Electrostatic environment at the active site of prolyl oligopeptidase is highly influential during substrate binding

The positive electrostatic environment of the active site of prolyl oligopeptidase was investigated by using substrates with glutamic acid at positions P2, P3, P4, and P5, respectively. The different substrates gave various pH rate profiles. The pKa values extracted from the curves are apparent parameters, presumably affected by the nearby charged residues, and do not reflect the ionization of a simple catalytic histidine as found in the classic serine peptidases like chymotrypsin and subtilisin. The temperature dependence of kcat/Km did not produce linear Arrhenius plots, indicating different changes in the individual rate constants with the increase in temperature. This rendered it possible to calculate these constants, i.e. the formation (k1) and decomposition (k-1) of the enzyme-substrate complex and the acylation constant (k2), as well as the corresponding activation energies. The results have revealed the relationship between the complex Michaelis parameters and the individual rate constants. Structure determination of the enzyme-substrate complexes has shown that the different substrates display a uniform binding mode. None of the glutamic acids interacts with a charged group. We conclude that the specific rate constant is controlled by k1 rather than k2 and that the charged residues from the substrate and the enzyme can markedly affect the formation but not the structure of the enzyme-substrate complexes.     

In silico prediction of peptides binding to multiple HLA-DR molecules accurately identifies immunodominant epitopes from gp43 of Paracoccidioides brasiliensis frequently recognized in primary peripheral blood mononuclear cell responses from sensitized individuals

One of the major drawbacks limiting the use of synthetic peptide vaccines in genetically distinct populations is the fact that different epitopes are recognized by T cells from individuals displaying distinct major histocompatibility complex molecules. Immunization of mice with peptide (181-195) from the immunodominant 43 kDa glycoprotein of Paracoccidioides brasiliensis (gp43), the causative agent of Paracoccidioidomycosis (PCM), conferred protection against infectious challenge by the fungus. To identify immunodominant and potentially protective human T-cell epitopes in gp43, we used the TEPITOPE algorithm to select peptide sequences that would most likely bind multiple HLA-DR molecules and tested their recognition by T cells from sensitized individuals. The 5 most promiscuous peptides were selected from the gp43 sequence and the actual promiscuity of HLA binding was assessed by direct binding assays to 9 prevalent HLA-DR molecules. Synthetic peptides were tested in proliferation assays with peripheral blood mononuclear cells (PBMC) from PCM patients after chemotherapy and healthy controls. PBMC from 14 of 19 patients recognized at least one of the promiscuous peptides, whereas none of the healthy controls recognized the gp43 promiscuous peptides. Peptide gp43(180-194) was recognized by 53% of patients, whereas the other promiscuous gp43 peptides were recognized by 32% to 47% of patients. The frequency of peptide binding and peptide recognition correlated with the promiscuity of HLA-DR binding, as determined by TEPITOPE analysis. In silico prediction of promiscuous epitopes led to the identification of naturally immunodominant epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous patient population exposed to P. brasiliensis. The combination of several such epitopes may increase the frequency of positive responses and allow the immunization of genetically distinct populations.     

Escape from Gregarine Parasites Affects the Competitive Interactions of an Invasive Mosquito

When a species is introduced into a new location, it may escape, at least temporarily, from its natural enemies. In field surveys, we found that when the exotic, invasive mosquito, Aedes albopictus, invades new sites, it initially experiences reduced infection by its gut parasite, Ascogregarina taiwanensis. To determine the effect of this escape from parasitism on the competitive ability of A. albopictus, we performed a laboratory competition experiment in which infected and uninfected A. albopictus larvae were reared in microcosms alone and in competition with larvae of the native mosquito, Ochlerotatus triseriatus. We analyzed the effect of parasitism by A. taiwanensis on A. albopictus performance when subjected to intra- and interspecific competition across a range of larval densities, as well as the effect of A. albopictus parasitism by A. taiwanensis on the competitive impact of A. albopictus on O. triseriatus. At a density of 30 O. triseriatus larvae, O. triseriatus survivorship was significantly reduced by the addition of 30 unifected A. albopictus, but not by addition of 30 infected A. albopictus, and not by addition of 15 A. albopictus whether infected or uninfected. Although estimated finite rate of population increase (') showed similar trends, and was significantly affected by treatments, no pairwise differences in rate of increase were significant. Infection by A. taiwanensis also significantly prolonged A. albopictus female development time and reduced the intraspecific competitive effect of increased density of A. albopictus, but did not affect A. albopictus survivorship, mass, or estimated finite rate of population increase. Thus, when A. albopictus escapes from this parasite as it colonizes new sites, this escape may give it a small, but significant, added competitive advantage over O. triseriatus, which may facilitate range expansion of A. albopictus and enhance A. albopictus's initial impact on resident species.     

Plasticity of insect reproduction: testing models of flexible and fixed development in response to different growth rates

We tested alternative developmental hypotheses describing when during an insect oviposition cycle reproductive tactics are determined. Newly eclosed adult females of the grasshopper Romalea guttata were raised on eight different feeding treatments consisting of a low food diet, a high food diet, and changes from high to low food, or low to high food, at different times during the first oviposition cycle. When initial food availability was high, a decline in food availability >7 days after adult eclosion produced no significant increase in time to oviposition compared to constant high food. In contrast, when initial food availability was low, an increase in food availability as late as day 14 produced a significant decrease in time to oviposition compared to constant low food. Thus, time to oviposition is determined by feeding rate early in the oviposition cycle, but the time of this determination is later when food availability is lower. Masses of individual eggs were unaffected by these treatments. When initial food availability was high, a decrease in food availability on day 21 produced no significant change in numbers of eggs in a clutch compared to constant high food. In contrast, when initial food availability was low, an increase in food availability after day 7 produced no significant change in number of eggs in a clutch compared to constant low food. Changes in egg production resulted from oocyte resorption, which appeared to become unresponsive to food availability between day 14 and day 21. Our results refute the hypothesis that reproductive tactics are continuously flexible. Development toward oviposition seems to be structured so that reproductive tactics become independent of feeding late during the first oviposition cycle. Reproductive tactics become unresponsive to food at different times for groups initially receiving low or high food, suggesting that a particular developmental state, rather than some absolute time, marks the shift to development that is unresponsive to␣food. Plasticity in reproductive tactics appears to be␣controlled by hormones in a manner similar to the hormonal control of plasticity of metamorphosis in other insects. Received: 21 September 1997 / Accepted: 10 March 1998     

Negative effects of habitat drying and prior exploitation on the detritus resource in an ephemeral aquatic habitat

Ephemeral aquatic habitats are characterized by cycles of drying and subsequent inundation, and by production of sequential non-overlapping cohorts of organisms. Both processes may alter the quantity or quality of resources, and may therefore affect survival and development of cohorts that subsequently colonize ephemeral habitats. We examined these effects of habitat drying and non-overlapping cohorts on experimental cohorts of the tree hole mosquito Aedes triseriatus, testing specifically whether the value of leaf litter as a food resource is altered by cycles of inundation and drying, or by exploitation by a prior non-overlapping cohort. We created four treatments of leaf litter: (1) no prior cohort, continuously wet; (2) no prior cohort, one␣wet/dry cycle; (3) prior cohort, continuously wet, and (4) prior cohort, one wet/dry cycle, and tested for effects on individual fitness components (survivorship, mean dry mass at, and median days to eclosion) and on population growth (estimated finite rate of increase –λ′). Both resource drying and the presence of a prior cohort negatively affected individual fitness components in tires, increasing days to eclosion, and decreasing mean dry mass at eclosion for both sexes. Resource drying also negatively affected estimated rates of increase (λ′) in tree holes. A prior cohort had no significant effects on λ′. These results indicate that intraspecific interactions among mosquito larvae may include amensalistic effects of earlier, non-overlapping cohorts, and that resource drying reduces resource quality. The latter effect indicates that enhanced production of A. triseriatus from recently filled containers is not due to resource drying per se, and may result from more complex community-level effects of habitat drying. Extreme cycles of drying and inundation seem likely to increase intraspecific resource competition among drought-adapted species like A. triseriatus. Received: 5 May 1997 / Accepted: 20 January 1998     

Reduction of ortho-aminobenzoyl-proline fluorescence and formation of pyrrolobenzodiazepine-5,11-dione

Summary Theortho-aminobenzoic acid (Abz) group is widely employed as a fluorescent marker for peptides used as substrates for the study of proteolytic enzyme activity. In fact, a direct correlation has been observed between fluorescence intensity and enzyme activity. An unusual behavior of the fluorescence properties of this group, which would lead to erroneous evaluation of the enzyme activity, was observed when it is bound directly to proline. Here we report a systematic NMR, fluorescence and X-ray diffraction study of the compounds obtained from Boc-Abz-Pro-NH₂, Boc-Abz-Pro-OH, as well as from various other Boc-Abz-Pro-X derivatives, after treatment with HCl or TFA under anhydrous conditions. We verified that, as recently reported, even under these synthetic conditions, deprotection of Boc-Abz-Pro-NH₂ or Boc-Abz-Pro-OH leads to the formation of the same product: pyrrolobenzodiazepine-5,11-dione. However, the formation of this compound was not detected with Abz-Pro-N(CH₃)₂, Abz-Pro-Leu-Gly-NH₂ or Abz-pyrrolidine. For all these compounds we observed an unusual behavior for the fluorescence quantum yield of Abz that can be explained as the consequence of a non-radiative deactivation process produced, specifically, by the amidation of the Abz carboxyl group with proline or a similar secondary amine such as pyrrolidine. In conclusion, these results indicate that Abz cannot be used as an internal fluorescence marker for proteolytic enzyme activity when bound directly to proline.     

Effects of habitat drying on size at and time to metamorphosis in the tree hole mosquito Aedes triseriatus

Models of complex life cycles predict that greater mortality of immature stages should induce earlier metamorphosis at smaller sizes. We tested for effects of one source of mortality, habitat drying, on size at and time to metamorphosis of the tree hole mosquito Aedes triseriatus. In a laboratory experiment, we manipulated two variables associated with drying, volume of water and solute concentration, and recorded mean mass at and median days to eclosion for males and females in replicate cohorts. We also tested for treatment effects on the correlation of mass at and time to eclosion. For females, decreasing volume consistently induced metamorphosis at smaller sizes than did constant volume. Decreasing volume also led to earlier metamorphosis of females than did constant volume, but only in one of two experimental runs. For both sexes, increasing concentration led to greater size at metamorphosis and, for males, earlier metamorphosis than did constant volume, but again only in one of two experimental runs. Correlations of size at and time to metamorphosis tended to be positive for females and negative for males, and this difference was significant. For both sexes, decreasing volume led to larger (more positive) correlations than did constant volume, but only in one of two experimental runs. The effects of decreasing volume on females are consistent with the predictions of models of complex life cycles, and suggest that A. triseriatus can perceive volume changes and modify metamorphosis to escape a deteriorating habitat. The effects of increasing concentration are opposite to those predicted, and are consistent with enhanced growth rates, possibly due to enhanced microbial growth, as solutes become concentrated due to drying. The responses of these mosquitoes to habitat drying are complex, and we suggest that habitat drying increases both mortality and growth rates, yielding no simple predictions of how habitat drying will affect these mosquitoes in natural tree holes.     

Some properties of 3-phosphoglycerate phosphatase from developing rice grain

Some properties of 3-P-glycerate phosphatase from developing caryopsis of rice (Oryza sativa L., variety IR26) were studied. The enzyme was found to be soluble and not bound to starch, and concentrated mainly in the pericarp-aleurone layer; its maximum activity was at 12 to 14 days after flowering. Contents of 3-P-glycerate and chlorophyll were highest in the grain at 7 to 8 days after flowering when starch synthesis was at a maximum. The enzyme was purified about 100-fold by precipitation with 50 to 80% ammonium sulfate, followed by chromatography through Sephadex G-200 and CM-Sephadex C-50. The pH optimum was from 5.7 to 6 and no cation was required for activity. The purified preparation had an apparent Km of 2.85 mm and was inhibited by Cu(2+), Hg(2+), Zn(2+), Fe(3+), molybdate, and F(-). The enzyme also exhibited high activity toward UTP, ATP, and p-nitrophenyl phosphate; moderate activity toward other phosphates; but no activity toward phytate. A molecular weight of about 23,000 was obtained for the 3-P-glycerate peak during gel filtration on Sephadex G-200, which corresponded to a value of 26,000 for the major protein fraction by thin layer gel filtration on Sephadex G-150. Zymograms of the whole extract and semipurified preparations showed two phosphatase bands with 3-P-glycerate as substrate.     

Conformations of synthetic tetradecapeptide renin substrate and of angiotensin I in aqueous solution.

The properties of aqueous solutions of synthetic renin substrate tetradecapeptide (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser) were examined through electrometric titrations, infrared and circular dichroism spectroscopy, and spectrofluorometry. Titration studies of angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) were also made, whose results indicated a flexible folded conformation similar to that previously proposed for the octapeptide angiotensin II, with a possible additional beta turn at the C terminus. The experimental results of the tetradecapeptide study, associated with Chou and Fasman calculations and with an analysis of structure-activity relationships in renin substrates and competitive inhibitors, led to the proposal of a beta turn involving the His-Pro-Phe-His sequence of the tetradecapeptide. This beta turn would be stabilized by beta-antiparallel interaction between residues 3-4 and 10-12 and by electrostatic attraction between the N-terminal ammonium and C-terminal carboxylate groups and would be destabilized below pH 5 by electrostatic repulsion between His6 and His9. The capacity to assume this conformation is related to structural requirements for renin substrates and competitive inhibitors.