Progress with schistosome transgenesis

Genome sequences for Schistosoma japonicum and Schistosoma mansoni are now available. The schistosome genome encodes ~13,000 protein encoding genes for which the function of only a minority is understood. There is a valuable role for transgenesis in functional genomic investigations of these new schistosome gene sequences. In gain-of-function approaches, transgenesis can lead to integration of transgenes into the schistosome genome which can facilitate insertional mutagenesis screens. By contrast, transgene driven, vector-based RNA interference (RNAi) offers powerful loss-of-function manipulations. Our laboratory has focused on development of tools to facilitate schistosome transgenesis. We have investigated the utility of retroviruses and transposons to transduce schistosomes. Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) can transduce developmental stages of S. mansoni including eggs. We have also observed that the piggyBac transposon is transpositionally active in schistosomes. Approaches with both VSVG-MLV and piggyBac have resulted in somatic transgenesis and have lead to integration of active reporter transgenes into schistosome chromosomes. These findings provided the first reports of integration of reporter transgenes into schistosome chromosomes. Experience with these systems is reviewed herewith, along with findings with transgene mediated RNAi and germ line transgenesis, in addition to pioneering and earlier reports of gene manipulation for schistosomes.     

Relationships between cytology, bacteriology and vaginal discharge scores and reproductive performance in dairy cattle

The objective was to compare three diagnostic approaches for intrauterine infection and inflammation: scoring of vaginal contents; quantification of percentage of nucleated cells that were polymorphonuclear leukocytes (PMN) following endometrial cytology; and intra-uterine bacteriology. Dairy cows (n = 303) were examined twice, Days 28 (D28) and 42 (D42), where Day 0 = day of calving. Associations between gross vaginal inflammation scores, uterine cytology, and bacteriology, and subsequent reproductive performance were examined using multivariable models. There was fair agreement at D28 (Kappa = 0.29), but only slight agreement at D42 (Kappa < 0.15), between PMN% and gross vaginal inflammation score. Cows were categorized as having PMN% in the highest quartile (H), or not (L), at both D28 and D42; therefore, cows were categorized as PMNLL, PMNLH, PMNHL, or PMNHH. Cows in the highest PMN% quartile at both time periods were slower to conceive (P < 0.001) than those in all other quartiles (mean ± SEM 32.2 ± 2.3, 37.0 ± 5.3, 40.8 ± 4.1, and 55.3 ± 7.3 d from start of breeding to conception for PMNLL, PMNLH, PMNHL, and PMNHH PMN% cows, respectively). Milk yield was greater (P = 0.001) in cows in the lower quartiles for PMN% at D28 and D42 (i.e., PMNLL) than those in the PMNHH and PMNHL categories, with PMNLH intermediate (P = 0.001). We concluded that PMN% was a better predictor of reproductive performance than either intra-uterine bacteriology or gross vaginal inflammation score. Cows in the highest quartile for PMN% at both D28 and D42 had lower pregnancy rates, took longer to conceive, and had a lower milk yield than those in the lower PMN% categories.     

Testing Moran's theorem in an agroecosystem

The abundance and reproductive effort of populations frequently fluctuate across space and time, a phenomenon known as spatial synchrony. Knowledge of the causes of this behavior underlies the ability to manage species, protect the health of humans and the environment, and increase agricultural sustainability. We used an agroecosystem to test Moran's theorem – spatial synchrony results from environmental entrainment. The controlled conditions of the agroecosystem allowed us to create a highly correlated environment while negating the effects of the alternative hypotheses: dispersal and trophic interactions. Under such conditions, synchrony of fruit production by 4288 trees was high over six years in a 32.5 ha pistachio orchard and occurred at similar temporal frequency as weather patterns demonstrating the Moran effect. The spatial synchrony of fruit production was less than the presumed synchrony of the environment supporting research from microcosms and observational studies showing the Moran effect is degraded by local mechanisms. Indeed even under the homogeneous environment of this system, synchrony declined significantly with distance among trees. We present evidence suggesting that the correlation of the local environment affects intrinsic dynamics to cause these patterns. Our findings demonstrate that the Moran effect is, at minimum, partially responsible for the synchronous fruit production in this system. Agroecosystems are often overlooked in basic ecological research; this experiment provides an example of their comparative advantages for the study of some ecological questions.     

Discovery of selective glucocorticoid receptor modulator MK-5932

A series of partial agonists of the Glucocorticoid Receptor were prepared targeting reduced transactivation activity, while maintaining significant transrepression activity. Incorporation of an ortho-aryl amide produced compounds with the desired in vitro profile. Bioreactors consisting of Suspension cultures of Sf21 cells co expressing a CYP3A4 and NADPH-cytochrome P450 oxireductase were used to prepare the major metabolites of these compounds and revealed that oxidative N-dealkylation provided a pathway for formation of metabolites that were more agonistic than the parent partial agonists. Oxidative N-dealkylation was blocked in a new series of compounds, however oxidation alone was capable of producing full agonist metabolites. Incorporation of an ortho-primary amide and utilization of fluorine to modulate agonism afforded partial agonist MK-5932. Synthesis of the major metabolites of MK-5932 using bioreactor technology revealed that no significant GR-active metabolites were formed. Orally administered MK-5932 displayed anti-inflammatory efficacy in a Rat Oxazolone-induced chronic dermatitis model, while sparing plasma insulin.     

DIETS OF COASTAL BOTTLENOSE DOLPHINS FROM THE U. S. MID-ATLANTIC COAST DIFFER BY HABITAT

We recorded 31 species in the stomachs of 146 coastal bottlenose dolphins ( Tursiops truncatus ) from North Carolina, U. S. A. Sciaenid fishes were the most common prey (frequency of occurrence = 95%). By mass, Atlantic croaker ( Micropogonias undulatus ) dominated the diet of dolphins that stranded inside estuaries, whereas weakfish ( Cynosicon regalis ) was most important for dolphins in the ocean. Inshore squid ( Loligo sp.) was eaten commonly by dolphins in the ocean, but not by those in the estuaries. There was no significant pattern in prey size associated with dolphin demography, but the proportion of the diet represented by croaker was higher for males than for females, and mature dolphins ate more croaker than did juveniles. Dietary differences between dolphins that stranded in the estuaries and those that stranded on ocean beaches support the hypothesis that some members of the population inhabit the ocean primarily while others reside principally in estuaries. The overwhelming majority of prey were soniferous species (75% of numerical abundance), which is consistent with the hypothesis that bottlenose dolphins use passive listening to locate noise-making fishes. However, spatiotemporal patterns in consumption of Sciaenid fishes did not coincide with their spawning, which is when peak sound production is thought to occur.     

N-Glycosylation regulates endothelial lipase-mediated phospholipid hydrolysis in apoE- and apoA-I-containing high density lipoproteins

Endothelial lipase (EL) is a member of the triglyceride lipase gene family with high phospholipase and low triacylglycerol lipase activities and a distinct preference for hydrolyzing phospholipids in HDL. EL has five potential N-glycosylation sites, four of which are glycosylated. The aim of this study was to determine how glycosylation affects the phospholipase activity of EL in physiologically relevant substrates. Site-directed mutants of EL were generated by replacing asparagine (N) 62, 118, 375, and 473 with alanine (A). These glycan-deficient mutants were used to investigate the kinetics of phospholipid hydrolysis in fully characterized preparations of spherical reconstituted high density lipoprotein (rHDL) containing apolipoprotein E2 (apoE2) [(E2)rHDL], apoE3 [(E3)rHDL], apoE4 [(E4)rHDL], or apoA-I [(A-I)rHDL] as the sole apolipoprotein. Wild-type EL hydrolyzed the phospholipids in (A-I)rHDL, (E2)rHDL, (E3)rHDL, and (E4)rHDL to similar extents. The phospholipase activities of EL N118A, EL N375A, and EL N473A were significantly diminished relative to that of wild-type EL, with the greatest reduction being apparent for (E3)rHDL. The phospholipase activity of EL N62A was increased up to 6-fold relative to that of wild-type EL, with the greatest enhancement of activity being observed for (E2)rHDL. These data show that individual N-linked glycans have unique and important effects on the phospholipase activity and substrate specificity of EL.     

Architecture of the VE-cadherin hexamer

Vascular endothelial-cadherin (VE-cadherin) is the major constituent of the adherens junctions of endothelial cells and plays a key role in angiogenesis and vascular permeability. The ectodomains EC1-4 of VE-cadherin are known to form hexamers in solution. To examine the mechanism of homotypic association of VE-cadherin, we have made a 3D reconstruction of the EC1-4 hexamer using electron microscopy and produced a homology model based on the known structure of C-cadherin EC1-5. The hexamer consists of a trimer of dimers with each N-terminal EC1 module making an antiparallel dimeric contact, and the EC4 modules forming extensive trimeric interactions. Each EC1-4 molecule makes a helical curve allowing some torsional flexibility to the edifice. While there is no direct evidence for the existence of hexamers of cadherin at adherens junctions, the model that we have produced provides indirect evidence since it can be used to explain some of the disparate results for adherens junctions. It is in accord with the X-ray and electron microscopy results, which demonstrate that the EC1 dimer is central to homotypic cadherin interaction. It provides an explanation for the force measurements of the interaction between opposing cadherin layers, which have previously been interpreted as resulting from three different interdigitating interactions. It is in accord with observations of native junctions by cryo-electron microscopy. The fact that this hexameric model of VE-cadherin can be used to explain more of the existing data on adherens junctions than any other model alone argues in favour of the existence of the hexamer at the adherens junction. In the context of the cell-cell junction these cis-trimers close to the membrane, and trans-dimers from opposing membranes, would increase the avidity of the bond.     

Relative contributions of leaf area ratio and net assimilation rate to change in growth rate depend on growth temperature: comparative analysis of subantarctic and alpine grasses

The present study shows that the relative contributions of leaf area ratio (LAR) and net assimilation rate (NAR) to variation among species in relative growth rate (RGR) depend on growth temperature. We grew three subantarctic and three alpine Poa species at daytime temperatures of 7, 12 and 17 degrees C, and analysed interspecific and temperature-related variation in RGRs by growth analysis. Variation in NAR accounted for most of the interspecific differences in RGR at low growth temperature, whereas variation in both NAR and LAR contributed strongly to interspecific differences in RGR at high growth temperature. For most species, the increase in RGR from 7 to 12 degrees C was attributable to an increase in LAR, whereas the increase in RGR from 12 to 17 degrees C was attributable to an increase in NAR. There were no differences between native subantarctic and alpine species in the plasticity of growth responses to temperature. However, Poa annua, a species introduced to the subantarctic, showed much greater growth plasticity than other species. There was little difference among species in tolerance of high-temperature extremes.