Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium.

Two rapid and simple in planta transformation methods have been developed for the model legume Medicago truncatula. The first approach is based on a method developed for transformation of Arabidopsis thaliana and involves infiltration of flowering plants with a suspension of Agrobacterium. The second method involves infiltration of young seedlings with Agrobacterium. In both cases a proportion of the progeny of the infiltrated plants is transformed. The transformation frequency ranges from 4.7 to 76% for the flower infiltration method, and from 2.9 to 27.6% for the seedling infiltration method. Both procedures resulted in a mixture of independent transformants and sibling transformants. The transformants were genetically stable, and analysis of the T2 generation indicates that the transgenes are inherited in a Mendelian fashion. These transformation systems will increase the utility of M. truncatula as a model system and enable large-scale insertional mutagenesis. T-DNA tagging and the many adaptations of this approach provide a wide range of opportunities for the analysis of the unique aspects of legumes.

Inhibition of the in vitro formation of dense cells and of irreversibly sickled cells by charybdotoxin, a specific inhibitor of calcium-activated potassium efflux.

Charybdotoxin, a specific inhibitor of the calcium-activated potassium channel, was found to inhibit the in vitro formation of irreversibly dehydrated cells and of irreversibly sickled cells, which occur as a result of repeated cycles of sickling and unsickling of sickle red blood cells. The degree of formation of dense cells was measured by Percoll-renografin density gradient centrifugation. 50% inhibition of the formation was achieved at a concentration of 30 nM of charybdotoxin. The approximate half-life of this compound in the circulation of the guinea pig was determined to be 4 h. Charybdotoxin did not inhibit the sickling of sickle cells under deoxygenation. The effects of charybdotoxin in preventing the irreversible changes of sickle cell membranes may be related to the inhibition of calcium-activated potassium efflux in sickle red blood cells.

Detection of malignant hyperthermia susceptibility using a spin label technique on red blood cells.

Using spin labelled red blood cells and electron paramagnetic resonance spectroscopy (EPR), we observed that halothane 3 mmol litre-1 produced a much greater decrease in the rotational correlation time of red blood cells from individuals who were at risk for malignant hyperthermia (MH), compared with normals. Subsequently we performed blind tests on 14 individuals whose MH susceptibility status had been determined on the basis of in vitro contracture studies. When compared with the results of the in vitro contracture studies, the EPR studies predicted correctly the patients' status in 13 of the 14 individuals.

Is Ca2+ -activated potassium efflux involved in the formation of ischemic brain edema?

A possible role of Ca2+ -activated potassium efflux in brain ischemia was studied using a rat focal cortical infarction model. Three days after ischemic insult, tissue contents of water, sodium, potassium and calcium ions were measured. Charybdotoxin, a specific inhibitor of Ca2+ -activated potassium efflux, was found to reduce the formation of ischemic brain edema when a dosage of 0.15 mg/kg was given by i.v. 20-30 min prior to the onset of ischemic insult.

Protoplast Fusion: EFFECT OF LOW TEMPERATURE ON THE MEMBRANE FLUIDITY OF CULTURED CELLS.

The relation between the composition of the phospholipid molecular species in a cell membrane and the velocity of protoplast fusion was studied using cells cultured at a low temperature (10 C). Cells cultured at a low temperature contained larger proportions of phospholipids of low phase transition point, the 1,2-dilinoleoyl-type, than those cultured at a normal temperature (25 C). When treated with polyethylene glycol 6000, protoplasts from cells cultured at 10 C fused and progressed to the fused sphere stage more rapidly than did those from cells cultured at 25 C.

Effect of temperature on membrane fluidity and protoplast fusion.

The fusion of plant protoplasts is greatly enhanced by a rise in temperature in the presence of the fusion-inducing agents polyethylene glycol or polyvinyl alcohol. Kinetic analysis of this reaction reveals that membrane fluidity is important in regulating the fusion process.