Drosophila ventral furrow morphogenesis: a proteomic analysis

Ventral furrow formation is a key morphogenetic event during Drosophila gastrulation that leads to the internalization of mesodermal precursors. While genetic analysis has revealed the genes involved in the specification of ventral furrow cells, few of the structural proteins that act as mediators of ventral cell behavior have been identified. A comparative proteomics approach employing difference gel electrophoresis was used to identify more than fifty proteins with altered abundance levels or isoform changes in ventralized versus lateralized embryos. Curiously, the majority of protein differences between these embryos appeared well before gastrulation, only a few protein changes coincided with gastrulation, suggesting that the ventral cells are primed for cell shape change. Three proteasome subunits were found to differ between ventralized and lateralized embryos. RNAi knockdown of these proteasome subunits and time-dependent difference-proteins caused ventral furrow defects, validating the role of these proteins in ventral furrow morphogenesis.     

Leaf axil sampling of midwest U.S. maize for mycotoxigenic Fusarium fungi using PCR analysis

PCR analysis was used to detect Fusarium species generically, as well as the mycotoxin-producing species F.␣subglutinans, F. proliferatum, and F. verticillioides in leaf axil and other maize tissues during ear fill in a multiyear study in central Illinois. The frequency of Fusarium detected varied from site to site and year to year. Fusarium was generically detected more frequently in leaf axil material than in leaf/husk lesions. In two growing seasons, the leaf axil samples were also tested for the presence of the mycotoxin producing species F. proliferatum, F. subglutinans, and F. verticillioides. Overall, F. proliferatum and F. verticillioides were detected less often than F. subglutinans. Fusarium was generically and specifically detected most commonly where visible fungal growth was present in leaf axil material. Disclaimer: The mention of firm names or trade products in this article does not imply that they are endorsed or recommended by the United States Department of Agriculture over other firms or similar products not mentioned.     

Indirect reduction of ear molds and associated mycotoxins in Bacillus thuringiensis corn under controlled and open field conditions: utility and limitations

In 1995, ears of a experimental inbred (CG59-2) containing a synthetic Bacillus thuringiensis Cry IA (b) gene driven by PEPC, pith and pollen promoters and artificially infested with Ostrinia nubilalis (Hübner) larvae in small plot studies were free from insect damage, whereas 40-50% of the corresponding non-Bt inbred ears were damaged. Bt inbred ears that were inoculated with Aspergillus flavus Link and Fusarium proliferatum T. Matsushima (Nirenberg) or exposed to natural mold inoculum after infestation with O. nubilalis were free of visible signs of mold, as compared with approximately 30-40% of the non-Bt ears similarly treated. Results in 1996 using the same inbred with a single allele dose of the Bt gene showed similar trends. Mean total fumonisin levels for non-Bt versus Bt inbred ears were not significantly different (2.8 versus 0.8 ppm, respectively) in 1996. In paired hybrid studies run in 0.4-ha (1-acre) fields, an event 176 Bt hybrid had significantly lower amounts of damage and signs of Fusarium spp. mold, but not fumonisin, compared with a corresponding non-Bt hybrid from 1996 to 1998. However, two hybrid pairs that contained either MON810 or Bt11 constructs examined in similar fields at the same site had lower levels of fumonisin in both 1997 (30- to 40-fold) and 1998. High intrafield variability in insect infestation and presence of Helicoverpa zea (Boddie) in Bt hybrids was apparently responsible for fewer significant differences in fumonisin levels in 1998. Similar trends for all three hybrid pairs were noted in small plot trials at another site. Incidence of other ear pests or insect predators varied as much among non-Bt hybrids as they did for Bt/non-Bt hybrid pairs.     

Dusky sap beetles (Coleoptera: Nitidulidae) and other kernel damaging insects in Bt and non-Bt sweet corn in Illinois

Bt and non-Bt sweet corn hybrids (Rogers 'Empire' Bt and non-Bt, respectively) were compared for distribution of kernel damaging insect pests in central Illinois in 1998 and 1999. The occurrence and damage by caterpillars [primarily Helicoverpa zea (Boddie)] were reduced by at least 80% in each year for the Bt compared with the non-Bt hybrid. However, the incidence of sap beetle adults (primarily Carpophilus lugubris Murray) was higher, and larvae, lower for the Bt versus non-Bt in 1999. The incidence of ears with more than five kernels damaged by sap beetles was higher for the Bt compared with non-Bt hybrid in 1998 (13.8 versus 5.5%), but nearly equivalent in 1999 (15.3 versus 15.1%, respectively). Distribution of predators on plants (primarily Coccinelidae) and harvested ears (primarily Orius spp.) were not significantly different on Bt versus non-Bt hybrids. Ears with husks flush with the ear tip or with ear tips exposed had significantly higher sap beetle damage for both hybrids, and the Bt hybrids had significantly higher incidence of exposed ear tips in both years. Sap beetle numbers determined by scouting were often proportional to numbers of beetles captured in baited traps, increasing and decreasing at about the same time. However, values determined with traps were typically less variable than when scouted, and time of sampling was typically four times more rapid for each trap than for each 10 plant scout sample when measured in 1999.     

Modeling Virus Adsorption in Batch and Column Experiments

Experiments with batch suspensions, recirculating columns and flow-through columns have been carried out involving a sandy soil and five bacteriophages: MS2, PRD1, X174, Q and PM2. In batch and recirculating column experiments, attachment and detachment rate coefficients were determined by fitting a two-parameter (attachment and detachment) model. In general, attachment and detachment rate coefficients were not found to be significantly different between the two kinds of experiments. There was one exception, however: MS2 appeared to detach faster in the presence of strong advective flow. In the case of flow-through column experiments, it is shown that a two-site model, with adsorption to equilibrium and kinetic sites, fits the breakthrough curves of all the phages, except PM2, satisfactorily. A one-site kinetic model was found to be appropriate for phage PM2. A small proportion of bacteriophages MS2, PRD1, and Q adsorbed to equilibrium sites, whereas a large proportion of X174 adsorbed to equilibrium sites. Such a distinction between adsorption to equilibrium and kinetic sites cannot be made in the case of batch or recirculating column experiments. Kinetic attachment rate coefficients were found to be significantly higher for the bacteriophages with presumably stronger negative charge. This may be ascribed to the presence of multivalent cations. Under these conditions, bacteriophage X174 appears to behave more conservatively than more negatively charged viruses, and may then be a better choice as a relatively conservative tracer for virus transport through the subsurface.     

Stability studies on the cathepsin L proteinase of the helminth parasite, Fasciola hepatica

Fasciola hepatica, the liver fluke, secretes a cathepsin L cysteine proteinase. The enzyme is active over the pH range 5-9 and is remarkably stable at 37 degrees C, pH 7.0, in contrast to mammalian cathepsin Ls that are active in the acidic pH range and are inactivated within 15 min at neutral pH. The liver fluke proteinase is also very tolerant of organic solvents, particularly dimethylformamide. However, it is completely inactivated by 1 mM Hg(2+) and adversely affected by other heavy metals and divalent cations. Addition of glycerol and EDTA enhanced the liver fluke enzyme's stability at 50 degrees C, while glucose and glycerol protected the enzyme from inactivation by repeated freeze-thawing. The high stability of liver fluke cathepsin L suggests that it may have potential for use in bioindustrial applications.     

Turnover of phosphatidylcholine in Saccharomyces cerevisiae. The role of the CDP-choline pathway

The regulation of phosphatidylcholine degradation as a function of the route of phosphatidylcholine (PC) synthesis and changing environmental conditions has been investigated in the yeast Saccharomyces cerevisiae. In the wild-type strains studied, deacylation of phosphatidylcholine to glycerophosphocholine is induced when choline is supplied to the culture medium and, also, when the culture temperature is raised from 30 to 37 degrees C. In strains bearing mutations in any of the genes encoding enzymes of the CDP-choline pathway for phosphatidylcholine biosynthesis (CKI1, choline kinase; CPT1, 1, 2-diacylglycerol choline phosphotransferase; PCT1, CTP:phosphocholine cytidylyltransferase), no induction of phosphatidylcholine turnover and glycerophosphocholine production is seen in response to choline availability or elevated temperature. In contrast, the induction of phosphatidylcholine deacylation does occur in a strain bearing mutations in genes encoding enzymes of the methylation pathway for phosphatidylcholine biosynthesis (i.e. CHO2/PEM1 and OPI3/PEM2). Whereas the synthesis of PC via CDP-choline is accelerated when shifted from 30 to 37 degrees C, synthesis of PC via the methylation pathway is largely unaffected by the temperature shift. These results suggest that the deacylation of PC to GroPC requires an active CDP-choline pathway for PC biosynthesis but not an active methylation pathway. Furthermore, the data indicate that the synthesis and turnover of CDP-choline-derived PC, but not methylation pathway-derived PC, are accelerated by the stress of elevated temperature.