A new tool to sense pH changes at the neuromuscular junction synaptic cleft.

Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shown that pH affects the opening of postsynaptic channels and therefore the production of tools that allow to study these behaviors should result of paramount value. We fused α-bungarotoxin, a neurotoxin derived from the snake Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to the pH sensitive GFP Super Ecliptic pHluorin, and efficiently expressed it in Pichia pastoris. This sensor allows synaptic changes in pH to be measured without the need of incorporating transgenes into animal cells. Here, we show that incubation of the mouse levator auris muscle with a solution containing this recombinant protein is enough to fluorescently label the endplate post synaptic membrane. Furthermore, we could physiologically alter and measure post synaptic pH by evaluating changes in the fluorescent signal of pHluorin molecules bound to acetylcholine receptors. In fact, using this tool we were able to detect a drop in 0.01 to 0.05 pH units in the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electrical stimulation. Further experiments will allow to learn the precise changes in pH during and after synaptic activation.

Collectins and galectins in the abomasum of goats susceptible and resistant to gastrointestinal nematode infection.

Originally described in cattle, conglutinin belongs to the collectin family and is involved in innate immune defense. It is thought that conglutinin provides the first line of defense by maintaining a symbiotic relationship with the microbes in the rumen while inhibiting inflammatory reactions caused by antibodies leaking into the bloodstream. Due to the lack of information on the similar lectins and sequence detection in goats, we characterized the goat conglutinin gene using RACE and evaluated the differences in its gene expression profile, as well as in the gene expression profiles for surfactant protein A, galectins 14 and 11, interleukin 4 and interferon-gamma in goats. We used Saanen and Anglo Nubian F2 crossbred goats monitored over a period of four months and characterized them as resistant (R) or susceptible (S) based on the average values of EPG counts. Goat conglutinin was similar to bovine conglutinin, but its gene expression varied among different tissues. However, as with bovine conglutinin, it was most highly expressed in the liver. Variation in conglutinin (R=24.3±3.9; S=23.5±2.6, p=0.059), protein surfactant A (R=23.8±5.2, S=24.4±2.3, p=0.16), galectin 14 (R=15.9±3.5, S=14.7±6.2, p=0.49) and galectin l1 gene expression (R=25.4±2.6, S=25.8±3.7, p=0.53) was not significant between groups. However, there were weak correlations between interleukin 4 and the protein surfactant A gene (r=0.459, p=0.02) and between interleukin 4 and galectin 11 (r=0.498, p=0.01). Strong correlation between interferon-gamma and galectin 14 (r=0.744, p=0.00) was observed. Galectin 14 was negatively correlated with the number of nematodes in the goat (r=-0.416, p=0.04) as well as the EPG count (r=-0.408, p=0.04). This is the first study to date that identifies the gene expression of conglutinin, surfactant protein A and galectins 14 and 11 in the goat abomasum. In conclusion, we present evidence that lectin is involved in the immune response to gastrointestinal nematodes, which suggests that collectins and galectins are involved in the molecular recognition of helminths.

Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr).

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD- nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.