Polyoma virus middle-T-transformed PECAM-1 deficient mouse brain endothelial cells proliferate rapidly in culture and form hemangiomas in mice

Wild-type mouse brain endothelial (bEND) cells transformed with the polyoma virus middle-T proliferate rapidly in culture and form hemangiomas in mice. These cells express high levels of platelet/endothelial cell adhesion molecule-1 (PECAM-1), a molecule shown to be important during hemangioma formation. In this study, we have examined the ability of polyoma virus middle-T-transformed mouse bEND cells prepared from PECAM-1-/- mice to proliferate in culture and form hemangiomas in mice. We show that these cells express a number of endothelial cell markers and share a similar morphology with PECAM-1+/+ bEND cells. PECAM-1-/- bEND cells exhibit a limited ability to form tubes in Matrigel and rapidly form hemangioma when injected into nude mice, very similar to PECAM-1+/+ bEND cells. These cells, however, have increased proliferation, slower migration, altered endothelial cell adhesion molecule expression, and are less adherent when compared to PECAM-1+/+ bEND cells. Therefore, lack of PECAM-1 expression impacts polyoma middle-T-transformed endothelial cell proliferative, adhesive, and migratory properties without impacting their ability to rapidly form hemangiomas in mice or poorly organize to capillary-like structures in Matrigel.     

Comparison of Dn4- and Dn7-carrying spring wheat genotypes artificially infested with Russian wheat aphid (Homoptera: Aphididae) biotype 1

Genetic resistance is a useful control strategy for managing Russian wheat aphid, Diuraphis noxia (Mordvilko), in wheat, Triticum aestivum L. In 2003, a Russian wheat aphid population (denoted as biotype 2) identified in Colorado was virulent to genotypes carrying the Dn4 Russian wheat aphid resistance gene, necessitating the rapid identification and deployment of new sources of resistance. Although the Dn7 gene had shown excellent resistance to Russian wheat aphid biotypes 1 and 2 in evaluations in the greenhouse, no information is available on the amount of protection provided by Dn7 under field conditions. The objective of this study was to compare the reaction of Dn4- and Dn7-carrying spring wheat genotypes under artificial infestation by Russian wheat aphid biotype 1 in the field. Irrigated field experiments were conducted in 2003 and 2004 in a split-split plot arrangement with six replications. The whole plot treatment was infestation level (control, 1x, and 10x Russian wheat aphids), and the subplot treatment was resistance source (Dn4- and Dn7-carrying genotypes). The sub-subplot treatment consisted of side-by-side planting of resistant and susceptible genotypes. The Dn4 subplot was significantly more damaged than the Dn7 subplot in 2003, but not in 2004. Interaction effects observed in 2004 suggested an advantage of Dn7 relative to Dn4 in terms of reduced Russian wheat aphid abundance and plant damage. Deployment of the Dn7 Russian wheat aphid resistance gene should provide protection in the field comparable with that provided by the Dn4 resistance gene for management of Russian wheat aphid biotype 1.     

Digestive organ sizes and enzyme activities of refueling western sandpipers (Calidris mauri): contrasting effects of season and age

We examined seasonal and age-related variation in digestive organ sizes and enzyme activities in female western sandpipers (Calidris mauri) refueling at a coastal stopover site in southern British Columbia. Adult sandpipers exhibited seasonal variation in pancreatic and intestinal enzyme activities but not in digestive system or organ sizes. Spring migrants had 22% higher total and 67% higher standardized pancreatic lipase activities but 37% lower total pancreatic amylase activity than fall migrants, which suggests that the spring diet was enriched with lipids but low in glycogen. Spring migrants also had 47% higher total intestinal maltase activity as well as 56% higher standardized maltase and 13% higher standardized aminopeptidase-N activities. Spring migrants had higher total enzymic capacity than fall migrants, due primarily to higher total lipase and maltase activities. During fall migration, the juvenile's digestive system was 10% larger than the adult's, and it was composed differently: juveniles had a 16% larger small intestine but a 27% smaller proventriculus. The juvenile's larger digestive system was associated with lower total enzymic capacity than the adult's due to 20% lower total chitinase and 23% lower total lipase activities. These results suggest that juvenile western sandpipers may process food differently from adults and/or have a lower-quality diet.     

Adhesion of Aureobasidium pullulans is controlled by uronic acid based polymers and pullulan

Aureobasidium pullulans is a potentially pathogenic microfungus that produces and secretes the polysaccharide pullulan and other biomacromolecules, depending on the microbe's physiological state. The role of these macromolecules in mediating adhesion and attachment were examined. Interfacial forces and adhesion affinities of A. pullulans were probed for early-exponential phase (EEP) and late-exponential phase (LEP) cells, using atomic force microscopy (AFM). Biochemical assays showed that A. pullulans produces both pullulan and a uronic acid based polymer. The pullulan is not produced until the LEP, and it can be removed by treatment with pullulanase. Both adhesion forces between the microbe and the AFM tip (silicon nitride) and attachment of the cells to quartz sand grains were controlled by the density of the uronic acid polymer. Uronic acid polymers doubled in density between the EEP and the LEP and were unaffected by the enzyme pullulanase. Retention to quartz in a packed column was quantified using the collision efficiency (alpha), the fraction of collisions between the microbes, and the sand grains, that result in attachment. Adhesion forces and retention on glass were well correlated, with these values being higher for EEP cells (F(adh) = 7.65 +/-4.67 nN; alpha = 1.15) than LEP (F(adh) = 2.94 +/- 0.75; alpha = 0.49) and LEP + pullulanase cells (F(adh) = 2.33 +/-2.01 nN; alpha = 0.43). Steric interactions alone do not describe the adhesion behavior of this fungus, but they do provide information regarding the length and density of the macromolecules studied.     

G350 of Escherichia coli RNase P RNA contributes to Mg2+ binding near the active site of the enzyme

G350 of Escherichia coli RNase P RNA is a highly conserved residue among all bacteria and lies near the known magnesium binding site for the RNase P ribozyme, helix P4. Mutations at G350 have a dramatic effect on substrate cleavage activity for both RNA alone and holoenzyme; the G350C mutation has the most severe phenotype. The G350C mutation also inhibits growth of cells that express the mutant RNA in vivo under conditions of magnesium starvation. The results suggest that G350 contributes to Mg(2+) binding at helix P4 of RNase P RNA.     

A complex role for distal-less in crustacean appendage development.

The developing leg of Drosophila is initially patterned by subdivision of the leg into proximal and distal domains by the activity of the homeodomain proteins Extradenticle (Exd) and Distal-less (Dll). These early domains of gene expression are postulated to reflect a scenario of limb evolution in which an undifferentiated appendage outgrowth was subdivided into two functional parts, the coxapodite and telopodite. The legs of most arthropods have a more complex morphology than the simple rod-shaped leg of Drosophila. We document the expression of Dll and Exd in two crustacean species with complex branched limbs. We show that in these highly modified limbs there is a Dll domain exclusive of Exd but there is also extensive overlap in Exd and Dll expression. While arthropod limbs all appear to have distinct proximal and distal domains, those domains do not define homologous structures throughout arthropods. In addition, we find a striking correlation throughout the proximal/distal extent of the leg between setal-forming cells and Dll expression. We postulate that this may reflect a pleisiomorphic function of Dll in development of the peripheral nervous system. In addition, our results confirm previous observations that branch formation in multiramous arthropod limbs is not regulated by a simple iteration of the proximal/distal patterning module employed in Drosophila limb development.     

Field Evaluation of a Semiautomated Method for Rapid and Simple Analysis of Recreational Water Microbiological Quality

An early warning system using a rapid enzymatic semiautomated method suitable for fecal coliform detection in recreational waters within 8 h was developed further and evaluated in this study. This rapid method was compared to the standard method followed in the United Kingdom. We used 1,011 samples originating from 206 different locations in Wales. When we assessed the presence or absence of fecal coliforms, targeting very low levels of contamination, we obtained 83.9% agreement between the rapid method and the lauryl sulfate broth-membrane filtration technique, whereas direct confirmation of the samples processed by the rapid method showed 89.3% agreement. Environmental enzymatic background activity was found to be the main limiting factor for this method. Owing to a specific and integrated handling of the results by the software of the instrument, the percentage of false-positive results (a consequence of enzymatic background) was successfully limited to 2.9% by the direct confirmation evaluation. However, 7.8% false-negative results due to “late-growers” had to be accepted in order to produce results within a working day. At present, the method can be used in a more conservative way to assess the environmental threshold of 100 CFU of fecal coliforms per 100 ml in recreational waters. The implications of our findings with regard to the applicability of rapid enzymatic methods are discussed.     

Heterogeneous nuclear ribonucleoproteins C1/C2 identified as autoantigens by biochemical and mass spectrometric methods

The antigenic specificity of an unusual antinuclear antibody pattern in three patient sera was identified after separating HeLa-cell nuclear extracts by two-dimensional (2D) gel electrophoresis and localizing the antigens by immunoblotting with patient serum. Protein spots were excised from the 2D gel and their contents were analyzed by matrix-assisted laser desorption-ionization (MALDI) or nanoelectrospray ionization time-of-flight (TOF) tandem mass spectrometry (MS) after in-gel digestion with trypsin. A database search identified the proteins as the C1 and C2 heterogeneous nuclear ribonucleoproteins. The clinical spectrum of patients with these autoantibodies includes arthritis, psoriasis, myositis, and scleroderma. None of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis had similar antibodies. High-resolution protein-separation methods and mass-spectrometric peptide mapping in combination with database searches are powerful tools in the identification of novel autoantigen specificities.