GD1b-Derived Gangliosides Modulate Fc��RI Endocytosis in Mast Cells

The role of the mast cell–specific gangliosides in the modulation of the endocytic pathway of FcεRI was investigated in RBL-2H3 cells and in the ganglioside-deficient cell lines, E5 and D1. MAb BC4, which binds to the α subunit of FcεRI, was used in the analysis of receptor internalization. After incubation with BC4-FITC for 30 min, endocytic vesicles in RBL-2H3 and E5 cells were dispersed in the cytoplasm. After 1 hr, the endocytic vesicles of the RBL-2H3 cells had fused and formed clusters, whereas in the E5 cells, the fusion was slower. In contrast, in D1 cells, the endocytic vesicles were smaller and remained close to the plasma membrane even after 3 hr of incubation. When incubated with BC4-FITC and subsequently imunolabeled for markers of various endocytic compartments, a defect in the endocytic pathway in the E5 and D1 cells became evident. In the D1 cells, this defect was observed at the initial steps of endocytosis. Therefore, the ganglioside derivatives from GD1b are important in the endocytosis of FcεRI in mast cells. Because gangliosides may play a role in mast cell–related disease processes, they provide an attractive target for drug therapy and diagnosis.     

Effect of inoculation sequence and nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 growing on human teeth in an artificial mouth

Human teeth in an artificial mouth were inoculated with Streptococcus mutans BHT, Streptococcus mitior LPA-1, or sequentially with both organisms. Incubation was continued for 90 h. Mixed populations were largest when a nutrient supplement containing 5–0% (w/v) sucrose was supplied. Fewer organisms were recovered from experiments with synthetic saliva only, or when a supplement containing 0–05% (w/v) glucose was available. The inoculation sequence determined the total viable count and a larger population resulted when Strep, mutans was the initial colonizer ( P < 0–01). Strep, mutans was always able to become established even when super-infected on to a 24 h plaque of Strep, mitior. The final proportion of Strep mutans was lower when it was the superinfecting organism and the sucrose ( P < 0–01) or glucose ( P < 0–05) nutrient supplement was provided. This work confirms the importance of inoculation sequence and presence of sugars in plaque accumulation and demonstrates the fundamental role of microbial interactions in this process.     

Plant community establishment in a restored wetland: Effects of soil removal

Question: This study investigated the establishment of wetland plant assemblages following soil removal and restored hydrology in a former agricultural field. The following questions were posed. Does plant community composition differ as a result of soil removal? Does soil removal reduce the frequency of non-wetland plants? Does soil removal reduce the frequency of non-native invasive plants? Location: The Panzner Wetland Wildlife Reserve (PWWR) in Summit County, northeastern Ohio, USA. Methods: During 2000–2001, restoration was conducted on two adjoining fields (3.9 ha total) by excavating the upper 40–50 cm of soil layer and establishing 12 10 m × 10 m undisturbed control plots. Preliminary data included seed bank composition and soil organic matter, estimated from three different soil depths on the control plots. In spring 2004, a 10 m × 10 m soil-removed plot was established adjacent to each control plot. Plant percent cover of all species was estimated within the center 5 m × 5 m of every plot. Above-ground biomass of all species from three 0.25-m² quadrats was collected. Environmental water measurements included water depth, temperature, dissolved oxygen, pH, and conductivity. Results: The top 10 cm of soil contained the most seeds, the highest species diversity, the greatest proportion of annual to perennial plants, and the lowest organic content. Obligate and facultative wetland plants were found in soil-removed plots while facultative upland and upland plants were found in control plots. The only plots with arable weeds were the control plots. However, plant communities on soil-removed plots in the North field, which had a higher elevation (ca. 15–20 cm), had a different species composition than soil-removed plots in the South field. Conclusions: The results of a controlled, replicated large-scale study on the effects of soil removal showed that soil removal altered both the biotic and abiotic environment, but that the proximity to the water table was the primary controlling factor in the assembly of plant communities.     

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G¹. This chimeric G specifically infects cells expressing the TVA receptor¹. The gene encoding TVA can been delivered by various methods^2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)² onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.We recently modified this technique to use VSV as a transsynaptic tracer⁹. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.⁴ and Beier et al.⁹ permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).     

Child Health Assessment and Screening Using a Volunteer Staff

A child health assessment and screening program, staffed by volunteers, has evaluated approximately 5,000 children in a general pediatric clinic. A sample of 500 children was studied to determine characteristics of the population served, quality of the work of the volunteers and the number of new problems identified. Use of well-trained volunteers, provided with adequate supervision and follow-up physical examination of the children, identified many new problems at minimal cost and proved an effective means of expanding quality health care.     

Effect of mercury on algal growth rates

In experiments with one freshwater (Chlorella pyrenoidosa) and three marine organisms (Phaeodactylum tricornutum, Cyclotella nana, and Chaetoceros galvestonensis), mecury was more toxic than the other metals tested (silver, cadmium, lead, and copper); and its toxicity is comparatively irreversible. Growth was monitored by changes in fluorescence of the cultures over a 3-day test period. The toxicity of the mercury varied inversely with the concentrations of nutrients present. Preliminary experiments indicate that mercury in the form of mercuric chloride is more than as dimethylmercury.