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Substantial Activity (substantial + activity)
Selected AbstractsExpression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensisFEBS JOURNAL, Issue 3 2002Evert Bokma Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the ,1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower kcat and a slightly higher Km than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with ,,2% residual activity. In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower kcat and an approximately twofold higher Km than the wild-type enzyme. More interestingly, it showed activity to higher pH values than the other variants. The X-ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that, compared with wild-type hevamine, the carbonyl oxygen atom of the N -acetyl group of the ,1 sugar residue has rotated away from the C1 atom of that residue. The combined structural and kinetic data show that Asp125,and Tyr183 contribute to catalysis by positioning the,carbonyl oxygen of the N -acetyl group near to the C1 atom. This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis. [source] Specificities of proteases for use in leather manufactureJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 3 2006Farhad Foroughi Abstract Proteases are used in leather manufacture in the processes of soaking, unhairing and bating of hides and skins. However proteases can be relatively non-specific in their usage, and for improved efficacy of enzyme biocatalysis within the industry, an analysis of specific activities of enzymes towards skin proteins was undertaken. Most commercial proteases for soaking showed substantial activity against the substrates elastin,Congo Red and Azocoll but little or no activity against keratin,azure and hide powder black. Enzymes used for unhairing in conjunction with 30% of the usual concentration of sulfide to effect chemical unhairing showed moderate activity against all substrates tested (selected as representative of skin proteins), while proteases used in bating showed activity against Azocoll and elastin,Congo Red but had no keratinase activity and little activity against hide powder black. Bating proteases and soaking proteases displayed similar activities at pH 8. Microbes isolated in the screening of organisms from putrefied skins included one fungal and two bacterial isolates whose extracellular enzymes had efficient unhairing activity without the addition of sulfide. Enzyme activities for these proteases included high activity measured against Azocoll with little or no activity against elastin,Congo Red, keratin,azure and hide powder black. Neither elastase nor keratinase activities were determined as being essential for unhairing. Copyright © 2005 Society of Chemical Industry [source] Contour-advective semi-Lagrangian algorithms for many-layer primitive-equation modelsTHE QUARTERLY JOURNAL OF THE ROYAL METEOROLOGICAL SOCIETY, Issue 596 2004A. R. Mohebalhojeh Abstract For an f -plane many-layer primitive-equation isopycnal model, three ,contour-advective semi-Lagrangian' (CASL) algorithms and a standard pseudo-spectral (PS) algorithm are compared as regards their representation of balance and imbalance during the evolution of a highly complex vortical flow with substantial activity in small horizontal and vertical scales. The three CASL algorithms employ (q,h,,), (q,,,,), and (q,,,,) as their prognostic variables, where q is the Rossby potential vorticity, h is layer thickness, , is horizontal divergence, , and , represent departures of vorticity from, respectively, geostrophic and Bolin,Charney balance. It is demonstrated that the CASL algorithm with (q,,,,) improves on the algorithm with (q,h,,) across nearly the whole range of applicability of the algorithms, i.e. practically up to the limit where overturning and diabatic effects may dominate. Unlike in the PS algorithm, the improvement is achieved without sacrificing the accuracy of the vortical part of the flow by excessive damping. Copyright © 2004 Royal Meteorological Society [source] A randomized study of docetaxel and dexamethasone with low- or high-dose estramustine for patients with advanced hormone-refractory prostate cancerBJU INTERNATIONAL, Issue 3 2006THOMAS NELIUS OBJECTIVE To test the combination of docetaxel with two different doses of estramustine in patients with hormone-refractory prostate cancer (HRPC), to improve response rates and to lower side-effects, as docetaxel-based chemotherapy is an increasing option for men with advanced HRPC, and alone or combined with estramustine, docetaxel improves median survival. PATIENTS AND METHODS In all, 72 patients with metastatic HRPC were randomly assigned to receive docetaxel (70 mg/m2 intravenously, on day 2 every 21 days) and estramustine (3 × 280 mg/day oral starting 1 day before docetaxel, for 5 consecutive days) for arm A, or estramustine (3 × 140 mg/day oral starting 1 day before docetaxel, for 3 consecutive days) for arm B. Premedication with oral dexamethasone at a total daily dose of 16 mg, in divided doses twice a day was administered in arm A on day 1,5 and in arm B on day 1,3. Initially, six cycles were administered. Chemotherapy was restarted after a significant increase in prostate-specific antigen (PSA) level. Patients were monitored for any measurable PSA response and toxicity. RESULTS Between the arms there was no statistically significant difference in time to progression and overall survival. However, treatment B had less treatment-related toxicity than A. Independent prognostic variables were baseline factors like PSA level, haemoglobin level, Eastern Cooperative Oncology Group performance status, and bone pain at presentation. CONCLUSIONS In this randomized phase II study the combination of docetaxel and estramustine had substantial activity in HRPC, with a significant incidence of severe toxicity, both haematological and not. Nevertheless, treatment-related toxicity was predictable and manageable. There was no better effect with a higher dose of estramustine with docetaxel than for a lower dose. There was a slight tendency to higher toxicity for high-dose estramustine but this was not statistically significant. The present results support the assertion that estramustine is not necessary in docetaxel-based treatment regimens. [source] Design of a pH-sensitive pore-forming peptide with improved performanceCHEMICAL BIOLOGY & DRUG DESIGN, Issue 1 2004D.H. Haas Abstract:, GALA is a 30 residue synthetic peptide designed to interact with membranes in a pH-sensitive manner, with potential applications for intracellular drug and gene delivery. Upon reduction of the pH from neutral to acidic, GALA switches from random coil to , -helix, inserts into lipid bilayers, and forms oligomeric pores of defined size. Its simple sequence and well-characterized behavior make the peptide an excellent starting point to explore the effects of sequence on structure, pH sensitivity, and membrane affinity. We describe synthesis and characterization of two derivatives of GALA, termed GALAdel3E and YALA. GALAdel3E has a deletion of three centrally located glutamate residues from GALA, while YALA replaces one glutamate residue with the unusual amino acid 3,5-diiodotyrosine. Both derived peptides retain pH sensitivity, showing no ability to cause leakage of an encapsulated dye from unilamellar vesicles at pH 7.4 but substantial activity at pH 5. Unlike GALA, neither peptide undergoes a conformational change upon reduction of the pH, remaining , -helical throughout. Interestingly, the pH at which the peptides activate is shifted, with GALA becoming active at pH ,5.7, GALAdel3E at pH ,6.2, and YALA at pH ,6.7. Furthermore, the peptides GALAdel3E and YALA show improved activity compared with GALA for cholesterol-containing membranes, with YALA retaining the greatest activity. Improved activity in the presence of cholesterol and onset of activity in the critical range between pH 6 and 7 may make these peptides useful in applications requiring intracellular delivery of macromolecules, such as gene delivery or anti-cancer treatments. [source] | ||||||||||