Fusion of Avena sativa mesophyll cell protoplasts by electrical breakdown

Studies with the light microscope were carried out on mesophyll cell protoplasts of Avena sativa which had been made to undergo fusion by reversible electrical breakdown of the cell membrane. In order to establish close membrane contact between the cells, an important prerequisite for fusion, a method known as dielectrophoresis was used. In an inhomogeneous alternating electrical field the protoplasts adhere to the electrodes and to each other in the direction of the field lines. The cells which were thus brought into close contact with each other could be made to fuse by the application of a field pulse of high amplitude (about 750 V/cm) and short duration (20–50 μs). The field strength required for fusion exceeds the value necessary for the electrical breakdown of the cell membrane. Fusion took place within some minutes and led to a high yield of fused protoplasts. The fusion of cells being in the electric field occured in a synchronous manner. In some of the fusion experiments part of the protoplasts of A. sativa were stained with neutral red. When these cells were fused with unstained protoplasts, the vacuoles from the different cells within the fused aggregate could be shown to remain separate for quite some time.     

Isolation of photosynthetic catalysts from cyanobacteria.

Methods are described for the isolation of ferredoxins I and II, cytochrome c-553, cytochrome f, cytochrome c-550 and plastocyanin from large quantities of various cyanobacteria. The amino acid composition of cytochrome c-550 is reported. There is a variation in the relative amounts of these proteins in different batches of cells which may relate to the nutritional status of the organisms.     

Loss of function of biomembranes and solubilization of membrane proteins during freezing

Isolated thylakoid membranes are damaged during freezing in dilute salt solutions, as shown by the inactivation of photochemical thylakoid reactions. After freezing, a number of membrane proteins were found in the particle-free supernatant. Up to 5% of the total membrane protein was solubilized by freezing, and the pattern of released proteins as seen in sodium dodecyl sulfate gel electrophoretograms was influenced by the nature of the solutes present. Membranes protected by sucrose did not release much protein during freezing. Concentrated salt solutions caused protein release also in the absence of freezing. Among the proteins released were ferredoxin—NADP⁺ reductase, plastocyanin and coupling factor CF₁. Subunits of CF₁ were found in different proportions in the supernatants of thylakoid suspensions after freezing in the presence of different salts. Cyclic photophosphorylation was largely inactivated before significant protein release could be detected.It is suggested that protein release is the final consequence of the non-specific suppression of intramembrane ionic interactions by the high ionic strength created in the vicinity of the membranes by the accumulation of salts during slow freezing. Salt effects on water structure and alterations of nonpolar membrane interactions by the incorporation of (protonated) lipophilic anions from organic salts into the membrane phase during freezing may also be involved.     

The protective action of disodium cromoglycate on alveolar macrophages in phagocytosis of fibrogenic silica.

Treatment of fibrogenic silica (DQ-12) with Disodium Cromoglycate (DSCG) prior to its in-vitro and in-vivo phagocytosis by alveolar macrophages prevents the destruction of the cells and the fibrosis of lung tissue which are a consequence of phagocytosis. However, the treatment of alveolar macrophages with DSCG before phagocytosis of the silica had no, or a negligible, protective effect on the cells. Acid phosphatase activity which was significantly enhanced above the control in cells phagocytosing the silica was returned to the range found in phagocytosis of inert dust when silica treated with DSCG was phagocytized. The inhibitor of DNA- dependent RNA synthesis actinomycin D caused an increase of acid phosphatase activity. DSCG did not depress the phagocytic ability of alveolar macrophages. It appears that the catabolic enzyme process predominant in cells phagocytosing DQ-12 was under control in cells phagocytosing DQ-12 treated with DSCG and that DSCG probably acted as a regulator of the factors permitting catabolism. From the results it is suggested that the equilibrium of the enzyme reactions which accompany phagocytosis was such that the integrity of the phagocytes was preserved.     

Distribution of RNA polymerase on Drosophila polytene chromosomes as studied by indirect immunofluorescence

Using indirect immunofluorescence visualization techniques we investigated the in situ distribution of RNA polymerase B on Drosophila melanogaster polytene chromosomes. The enzyme was found at many sites distributed throughout the genome in a pattern clearly distinct from that observed for histone H1, but it was especially concentrated in puffs induced by heat shock.     

Two enzymes in Streptomyces griseus forb the synthesis of dTDP-L-dihydrostreptose from dTDP-6-deoxy-D-xylo-4-hexosulose.

The biosynthesis of dTDP-L-dihydrostreptose from dTDP-6-deoxy-D-$\text{xylo}$-4-hexosulose requires two enzymes: dTDP-4-keto-L-rhamnose-3,5-epimerase and a NADPH-dependent dTDP-“dihydrostreptose synthase”. These enzymes could be separated on a Sephadex G-100 column.     

The mechanism of electrical breakdown in the membranes ofValonia utricularis

Summary The dielectric breakdown in the membranes of cells ofValonia utricularis was investigated using intracellular electrodes and 500-sec current pulses. Electrical breakdown, which occurs when the membrane potential reaches a well-defined critical value, is not associated with global damage to the cell or its membranes (the membrane reseals in <5 sec). It was thus possible to investigate the effect of temperature on dielectric breakdown in single cells. It was found that the critical potential for breakdown was strongly dependent on temperature, decreasing from 1000 mV at 4°C to 640 mV at 30°C. The decrease in the breakdown potential with increasing temperature and the very short rise-time of the breakdown current (1 sec) suggests that the Wien field dissociation does not play a major role in the breakdown process. It is shown that the nonlinearI–V characteristics observed at different temperatures can be accurately accounted for with no adjustable parameters, by considerations of the mechanical compression of the membrane due to stresses induced by the electric field. Electrical breakdown on this scheme results from an electromechanical instability in the membrane. On this basis the present results indicate that the elastic modulus of the region of the membrane where breakdown occurs, decreases by a factor of 2 with increasing temperature from 4 to 30°C. On the assumption of a thickness of 4.0 nm and a dielectric constant of 5, the elastic modulus is estimated to have a value of 5×10⁶ Nm^–2 at 20°C.     

Changes in DNA secondary structure afterγ-irradiation

Native calf thymus DNA was gamma-irradiated at 500 mug/ml in 0.01 M NaCl in the presence or absence of oxygen. By irradiation, an increasing amount of DNA becomes reactive with a water-soluble carbodiimide-derivative (CMEC). In the DNA sections reactive with CMEC the nucleotide strands are separated, a phenomenon previously described as radiation-induced denaturation. The dose-effect curve for the formation of denatured DNA shows an upward-bent form; a distinct oxygen effect of about 2 is observed. By a comparative study with DNA samples, degraded partially with DNAse I, it was shown that a minor part of the radiation-induced denaturation results from the formation of the radiation-induced single strand breaks, whereas the major part is a local denaturation independent of the strand breaks. In these locally denatured regions 20 to 50 nucleotide pairs are separated.