Chemical synthesis,expression and mutagenesis of a gene encoding β-cryptogein,an elicitin produced by Phytophthora cryptogea

Elicitins are 10 kDa holoproteins secreted by Phytophthora fungi, that elicit an incompatible hypersensitive reaction, leading to resistance against fungal and bacterial plant pathogens. Comparison of primary sequences of -elicitins and -elicitins indicated several potential necrotic activity-determining residues. All of the highly necrotic -elicitins have a hydrophilic residue (usually lysine) at position 13, whereas in the less necrotic -elicitins this residue is replaced by a valine. Here, we report the synthesis and expression of a gene encoding a highly necrotic elicitin, -cryptogein, and we show that the substitution of Lys-13 of this recombinant protein by a valine leads to a drastic alteration to the necrotic activity of the recombinant protein.     

The efficiency of patch sampling for determination of relative tick burdens in comparison with total tick counts

Quanttative data on host tick burdens ar fundamental for the initiation of control strategies and effective management of wildlife populations, but the methods of live sampling employed for domestic animals are unsuitable for sampling wild animals. Despite advances in the use of destructive methods (the scrub and digestion techniques) to obtain measures of the total tick burden on wildlife, these methods are too involved for many field workers, who often need only measures of relative tick burden. Recently, patch sampling methods have been introduced whereby only certain predilection sites are sampled, the presumption being that the number of ticks collected gives an indiccation of the relative degree of infestation. We examined the validity of patch sampling as a measure of relative tick burden by comparing adult ticks collected from the ears, head, neck, foreleg and perianal region of impala (aepyceros melampus) with total tick burdens of the same animals derived from the digestion technique. Adult ticks from patch sampling were positively and significantly correlated with total adults and total ticks (larvae, nymphs, and adults) on impala, with ticks patch sampled from the neck showing the highest correlation with the total tick burden. Comparison of relative tick loads from patch sampling with absolute tick loads from digestion for three classes of impala (females, bachelor males and territorial males) gave qualitatively similar results. We conclude that, when measures of relative tick load are sufficient and destructive sampling is not feasible, patch sampling can provide reliable information on relative tick burdens that are positively correlated with the total tick burden.     

Purification and structural characterization of insulin from a caecilian, Typhlonectes natans (Amphibia: Gymnophiona)

Despite the important position of amphibia in phylogeny, efforts at the structural characterization of amphibian neurohormonal peptides have largely been confined to the Anurans (frogs and toads). Insulin was purified from an extract of the pancreas of the caecilian, Typhlonectes natans. The primary structure of the peptide was established as:

A-chain: Gly-Ile-Val-Glu-Lys⁵-Cys-Cys-Leu-Ser-Thr¹⁰-Cys-Ser-Leu-Tyr-Glu¹⁵-Leu-Glu-Ser-Tyr-Cys²⁰-Asn

B-chain: Ile-Ala-Asn-Gln-His⁵-Leu-Cys-Gly-Ser-His¹⁰-Leu-Val-Glu-Ala-Leu¹⁵-Tyr-Leu-Val-Cys-Ala²⁰-Asp-Arg-Gly-Phe- Phe²⁵-Tyr-Thr-Pro-Lys-Ser³⁰

This amino acid sequence contains several unusual substitutions (Gln → Lys at A5, His → Leu at A8, Gln → Glu at A15, and Gly → Ala at B20) that are not present in other amphibian insulins. The structure of insulin appears to be less well conserved among the different orders of amphibia, compared with reptiles and birds.     

Maintenance of two genetic entities by habitat selection

Summary In the laboratory, the two species of copepodsLepeophtheirus thompsoni andLepeophtheirus europaensis, ectoparasites of flatfishes, can meet and mate on at least one host species. In the wild however, these two species are found isolated on their sympatric hosts. Habitat selection theoretically represents a powerful enough mechanism to explain the maintenance of genetic heterogeneity in the wide sense. In this paper, the host colonization process is studied for both parasite species. It is shown that each parasite can develop and reach adult age on each host species. However,L. thompsoni is highly selective; it almost totally refuses to colonize hosts other than its natural one.Lepeophtheirus europaensis, on the contrary, readily infests turbot and brill in single-host experiments, but strongly prefers the brill when it has a choice. It appears that these two genetic entities are sympatrically maintained due to strong habitat selection. Such a pattern could theoretically only occur in a soft-selection context (density dependence). This point is discussed with respect to the different patterns in host use found in the geographical distribution of these parasites.     

Refuge evolution and the population dynamics of coupled host—parasitoid associations

Summary We have investigated the theoretical consequences of character evolution for the population dynamics of a host—parasitoid interaction, assuming a monophagous parasitoid. In the purely ecological model it is assumed that hosts can escape parasitism by being in absolute refuges. A striking property of this model is a threshold effect in control of the host by the parasitoid, when host density dependence is weak. The approximate criteria for the parasitoid to regulate the host to low densities are (1) that the parasitoid's maximum population growth rate should exceed the host's and (2) that the maximum growth rate of the host in the refuge should be less than unity. We then use this ecological framework as a basis for a model which considers evolutionary changes in quantitative characters influencing the size of the absolute refuge. For each species, an increase in its refuge-determining character comes at a cost to maximum population growth rate. We show that refuge evolution can substantially alter the population dynamics of the purely ecological model, resulting in a number of emergent and sometimes counter-intuitive properties. In general, when the host has a high carrying capacity, systems are polarized either with low or minor refuge and top-down control of the host by the parasitoid or with a refuge and bottom-up control of the host by a combination of its own density dependence and the parasitoid. A particularly tantalizing result is that co-evolutionary dynamics can modify ecologically unstable systems into ones which are either stable or quasi-stable (with bouts of unstable dynamics, punctuating long-term periods of quasi-stable behaviour). We present five quantitative criteria which must all be met for the parasitoid to be the agent responsible for control of the host at a co-evolutionary equilibrium. The apparent stringency of this full set of requirements supports the empirically-based suggestion that monophagous parasitoid-driven systems should be less common in nature than those driven by multiple forms of density dependence. Further, we apply our theory to the question of whether exploiters may harvest their victims at maximum sustainable yields and to the evolutionary stability of biological control. Finally, we present a series of testable predictions of our theory and methods useful for testing them.     

Radiative properties of hardwood leaves to ultraviolet irradiation

Spectral reflectance and transmittance of leaves to ultraviolet irradiation were determined under laboratory conditions for seven species of hardwood trees, namely red oak (Quercus rubra, L), black oak (Q. velutina, Lamarch), white oak (Q. alba, L.), sugar maple (Acer saccharum), Norway maple (A. plantanoides), hickory (Carya tomemtosa), sweetgum (Liquidambar styraciflua), and black oak litter. The experimental system consisted of a solar simulator, an integrating sphere, and a spectroradiometer. Experiments were repeated three to five times for both adaxial and abaxial surfaces of fresh leaves chosen at randomly. The spectral distributions and simple averages of the radiative properties in the wavelength ranges of ultraviolet-B (UV-B, 280–320 nm) and ultraviolet-A (UV-A, 320–400 nm) were determined. The spectral distributions of reflectance were similar between adaxial and abaxial surfaces, although the magnitude varied among tree species. Leaf reflectance was very low for the ultraviolet spectrum in general and varied among species and between adaxial and abaxial surfaces. It was generally higher over the UV-A waveband compared to UV-B, and higher on the abaxial than adaxial surface. The broadband reflectance in the UV-A range (over all species) was 5.0 and 3.9% for abaxial and adaxial surface, respectively, compared to 3.5 and 2.8% in UV-B. The transmittance through leaves was extremely small in the UV-B (<0.1%) and nearly zero in the UV-A spectral range. Consequently, the absorptance of ultraviolet radiation by leaves, as determined from the measured reflectance and transmittance, was quite high, being more than 90% for all the combinations of species and wavebands examined. The reported results are useful for studies requiring spectral radiative properties of the examined leaves with respect to ultraviolet irradiation.     

Substance P-related antagonists inhibit vasopressin and bombesin but not 5′-3-O-(thio) triphosphate-stimulated inositol phosphate production in swiss 3T3 cells

The substance P (SP) analogues [DArg¹, DPhe⁵, DTrp^7,9, Leu¹¹] SP (AntD) and [Arg⁶, DTrp^7,9, MePhe⁸] SP (6–11) (AntG) inhibit the action of many different neuropeptides including SP. These analogues might be useful in the treatment of small cell lung cancer but their mechanism of action is unclear. Here, we analyzed the effect of AntD and AntG on neuropeptide vs. guanosine 5′-3-O-(thio) triphosphate (GTPγS) stimulated inositol phosphate generation in permeabilized Swiss 3T3 cells. AntD inhibited vasopressin and bombesin stimulated inositol phosphate formation (IC₅₀ of 0.75 μM and 2 μM, respectively). Similarly, AntG inhibited vasopressin-stimulated inositol phosphate generation with an IC₅₀ of 1 μM. Strikingly, neither AntD up to 10 μM nor AntG up to 20 μM was able to inhibit GTPγS-stimulated inositol phosphate generation. Dose-response curves of neuropeptide-induced inositol phosphate generation were dramatically displaced to the right by either 10 μM AntD or 20 μM AntG. However, neither antagonist affected the dose response of GTPγS-stimulated inositol phosphate generation. Furthermore, 20 μM AntD had no effect on AIF^?₄-induced inositol phosphates in COS-1 cells transfected with G_αq. AntD inhibited [³H]vasopressin binding competitively in intact Swiss 3T3 cells and both AntD and AntG inhibited [³H]vasopressin binding in Swiss 3T3 and rat liver membranes. Scatchard analysis revealed that AntD inhibited vasopressin binding by reducing receptor affinity without affecting receptor number in both intact and membrane preparations of Swiss 3T3 cells. The results strongly suggest that SP analogues AntD and AntG block the action of the Ca²⁺ mobilizing neuropeptides at the receptor level, rather than inhibiting G protein-stimulated inositol phosphate production. © 1995 Wiley-Liss, Inc.     

Contribution of increased glutathione content to mechanisms of oxidative stress resistance in hydrogen peroxide resistant hamster fibroblasts

An H₂O₂-resistant variant (OC14) of the HA1 Chinese hamster fibroblast cell line, which demonstrates cross resistance to 95% O₂ and a 2-fold increase in total glutathione content, was utilized to investigate mechanisms responsible for cellular resistance to H₂O₂- and O₂-toxicity. OC14 and HA1 cells were pretreated with buthionine sulfoximine (BSO) to deplete total cellular glutathione. Following BSO pretreatment, cells were either placed in 250 μM BSO to maintain the glutathione depleted condition and challenged with 95% O₂, or challenged with hydroged peroxide in the absence of BSO. Total glutathione and the activities of CuZn superoxide dismutase, Mn superoxide dismutase, catalase, glutathione peroxidase, and glutathione transferase were evaluated immediately following the BSO pretreatment as well as following 39 to 42 hr of exposure to 250 μM BSO. BSO treatment did not cause significant decreases in any cellular antioxidant tested, except total glutathione depletion resulted in significant (P < 0.05) sensitization to O₂-toxicity and H₂O₂-toxicity in both cell lines at every time point tested. However, glutathione depletion did not completely abolish the resistance to either O₂- or H₂O₂-toxicity demonstrated by OC14 cells, relative to HA1 cells. Also, glutathione depletion did not effect the ability of OC14 cells to metabolize extracellular H₂O₂. These data indicate that glutathione dependent processes significantly contribute to cellular resistance to acute H₂O₂- and O₂-toxicity, but are not the only determinants of resistance in cell lines. The contribition of aldehydes formed by lipid peroxidation in mechanisms involved with the sensitization to O₂-toxicity in glutathione depleted cells was tested by measuring the lipid peroxidation byproduct, 4-hydroxy-2-nonenal (4HNE), bound in Schiff-base linkages or in its free form in cell homogenates at 49 hr of 95% O₂-exposure. No significant increase in 4HNE was detected in glutathione depleted cells relative to glutathione competent cells, indicating that glutathione depletion does not sensitize these cells to O₂-toxicity by altering the intracellular accumulation of free or Schiff-base bound 4HNE. © 1995 Wiley-Liss Inc.     

Striga hermonthica decreases photosynthesis in Zea mays through effects on leaf cell structure

Maize seedlings were grown in pots either with or without preconditionedseeds of the parasitic weed, Striga hermonthica. After between4 and 8 weeks, net photosynthesis in the leaves of maize plantsinfected with Striga decreased compared to leaves of uninfectedcontrol plants. The activities of four enzymes of photosyntheticmetabolism were, however, little affected by infection. A pulse-chaseexperiment using ¹⁴CO₂ showed that C₄ acids were the main earlyproducts of assimilation even when the rate of photosynthesiswas much decreased by infection, but more radio-activity appearedin glycine and serine than in leaves of healthy maize plants.Leaves of infected maize required longer to reach a steady rateof photosynthesis upon enclosure in a leaf chamber than leavesof uninfected plants after similar treatment. Electron microscopy of transverse sections of the leaves ofinfected maize indicated that the cell walls in the bundle sheathand vascular tissue were less robust than in leaves of healthyplants. The results suggest that infection with Striga causesan increase in the permeability of cell walls in the bundlesheath, leakage of CO₂ from the bundle sheath cells and decreasedeffectiveness of C₄ photosynthesis in host leaves. Key words: Zea mays, Striga hermonthica, photosynthesis, photorespiration, enzyme activity