Ecotropic murine leukemia virus-induced fusion of murine cells.

Extensive fusion occurs upon cocultivation of murine fibroblasts producing ecotropic murine leukemia viruses (MuLVs) with a large variety of murine cell lines in the presence of the polyene antibiotic amphotericin B, the active component of the antifungal agent Fungizone. The resulting polykaryocytes contain nuclei from both infected and uninfected cells, as evidenced by autoradiographic labeling experiments in which one or the other parent cell type was separately labeled with [3H]thymidine and fused with an unlabeled parent. This cell fusion specifically requires the presence of an ecotropic MuLV-producing parent and is not observed for cells producing xenotropic, amphotropic, or dualtropic viruses. Mouse cells infected with nonecotropic viruses retain their sensitivity toward fusion, whereas infection with ecotropic viruses abrogates the fusion of these cells upon cocultivation with other ecotropic MuLV-producing cells. Nonmurine cells lacking the ecotropic gp70 receptor are not fused under similar conditions. Fusion is effectively inhibited by monospecific antisera to gp70, but not by antisera to p15(E), and studies with monoclonal antibodies identify distinct amino- and carboxy-terminal gp70 regions which play a role in the fusion reaction. The enhanced fusion which occurs in the presence of amphotericin B provides a rapid and sensitive assay for the expression of ecotropic MuLVs and should facilitate further mechanistic studies of MuLV-induced fusion of murine cells.     

Variation in the transition from inside to outside work in the red harvester ant Pogonomyrmex barbatus

Summary. In laboratory colonies of the red harvester ant, Pogonomyrmex barbatus, we observed the sequence of tasks performed by marked individuals. Observations of about 760 ants in three laboratory colonies indicate that ants often move from inside to outside work. However, there was a great deal of variation in the sequence. If trends in sequence were weak because ants do move from inside to outside work but the duration of our observations was too short to see the transition, ants should be observed to stay either inside or outside. There was no significant tendency for ants to persist in inside or outside work, indicating the variability in sequence is real. Ants tended to perform midden work before they died. Foraging activity is low in laboratory colonies, and it may be that ants that would be foragers in the field end up as midden workers in the laboratory. High variability in task sequence, in uniform laboratory conditions, contrasts with the apparently more consistent sequence from inside to outside work in the field. This suggests that requirements imposed by variable external conditions and colony needs in the field have a strong influence on task sequence.Received 7 May 2004; revised 11 November 2004; accepted 18 November 2004.     

Time courses of calcium and calcium-bound buffers following calcium influx in a model cell.

Fixed and diffusible calcium (Ca) buffers shape the spatial and temporal distribution of free Ca following Ca entry through voltage-gated ion channels. This modeling study explores intracellular Ca levels achieved near the membrane and in deeper locations following typical Ca currents obtained with patch clamp experiments. Ca ion diffusion sets an upper limit on the maximal average Ca concentration achieved near the membrane. Fixed buffers restrict Ca elevation spatially to the outermost areas of the cell and slow Ca equilibration. Fixed buffer bound with Ca near the membrane can act as Ca source after termination of Ca influx. The relative contribution of fixed versus diffusible buffers to shaping the Ca transient is determined to a large extent by the binding rate of each buffer, with diffusible buffer dominating at equal binding rates. In the presence of fixed buffers, diffusible buffers speed Ca equilibration throughout the cell. The concentration profile of Ca-bound diffusible buffer differs from the concentration profile of free Ca, reflecting theoretical limits on the temporal resolution which can be achieved with commonly used diffusible Ca indicators. A Ca indicator which is fixed to an intracellular component might more accurately report local Ca concentrations.     

Nitrogen and oxygen isotopes in nitrate in the groundwater and surface water discharge from two rural catchments: implications for nitrogen loading to coastal waters

In Prince Edward Island, Canada, widespread intensive potato production has contributed to elevated nitrate concentrations in groundwater and streams, and eutrophic or anoxic conditions occur regularly in several estuarine systems. In this research, the stable isotopes of nitrogen and oxygen in nitrate in intertidal groundwater discharge and stream water were used, in conjunction with water quality and quantity data and land use information, to better understand the characteristics of nitrate delivered to two small estuaries with contrasting land use in their contributory catchments. Most of the water samples collected during the two-year study had isotopic signatures that fell in the range expected for nitrate derived from ammonium-based fertilizers (26.5 % of the samples) or in the overlapping range formed between ammonium-based fertilizers and nitrate derived from soil (64 % of the samples). Overall, isotopic signatures spanned over relatively narrow ranges, and correlations with other water quality parameters, or catchment characteristics, were weak. Nitrate in groundwater discharge and surface water in the Trout River catchment exhibited significantly different isotopic signatures only for the nitrogen isotope, while in the McIntyre Creek catchment groundwater discharge and surface water had similar isotopic signatures. When the isotopic results for the waters from the two catchments were compared, the surface waters were found to be similar, while the isotopic signatures of nitrate in groundwater were distinct only for the nitrogen isotope. Denitrification in the two study catchments was not evident based on the isotopic results for nitrate; however, in the case of the Trout River catchment, where a small freshwater pond exists, an average nitrate load reduction of 14 % was inferred based on a comparison of nitrate loads entering and leaving the pond. Overall, it appears that natural attenuation processes, occurring either in the streams or groundwater flow systems, do not significantly reduce nitrate loading to these estuaries.     

Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy

Nitric oxide (NO) has been demonstrated to mediate events during ovulation, pregnancy, blastocyst invasion and preimplantation embryogenesis. However, less is known about the role of NO during postimplantation development. Therefore, in this study, we explored the effects of NO during vascular development of the murine yolk sac, which begins shortly after implantation. Establishment of the vitelline circulation is crucial for normal embryonic growth and development. Moreover, functional inactivation of the endodermal layer of the yolk sac by environmental insults or genetic manipulations during this period leads to embryonic defects/lethality, as this structure is vital for transport, metabolism and induction of vascular development. In this study, we describe the temporally/spatially regulated distribution of nitric oxide synthase (NOS) isoforms during the three stages of yolk sac vascular development (blood island formation, primary capillary plexus formation and vessel maturation/remodeling) and found NOS expression patterns were diametrically opposed. To pharmacologically manipulate vascular development, an established in vitro system of whole murine embryo culture was employed. During blood island formation, the endoderm produced NO and inhibition of NO (L-NMMA) at this stage resulted in developmental arrest at the primary plexus stage and vasculopathy. Furthermore, administration of a NO donor did not cause abnormal vascular development; however, exogenous NO correlated with increased eNOS and decreased iNOS protein levels. Additionally, a known environmental insult (high glucose) that produces reactive oxygen species (ROS) and induces vasculopathy also altered eNOS/iNOS distribution and induced NO production during yolk sac vascular development. However, administration of a NO donor rescued the high glucose induced vasculopathy, restored the eNOS/iNOS distribution and decreased ROS production. These data suggest that NO acts as an endoderm-derived factor that modulates normal yolk sac vascular development, and decreased NO bioavailability and NO-mediated sequela may underlie high glucose induced vasculopathy.     

Life cycle of Amblyomma cooperi (Acari: Ixodidae) using capybaras (Hydrochaeris hydrochaeris) as hosts

The life cycle of Amblyomma cooperi was evaluated under laboratory conditions testing different host species. Larval infestations were performed on chickens (Gallus gallus) and capybaras (Hydrochaeris hydrochaeris). Nymphal infestations were performed on G. gallus, H. hydrochaeris, guinea pigs (Cavia porcellus) and wild mice (Calomys callosus). Infestations by adult ticks were performed only on capybaras. All free-living stages were observed in darkness at 27 degrees C and RH 85%. Capybaras were significantly (p < 0.05) the most suitable hosts for immature ticks, with the highest larval (63.6%) and nymphal (48%) recovery. Larval and nymphal feeding and premolt periods were significantly different (p < 0.05) between ticks fed on different host species. Male nymphs showed premolt period significantly shorter (p < 0.05) than female nymphs. The overall sex ratio of adult ticks was 0.92:1 (M:F). Infestations by adult ticks on capybaras yielded more than 76% of engorged female recovery. Only three out of 33 engorged females fed on capybaras did not lay fertile eggs. The life cycle of A. cooperi in laboratory, reported for the first time, was completed in an average period of 189.4 days. During the premolt period, all A. cooperi engorged nymphs secreted distinct blackish drops, which seem to be inherent to this species. Our results, associated data in the literature, confirm the high suitability of capybaras for the adult stage of A. cooperi and also indicate this animal species as a primary host for immature stages of A. cooperi in nature. On the other hand, the results of larval and nymphal infestation on chickens and guinea pigs suggest that birds and wild guinea pigs, which are also present in the distribution area of A. cooperi in South America, could be potentially infested by A. cooperi immature stages in nature.     

Distinct mechanisms of neutralization by monoclonal antibodies specific for sites in the N-terminal or C-terminal domain of murine leukemia virus SU

The epitope specificities and functional activities of monoclonal antibodies (MAbs) specific for the murine leukemia virus (MuLV) SU envelope protein subunit were determined. Neutralizing antibodies were directed towards two distinct sites in MuLV SU: one overlapping the major receptor-binding pocket in the N-terminal domain and the other involving a region that includes the most C-terminal disulfide-bonded loop. Two other groups of MAbs, reactive with distinct sites in the N-terminal domain or in the proline-rich region (PRR), did not neutralize MuLV infectivity. Only the neutralizing MAbs specific for the receptor-binding pocket were able to block binding of purified SU and MuLV virions to cells expressing the ecotropic MuLV receptor, mCAT-1. Whereas the neutralizing MAbs specific for the C-terminal domain did not interfere with the SU-mCAT-1 interaction, they efficiently inhibited cell-to-cell fusion mediated by MuLV Env, indicating that they interfered with a postattachment event necessary for fusion. The C-terminal domain MAbs displayed the highest neutralization titers and binding activities. However, the nonneutralizing PRR-specific MAbs bound to intact virions with affinities similar to those of the neutralizing receptor-binding pocket-specific MAbs, indicating that epitope exposure, while necessary, is not sufficient for viral neutralization by MAbs. These results identify two separate neutralization domains in MuLV SU and suggest a role for the C-terminal domain in a postattachment step necessary for viral fusion.     

Elevated glucose inhibits VEGF-A-mediated endocardial cushion formation: modulation by PECAM-1 and MMP-2

Atrioventricular (AV) septal defects resulting from aberrant endocardial cushion (EC) formation are observed at increased rates in infants of diabetic mothers. EC formation occurs via an epithelial-mesenchymal transformation (EMT), involving transformation of endocardial cells into mesenchymal cells, migration, and invasion into extracellular matrix. Here, we report that elevated glucose inhibits EMT by reducing myocardial vascular endothelial growth factor A (VEGF-A). This effect is reversed with exogenous recombinant mouse VEGF-A165, whereas addition of soluble VEGF receptor-1 blocks EMT. We show that disruption of EMT is associated with persistence of platelet endothelial cell adhesion molecule-1 (PECAM-1) and decreased matrix metalloproteinase-2 (MMP-2) expression. These findings correlate with retention of a nontransformed endocardial sheet and lack of invasion. The MMP inhibitor GM6001 blocks invasion, whereas explants from PECAM-1 deficient mice exhibit MMP-2 induction and normal EMT in high glucose. PECAM-1-negative endothelial cells are highly motile and express more MMP-2 than do PECAM-1-positive endothelial cells. During EMT, loss of PECAM-1 similarly promotes single cell motility and MMP-2 expression. Our findings suggest that high glucose-induced inhibition of AV cushion morphogenesis results from decreased myocardial VEGF-A expression and is, in part, mediated by persistent endocardial cell PECAM-1 expression and failure to up-regulate MMP-2 expression.     

Use of CC traps with different trap base colors for silverleaf whiteflies (Homoptera: Aleyrodidae), thrips (Thysanoptera: Thripidae), and leafhoppers (Homoptera: Cicadellidae)

During 1996, 1997, and 1999, studies were conducted in cotton, sugar beets, alfalfa, yardlong bean, and peanut fields to compare insect catches in CC traps equipped with different trap base colors. The studies were conducted in southwestern United States, China, and India. The nine colors, white, rum, red, yellow, lime green, spring green, woodland green (dark green), true blue, and black, varied in spectral reflectance in the visible (400-700 nm) and near-infrared (700-1050 nm) portions of spectrum. Lime green, yellow, and spring green were the three most attractive trap base colors for silverleaf whitefly, Bemisia argentifolii Bellows & Perring, and leafhopper, Empoasca spp. adults. The three trap base colors were moderately high in the green, yellow, and orange spectral regions (490-600 nm), resembling the spectral reflectance curve of the abaxial (underleaf) surfaces of green cotton leaves. True blue and white were the most attractive trap base colors for western flower thrips, Frankliniella occidentalis (Pergande), adults. The true blue and white trap bases were moderately high in the blue spectral region (400-480 nm).     

Does Leaf Position within a Canopy Affect Acclimation of Photosynthesis to Elevated CO2? : Analysis of a Wheat Crop under Free-Air CO2 Enrichment

Previous studies of photosynthetic acclimation to elevated CO₂ have focused on the most recently expanded, sunlit leaves in the canopy. We examined acclimation in a vertical profile of leaves through a canopy of wheat (Triticum aestivum L.). The crop was grown at an elevated CO₂ partial pressure of 55 Pa within a replicated field experiment using free-air CO₂ enrichment. Gas exchange was used to estimate in vivo carboxylation capacity and the maximum rate of ribulose-1,5-bisphosphate-limited photosynthesis. Net photosynthetic CO₂ uptake was measured for leaves in situ within the canopy. Leaf contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), light-harvesting-complex (LHC) proteins, and total N were determined. Elevated CO₂ did not affect carboxylation capacity in the most recently expanded leaves but led to a decrease in lower, shaded leaves during grain development. Despite this acclimation, in situ photosynthetic CO₂ uptake remained higher under elevated CO₂. Acclimation at elevated CO₂ was accompanied by decreases in both Rubisco and total leaf N contents and an increase in LHC content. Elevated CO₂ led to a larger increase in LHC/Rubisco in lower canopy leaves than in the uppermost leaf. Acclimation of leaf photosynthesis to elevated CO₂ therefore depended on both vertical position within the canopy and the developmental stage.