Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

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.     

An order-specific monoclonal antibody to Diptera reveals the impact of alternative prey on spider feeding behavior in a complex food web

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.     

Exclosure fences around nests of imperiled Florida Grasshopper Sparrows reduce rates of predation by mammals

Increasing nest survival by excluding predators is a goal of many bird conservation programs. However, new exclosure projects should be carefully evaluated to assess the potential risks of disturbance. We tested the effectiveness of predator exclosure fences (hereafter, fences) for nests of critically endangered Florida Grasshopper Sparrows (Ammodramus savannarum floridanus) at a dry prairie site (Three Lakes; 2015–2018) and a pasture site (the Ranch; 2015–2016) in Osceola County, Florida, USA. We installed fences at nests an average of 8 days after the start of incubation, and nest abandonment after fence installation was rare (2 of 149 installations). Predation was the leading cause of failure for unfenced nests at both sites (48–73%). At Three Lakes, nest cameras revealed that mammals and snakes were responsible for 61.5% and 38.5% of predation events, respectively, at unfenced nests. Fences reduced the daily probability of predation (0.016 for fenced nests vs. 0.074 for unfenced nests). The probability that a fenced nest would survive from discovery to fledging was more than double that of unfenced nests (60.4% vs. 27.7%). However, we found no difference in daily nest survival at the Ranch between the year before nests were fenced (2015; 0.874) and the year when all but one nest were fenced (2016; 0.867) because red imported fire ants (Solenopsis invicta) were responsible for 86% of predation events at fenced nests at the Ranch. The use of cameras at fenced nests revealed that site‐specific differences in nest predators explained variation in fence efficiency between sites. Our fence design may be useful for other species of grassland birds, but site‐specific predator communities and species‐specific response of target bird species to fences should be assessed before installing fences at other sites.     

Diminished Reovirus Capsid Stability Alters Disease Pathogenesis and Littermate Transmission

Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.     

Effect of alkylresorcin on biological membranes during activation of lipid peroxidation

The effect of alkyl resorcin isolated from the cells of Azotobacter chroococcum and of its structural analog devoid of the alkyl chain (resorcin) on liver microsomes and brain synaptosomes of the rat as well as on rabbit skeletal muscle sarcoplasmic reticulum fragments during activation of lipid peroxidation was studied. Alkyl resorcin was shown to produce a much more potent antioxidant effect as compared with resorcin, since it inhibited lipid peroxidation in all the three types of membranes under study at much lower concentrations. Both alkyl resorcin and resorcin which inhibit lipid peroxidation prevented lipid peroxidation-induced structural-functional damages of synaptosomal and sarcoplasmic reticulum fragment membranes. Unlike resorcin, alkyl resorcin exerted an additional effect on brain synaptosomal membranes which consisted in the stabilization of barrier functions of membranes during incomplete inhibition of lipid peroxidation. The cumulative data suggest that stabilization necessitates the presence of both resorcin radical and alkyl chain in the alkyl resorcin molecule.     

Flavone-8-acetic acid augments systemic natural killer cell activity and synergizes with IL-2 for treatment of murine renal cancer

The investigational drug flavone-8-acetic acid (FAA) potently augments NK activity in the spleen, liver, lungs, and peritoneum in a dose-dependent manner after i.v. or i.p. administration. Augmented NK activity peaks by 24 h after FAA injection and returns to normal after 6 days. Combined treatment of established murine renal cancer with FAA and rIL-2 results in up to 80% long term survival whereas FAA or rIL-2 alone were unable to induce any long term survivors. The optimal dose of rIL-2 required for use with FAA was in the range of 10,000 to 30,000 U/day. Further studies demonstrated that the regimen of FAA plus rIL-2 administration that was effective in treating established murine renal cancer also induced a more potent augmentation of NK activity than did either FAA or rIL-2 alone. Subsequent studies revealed that the therapeutic effectiveness of FAA plus rIL-2 was significantly reduced when tumor-bearing mice were treated with anti-asialo GM1 serum. These results are consistent with a role for augmented NK activity in the therapeutic effects of FAA plus rIL-2 murine renal cancer. In addition, these studies demonstrate that FAA and rIL-2 is a useful approach for cancer treatment in that subtoxic doses of rIL-2 can be used and significant anti-tumor efficacy occurs even without accompanying adoptive immunotherapy.     

Mechanism of stabilization of synaptosomes with alpha-tocopherol during exposure to phospholipase A2

Changes in potential-dependent fluorescence were studied, using fluorescent probe di-S-C3-(5), in synaptosome suspensions exposed to phospholipase A2, alpha-tocopherol and its derivatives. Phospholipase A2 increased potential-dependent fluorescence, i.e. depolarization of synaptosome membranes. The damaging phospholipase A2 effect was prevented and/or abolished by alpha-tocopherol added to synaptosome suspensions before and after phospholipase A2. Alpha-tocopherol derivatives (2,2,5,7,8-pentamethyl-6-hydroxychromane and alpha-tocopheryl-acetate as well as 4-methyl-2,6-di-tert-butylphenol) failed to exert a protective effect on synaptosome membranes modified by phospholipase A2. It is suggested that alpha-tocopherol effect is determined by its interaction with fatty acids, with 6-hydroxy groups of chromanol nucleus and phytol chain being essential for the complex formation.     

Voting with your feet: Payoff biased migration and the evolution of group beneficial behavior

Human migration is nonrandom. In small scale societies of the past, and in the modern world, people tend to move to wealthier, safer, and more just societies from poorer, more violent, less just societies. If immigrants are assimilated, such nonrandom migration can increase the occurrence of culturally transmitted beliefs, values, and institutions that cause societies to be attractive to immigrants. Here we describe and analyze a simple model of this process. This model suggests that long run outcomes depend on the relative strength of migration and local adaptation. When local adaption is strong enough to preserve cultural variation among groups, cultural variants that make societies attractive always predominate, but never drive alternative variants to extinction. When migration predominates, outcomes depend both on the relative attractiveness of alternative variants and on the initial sizes of societies that provide and receive immigrants.