Electrofusion Research Articles

Membrane Permeability Changes and Ultrastructural Abnormalities Observed During Protoplast Fusion

Summary A comparative study between polyethylene glycol (PEG) fused and electrofused protoplasts of pea has shown that the chemical fusion method was inferior, and PEG fusion products of about 15 % were possible, but over 50% of the initial protoplast mix was lost or damaged. The PEG treated protoplasts had decreased viability, respiratory O 2 consumption was lower, and PEG markedly decreased O 2 evolution in the light, the rate of CO 2 fixation, protein and chlorophyll levels. Membrane damage was present in PEG treated protoplasts, as evident by the large amount of efflux of intracellular potassium (K) (>70%) and CO 2 fixation products (>40%). The best electro fusion protocol (achieved by trial and error) produced 25—30% fusion products, and higher protoplast viability than PEG. Protoplast membrane damage due to electric currents was lower, and respiratory O 2 consumption, O 2 evolution and CO 2 fixation in the light were close to, and in some cases higher than in freshly isolated (control) protoplasts. Protein and chlorophyll content of electrofused protoplasts were close to controls, and the loss of intracellular K was now about 10%. Electron micrographs of isolated protoplasts showed considerable accumulation of osmiophilic deposits near membranes, and especially at membrane fusion sites. PEG mediated fusion of protoplasts caused greater protoplast distortion and membrane disruption compared to electrofusion, but PEG fusion caused increased vesicle formation near membranes in close contact. Only PEG treatment induced a high percentage (40%) of inter-organelle fusion even within intact protoplasts, but these were predominantly only part fusion of the outer membranes.

Analysis of the electrofusion joining process in polyethylene gas piping systems

Electrofusion (EF) is a semi-automated technology for joining polyethylene gas distribution pipes. To design reliable EF joints, a computational model for the EF joining process needs to be established. In the present work, a simulation method for the EF joining process, in which the thermomechanical coupling behavior is taken into account, has been developed employing ADINA and ADINA-T. The method can be used to simulate the closure process of the clearance between the joint and the pipe. In addition, the histories of the interface pressure and the temperature distributions, the parameters which have a critical importance on the quality of the joint, can be obtained. First, the computed results were compared with the experimental data from a small scale EF joint and good agreement was obtained. Next, the method was applied to design a large scale EF joint and the design optimization was carried out, resulting in the successful development of a reliable joint.

Fusion and metabolism of plant cells as affected by microgravity.

Plant cell protoplasts derived from leaf tissue of two different tobacco species (Nicotiana tabacum., N. rustica L.) were exposed to short-term (sounding rocket experiments) and long-term (spacelab) microgravity environments in order to study both (electro) cell fusion and cell metabolism during early and later stages of tissue regeneration. The period of exposure to microgravity varied from 10 min (sounding rocket) to 10 d (space shuttle). The process of electro fusion of protoplasts was improved under conditions of microgravity: the time needed to establish close membrane contact between protoplasts (alignment time) was reduced (5 as compared to 15 s under 1 g) and numbers of fusion products between protoplasts of different specific density were increased by a factor of about 10. In addition, viability of fusion products, as shown by the ability to form callus, increased from about 60% to more than 90%. Regenerated fusion products obtained from both sounding-rocket and spacelab experiments showed a wide range of intermediate properties between the two parental plants. This was verified by isozyme analysis and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). In order to address potential metabolic responses, more general markers such as the overall energy state (ATP/ADP ratio), the redox charge of the diphosphopyridine nucleotide system (NADH/NAD ratio), and the pool size of fructose-2,6-bisphosphate (Fru 2,6 bisp), a regulator of the balance between glycolysis and gluconeogenesis, were determined. Responses of these parameters were different with regard to short-term and long-term exposure. Shortly after transition to reduced gravitation (sounding rocket) ratios of ATP/ADP exhibited strong fluctuation while the pool size of NAD decreased (indicating an increased NADH/NAD ratio) and that of Fru 2,6 bisp increased. As similar changes can be observed under stress conditions, this response is probably indicative of a metabolic stress compensation. Samples taken for up to 7 d of exposure to microgravity showed the opposite effect. Here, the ratios of ATP/ADP and of NADH/NAD, and the pool size of Fru 2,6 bisp were decreased. We take this an an indication of metabolic relaxation, i.e. decreased metabolic turnover. As rates of protoplast regeneration and cell division were obviously similar to 1-g controls, we conclude that under conditions of microgravity regenerating tobacco mesophyll protoplasts need less metabolic energy for the same effort.

Comparison of three human-murine heteromyeloma cell lines for formation of human hybridomas after electrofusion with human peripheral blood lymphocytes from meningococcal cases and carriers

Peripheral blood lymphocytes from meningitis patients and healthy meningococcal carriers were fused by electrofusion with the three human-murine heteromyeloma cell lines CB-F7, K6H6B5 and H7NS. 934 hybridomas producing human immunoglobulins were obtained in 30 fusions. Heteromyeloma K6H6B5 yielded a significantly higher proportion of hybridomas producing IgG antibodies than did the two other cell lines. CB-F7 and K6H6B5 yielded comparable numbers of hybridomas whose supernatants reacted with homologous bacteria, whereas the cell line H7NS was less efficient.

Design of electrofusion joints and evaluation of fusion strength using fusion simulation technology

AbstractFusion simulation is one of the key techniques in designing and producing electrofusion (EF) joints for gas distribution and in evaluating fusion joint integrity. This paper describes the result of a numerical simulation of a thermal fusion process, using the finite element method. A nonlinear heat transfer computer program was used to obtain the temperature profile of a large electrofusion joint at fusion. The effects of applied voltage, heating time, wire pitch, and ambient temperature were examined for designing a 150‐mm EF joint. A method to shorten the cooling time was also investigated. The fusion condition range suitable for a 150‐mm EF joint was found to be slightly narrower than that suitable for a 50‐mm EF joint. Examination of the effect of wire pitch revealed that if the pitch is extremely large, thermal degradation starts in the resin close to the wire before the fusion‐interface strength reaches the maximum value. We have developed a program to simulate the process of closing the gap between the pipe and the joint due to resin expansion and melting after the power is supplied.

Effect of Design Factors of Electrofusion Joints on Fusion Strength

This paper describes the effect, on fusion strength, of design factors of electrofusion (EF) joints, which are new-type fusion joints for polyethylene gas pipes.A comprehensive evaluation method for EF joint integrity has been developed. First, fusion interface temperature and welding voltage and current were continuously measured while block specimens were being fused. Then, wire movement was observed and the width of the molten layers was measured with a polarzing microscope.A determination of the fusion mechanism of EF joints is attempted, and the influence of various design factors on fusion strength is discussed. The relationships between the fusion joint strength and input energy or interface temperature when wire arrangement, conditions of welding current and voltage, and wire material were varied, were clarified using block specimens.Wire temperature during application of welding current was measured. The thermal degradation of polyethylene resin in the vicinity of the wires by FT-IR and GPC analysis was chemically examined.