Inbreeding and incompatibility in Trichogramma nr. brassicae: evidence and implications for quality control

Inbreeding effects and incompatibility relationships were examined in strains of the egg parasitoid Trichogramma nr brassicae (Hymenoptera: Trichogrammatidae) from southeastern Australia. Crosses between strains provided weak evidence of incompatibility in a few cases. However sex ratio in crosses within strains tended to be more female-biased than in crosses between strains. Inbreeding was imposed for four generations (F>0.59) of sib mating. The fitness of inbred strains was compared to that of outbred strains generated by crossing the inbred strains. No effects of inbreeding were found for any of the four female traits examined (fecundity, body length, head width and hind tibia length), indicating that T. nr. brassicae is not subjected to inbreeding depression. Inbreeding effects were also not found for male mating success as expected for the haploid sex. There were differences among strains for all traits apart from fecundity, indicating heritable variation. Strain differences for fitness measures were uncorrelated with wasp size. The potential use of inbreeding in the quality control of Trichogramma for mass-release is discussed. Inbreeding may be a useful tool in minimising the effects of laboratory adaptation, thereby extending the useful life of a strain.     

LIFE-HISTORY ADAPTATION AND REPRODUCTIVE ISOLATION IN A GRASSHOPPER HYBRID ZONE

Patterns of life-history adaptation and reproductive isolation were investigated in the acridid grasshoppers Melanoplus sanguinipes and M. devastator, which hybridize along an altitudinal gradient in the Sierra Nevada of California. Melanoplus sanguinipes females crossed with M. devastator males produced eggs that were approximately half as viable as eggs from other crosses. Diminished viability was not attributable either to infection by Wolbachia pipientis or to failure of sperm transfer. When offered an opportunity to choose a mate, females from all populations discriminated against males of the other species, whereas in no-choice tests measuring copulation duration only females from the tails of the clines showed preferences. Melanoplus sanguinipes, found at high elevations where the growing season is short, exhibited faster egg hatch, faster larval development, smaller adult body sizes, and smaller clutch sizes than M. devastator. Melanoplus devastator, from California's Central Valley, endured a hot and dry summer in a reproductive diapause that was absent in M. sanguinipes. Clines in reproductive diapause and clutch size coincided with the region of reproductive incompatibility. Development time, body size, and hatch time also changed across the hybrid zone, but the regions of largest transitions in these traits were either difficult to locate using the limited populations studied here or were not coincident with the zone's center. A method is described for combining ecological and phylogenetic analyses to address the unknown issue of whether life-history divergence has conributed to reproductive isolation in this system.     

Computed three-dimensional structures for theras-binding domain of theraf-p74 protein complexed withras-p21 and with its suppressor protein, rap-1A

The three-dimensional structures of theras-p21 protein and its protein inhibitor, rap-1A, have been computed bound to theras-binding domain, RBD (residues 55–131), of theraf-p74 protein, a critical target protein ofras-p21 in theras-induced mitogenic signal transduction pathway. The coordinates of RBD have been reconstructed from the stereoview of an X-ray crystal structure of this domain bound to rap-1A and have been subjected to energy minimization. The energy-minimized structures of bothras- p21 and rap-1A, obtained in previous studies, have been docked against RBD, using the stereo figure of the RBD-rap-1A complex, based on a six-step procedure. The final energy-minimized structure of rap-1A-RBD is identical to the X-ray crystal structure. Comparison of theras-p21- and rap-1A-RBD complexes reveals differences in the structures of effector domains ofras-p21 and rap-1a, including residues 32–47, a domain that directly interacts with RBD, 60–66, 96–110, involved in the interaction ofras-p21 withjun kinase (JNK) andjun protein, and 115–126, involved in the interaction of p21 with JNK. The structure of the RBD remained the same in both complexes with the exception of small deviations in its-2 binding loop (residues 63–71) and residues 89–91, also involved in binding to rap-1A. The results suggest that the binding of these two proteins to RBD may allow them to interact with other cellular target proteins such as JNK andjun.     

Water relations and leaf chemistry of Chrysothamnus nauseosus ssp. consimilis (Asteraceae) and Sarcobatus vermiculatus (Chenopodiaceae)

At Mono Lake, California, we investigated field water relations, leaf and xylem chemistry, and gas exchange for two shrub species that commonly co-occur on marginally saline soils, and have similar life histories and rooting patterns. Both species had highest root length densities close to the surface and have large tap roots that probably reach ground water at 3.4-5.0 m on the study site. The species differed greatly in leaf water relations and leaf chemistry. Sarcobatus vermiculatus had a seasonal minimum predawn xylem pressure potential (ψ_pd) of -2.7 MPa and a midday potential (ψ_md) of -4.1 MPa. These were significantly lower than for Chrysothamnus nauseosus, which had a minimum ψ_pd of -1.0 MPa and ψ_md of -2.2 MPa. Sarcobatus had leaf Na of up to 9.1 % and K up to 2.7 % of dry mass, and these were significantly higher than for Chrysothamnus which had seasonal maxima of 0.4% leaf Na and 2.4 % leaf K. The molar ratios of leaf K/Na, Ca/Na, and Mg/Na were substantially lower for Sarcobatus than for Chrysothamnus. Xylem ionic contents indicated that both species excluded some Na at the root, but that Chrysothamnus was excluding much more than Sarcobatus. The higher Na content of Sarcobatus leaves was associated with greater leaf succulence, lower calculated osmotic potential, and lower xylem pressure potentials. Despite large differences in water relations and leaf chemistry, these species maintained similar diurnal patterns and rates of photosynthesis and stomatal conductance to water vapor diffusion. Sarcobatus ψ_pd may not reflect soil moisture availability due to root osmotic and hydraulic properties.     

Effects of chlorogenic acid-and tomatine-fed caterpillars on the behavior of an insect predator

If generalist insect predators are a selective force contributing to patterns of feeding specialization by insect herbivores, then predators should be deterred from eating allelochemical-fed prey. The attack and feeding behaviors of naive predators (Podisus maculiventris stinkbugs) reared on control caterpillars (Manduca sexta) fed plain diet were compared to experienced predators reared on caterpillars fed tomato allelochemicals. Tomatine-fed prey were found more quickly by both naive and tomatine-experienced predators, and chlorogenic acid-experienced predators were more stimulated to begin searching for prey. However, experienced predators were less likely to attack both chlorogenic acidfed and tomatine-fed caterpillars than were naive predators. These results indicate that allelochemical-fed prey were easier for predators to locate, but allelochemical-containing prey often deterred predation by experienced predtors.     

Multidrug resistance evaluation by confocal microscopy in primary urothelial cancer explant colonies

Assessing functional multidrug resistance (MDR) status in clinical biopsy material using drug autofluorescence has potential applications to clinical management. The small size of many cystoscopy specimens has led us to develop, as an alternative to flow cytometry, a protocol for studying epirubicin accumulation in adherent colonies of primary bladder cancer cells viewed live andin situ by confocal microscopy. The limitations to quantitation inherent in this technique are compensated for by preservation of cellular organisation and the elimination of non-malignant cells. Biopsy material is disaggregated and explanted into culture-grade petri dishes. After incubation for three to seven days plaques of epithelial cells have developed. Classical patterns of sensitive and resistant drug distribution are observed. Cells of the rolled edges of the colony accumulate more drug than those of the inner epithelial monolayer. Some central areas of larger colonies give the appearance of drug arrested at the intercellular junctions to give a fenestrated pattern. These observations contribute to the understanding of mechanisms in MDR as well as forming the basis for a clinical urological MDR evaluation protocol.     

Comparison of the computed three-dimensional structures of oncogenic forms (bound to GDP) of theras-Gene-Encoded p21 protein with the structure of the normal (non-transforming) wild-type protein

Theras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions in the polypeptide chain. The most commonly found oncogenic forms contain Val in place of Gly 12 or Leu in place of Gln 61. To determine the effects of these substitutions on the three-dimensional structure of the whole p21 protein, we have performed molecular dynamics calculations on each of these three proteins bound to GDP and magnesium ion to compute the average structures of each of the three forms. Comparisons of the computed average structures shows that both oncogenic forms with Val 12 and Leu 61 differ substantially in structure from that of the wild type (containing Gly 12 and Gln 61) in discrete regions: residues 10–16, 32–47, 55–74, 85–89, 100–110, and 119–134. All of these regions occur in exposed loops, and several of them have already been found to be involved in the cellular functioning of the p21 protein. These regions have also previously been identified as the most flexible domains of the wild-type protein and have been bound to be the same ones that differ in conformation between transforming and nontransforming p21 mutant proteins neither of which binds nucleotide. The two oncogenic forms have similar conformations in their carboxyl-terminal domains, but differ in conformation at residues 32–47 and 55–74. The former region is known to be involved in the interaction with at least three downstream effector target proteins. Thus, differences in structure between the two oncogenic proteins may reflect different relative affinities of each oncogenic protein for each of these effector targets. The latter region, 55–74, is known to be a highly mobile segment of the protein. The results strongly suggest that critical oncogenic amino acid substitutions in the p21 protein cause changes in the structures of vital domains of this protein.     

Conformation of the transmembrane domain of the c-erbB-2 oncogene-encoded protein in its monomeric and dimeric states

The human c-erbB-2 oncogene is homologous to the ratneu oncogene, both encoding transmembrane growth factor receptors. Overexpression and point mutations in the transmembrane domain of the encoded proteins in both cases have been implicated in cell transformation and carcinogenesis. In the case of theneu protein, it has been proposed that these effects are mediated by conformational preferences for anα-helix in the transmembrane domain, which facilitates receptor dimerization, an important step in the signal transduction process. To examine whether this is the case for c-erbB-2 as well, we have used conformational energy analysis to determine the preferred three-dimensional structures for the transmembrane domain of the c-erbB-2 protein from residues 650 to 668 with Val (nontransforming) and Glu (transforming) at position 659. The global minimum energy conformation for the Val-659 peptide from the normal, nontransforming protein was found to contain several bends, whereas the global minimum energy conformation for Glu-659 peptide from the mutant, transforming protein was found to beα-helical. Thus, the difference in conformational preferences for these transmembrane domains may explain the difference in transforming ability of these proteins. The presence of higher-energyα-helical conformations for the transmembrane domain from the normal Val-659 protein may provide an explanation for the presence of a transforming effect from overexpression of c-erbB-2. In addition, docking of the oncogenic sequences in theirα-helical and bend conformations shows that the all-α-helical dimer is clearly favored energetically over the bend dimer.     

Maintenance of Weight Loss: A Needs Assessment

This study identified facilitators and obstacles to maintenance of weight loss following a very-low-calorie-diet and behavior modification program. A survey was mailed to a random sample of 178 program completers and received a 61% response rate; the most frequent follow-up period was more than 2 years. Twenty-nine percent reported weighing the same (within 10 lbs) or less than the end of their participation in the treatment program (maintainers), while 71% reported their present weight was a mean of 65% higher than their initial weight loss (regainers). Maintainers were significantly more likely to report engaging in regular aerobic exercise, attending a maintenance support group, and confidence in their ability to manage their weight in the future, while regainers were more likely to report stress and motivation as frequent weight management obstacles. Respondents consistently identified the need for low/no cost ongoing support. Maintainers and relapsers reported similar challenges in managing their weight, yet with different results, suggesting the need to identify subgroups for which different post-treatment support options could be applied.     

Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.

SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that is mediated, in part, by plasmin. SPARC peptides released specifically by plasmin induce angiogenesis in vivo. We therefore propose that SPARC acts as an intrinsic regulator of angiogenesis in vivo.