by Kasia G. Gora.
Ph.D.
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Al-Yousuf, Karamallah. "Investigating the anti-melanoma activity of combinatorial paclitaxel and MEK inhibitor." Thesis, University of Dundee, 2018. https://discovery.dundee.ac.uk/en/studentTheses/1be821d2-2436-4c31-8485-e62b86d6d33b.
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Melanoma has increased considerably over the past forty years. Traditional and newly-developed anti-melanoma drugs do not produce a satisfactory therapeutic response for the treatment of late stage melanoma. Nanomedicine is a potent tool for clinicians to circumvent many of the existing shortcomings in cancer therapy. Combination of more than one drug may maximise the therapeutic response and minimise undesirable effects of the chemotherapeutic agents in cancer patients. I developed a novel combinatorial approach that can be loaded into targeted anti-melanoma nanocarriers. Our group have been working on the development of a theranostic superparamagnetic iron oxide nanoparticle (SPION), designed to accommodate the simultaneous encapsulation of two anti-melanoma compounds, with the addition of a melanoma cell-specific targeting moiety (α-MSH) attached to the surface of the nanoparticle (NP). The selection of PTX and SEL for being loaded on α-MSH-SPION takes into consideration the anti-melanoma potency, mechanism(s) of action, physicochemical properties of each drug and their ability for embedding in the hydrophobic pocket of the NP. The synergistic ratio of PTX-SEL combination exerted limited cytotoxic effect towards normal skin cells, but was potent in melanoma cells. Also, we have analysed the drug combination in vivo, where the combinatorial therapy had a statistically significant effect in blocking tumour growth. In a dose- and time-dependent manner, PTX-SEL co-treatment increased oxidative stress in melanoma cells. The pro-oxidant effect of PTX-SEL is specific to melanoma rather than normal cells. These effects accompanied by mitochondrial dysfunction and increased mitochondrial ROS production indicating that mitochondria are the key source of PTX-SEL-induced-ROS production. In addition, our findings show that antioxidants antagonise the drug combination-induced melanoma cell death. Overall, our findings establish the PTX-SEL drug combination is potent and selective anti-melanoma approach with mechanistic rationale offering therapeutic benefit when loaded to α-MSH-SPIONs that can wide the horizons of clinical pharmacology field.
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Sancho, Elena. "Conformation and activity of plasminogen activator inhibitor type 1 (PAI-1)." Thesis, University of Aberdeen, 1994. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU552562.
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Plasminogen activator inhibitor type 1 (PAI-1) is a unique member of the serpins in that it can adopt various conformations. It is synthesized as an active inhibitor which rapidly converts to a latent form that is not inhibitory. Conformational studies on this protein have been made difficult by this property. The production of recombinant PAI-1 was studied. The expression of r-PAI-1 by the bacterial strain MSD 1005 accounted for some 15-20% of the total cell protein, although some was produced in the form of inclusion bodies. As an attempt to improve the yields of PAI-1 antigen and activity, the cells were grown at low temperatures or under osmotic-stress conditions in the presence of betaine. These did not affect the distribution of r-PAI-1 between the soluble phase and inclusion bodies or the specific activity of the r-PAI-1. A quick protocol for the purification of r-PAI-1 from the strain MSD 1005 was developed. Approximately 26 mg of r-PAI-1 could be purified from 1.51 of bacterial culture in one step by ion exchange chromatography. The specific activity of the purified r-PAI-1 was found to be close to the theoretical maximum and to be unusually stable. The stability of the r-PAI-1 was found to be dependent on the low pH and the high NACl concentration used during the purification protocol. The effect of arginine was found to be comparable to that of NaCl. Vitronectin or aprotinin had no effect on the specific activity of r-PAI-1. Active, denaturant activated and latent r-PAI-1 were compared in terms of their binding to heparin and to a panel of monoclonal antibodies. Complex formation with t-PA was also assessed. Active and denaturant activated PAI-1 were indistinguishable in these studies.
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Grundy, Martin. "Activity of the aurora kinase B inhibitor AZD1152 in acute myeloid leukaemia." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12758/.
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Aurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies including those of leukaemic origin. Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder of haematopoietic progenitor cells whose prognosis is particularly poor and where standard induction therapy has changed little over the past thirty years. This thesis evaluated the effects of AZD1152-hQPA (barasertib-hQPA), a highly selective inhibitor of aurora-B kinase, in AML cell lines and primary samples. Inhibition of phospho-Histone H3 (pHH3) on serine 10 can be used as a biomarker for AZD1152-hQPA activity and an assay was optimized to measure pHH3 in our cell lines and primary samples. AZD1152-hQPA inhibited pHH3 in our cell lines resulting in polyploid cells, apoptosis, and cell death, irrespective of cellular p53 status. Over-expression of the ATP-binding cassette (ABC) drug transporter proteins P-glycoprotein (Pgp) and Breast cancer resistance protein (BCRP) is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in AML. A cell line which over-expresses Pgp was developed by selecting for daunorubicin (DNR) resistance in OCI-AML3 cells. Pgp and also BCRP expressing AML cell lines were found to be resistant to AZD1152-hQPA and it was found that AZD1152-hQPA is effluxed by these transporters. pHH3 inhibition by low dose AZD1152-hQPA was seen in all of the primary samples tested with Pgp and BCRP positive samples being less sensitive. However, 50% inhibition of pHH3 by AZD1152-hQPA was achieved in 94.6% of these samples. The FLT3-ITD-expressing MOLM-13 and MV4-11 cell lines were particularly sensitive to AZD1152-hQPA. Internal tandem duplications (ITDs) within the FLT3 tyrosine kinase receptor are found in approximately 25% of AML patients and are associated with a poor prognosis. It was demonstrated that AZD1152-hQPA directly targets phosphorylated FLT3 in the FLT3-ITD cell lines along with inhibiting its downstream target pSTAT5. FLT3-ITD primary samples were particularly sensitive to clonogenic inhibition and pSTAT5 down-regulation after treatment with AZD1152-hQPA compared with FLT3 wild-type (WT) samples.
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Sampathkumar, Yamuna. "Thermal processing effects on total phenolic content, antioxidant activity, trypsin inhibitor activity and in-vitro protein digestibility of lentils." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107918.
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Heat pre-treatment of nutrient-rich lentil seeds prior to their processing into flour may enhance its use by reducing processing and preparation times in value added products. In this study, changes in trypsin inhibitor content, total phenolic content, antioxidant activity, and in-vitro protein digestibility of flours prepared from hulled red lentils and unhulled green lentils were determined subsequent to various processing methods such as oven roasting (OR), boiling and microwave heating (MH). The increasing interest in the phenolic content of plant based food-stuffs made us to assess two different lentil cultivars processed under fixed temperature and time combination. Total phenolic content and antioxidant activity (TAC) of 70% acetone lentil extracts were assayed spectrophotometrically at 760nm using Folin-Ciocalteu and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity methods, respectively. Significant differences in phenolic content and antioxidant activity were noted between hulled red and unhulled green lentil varieties. MH (5 min) led to a significant increase (P ≤ 0.05) in total phenolic content in unhulled green lentil flours (GLF) [8.85 mg GAE/g dry weight (DW) while raw flour comparatively showed lower phenolic content [7.5 mg GAE/g DW]. A similar increase was found after oven-roasting this material for 20 min. The TAC of untreated unhulled green lentil range was around 86%, and it was higher than the value obtained for the flour from untreated hulled red lentils. The increase in TPC of OR samples and microwave-heated samples over untreated ones may reflect reductions in TAC. Flour samples obtained from boiled lentils showed a sharp decrease in TPC and TAC values, which may be attributable to a number of factors in the matrix.Though lentils are rich in protein, their anti-nutritional components, the length of the time required for their preparation, as well as their unfavorable flavor, and reduced protein digestibility have limited their frequency of use for human consumption. By applying heat, anti-nutritional factors such as trypsin inhibitors can be largely removed. Our results show that MH treatment produced significant reduction (P≤0.05) in trypsin inhibitor activity when compared to OR or boiling methods. In- vitro protein digestibility (IVPD) was improved after processing. Longer processing times associated with OR or boiling methods improved IVPD to a greater extent than MH.Keywords: TAC- Total antioxidant activity; GA- Gallic Acid equivalent; Lentils; TPC- Total phenolic content; IVPD- In-Vitro Protein digestibility; MH- Microwave heating; OR- Oven roasting; GLF- Green lentil flours; RLF- Red lentil floursLe prétraitment thermique de lentilles avant de les moudre en farine peut faciliter son utilisation dans la préparation de produits à valeur ajoutée. Cette étude porte sur l'évaluation des effets des prétraitments thermiques par chauffage par torréfaction(CT), par chauffage par microondes (CM), ou par l'eau bouillante (EB), sur la teneur en composés phénoliques totaux (CPT), sur l'activité anti-oxydante totale (AAOT), sur les teneurs en inhibiteur de trypsine, et sur la digestibilité in-vitro des protéines (DIVP). Les essais ont été faits sur des farines obtenues à partir de lentilles rouges décortiquées et de lentilles vertes non decortiquées après l'application des prétraitment thermiques. Les deux varieties de lentilles ont été prétraitées selon des combinaisons déterminées de températures et de durées de traitment. Les teneurs en CPT et l'AAOT ont été évaluées par spectrophotométrie à 760 nm en utilisant la méthode de Folin-Ciocalteu et la méthode DPPH (1, 1- diphényl-2-picrylhydrazyle) de piégeage des radicaux libres. Les résultats ont démontré des différences significatives entre les deux types de lentilles étudiées. Le prétraitement CM (5 min) a conduit à une augumentation significative (P≤0.05) de la teneur en CPT dans les échantillons de farine de lentilles vertes [8.85 mg GAE/g de poids sec (ps) lorsque comparé aux échantillons de farine de lentilles vertes non-traitées [7.5 JEU mg/g ps]. Une tendance similaire a été observée auprés des farines de lentilles vertes torréfiées au four pendant 20 min. L'AAOT des farines de lentilles vertes non-traitées était d'environ 86% et elle était supérieure à celle obtenue à partir de lentilles rouges non-traitées. Les teneurs en CPT observées dans les échantillons traités soit par CT soit par CM étaient plus élevées que celles des échantillons non traitées et peuvent refléter des réductions en AAOT obetnues. Les teneurs en CPT et en AAOT des échantillons obtenues à la suite du prétraitment EB étainet nettement inférieures et peut être attributées à plusieurs facteurs dans la matrice. Bien que les lentilles soit riches en protéines, plusiers facteurs limitent leurs utilisation pour la consommation humaine. Ces principaux facteurs sont : la présence de composants antinutritionnels, le temps nécessaire à la préparation, les saveurs désagréables, et la digestibilité réduites des protéines. On peut réduire l'impacte des facteurs antinutritionnels comme les inhibiteurs de la trypsine par des traitements thermiques. Les résultats ont demontré que l'utilisation du pretraitment CM permettait de réduire significativement (P≤0.05) l'activité des inhibiteurs de trypsine lorsque comparés aux prétraitments CT et EB. Il a aussi été démontré que l'augmentation de la dureé des prétraitments thermiques par CT ou par EB permettait d'accroitre la DIVP. Dans tous les cas étudiés, l'application d'un prétraitment thermique sur les lentilles a permis d'améliorer la DIVP.Mots clés : Lentilles vertes, lentilles rouges, farines, traitements thermiques, composés phénoliques totaux (CPT), activité anti-oxydante totale (AAOT), digestibilité in-vitro des protéines (DIVP), équivalent en acide gallique, chauffage par torréfaction (CT), chauffage par microondes (CM), chauffage par trempage dans l'eau bouillante (EB).
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Mendis, Mihiri Marini. "Variability in Arabinoxylan, Xylanase Activity and Xylanase Inhibitor Levels in Hard Spring Wheat." Thesis, North Dakota State University, 2012. https://hdl.handle.net/10365/26806.
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Arabinoxylans (AX), xylanase, and xylanase inhibitors have an important role in many cereal food processing applications. The effect of growing location (L), genotype (G), and their interactions (L ×G) on AX, apparent xylanase and apparent xylanase inhibition activities of National Science Foundation Grant (# HRD-0811239) to the NDSU Advance FORWARD program
North Dakota Wheat Commission
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Zwahlen, Hugo. "Effects of low-dose "Factor VIII Inhibitor Bypassing Activity (FEIBA)" in resistant heamophilia /." [S.l : s.n.], 1986. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Ren, Zhongyuan. "Small molecules regulated bone resorption and enzyme activity in osseous cells." Thesis, Lyon 1, 2014. http://www.theses.fr/2014LYO10291/document.
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La Cathepsine K est parmi la plus efficace des collagénases de mammifère pour cliver la triple hélice de collagène de type-1. Nous avons développé une série d'azanitriles, (CKI-8 and CKI-13) inhibiteurs de cathepsine K. CKI-8 (un isomère de CKI-13) et CKI-13 ne sont pas toxiques sur les osteoblastes Saos-2 et les cellules RAW 264.7 jusqu' à une concentration de 1000 nM, tandis qu'ils ne le sont pas jusqu'à une concentration de 100 nM sur les osteoclastes. CKI-8 n'affecte pas l'activité de la phosphatase alkaline ainsi que la minéralisation induite par les Saos-2 et par les osteoblastes primaires. CKI-13 diminue de 35 % la minéralisation induite par les Saos-2 tandis qu'il n'affecte pas la minéralisation induite par les osteoblastes primaires. L'addition de CKI-13 diminue l'activité de la phosphatase alkaline d'environ 20% (Saos-2) et de 40 % (osteoblastes primaires). La résorption osseuse sur des tranches d'os d'origine bovine est diminuée avec 10 nM de CKI-13, 100 nM de CKI- 8 et 100 nM d'inhibiteur commercial E64. CKI-8 et CKI-13 diminuent la mobilité des osteoclastes. Nous avons développé un dosage d'hydrolyse de PPi par la phosphatase alkaline au moyen de l'IR, ayant l'avantage de fonctionner sur des vésicules matricielles et des cellules avec des substrats naturels à un pH physiologique. La bande de PPi localisée à 1107 cm-1 (∑= 2158 ± 211 M-1.cm-1) et celles de Pi localisées à 1076 cm-1 (∑= 1346 ± 116 M-1.cm- 1) et à 991 cm-1 (∑= 493 ± 49 M-1.cm-1) ont servis à mesurer les concentrations du substrat et du produitCathepsin K is among the most potent mammalian collagenase, capable of cleaving the triple helix in type-I collagen. We developed a series of azanitriles (CKI-8 and CKI-13) which are inhibitors of cathepsin K. CKI-8 (an isomer of CKI-13) and CKI-13 did not induce significant toxicity on osteoblasts Saos-2 and RAW 264.7 cells up to 1000 nM, while they were not toxic on mature osteoclasts up to 100 nM. Commercial E64 inhibitor was not toxic in primary osteoclast cells up to 1000 nM. CKI-8 did not affect alkaline phosphatase activity as well the mineralization induced by Saos-2 cells and by primary osteoblasts. CKI-13 decreased by 35% the mineralization induced by Saos-2 cells while it did not on mineralization induced by primary osteoblasts. Addition of CKI-13 decreased alkaline phosphatase activity by around 20% (Saos-2 cells) and 45% (primary osteoblasts). Bone resorption on bovine slices decreased significantly with 10 nM of CKI-13, with 100 nM of CKI-8 and commercial inhibitor E64. Our findings indicated that CKI-8 and CKI-13 inhibited bone resorption and affected the mobility of osteoclast. To monitor directly the PPi hydrolytic activity by alkaline phosphatase, we developed an infrared (IR) assay taking the advantage to use natural substrate under physiological pH in matrix vesicles and in living cells. PPi band located at 1107 cm-1 (∑= 2158 ± 211 M-1.cm-1) and Pi bands located at 1076 cm-1 (∑= 1346 ± 116 M-1.cm-1) and at 991 cm-1 (∑= 493 ± 49 M-1.cm-1) served to measure the substrate and the product concentrations
Books on the topic "Inhibitor Activity"
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Roué, Gael. Activity of the Novel BCR Kinase Inhibitor IQS019 in B-NHL. Saarbrücken: LAP LAMBERT Academic Publishing, 2017.
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G, Cory Joseph, and Cory Ann H, eds. Inhibitors of ribonucleoside diphosphate reductase activity. New York: Pergamon Press, 1989.
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Merton, Sandler, and Smith H. J. 1930-, eds. Design of enzyme inhibitors as drugs. Oxford: Oxford University Press, 1994.
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Merton, Sandler, and Smith H. J. 1930-, eds. Design of enzyme inhibitors as drugs. Oxford [England]: Oxford University Press, 1989.
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Gupta, Satya Prakash. Matrix metalloproteinase inhibitors: Specificity of binding and structure-activity relationships. Basel: Springer, 2012.
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1935-, Tipper Donald J., ed. Antibiotic inhibitors of bacterial cell wall biosynthesis. Oxford [Oxfordshire]: Pergamon Press, 1987.
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Kiss, S., and M. Simihăian. Improving Efficiency of Urea Fertilizers by Inhibition of Soil Urease Activity. Dordrecht: Springer Netherlands, 2002. http://dx.doi.org/10.1007/978-94-017-1843-1.
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Atwell, Mark M. Inhibition of secretary activity in cells isolated from the rat stomach. Birmingham: Aston University, Department of Pharmaceutical Sciences, 1990.
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M, Simihǎian, ed. Improving efficiency of urea fertilizers by inhibition of soil urease activity. Dordrecht: Kluwer Academic Publishers, 2002.
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J, Cragoe Edward, Kleyman Thomas R, and Simchowitz Louis, eds. Amiloride and its analogs: Unique cation transport inhibitors. New York, N.Y: VCH, 1992.
Find full textBook chapters on the topic "Inhibitor Activity"
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Kruithof, E. K. O. "Plasminogen activator inhibitor activity assay." In ECAT Assay Procedures A Manual of Laboratory Techniques, 147–50. Dordrecht: Springer Netherlands, 1992. http://dx.doi.org/10.1007/978-94-011-2992-3_18.
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Turecek, Peter, and Hans Peter Schwarz. "Factor Eight Inhibitor Bypassing Activity." In Production of Plasma Proteins for Therapeutic Use, 49–63. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2012. http://dx.doi.org/10.1002/9781118356807.ch4.
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Darragh, A., M. Kenny, R. Lambe, T. Lenehan, I. Brick, and C. Maulet. "Indalpine, A New Serotonin Uptake Inhibitor." In Biological Psychiatry, Higher Nervous Activity, 347–52. Boston, MA: Springer US, 1985. http://dx.doi.org/10.1007/978-1-4684-8329-1_50.
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Kluft, C., and P. Meijer. "Plasmin inhibitor activity (previously α2-antiplasmin)." In Laboratory Techniques in Thrombosis - a Manual, 257–64. Dordrecht: Springer Netherlands, 1999. http://dx.doi.org/10.1007/978-94-011-4722-4_28.
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Kaspari, Marion, and Elke Bogner. "Antiviral Activity of Proteasome Inhibitors/Cytomegalovirus." In Viral Proteases and Antiviral Protease Inhibitor Therapy, 71–81. Dordrecht: Springer Netherlands, 2009. http://dx.doi.org/10.1007/978-90-481-2348-3_4.
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Chelliah, Ramachandran, Eric Banan-MwineDaliri, and Deog-Hwan Oh. "Screening of Actinobacteria for Enzyme Inhibitor Activity." In Methods in Actinobacteriology, 475–78. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-1728-1_69.
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Walker, Mark C., and James K. Gierse. "In Vitro Assays for Cyclooxygenase Activity and Inhibitor Characterization." In Methods in Molecular Biology, 131–44. Totowa, NJ: Humana Press, 2010. http://dx.doi.org/10.1007/978-1-59745-364-6_11.
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Mirsky, Arthur. "The Insulinase and Insulinase-Inhibitor Activity of the Liver." In Novartis Foundation Symposia, 263–77. Chichester, UK: John Wiley & Sons, Ltd., 2008. http://dx.doi.org/10.1002/9780470718858.ch19.
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Maxwell, Robert A., and Helen L. White. "Tricyclic and Monoamine Oxidase Inhibitor Antidepressants: Structure-Activity Relationships." In Handbook of Psychopharmacology, 83–155. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4613-4045-4_3.
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Lacroix-Desmazes, S., A. Moreau, M. P. Horn, M. D. Kazatchkine, and S. V. Kaveri. "Factor VIII Inhibitor with Catalytic Activity towards Factor VIII." In Catalytic Antibodies, 102–14. Basel: KARGER, 2000. http://dx.doi.org/10.1159/000058800.
Full textConference papers on the topic "Inhibitor Activity"
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Liu, Keshun, and Mike Woolman. "Developing an optimized method for measuring chymotrypsin inhibitor activity in protein products." In 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/yucc6741.
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Protease inhibitors of protein nature, such as trypsin inhibitors and chymotrypsin inhibitors, are rich in seeds of legume crops. Soybeans contain Kunitz inhibitor and Bowman-Birk inhibitor. The former mainly inhibits trypsin, while the latter inhibits both trypsin and chymotrypsin. Other legumes contain similar types. Historically, trypsin inhibitor activity in legume products has been of primary interest for measurement due to its antinutritional implication. However, Bowman-Birk inhibitor has been shown therapeutic. It is also more resistant to heat than Kunitz inhibitor. As increasing volumes of plant proteins are being used for food or feed in recent years, there is a growing interest in monitoring chymotrypsin inhibitor activity (CIA) in these products as well. Yet, reported methods for CIA assay vary greatly, with no standard method being available. Three years ago, at our USDA lab, we developed an improved method for measuring trypsin inhibitor activity, which was later adopted as AOCS Official Method, Ba 12a-2020. This presentation reports our new effort in developing a method for measuring CIA, using N-benzoyl-L-tyrosine p-nitroanilide (BTpNA) as a substrate. Unlike the substrate for measuring trypsin inhibitor activity, BTpNA is not water soluble, an organic solvent that is miscible with water must be present. Therefore, the assay system for measuring CIA was much more complicated than that for measuring trypsin inhibitor activity. This made the method development more difficult than originally thought. After investigating effects of many assay parameters, such as organic solvent, the sequence of adding reagents, % chymotrypsin inhibition, etc., an optimized method for CIA measurement was finally developed. It featured dimethylformamide as the organic solvent, the enzyme-last sequence, 5 mL total assay volume, and calculation of the inhibitor activity based on % chymotrypsin inhibition. The proposed method was reliable and robust and could be standardized for measuring CIA in various protein products.
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de Agostini, A., F. Barja, S. Carrel, P. C. Harpel, and M. Schapira. "C1 -INHIBITOR: STRUCTURE-ACTIVITY RELATIONSHIPS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642903.
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Cl-inhibitor [C1 -In] and other protease inhibitors of the serpin superfamily inactivate serine proteases by forming bimolecular enzyme-inhibitor complexes, a reaction that is associated with changes in the inhibitor conformation. To determine the significance of these changes, we have examined the influence of various treatments on the binding to C1-In of monoclonal antibody 4C3. This antibody was previously shown to bind to an epitope created during the reaction of C1-In with the Arg-specific protease plasma kallikrein [K]: the site for 4C3 was expressed on the K-C1-In complex, on C1-In cleaved at position Pi and released from K-C1-In [C1-In*], but not on unreacted C1-In. The binding of 4C3 to the various forms of C1-In was now measured by radioimmunoassay and Western blot. Following inactivation by C1-In of the Arg-specific enzymes factor XII active fragment [Xllf] or C1s, the binding site for 4C3 was detectable on XIIf-CI-In, C1s-C1-In and C1-In*. However, when K or Xllf were incubated with heat-inactivated C1-In, bo+h enzymes remained active, no complex was formed, and the site for 4C3 was not created. When C1-In was cleaved by neutrophil elastase [E] (a Met-orVal-specific protease that is not inhibited by C1 -In), the 1st cleavage product C1-In’ retained inhibitory activity (as shown by its ability to form a complex with Xllf) but did not bind 4C3. However, subsequent cleavage of C1-In’ by E at position P3 yielded C1-In’, a product which was inactive but bound 4C3. Thus, identical conformational changes of C1-In (as assessed by the emergence of the site for 4C3) are seen when Cl-In inactivates its target enzymes while being cleaved at Pi or when the inhibitor is catalytically inactivated by cleavage at P3. Therefore, these changes are necessary but not sufficient for observing enzyme inactivation.
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Donaldson, V. H., and M. D. B. H. Mitchell. "INTERACTIONS OF DYSFUNCTIONAL Cl-INHIBITORS FROM PATIENTS WITH TYPE II HEREDITARY ANGIONEUROTIC EDEMA (HANE) WITH ACTIVATED HAGEMAN FACTOR (FACTOR XIIa)." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643302.
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Type II HANE is characterized by a deficiency of Cl-inhibitor (Cl-INH) activity in serum which is associated with a dysfunctional inhibitor protein having a normal or increased quantity o|_the antigenic properties of normal serum Cl-inhibitor. Dysfunctional Cl-INH proteins were purified from members_of eight different kindred with Type II HANE and compared to normal Cl-inhibitor with respect to their inhibitory activity directed against the amidolytic and clot-promoting properties of purified activated Hageman factor. All but one dysfunctional Cl-inhibitor blocked the amidolytic activity of ellagic acid-activated Hageman factor; all eight blocked the clot-promoting activity of Hageman factor activated in solutions of sulfatides and BSA. The inhibition _of amidolytic activity was equal to or greater than that of normal Cl-INH (Donaldson, et al., 3. Clin. Invest. 75:124,1985). The impairment of the specific Hageman factor coagulant activity of activated Hageman factor by six^f the eight dysfunctional inhibitors was less than that of the normal Cl-inhibitor, although readily measured. Dysfunctional Cl-inhibitor proteins were also heterogeneous with respect to their formation of stable complexes and their susceptibility to cleavage by Hageman factor activated with BSA-sulfatides when analyzed in SDS-gel electrophoresis. Although these observatons cannot be directly applied to in vivo pathophysiologic changes in plasma, dysfunctional Cl-inhibitors do have the potential of regulating activated Hageman factor.
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Booth, N. A., A. Reith, and B. Bennett. "A PLASMINOGEN ACTIVATOR INHIBITOR (PAI-2) CIRCULATES IN TWO HIGH MOLECULAR WEIGHT FORMS IN PREGNANCY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644459.
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Normal vascular endothelium and platelet α-granules contain an inhibitor of plasminogen activator (PAI-1) of about 48000 molecular weight, which is released by stimuli such as thrombin. An immunologically distinct inhibitor (PAI-2) of about 47000 molecular weight has been purified from placenta and from a histiocytic cell line U-937. The level of PA-inhibition in plasma is raised in late pregnancy and this may be due to increases in PAI-1 or in PAI-2 or in both.Using SDS-PAGE and zymography on fibrin/plasminogen /u-PA detector gels, we have found that normal plasma contains a band of inhibition of apparent molecular weight 40000, which can be neutralised by antiserum raised against PAI-1. Pregnancy plasma contained this band as well as additional inhibitor bands of apparent molecular weights 75000 and 130000. The novel high molecular weight PA-inhibitors were detectable by zymography at about 12 weeks gestation. They were specific for plasminogen activator and did not inhibit plasmin. They were inhibited by antiserum raised against PAI-2 from U-937 cells (a gift from Dr EKO Kruithof) and thus are immunologically related to PAI-2. They may represent circulating complexes of PAI-2 with another protein or aggregates of PAI-2, which retain inhibitory activity after SDS-PAGE. PAI-2 appears to represent a pregnancy associated protein that circulates in a number of different molecular weight forms.
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Saeki, T., K. HARADA, T. Youshimura, Y. Nakamura, T. Fujimori, K. E. Katayama, Y. Yamagish, I. Yamastu, and I. Ikeda. "MODE OF ACTION OF A NOVEL ANTI-PLATELET AGENT (E-5510)." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643427.
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A novel anti-platelet agent, 4-cyano-5,5-bis(4-methoxy-phenyl)-4-pentenoic acid(E-5510), has been shown to inhibit platelet aggregation and secretion induced by thrombin as well as by other inducers such as ADP, collagen and PAF. Although E-5510 can act as a cyclooxygenase inhibitor,inhibition of cyclooxygenase may not be the primary mode of action since this compound effectively inhibits thrombin-induced platelet activation. In this paper, effects^of E-5510 on arachidonic acid (AA) metabolism, intracellular Ca++ and cAMP in human platelets are examined.(1) Effect on AA metabolism. Platelets prelabeled with [14 C]-AA were stimulated with thrombin. E-5510 inhibited not only thromboxane A2 and HHT generation but also 12-HETE generation in a dose-dependent fashion. The total AA released was also reduced by E-5510. An almost 50% reduction was obtained by 10 uM of this compound. On the other hand, a cyclooxygenase inhibitor such as U-53059 increased 12-HETE generation in a dose-dependent fashion. In addition to the inhibition of AA metabolism, E-5510 exerted inhibitory effects on phosphatidic acidgeneration, which suggests the possible inhibition of phospholipase C activity by this compound.(2) Effect on intracellular Ca++ and protein phosphorylation. Intracellular Ca++ mobilization was examined using Fura-2 loaded human platelet suspension. The increase in intracellular Ca induced by thrombin was inhibited by E-5510 and the increase in phosphorylation of 40 K protein was also suppressed by this compound after the stimulation of human platelets by thrombin.(3) Effect on cAMP. Platelets were incubated with 10-100 uM of E-5510 and the cAMP content in human platelets was measured. E-5510 increased the cAMP content in a dose-dependent fashion. In platelet homogenate, E-5510 inhibited phosphodiesterase activity with an IC50 of 10 uM.These results suggest that E-5510 may inhibit platelet aggregation and secretion through the multiple modes of action, such as inhibition of phospholipase C, phosphodiesterase and cyclooxygenase, in the process of platelet activation.
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Tans, G., T. Janssen-Claessen, J. Rosing, and J. H. Griffin. "APPLICATION OF SPECIFIC SERINE PROTEASE INHIBITORS IN ASSAYS FOR ACTIVATED CONTACT FACTORS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643301.
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We developed amidolytic assays to determine human Factor Xlla, Factor XIa and plasma kallikrein in mixtures containing variable amounts of each enzyme. The commercially available chromogenic substrates pro-phe-arg-pNA (S2302 or chromozym PK), glu-pro-arg-pNA (S2366), ile-glu-(piperidyl)-gly-arg-pNA (S2337), and ile-glu-gly-arg-pNA (S2222) were tested for their suitability as substrates in these assays. 8-Factor Xlla, Factor XIa and plasma kallikrein each exhibit considerable activity towards a number of these substrates. This precludes direct quantitation of the individual enzymes when large amounts of other activated contact factors are present. Several serine protease inhibitors were tested on their ability to selectively inhibit those contact factors that may interfere with the factor tested for. Soybean trypsin inhibitor efficiently inhibits kallikrein, inhibits Factor XIa at moderate concentrations, but did not affect the amidolytic activity of Factor Xlla. Therefore, this inhibitor can be used to abolish a kallikrein and Factor XIa contribution in a Factor Xlla assay. We also report the rate constants of inhibition of contact activation factors by three different chloromethylketones. D-phe-pro-arg-CH 2 Cl was moderately active against contact factors (k - 2271 M-ls-1 at pH 8.3) but showed no differences in specif ity. D-phe-phe-arg-CH2 Cl was a very efficient inhibitor of kallikrein (k = 118,000 M-ls-1 at pH 8.3) whereas it slowly inhibited Factor Xlla (k = 1389 M-ls-1) and Factor XIa (k = 110 M-ls-1). Also dansyl-glu-gly-arg-CH2Cl was more reactive towards kallikrein (k 15,662 M-ls-1) than towards Factor Xlla (k = 462 M-ls-1) and Factor XIa (k = 63 M-ls-1). Since phe-phe-arg-CH2Cl is highly specific for kallikrein it can be used in a Factor XIa assay to selectively inhibit kallikrein. Based on the catalytic efficiencies of chromogenic substrate conversion and the inhibition characteristics of serine protease inhibitors and chloromethyl ketones we were able to develop quantitative assays for Factor Xlla, Factor XIa and kallikrein in mixtures of contact activation factors.
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Jansen, J. W. C. M. "EFFECTS OF INHIBITORS ON COLLAGEN INDUCED PLATELET AGGREGATION IN SIX DIFFERENT SPECIES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643445.
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One approach to the development of antithrombotics is inhibition of platelet aggregation. The pharmacological approach often used is to test compounds on collagen induced platelet aggregation measured in platelet rich plasma. Therefore we have compared inhibitors with different mechanism of action on aggregation of platelets from six different species commonly used in pharmacological studies. Aggregation was induced with submaximal amounts of collagen (Hormone Chemie).Inhibitors of the cyclooxygenase system, aspirin and indomethacin, were very potent in inhibiting aggregation of platelets from humans guinea pig and dog (IC50 20-60 and 1-3 ¼M resp.). Aggregation of pig and rat platelets was poorly inhibited by both of these compounds (IC5: 700-900 ¼M), whereas platelets from mice showed intermediate sensitivety (IC50 ca.100 ¼M).The combined lipoxygenase/cyclooxygenase inhibitor BW755C, was extremely active on platelets of guinea pig (IC50 1 ¼M) and was poorly active in mice platelets (IC50 300 ¼M). In the other species the inhibitory activity ranged from 20-80 ¼M.The phosphodiesterase inhibitors, papaverine and BL3459 inhibited aggregation in all species (IC50 50-100 and 1-5 ¼M resp.). Dipyridamole inhibited aggregation also in all species but with lower activity (IC50 > 100 ¼M).Conclusion: remarkable species differences are present with respect to inhibition of collagen induced platelet aggregation by the various compounds e.g. rat and porcine platelet aggregation was hardly inhibited by cyclooxygenase inhibitors. The effects of the compounds on human platelets are comparable to the effects on canine plateletes.
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Agarwal, Kailash C. "NEW INSIGHTS INTO THE ANTIPLATELET ACTIVITY OF FORSKOLIN: ROLE OF PLASMA ADENOSINE AND SPECIES DIFFERENCES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643584.
Full textAbstract:
Forskolin stimulates adenylate cyclase by interacting with the catalytic subunit and inhibits platelet aggregation. This inhibition is greatly potentiated by adenosine (Ado) which stimulates adenylate cyclase through membrane-bound Ado receptors. Forskolin is 2-4 fold more potent as an inhibitor of collagen-induced rat platelet aggregation as compared to human platelets (IC50 values, in rat PRP, 0.5-0.8 μM; in human PRP, 1.5-2 μM). However, if the blood is pretreated with adenosine deaminase (ADA), an enzyme that degrades Ado to inosine, the inhibitory action of forskolin is greatly reduced producing similar effects both in human and rat PRPs (IC50, 2-3 μM) and whole blood (IC50, 4.6 μM). Both 5’-methylthioadenosine (MTA, 50-100 μM), an antagonist of Ado receptors, and 2’,5’-dideoxyadenosine (DDA, 100 μM), an inhibitor of adenylate cyclase, reverse the inhibition of platelet aggregation in rat PRP, whereas, no reversal is seen in human PRP. When Ado in the rat plasma is degraded by ADA pretreatment, DDA or MTA shows no reversal as seen in human PRP. The inhibitory action of forskolin (1-2 μM), which is only weakly inhibitory alone (<20%) in human whole blood, can be greatly potentiated (100% inhibition) by the inhibitors of nucleoside transport, dipyridamole (10 μM) or dilazep (2 μM). Only slight potentiation is seen in rat whole blood suggesting that rat plasma Ado levels are not affected significantly perhaps due to weakly active erythrocytic nucleoside transport system. Sato and Ui (In: Physiology and Pharmacology of Adenosine, Daly et al, Eds. Raven Press, 1983, 1-11), have shown that rat plasma contains much higher Ado levels (7.55 ± 0.51 pM) than human plasma (0.29 ± 0.08 μM). These studies demonstrate that plasma adenosine plays an important role in the forskolin antiplatelet activity which can be greatly potentiated in human whole blood by the clinically used drugs, dipyridamole and dilazep. (Supported by US PHS Grant CA 07340).
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Beretz, A., F. Lanza, A. Stierlé, and J.-P. Cazenave. "CYCLIC NUCLEOTIDE PHOSPHODIESTERASE INHIBITORS PREVENT AGGREGATION AND SECRETION OF HUMAN PLATELETS BY RAISING CYCLIC AMP AND REDUCING CYTOPLASMIC FREE CALCIUM MOBILIZATION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643586.
Full textAbstract:
Drugs that raise platelet cyclic AMP (cAMP) are potent inhibitors of platelet activation. We have studied the effects of 5 inhibitors of cyclic nucleotide phosphodiesterase (PDE) of different chemical structures (quercetin, Ro 15-2041, HL-725, cilostamide and MY-5445), which are all potent inhibitors of platelet function. The concentrations that inhibit by 50 % crude cAMP-PDE activity (IC50) from human platelets are: 0.06 μM(HL-725), 0.15 μM(Ro 15-2041 ), 0.23 μM(cilostamide), 6.9 μM(MY-5445) and 44.4 μM(quercetin). We measured on the same preparation of washed human platelets loaded with quin2, the aggregation and the increase in intracellular Ca2+ ([Ca2+]i) induced by 5 μM ADP alone or in the presence of PDE inhibitors.PGE1 (2 nM) potentiates significantly (1.6 to 3.3 fold) the inhibitory effects of PDE inhibitors on [Ca2+]i rises and platelet aggregation. Adrenaline, an inhibitor of adenylate cylase, prevents the effect of PDE inhibitors on ADP-induced [Ca2+]i rise and platelet aggregation. These results suggest that these compounds inhibit [Ca2+]i mobilization and subsequent ADP-induced aggregation through a rise in cAMP, because both effects are potentiated by PGE1 and inhibited by adrenaline. The inhibitor concentrations which potentiate the action of PGE1, on [ Ca2+]i levels also potentiate the rise in platelet cAMP induced by PGE-<. These results suggest that PDE inhibitors inhibit platelet aggregation Ly raising cAMP levels and subsequently inhibiting [Ca2+]i mobilization.
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Iwamoto, M., N. Sugiyama, T. Sasaki, and Y. Abiko. "DOMAIN OF BINDING ACTIVITY WITH PLASMIN KRINGLE IN SYNTHESIZED C-TERMINAL PEPTIDES , OF α2-PLASMIN INHIBITOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644612.
Full textAbstract:
The inhibitory reaction between plasmin and α2-plasmin inhibitor (α2-PI) proceeds with two steps, a very fast reversible reaction followed by a slower irreversible transition. The first step is dependent on the interaction between lysine binding site (LBS) of plasmin and the corresponding complementary site of α2-PI (kringle binding site(KBS)). It has been reported that KBS is located in a C-terminal tryptic fragment (T-11; J. Biochem. 99, 1699 (1986)).In order to investigate which amino acid residues of T-ll play important roles in binding of plasmin kringle, we tested inhibitory activity of synthesized peptides on the apparent rate constant in the reaction between α2-PI and plasmin. 50% inhibition concentrations of T-ll, peptide I, II, III and IV were 7, 18, 13, 35 and 250pM respectively, indicating that Leu9-Lysl0 is an important part for binding of T-ll to LBS. Peptide III lost its activity by depletion or amidation of the C-terminal lysine residue.In the system consisted of α2-PI and miniplasmin which lacked kringle 1-4, peptide I did not inhibit the interaction between them. Furthermore, peptide II competitively inhibited the binding of tranexamic acid to kringle 1-3 (Ki 0.85μM).These findings suggest that the C-terminal part is involved in the high affinity binding of α2-PI to plasmin kringle and that LyslO in T-ll and C-terminal carboxyl residue play crucial roles in binding to LBS of kringle.
Reports on the topic "Inhibitor Activity"
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Shea, Lonnie D. Identification of a PARP Inhibitor Sensitivity Signature in Breast Cancer Using a Novel Transcription Factor Activity Array. Fort Belvoir, VA: Defense Technical Information Center, March 2012. http://dx.doi.org/10.21236/ada559941.
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Olszewski, Neil, and David Weiss. Role of Serine/Threonine O-GlcNAc Modifications in Signaling Networks. United States Department of Agriculture, September 2010. http://dx.doi.org/10.32747/2010.7696544.bard.
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Significant evidence suggests that serine/threonine-O-linked N-acetyl glucosamine0-(GlcNAc) modifications play a central role in the regulation of plant signaling networks. Forexample, mutations in SPINDLY,) SPY (an O-GlcNAc transferase,) OGT (promote gibberellin GA) (signal transduction and inhibit cytokinin responses. In addition, mutating both Arabidopsis OGTsSEC (and SPY) causes embryo lethality. The long-term goal of this research is to elucidate the mechanism by which Arabidopsis OGTs regulate signaling networks. This project investigated the mechanisms of O-GlcNAc regulation of cytokinin and gibberellin signaling, identified additional processes regulated by this modification and investigated the regulation of SEC activity. Although SPY is a nucleocytoplasmic protein, its site of action and targets were unknown. Severalstudies suggested that SPY acted in the nucleus where it modified nuclear components such as the DELLA proteins. Using chimeric GFP-SPY fused to a nuclear-export signal or to a nuclear-import signal, we showed that cytosolic, but not nuclear SPY, regulated cytokinin and GA signaling. We also obtained evidence suggesting that GA and SPY affect cytokinin signaling via a DELLA-independent pathway. Although SEC and SPY were believed to have overlapping functions, the role of SEC in cytokinin and GA signaling was unclear. The role of SEC in cytokinin and GA responses was investigated by partially suppressing SPY expression in secplants using a synthetic Spymicro RNA miR(SPY). The possible contribution of SEC to the regulation of GA and cytokinin signaling wastest by determining the resistance of the miR spy secplants to the GA biosynthesis inhibitor paclobutrazol and to cytokinin. We found that the transgenic plants were resistant to paclobutrazol and to cytokinin, butonlyata level similar to spy. Moreover, expressing SEC under the 35S promoter in spy mutant did not complement the spy mutation. Therefore, we believe that SEC does not act with SPY to regulate GA or cytokinin responses. The cellular targets of Spy are largely unknown. We identified the transcription factor TCP15 in a two-hybrid screen for SPY-interacting proteins and showed that both TCP15 and its closely homolog TCP14 were O-GlcNAc modified by bacterially-produced SEC. The significance of the interaction between SPY and these TCPs was examined by over-expressing the minwild-type and spy-4plants. Overexpression of TCP14 or TCP15 in wild-type background produced phenotypes typical of plants with increased cytokinin and reduced GA signaling. TCP14 overexpression phenotypes were strongly suppressed in the spy background, suggesting that TCP14 and TCP15 affect cytokinin and GA signaling and that SPY activates them. In agreement with this hypothesis, we created a tcp14tcp15 double mutant and found that it has defects similar to spyplants. In animals, O-GlcNAc modification is proposed to regulate the activity of the nuclear pore. Therefore, after discovering that SEC modified a nucleoporinNUP) (that also interacts with SPY, we performed genetic experiments exploring the relationship between NUPs and SPY nupspy double mutants exhibited phenotypes consistent with SPY and NUPs functioning in common processes and nupseeds were resistant to GA biosynthesis inhibitors. All eukaryotic OGTs have a TPR domain. Deletion studies with bacterially-expressed SEC demonstrated SEC'sTPR domain inhibits SEC enzymatic activity. Since the TPR domain interacts with other proteins, we propose that regulatory proteins regulate OGT activity by binding and modulating the inhibitory activity of the TPR domain.
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Farazi, Mena, Michael Houghton, Margaret Murray, and Gary Williamson. Systematic review of the inhibitory effect of extracts from edible parts of nuts on α-glucosidase activity. INPLASY - International Platform of Registered Systematic Review and Meta-analysis Protocols, August 2022. http://dx.doi.org/10.37766/inplasy2022.8.0061.
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Review question / Objective: The aim of this review is to examine inhibitory effect of functional components in extracts from edible nuts on α-glucosidase activity. At the end of this review the following questions will be addressed by summarizing data of in-vitro studies: which nut extract has the strongest inhibitory effect? Which functional component (e.g. polyphenols) has the strongest inhibitory effect against α-glucosidase? Are there any differences between inhibition of α-glucosidase from different sources (e.g. yeast and mammalian)? Condition being studied: Any papers looking at inhibition of α-glucosidase activity (a carbohydrate digestive enzyme; includes sucrase, maltase and isomaltase activities) by extracts of edible parts of nut will be included in this review. Papers looking at other parts of nut plants and other enzymes will be excluded.
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Ori, Naomi, and Mark Estelle. Specific mediators of auxin activity during tomato leaf and fruit development. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597921.bard.
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The plant hormone auxin is involved in numerous developmental processes, including leaf and fruit development. The tomato (Solanumlycopersicum) gene ENTIRE (E) encodes an auxin-response inhibitor from the Aux/IAA family. While most loss-offunction mutations in Aux/IAA genes are similar to the wild type due to genetic redundancy, entire (e) mutants show specific effects on leaf and fruit development. e mutants have simple leaves, in contrast to the compound leaves of wild type tomatoes. In addition, e plants produce parthenocarpic fruits, in which fruit set occurs independently of fertilization. The aim of this research program was to utilize the e mutation to identify and characterize genes that mediate the specific effect of auxin in leaf and fruit development. The specific objectives of the project were to: 1. Characterize and map modifiers of the e leaf phenotype. 2. Characterize and map suppressors of the e fruit phenotype. 3. Dissect the developmental specificity of the E gene. 4. Examine the effect of fruit-overexpression of identified genes on fruit set and seed production. To identify mediators of auxin in leaf development, we mainly focused on one mutant, crawling elephant (crel, previously called t282), which showed substantial suppression of the e phenotype and other auxin-relatedphenotypes. We have identified the CREL gene as a homolog of the Arabidopsis VRN5 gene, involved in recruiting polycomb silencing complexes to specific targets. We showed that CREL affects auxin sensitivity in tomato. Suppressors of the e fruit phenotype have been further characterized and selected for more profound effects. Expression profiling by RNAseq was used to analyze the effect of e as well as crel on gene expression in leaves and fruits. This analysis has identified putative E and CREL targets. We have initiated studies to assess the role of some of these targets in flower and fruit development. The research has identified potential mediators of auxin response in leaf, flower and fruit development.
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Moran, Nava, Richard Crain, and Wolf-Dieter Reiter. Regulation by Light of Plant Potassium Uptake through K Channels: Biochemical, Physiological and Biophysical Study. United States Department of Agriculture, September 1995. http://dx.doi.org/10.32747/1995.7571356.bard.
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The swelling of plant motor cells is regulated by various signals with almost unknown mediators. One of the obligatory steps in the signaling cascade is the activation of K+-influx channels -K+ channels activated by hyperpolarization (KH channels). We thus explored the regulation of these channels in our model system, motor cell protoplasts from Samanea saman, using patch-clamp in the "whole cell" configuration. (a) The most novel finding was that the activity of KH channels in situ varied with the time of the day, in positive correlation with cell swelling: in Extensor cells KH channels were active in the earlier part of the day, while in Flexor cells only during the later part of the day; (b) High internal pH promoted the activity of these channels in Extensor cells, opposite to the behavior of the equivalent channels in guard cells, but in conformity with the predicted behavior of the putative KH channel, cloned from S. saman recently; (c) HIgh external K+ concentration increased (KH channel currents in Flexor cells. BL depolarized the Flexor cells, as detected in cell-attached patch-clamp recording, using KD channels (the K+-efflux channels) as "voltage-sensing devices". Subsequent Red-Light (RL) pulse followed by Darkness, hyperpolarized the cell. We attribute these changes to the inhibition of the H+-pump by BL and its reactivation by RL, as they were abolished by an H+-pump inhibitor. BL increased also the activity KD channels, in a voltage-independent manner - in all probability by an independent signaling pathway. Blue-Light (BL), which stimulates shrinking of Flexor cells, evoked the IP3 signaling cascade (detected directly by IP3 binding assay), known to mobilize cytosolic Ca2+. Nevertheless, cytosolic Ca2+ . did not activate the KD channel in excised, inside-out patches. In this study we established a close functional similarity of the KD channels between Flexor and Extensior cells. Thus the differences in their responses must stem from different links to signaling in both cell types.
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Prusky, Dov, Noel Keen, and Rolf Christoffersen. Involvement of Epicatechin in the Regulation of Natural Resistance of Avocado Fruit against Postharvest Pathogens. United States Department of Agriculture, January 1997. http://dx.doi.org/10.32747/1997.7613028.bard.
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In this project it was found that the activation of the mechanism of resistance in avocado fruits to Colletotrichum gloeosporioides depends on the increase of the level of the preformed antifungal diene. This increase is regulated by the synthesis of the flavonoid epicatechin present in the fruit peel. Epicatechin is an inhibitor of the enzyme lipoxygenase whose activity catalyze the breakdown of the antifungal diene. Increase in epicatechin concentration inhibits the breakdown of the antifungal compound and since the compound is continuously synthesized, both combined processes result in the increase of the antifungal level. Biotic and abiotic elicitors affecting the mechanism of resistance, all activate the synthesis of epicatechin. As abiotic elicitors were tested wounding, ethylene and CO2 treatments. As biotic elicitors were tested challenge inoculation with C. gloeosporioides, Colletotrichum magna (a non pathogen of avocado) and also non pathogenic strain of C. magna. In all the cases activation of the key enzymes of the phenylpropanoic pathway is followed by an enhance in the level of epicatechin and the antifungal diene. In order to determine the level of regulation by the different elicitors of the mechanism, the genes encoding for key enzymes of the phenylpropanoic pathway were cloned and it was found that the different elicitors regulate the expression of those genes at a translational level. Modulation of the mechanism of resistance could also be done by activation of lipoxygenase gene expression. For this purpose lipoxygenase from avocado was cloned and its over-expression, under the effect of methyl jasmonate, determined.
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Chamovitz, Daniel A., and Zhenbiao Yang. Chemical Genetics of the COP9 Signalosome: Identification of Novel Regulators of Plant Development. United States Department of Agriculture, January 2011. http://dx.doi.org/10.32747/2011.7699844.bard.
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This was an exploratory one-year study to identify chemical regulators of the COP9 signalosome. Chemical Genetics uses small molecules to modify or disrupt the function of specific genes/proteins. This is in contrast to classical genetics, in which mutations disrupt the function of genes. The underlying concept is that the functions of most proteins can be altered by the binding of a chemical, which can be found by screening large libraries for compounds that specifically affect a biological, molecular or biochemical process. In addition to screens for chemicals which inhibit specific biological processes, chemical genetics can also be employed to find inhibitors of specific protein-protein interactions. Small molecules altering protein-protein interactions are valuable tools in probing protein-protein interactions. In this project, we aimed to identify chemicals that disrupt the COP9 signalosome. The CSN is an evolutionarily conserved eight-subunit protein complex whose most studied role is regulation of E3 ubiquitinligase activity. Mutants in subunits of the CSN undergo photomorphogenesis in darkness and accumulate high levels of pigments in both dark- and light-grown seedlings, and are defective in a wide range of important developmental and environmental-response pathways. Our working hypothesis was that specific molecules will interact with the CSN7 protein such that binding to its various interacting proteins will be inhibited. Such a molecule would inhibit either CSN assembly, or binding of CSN-interacting proteins, and thus specifically inhibit CSN function. We used an advanced chemical genetic screen for small-molecule-inhibitors of CSN7 protein-protein interactions. In our pilot study, following the screening of ~1200 unique compounds, we isolated four chemicals which reproducibly interfere with CSN7 binding to either CSN8 or CSN6.
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Granot, David, and Richard Amasino. Regulation of Senescence by Sugar Metabolism. United States Department of Agriculture, January 2003. http://dx.doi.org/10.32747/2003.7585189.bard.
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Research objectives a. Analyze transgenic plants that undergo rapid senescence due to increased expression of hexokinase. b. Determine if hexokinase-induced senescence accelerates natural senescence using senescence specific promoters that drive expression of a reporter gene (GUS) and a cytokinin producing gene (IPT - isopentyl transferase). c. Isolate and analyze plant genes that suppress sugar-induced cell death (SICD) in yeast, genes that potentially are involved in programmed cell death and senescence in plants. Background to the topic Leaf senescence is a regulated process of programmed cell death (PCD) in which metabolites are recycled to other active parts of the plant. Senescence associated genes (SAGs) are expressed throughout leaf senescence. Sugar flux and metabolism is thought to playa fundamental regulatory role in senescence. We found that transgenic tomato plants with high hexokinase activity, the initial enzymatic step of sugar (hexose) metabolism, undergo rapid leaf senescence, directly correlated with hexokinase activity. These plants provide a unique opportunity to analyze the regulatory role of sugar metabolism in senescence, and its relation to cytokinin, a senescence-inhibiting hormone. In addition, we found that sugar induces programmed cells death of yeast cells in direct correlation to hexokinase activity. We proposed to use the sugar induced cell death (SICD) to isolate Arabidopsis genes that suppress SICD. Such genes could potentially be involved in senescence induced PCD in plants. Major conclusions The promoters of Arabidopsis senescence-associated genes, SAG12 and SAGI3, are expressed in senescing tomato leaves similar to their expression in Arabidopsis leaves, indicating that these promoters are good senescence markers for tomato plants. Increased hexokinase activity accelerated senescence and induced expression of pSAG12 and pSAG13 promoters in tomato plants, suggesting that sugar regulate natural senescence via hexokinase. Expression of IPT, a cytokinin producing gene, under pSAG12 and pSAG13 promoters, delayed senescence of tomato leaves. Yet, senescence accelerated by hexokinase was epistatic over cytokinin, indicating that sugar regulation of senescence is dominant over the senescence-inhibiting hormone. A gene designated SFP1, which is similar to the major super family monosaccharide transporters, is induced during leaf senescence in Arabidopsis and may be involved in sugar transport during senescence. Accordingly, adult leaves accumulate sugars that may accelerate hexokinase activity. Light status of the entire plant affects the senescence of individual leaves. When individual leaves are darkened, senescence is induced in the covered leaves. However, whole adult plant placed in darkness show delayed senescence. In a search for Arabidopsis genes that suppress SICD we isolated 8 cDNA clones which confer partial resistance to SICD. One of the clones encodes a vesicle associated membrane protein - VAMP. This is the first evidence that vesicle trafficking might be involved in cell death. Implications Increased hexokinase activity accelerates senescence. We hypothesized that, reduced hexokinase activity may delay senescence. Preliminary experiments using a hexokinase inhibitor support this possible implication. Currently we are analyzing various practical approaches to delay leaf senescence via hexokinase inhibition. .
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Elroy-Stein, Orna, and Dmitry Belostotsky. Mechanism of Internal Initiation of Translation in Plants. United States Department of Agriculture, December 2010. http://dx.doi.org/10.32747/2010.7696518.bard.
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Original objectives Elucidation of PABP's role in crTMV148 IRES function in-vitro using wheat germ extract and krebs-2 cells extract. Fully achieved. Elucidation of PABP's role in crTMV148 IRES function in-vivo in Arabidopsis. Characterization of the physical interactions of PABP and other potential ITAFs with crTMV148 IRES. Partly achieved. To conduct search for additional ITAFs using different approaches and evaluate the candidates. Partly achieved. Background of the topic The power of internal translation via the activity of internal ribosomal entry site (IRES) elements allow coordinated synthesis of multiple gene products from a single transcription unit, and thereby enables to bypass the need for sequential transformation with multiple independent transgenes. The key goal of this project was to identify and analyze the IRES-trans-acting factors (ITAFs) that mediate the activity of a crucifer-infecting tobamovirus (crTMV148) IRES. The remarkable conservation of the IRES activity across the phylogenetic spectrum (yeast, plants and animals) strongly suggests that key ITAFs that mediate its activity are themselves highly conserved. Thus, crTMV148 IRES offers opportunity for elucidation of the fundamental mechanisms underlying internal translation in higher plants in order to enable its rational manipulation for the purpose of agricultural biotechnology. Major conclusions and achievements. - CrTMV IRES requires PABP for maximal activity. This conclusion was achieved by PABP depletion and reconstitution of wheat germ- and Krebs2-derived in-vitro translation assays using Arabidopsis-derived PABP2, 3, 5, 8 and yeast Pab1p. - Mutations in the internal polypurine tract of the IRES decrease the high-affinity binding of all phylogenetically divergent PABPs derived from Arabidopsis and yeast in electro mobility gel shift assays. - Mutations in the internal polypurine tract decrease IRES activity in-vivo. - The 3'-poly(A) tail enhances crTMV148 IRES activity more efficiently in the absence of 5'-methylated cap. - In-vivo assembled RNPs containing proteins specifically associated with the IRES were purified from HEK293 cells using the RNA Affinity in Tandem (RAT) approach followed by their identification by mass spectroscopy. - This study yielded a list of potential protein candidates that may serve as ITAFs of crTMV148 IRES activity, among them are a/b tubulin, a/g actin, GAPDH, enolase 1, ribonuclease/angiogenin inhibitor 1, 26S proteasome subunit p45, rpSA, eEF1Bδ, and proteasome b5 subunit. Implications, both scientific and agriculture. The fact that the 3'-poly(A) tail enhances crTMV148 IRES activity more efficiently in the absence of 5'-methylated cap suggests a potential joint interaction between PABP, the IRES sequence and the 3'-poly(A). This has an important scientific implication related to IRES function in general.
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Grafi, Gideon, and Brian Larkins. Endoreduplication in Maize Endosperm: An Approach for Increasing Crop Productivity. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575285.bard.
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The focus of this research project is to investigate the role of endoreduplication in maize endosperm development and the extent to which this process contributes to high levels of starch and storage protein synthesis. Although endoreduplication has been widely observed in many cells and tissues, especially those with high levels of metabolic activity, the molecular mechanisms through which the cell cycle is altered to produce consecutive cycles of S-phase without an intervening M-phase are unknown. Our previous research has shown that changes in the expression of several cell cycle regulatory genes coincide with the onset of endoreduplication. During this process, there is a sharp reduction in the activity of the mitotic cyclin-dependent kinase (CDK) and activation of the S-phase CDK. It appears the M-phase CDK is stable, but its activity is blocked by a proteinaceous inhibitor. Coincidentally, the S-phase checkpoint protein, retinoblastoma (ZmRb), becomes phosphorylated, presumably releasing an E2F-type transcriptional regulator which promotes the expression of genes responsible for DNA synthesis. To investigate the role of these cell cycle proteins in endoreduplication, we have created transgenic maize plants that express various genes in an endosperm-specific manner using a storage protein (g-zein) promoter. During the first year of the grant, we constructed point mutations of the maize M-phase kinase, p34cdc2. One alteration replaced aspartic acid at position 146 with asparagine (p3630-CdcD146N), while another changed threonine 161 to alanine (p3630-CdcT161A). These mutations abolish the activity of the CDK. We hypothesized that expression of the mutant forms of p34cdc2 in endoreduplicating endosperm, compared to a control p34cdc2, would lead to extra cycles of DNA synthesis. We also fused the gene encoding the regulatory subunit of the M- phase kinase, cyclin B, under the g-zein promoter. Normally, cyclin B is expected to be destroyed prior to the onset of endoreduplication. By producing high levels of this protein in developing endosperm, we hypothesized that the M-phase would be extended, potentially reducing the number of cycles of endoreduplication. Finally, we genetically engineered the wheat dwarf virus RepA protein for endosperm-specific expression. RepA binds to the maize retinoblastoma protein and presumably releases E2F-like transcription factors that activate DNA synthesis. We anticipated that inactivation of ZmRb by RepA would lead to additional cycles of DNA synthesis.