Subtractive expressed sequence tag analysis and screening of cDNA libraries derived from Brassica napus leaves subjected to mechanical wounding, flea beetle feeding or cold temperatures revealed eight genes encoding NAC-domain transcription factors. The genes were found to be differentially regulated in response to biotic and abiotic stresses including wounding, insect feeding, Sclerotinia sclerotiorum infection, cold shock and dehydration. Five BnNAC proteins were orthologous to Arabidopsis thaliana ATAF1 or ATAF2 and gave rise to developmental abnormalities similar to the A. thaliana nam and cuc mutants when expressed ectopically in A. thaliana. Transgenic lines expressing BnNAC14, exhibited large leaves, thickened stems and hyper-developed lateral root systems similar to that observed with A. thaliana NAC1, but also were delayed in bolting and lacked an apical dominant tap root. Several of the BnNAC proteins were capable of activating gene expression in yeast and recognized an element within the CaMV35S promoter. A yeast two-hybrid screen revealed that BnNAC14 interacted with other select BnNAC proteins in vitro and identified an additional BnNAC gene, BnNAC485. The protein interaction and transcriptional activation domains were mapped by deletion analysis.

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.

One- and two-dimensional gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify cDNA encoding a chitin deacetylase (McCDA1) and three insect intestinal lipases (McIIL1, McIIL2 and McIIL3) associated with the Mamestra configurata (bertha armyworm) peritrophic matrix. Recombinant McCDA1 was active and chitin deacetylase activities were detected in the midgut. McCDA1 and the McIIL genes were expressed exclusively in the midgut; however, McCDA1 and McIIL2 were expressed in all larval stages, whereas McIIL1 was expressed mainly in feeding larvae and McIIL3 primarily during the moult.

Insect intestinal mucins (McIIM2-4) expressed in the midgut of feeding, starved and moulting Mamestra configurata larvae were identified. McIIM2 and McIIM4 were associated with the peritrophic matrix (PM). PMs from feeding and starved larvae were translucent and contained organized chitin bundles perpendicular to their long axis, whereas PM from moulting larvae consisted of an inner opaque mass surrounded by an outer translucent sleeve. Serine protease genes (McSP1, McSP2, McSP25 and McSP29) were also expressed in these larvae and several serine proteases were associated with the PM. Serine protease activity was also detected in the midgut of feeding, starved and moulting larvae.

Chronic myocardial ischemia and 2-[18F]fluoro-2-deoxy-D-glucose (FDG) uptake were studied with positron emission tomography in 12 swine instrumented with an external constrictor on the left anterior descending coronary artery (LAD). Serial changes in function (by echocardiography), blood flow (with H215O) and FDG were determined weekly. At 1 wk, function was normal and FDG uptake in the LAD and non-LAD regions was 0.43 +/- 0.12 and 0.45 +/- 0.11 mumol. min-1.g-1, respectively (not significant). At approximately 5 wk, LAD wall thickening decreased to 18 +/- 5 from 27 +/- 8% (P< 0.05), whereas LAD and non-LAD blood flows were 0.68 +/- 0.28 and 1.03 +/- 0.25 ml.min-1.g-1, respectively (P < 0.05). At that time, FDG uptake in LAD and non-LAD regions was 0.60 +/- 0.43 and 0.49 +/- 0.30 mumol.min-1.g-1, respectively (P < 0.05). By the use of transmural biopsies (n = 6), ATP and creatine phosphate in the LAD region were 3.62 +/- 0.73 and 5.91 +/- 1.44 mumol/g wet wt, respectively, and neither differed from values in remote regions. In this model of chronic ischemia, hypoperfused dysfunctional regions were characterized by enhanced glucose uptake and preserved bioenergetics. This supports the concept that the myocardium adapts to chronic ischemia.

No abstract

Field collections of over-wintering and summer adults and nymphs of Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), Lygus borealis (Kelton), and Lygus elisus Van Duzee were made weekly in five fields in Saskatchewan in 1998 and 1999. The crops sampled were alfalfa, Medicago sativa L. (Leguminosae), canola, Brassica napus L. (Cruciferae), and mustard, Sinapis alba L. (Cruciferae), at Vonda, and alfalfa and canola at Saskatoon. In alfalfa, the most abundant Lygus spp. found in May and June were over-wintering adult L. lineolaris and (or) L. borealis; the predominant species in mid-June to early July was L. borealis; and the population from mid-July to late August was dominated by L. lineolaris. In canola, adult populations of Lygus spp. were not found until mid-June. The predominant species, L. lineolaris, probably over-wintering adults, was first detected in canola at the early bud stage in late June to early July; high numbers of L. lineolaris adults occurred in canola in mid-August. Populations of Lygus spp. in organic mustard were negligible. Dissections of field-collected Lygus spp. nymphs revealed parasitism in up to 70% of the midsummer population in alfalfa. In contrast, less than 1% of the late-season Lygus spp. population, primarily L. lineolaris in canola and L. lineolaris and L. borealis in alfalfa, was parasitized.

The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double-stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase-B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase-B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double-stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.