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Yongbo Hu Erin Webb Jasbir Singh Barry A Morgan James A Gainor Thomas D Gordon Teruna J Siahaan 《The Journal of biological chemistry》2002,277(10):8366-8371
The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest. 相似文献
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An order-specific monoclonal antibody to Diptera reveals the impact of alternative prey on spider feeding behavior in a complex food web 总被引:1,自引:0,他引:1
James D. Harwood Mark R. Bostrom Erin E. Hladilek David H. Wise John J. Obrycki 《Biological Control》2007,41(3):397-407
Generalist predators have the capacity to restrict pest population growth, especially early in the season before densities increase. However, their polyphagous feeding habits sometimes translate into reduced pest consumption when they target alternative prey. An order-specific monoclonal antibody was developed to examine the strength of trophic connections between Diptera, a major category of non-pest prey, and linyphiid spiders in alfalfa. We report the development and characterization of a monoclonal antibody with order-level specificity to Diptera. This antibody elicited strong absorbance to 22 Diptera from 13 families, no false-positive reactivity to non-dipteran invertebrates, and antigen detection periods following prey consumption that were comparable between spiders. Over 900 field-collected females of the linyphiid spiders Erigone autumnalis and Bathyphantes pallidus were screened for Diptera antigen. Significantly more B. pallidus screened positive for Diptera (40%) compared to E. autumnalis (16%), indicating differential reliance on these prey. In parallel with the collection of spiders for gut-content analysis, prey availability was estimated at web sites. The two spiders exhibited different feeding responses to prey availability. Consumption of Diptera by B. pallidus was strongly correlated with Diptera abundance whilst the availability of other potential prey did not influence predation rates. Conversely, E. autumnalis did not prey upon Diptera in proportion to availability, but increased Collembola activity-density reduced dipteran consumption. Integration of molecular gut-content analysis with precise sampling of prey demonstrated how two closely related linyphiid spiders exhibit different feeding responses to the availability of prey under natural field conditions. Elucidating the feeding preferences of natural enemies is critical to effective incorporation of biological control by generalist predators in the management of agricultural pests. 相似文献
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J M Katz W G Laver D O White E M Anders 《Journal of immunology (Baltimore, Md. : 1950)》1985,134(1):616-622
The recognition of influenza virus hemagglutinin (HA) by T lymphocytes was examined by assaying the T cell proliferative response of influenza virus-primed T cells to purified HA of different influenza A subtypes or to isolated heavy (HA1) or light (HA2) polypeptide chains of the HA molecule. The proliferative response to HA was dependent on the activation of an Ly-1+2- subset of T cells and required the presence of nylon wool-adherent, radiation-resistant accessory cells. T cells from mice primed by infection with one strain of type A influenza virus cross-reacted with other purified HA not only of the same subtype as the priming virus but also of serologically distinct subtypes of influenza A (but not B) virus. The response of virus-primed T cells to the homologous HA or to HA of the same subtype was shown to involve recognition of determinants on both the HA1 and the HA2 chains. The recognition of HA of different subtype by cross-reactive T cells appeared to be directed predominantly to determinants on HA2. Because the antibody response to influenza virus HA is not cross-reactive between subtypes and is directed predominantly to determinants on HA1, the present results indicate that at least some of the determinants on HA recognized by T cells are different from those recognized by B cells and that the HA2 chain may be involved primarily in stimulation of T cell rather than B cell immunity. 相似文献
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Pollution of freshwater and estuaries 总被引:1,自引:1,他引:0
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Sport hunting has reportedly multiple benefits to economies and local communities; however, few of these benefits have been quantified. As part of their lease agreements with the Zambia Wildlife Authority, sport hunting operators in Zambia are required to provide annually to local communities free of charge i.e., provision a percentage of the meat obtained through sport hunting. We characterized provisioning of game meat to rural communities by the sport hunting industry in Zambia for three game management areas (GMAs) during 2004–2011. Rural communities located within GMAs where sport hunting occurred received on average > 6,000 kgs per GMA of fresh game meat annually from hunting operators. To assess hunting industry compliance, we also compared the amount of meat expected as per the lease agreements versus observed amounts of meat provisioned from three GMAs during 2007–2009. In seven of eight annual comparisons of these GMAs, provisioning of meat exceeded what was required in the lease agreements. Provisioning occurred throughout the hunting season and peaked during the end of the dry season (September–October) coincident with when rural Zambians are most likely to encounter food shortages. We extrapolated our results across all GMAs and estimated 129,771 kgs of fresh game meat provisioned annually by the sport hunting industry to rural communities in Zambia at an approximate value for the meat alone of >US$600,000 exclusive of distribution costs. During the hunting moratorium (2013–2014), this supply of meat has halted, likely adversely affecting rural communities previously reliant on this food source. Proposed alternatives to sport hunting should consider protein provisioning in addition to other benefits (e.g., employment, community pledges, anti-poaching funds) that rural Zambian communities receive from the sport hunting industry. 相似文献
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