Effect of different foods on development,reproduction and survival ofPhytoseiulus longipes [Acarina: Phytoseiidae]

The effect of different prey mite species on development of the immature stages and on survival and oviposition of adult predator femalePhytoseiulus longipes Evans was studied.P. longipes completed its development onTetranychus pacificus McGregor,Oligonychus punicae (Hirst), andPanonychus citri (McGregor), but not onPetrobia harti (Ewing). Females oviposited while feeding onT. pacificus andO. punicae, but not onP. citri orP. harti. The highest percent survival was observed onT. pacificus, followed byO. punicae andP. citri. The longevity ofP. longipes onP. harti (vial arenas) and on pollen (leaf arenas) was the same as that on leaves with no food. The provision of 10% sucrose solution and clover honey doubled the longevity ofP. longipes females compared with no food.     

Structure-activity relationships in flexible protein domains: regulation of rho GTPases by RhoGDI and D4 GDI

The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-diphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions of both RhoGDI and D4GDI using a series of NMR experiments which include (15)N relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro and in vivo functional assays have been carried out with N-terminally truncated proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abolish the ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-bound GTPase, but differ in their ability to regulate GTP-bound forms; these functional differences are attributed to the conformational differences of the N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitors. Therefore, the two transient helices, appear to be associated with different biological effects of RhoGDI, providing a clear example of structure-activity relationships in a flexible protein domain.     

Prey stage preference and functional response of Euseius hibisci to Tetranychus urticae (Acari: Phytoseiidae, Tetranychidae)

The aims of this study were: (a) determine the prey stage preference of female Euseius hibisci (Chant) (Phytoseiidae) at constant densities of different stages of Tetranychus urticae Koch (Tetranychidae), (b) assess the functional response of the predator females to the varying densities of eggs, larvae, or protonymphs of T. urticae, and (c) estimate the functional response of E. hibisci when pollen of Ligustrum ovalifolium was present as well. We conducted experiments on excised pieces of strawberry leaf arenas (Fragaria ananassa) under laboratory conditions of 25 ± 2 °C, 60 ± 5% RH and 12 h photophase. Our results indicated that the predator consumed significantly more prey eggs than other prey stages. Consumption of prey deutonymphs and adults was so low that they were excluded from the non-choice functional response experiments. The functional response on all food items was of type II. The two parameters of the functional response were estimated for each prey type by means of the adjusted non-linear regression model. The highest estimated value a (instantaneous rate of discovery) and the lowest value of T_h (handling time, including digestion) were found for the predator feeding on prey eggs, and a was lowest and T_h highest when fed protonymphs. Using the jack-knife method, the values for the functional response parameters were estimated. The values of a and T_h produced by the model were similar among all prey types except for the eggs, which were different. Using pollen simultaneously with prey larvae decreased the consumption of the latter over the full range of prey densities The suitability of this predator for biological control of T. urticae on strawberry is discussed.     

Trehalose favors a cutinase compact intermediate off-folding pathway

The folding of cutinase, an enzyme displaying lipolytic activity, has been studied in the presence of trehalose. Equilibrium unfolding data show that trehalose increases the free energy change between folded and unfolded states. Unfolding kinetics reveal the presence of an intermediate which is ca. 60% folded in terms of solvent exposure. Trehalose stabilizes this intermediate relative to the folded state. In contrast, the intermediate revealed by folding kinetics is more compact than the transition state, as shown by the positive slope observed at low denaturant concentration in the chevron plot, as well as the decrease in the observable rate constant for folding with the increase in trehalose concentration. This intermediate displays more than 50% of area buried from the solvent (relative to the native state) compared to around 40% for the transition state for folding and therefore appears to be off the folding pathway. Trehalose stabilizes and guanidine hydrochloride destabilizes this compact intermediate. Both unfolding and folding kinetics show that compact conformational states are stabilized by trehalose, in agreement with current models on the effect of compatible solutes. This effect occurs even for compact states that decelerate the folding as in the case of the intermediate revealed by folding kinetics.     

L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme.     

Overexpression of NRPS4 leads to increased surface hydrophobicity in fusarium graminearum

The plant pathogen Fusarium graminearum is the infamous cause of Fusarium head blight worldwide resulting in significant losses of yield and reduced grain feed quality. It also has the potential to produce a range of small bioactive peptides produced by the non ribosomal peptide synthetases (NRPSs). Most of these are unknown as F. graminearum contains 19 NRPS encoding genes, but only three have been assigned products. For the first time, we use deletion and overexpression mutants to investigate the functions and product of NRPS4 in F. graminearum. Deletion of NRPS4 homologues in Alternaria brassicicola and Cochloibolus heterostrophus has been shown to result in mutants unable to repel water. In a time study of surface hydrophobicity we observed that water droplets could penetrate 7 d old colonies of the NRPS4 deletion mutants. Loss in ability to repel water was first observed on 13 d old cultures of the wild type strain, whereas the overexpression strain remained water repellant throughout the 38 d time study. The conidia of both mutants were examined and those of the overexpression mutant showed distinct morphological differences in form of collapsed cells. These observations might suggest that the peptide product of NRPS4 could be an architectural factor in the cell walls of Fusarium or an indirect regulator of hydrophobicity.     

A comparative study of development and demographic parameters of <Emphasis Type="Italic">Tetranychus merganser</Emphasis> and <Emphasis Type="Italic">Tetranychus kanzawai</Emphasis> (Acari: Tetranychidae) at different temperatures

We investigated the effect of temperature on development and demographic parameters such as the intrinsic rate of natural increase (r _m) of the two spider mite species Tetranychus merganser Boudreaux and T. kanzawai Kishida at eleven constant temperatures ranging from 15 to 40°C at intervals of 2.5°C. Both male and female T. merganser and T. kanzawai completed development from egg to adult at temperatures ranging from 15 to 37.5°C. The longest developmental duration of immature stages was found at 15°C and the shortest developmental duration was found at 35°C for both species. Using linear and non-linear developmental rate models, the lower thermal thresholds for egg-to-adult (female and male) and egg-to-egg development were estimated as 12.2–12.3°C for T. merganser and as 10.8°C for T. kanzawai. The highest developmental rates were observed at around 35°C, whereas the upper developmental thresholds were around 40°C for both species. In fact, at 40°C, a few eggs of either species hatched, but no larvae reached the next stage. The r _m-values of T. merganser ranged from 0.072 (15°C) to 0.411 day⁻¹ (35°C), whereas those of T. kanzawai ranged from 0.104 (15°C) to 0.399 (30°C). The r _m-values were higher for T. kanzawai than for T. merganser at temperatures from 15 to 30°C, but not at 35°C (0.348 day⁻¹). Total fecundity of T. merganser was also higher than that of T. kanzawai at 35°C. These results indicate that higher temperatures favor T. merganser more than T. kanzawai.     

Direct measurement of the pKa of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: implications for the catalytic mechanism.

The ionization state of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [13Cgamma] aspartic acid and measuring the 13C chemical shifts in the apo, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pKa greater than approximately 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pKa value of less than 4 in this complex. Such a low pKa value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pKa = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 (13)Cgamma resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process.     

Comparative life-history studies on the predaceous mitesTyphlodromus annectens andT. porresi (Acari: Mesostigmata: Phytoseiidae)

Some aspects of the biologies ofTyphlodromus annectens De Leon andT. porresi McMurtry were studied at 25°C. The tetranychid mitesOligonychus punicae (Hirst),Panonychus citri (McGregor) andTetranychus pacificus McGregor were all favorable foods for development ofTy. annectens, whereas a low survival rate of immatures and no oviposition occurred on pollen.Tetranychus pacificus appeared to be a less favorable prey than the other two tetranychids forTy. porresi. Typhlodromus annectens, a very small species, andTy. occidentalis, a medium-sized species used for comparison, consumed significantly moreTe. pacificus larvae than did the medium-sizedTy. porreso. Comparing weights of fed and starved individuals suggested thatTy. occidentalis consumed the greatest volume of food material per time unit. The results are compared with those of previous studies of species in theoccidentalis species group ofTyphlodromus.     

Sphaeroplast and electroporation-mediated transformation of Lactobacillus plantarum

The model described is closely imitative of the natural estuarine environment, yet is simple in construction and easy to maintain. The system is self contained and requires no additional nutrient input. The current model has been in continuous operation for 10 months without replacement of the original sediment, and the sediment structure and major sediment meiofauna have been maintained.