Comparison of rates of penetration through insect cuticle of amphiphylic analogs of insect pyrokinin neuropeptides

Rates of penetration through the cuticle of amphiphylic analogs, synthesized by addition of 6-phenylhexanoic acid or 9-fluoreneacetic acid or 1-pyrenebutyric acid to the amino terminus of the pentapeptide Phe-Thr-Pro-Arg-Leu-amide, were assessed by quantitative analysis using reversed phase liquid chromatography. The analogs effectively penetrated the cuticle of both the adult American cockroach and tobacco budworm moth. However, the amounts of analogs that penetrated the cuticle of the cockroach were significantly lower and the rates of penetration were slower than for moth cuticle. Penetration of the analogs through the cuticle was dependent upon the size of the lipidic attachment to the pentapeptide. The 6-phenylhexanoic acid analog penetrated most rapidly followed by the 9-fluoreneacetic acid analog and the 1-pyrenebutyric acid analog penetrated slowest. All of the analogs exhibited an initial rapid period of penetration lasting 2-3 h followed by the establishment of a steady slow release state which lasted between 9-24 h and was dependent upon both the size and surface area of the aromatic lipidic portion of the analog and species of insect to which the analog was applied. The results confirmed the hypothesis that the insect cuticle could be employed as a slow release device for delivery of analogs of insect neuropeptides.     

Heterogeneous patterns of variation among multiple human x-linked Loci: the possible role of diversity-reducing selection in non-africans

Studies of human DNA sequence polymorphism reveal a range of diversity patterns throughout the genome. This variation among loci may be due to natural selection, demographic influences, and/or different sampling strategies. Here we build on a continuing study of noncoding regions on the X chromosome in a panel of 41 globally sampled humans representing African and non-African populations by examining patterns of DNA sequence variation at four loci (APXL, AMELX, TNFSF5, and RRM2P4) and comparing these patterns with those previously reported at six loci in the same panel of 41 individuals. We also include comparisons with patterns of noncoding variation seen at five additional X-linked loci that were sequenced in similar global panels. We find that, while almost all loci show a reduction in non-African diversity, the magnitude of the reduction varies substantially across loci. The large observed variance in non-African levels of diversity results in the rejection of a neutral model of molecular evolution with a multi-locus HKA test under both a constant size and a bottleneck model. In non-Africans, some loci harbor an excess of rare mutations over neutral equilibrium predictions, while other loci show no such deviation in the distribution of mutation frequencies. We also observe a positive relationship between recombination rate and frequency spectra in our non-African, but not in our African, sample. These results indicate that a simple out-of-Africa bottleneck model is not sufficient to explain the observed patterns of sequence variation and that diversity-reducing selection acting at a subset of loci and/or a more complex neutral model must be invoked.     

Natural selection on protein polymorphism in the rodent genus Peromyscus: evidence from interlocus contrasts

The effects of natural selection are generally locus-specific, whereas migration, drift, and inbreeding are expected to have relatively uniform effects across the entire genome. This suggests that multilocus surveys of multiple populations can be used to distinguish selection from demographic effects. The purpose of this study was to test for evidence of selection on protein polymorphism in natural populations of mice in the genus Peromyscus. We analyzed published data from geographic surveys of allozyme variation and used a coalescent-based simulation model to identify specific loci that deviated from neutral expectations. Observed F(ST) values generally exhibited a remarkably close fit to the expected neutral distributions, indicating that the majority of loci are simply tracking stochastic demographic processes. A smaller number of loci exhibited highly significant departures from the expectations of the neutral model and thus appear to be tracking the direct or indirect effects of selection. Most departures from neutrality were characterized by F(ST) values that far exceeded neutral expectations and were therefore attributable to spatially varying selection. Interestingly, the albumin locus was implicated as a candidate gene for local adaptation in four different species of Peromyscus. The results also demonstrate that selection can severely bias marker-based estimates of neutral parameters.     

Diuretic and myotropic activities of N-terminal truncated analogs of Musca domestica kinin neuropeptide

Musca kinin (Musdo-K; NTVVLGKKQRFHSWG-NH(2)) and N-terminal truncated analogs of 4-14 residues in length were assayed for diuretic and myotropic activity on housefly Malpighian tubules and hindgut, respectively. The pentapeptide was the minimum sequence required for biological activity, but it was > 5 orders of magnitude less potent than the intact peptide. The pharmacological profiles of the different analogs in the two assays were very similar, suggesting the same receptor is present on both tissues. Potency was little affected by the deletion of Asn(1), but was reduced > 10-fold after the removal of Thr(2). Deletion of the next 5 residues had relatively little effect, but after the second lysyl residue (Lys(8)) was removed potency fell by one to two orders of magnitude. There was a similar drop in potency after the removal of Arg(10), and at 100 microM the pentapeptide had only 20% of the diuretic activity of the intact peptide. The importance of Arg(10) was confirmed by comparing dose-response curves for Musdo-K [6-15] and Acheta kinin-V (AFSHWG-NH(2)) in the diuretic assay; the substitution of arginine by alanine produced a significant reduction in potency and some loss of activity.     

Synthesis of (S)-3-(1-hydroxy-p-carboran-12-yl)alanine, a novel hydrophobic tyrosine-mimetic for peptides

A new, p -carborane containing analog of tyrosine, 3-[1-hydroxy-1,12-dicarba-closo-dodecaboran (12)-12-yl]alanine, was prepared from protected 3-[1-hydroxy-1,12-dicarba-closo-dodecaboran (12)-12-yl]propionic acid in five steps using Oppolzer's sultam methodology for asymmetric hydroxyamination as the key step. The tyrosine mimetic can function as a hydrophobic surrogate for tyrosine residues in insect and mammalian neuropeptides to enhance the lipophilicity, and therefore, the cuticle and/or tissue permeability properties of mimetic analogs. As an amino acid, insertion of the mimic is not limited to the N-terminus but can replace a tyrosine residue at any position within a peptide sequence.     

Inhibition of digestive enzyme release by neuropeptides in larvae of Opisina arenosella (Lepidoptera: Cryptophasidae)

Leucokinins are a group of structurally related neuropeptides stimulating gut motility and fluid secretion by Malpighian tubule in insects. For studying effect of neuropeptides on digestive enzyme release, empty midgut tubes of larvae of Opisina arenosella ligated at both ends with hair were incubated with Leucokinins (LK I-VIII), LK analogues and Leucopyrokinin (LPK) in a bioassay apparatus at 37 degrees C for 30 min. The lumen contents were subsequently analyzed for digestive enzyme levels. The neuropeptides LK III, FFSWG amide, 122 A[1] WP-2, LPK and 434 [phi2] WP-1 inhibited the release of digestive enzymes, protease and amylase while LK VIII, unique in having tyrosine residue, stimulated protease release. The minimum sequence of amino acids at the C-terminal required for activity of LK peptides was found to be FXSWGamide (X=Asn, His, Ser, or Trp). The N-terminal pyroglutamate residue and proline at the C-terminal may contribute to the inhibitory effect of LPK on digestive enzyme release. The present study reveals for the first time an inhibitory effect for leucokinins and pyrokinin on the release of digestive enzymes from the insect midgut.     

Interactions in a tritrophic acarine predator-prey metapopulation system III: effects of Tetranychus urticae (Acari: Tetranychidae) on host plant condition

Spider mites are serious pests on many economically important plant species, because they may reduce plant productivity and, at high mite densities, overexploit and even kill the host plants. We have conducted a series of greenhouse experiments to quantify the effects of two-spotted spider mites (Tetranychus urticae) on host plants (Phaseolusvulgaris). The average amount of chlorophyll per cm² leaf area was used as a measure of plant condition. It was shown that chlorophyll concentration decreases with plant age and intensity of spider mite feeding. Damage caused by spider mites was assessed visually, using the Leaf Damage Index (LDI) defined by, and a mathematical relationship between the visual measurements and the amount of chlorophyll/cm² was fitted to data. The relationship may serve as a short-cut to estimate overall plant injury, expressed as the relative loss of chlorophyll/cm² leaf area caused by spider mites (D). D takes values between 0 (no injury) and 1 (all leaves dead). A highly significant positive relationship between the instantaneous spider mite density and D was found, even though D is expected to reflect the cumulated density of mites (mite-days). A model of plant growth incorporating information about plant age and D predicts that plant area has a maximum when plant age is about 60 days, and that plant area decreases exponentially with an increase in D. This revised version was published online in July 2006 with corrections to the Cover Date.     

Laser-induced fluorescence (LIF) spectra of herbaceous and woody pre- and post-digested plant material

Filtrate from pre- and post-digested plant material was exposed to 355-nm pulsed laser light and the subsequent laser-induced fluorescence (LIF) was recorded. Similarities and differences among spectra from 20 materials are discussed. Each material was replicated once, dried, ground, and exposed to chloroform (CHCl₃) for 24 h. The material represented aged (1 to 18 years old) plants from different herbaceous (grasses and forbs) and woody plant life forms. Mean peak fluorescence recorded among materials differed (P<0.0001) in both wavelength and peak amplitude (counts) across the spectral range (387 to 788 nm). Peak fluorescence was evaluated within each of three arbitrary color categories, blue near 455 nm and red near 674 nm, while only 16 of the materials produced a green peak near 528 nm. In general, the blue and green fluorescence peaks were broad while the red peak was narrow. Mean peak counts were largest in the red range. Varying amounts of laser beam absorption occurred among the materials evaluated due to different concentrations of filtrate and different absorption efficiencies; therefore, amplitude data (counts) were not used to determine statistical differences among materials. To overcome difficulties attributed to the raw count data, red/blue, red/green and blue/green count ratios within replicates were calculated. Using all three count ratios in a multivariate analysis of variance, the 16 materials could be separated into nine different (P<0.05) material groupings. The LIF technique may provide a reliable means to separate ground pre- and post-digested plant materials following further research into determining what fluorophores are producing the spectral signatures and how sample preparation affect peak wavelengths.     

Isolation of human platelet plasma membranes with polylysine beads

Human platelet plasma membranes were isolated with polylysine beads according to the technique developed by Jacobson and Branton (1977, Science [Wash. D. C.] 195:302--304). Lactoperoxidase-catalyzed surface iodination revealed that ninefold greater 125I specific activity was associated with the membranes isolated on beads than with whole platelets. Enrichment in the bead membrane preparation of the activities of membrane marker enzymes, bis(p-nitrophenyl)phosphate phosphodiesterase and Na,K-ATPase, was 8.0 and 4.4, respectively. Contamination with enzymes of other organelles, cytochrome oxidase and beta-glucuronidase, was relatively low as compared with membranes isolated by sucrose gradient centrifugation. Analysis by SDS polyacrylamide gel electrophoresis showed that a full complement of surface glycoproteins was present on the membranes isolated with polylysine beads. The polylysine bead technique is a rapid, reproducible and efficient method for the preparation of relatively pure platelet plasma membranes.