Protoplast formation

The balance of evidence is against the penetration of penicillin inside the cell [Cooper, 5], and the bound penicillin forms part of a lipoprotein complex loosely associated with the membrane, which is 95 % released in a high-molecular weight form on protoplast formation [Duersken, 14], Radioactive penicillin is not associated with ribosomes, messenger RNA, purified cell wall, or purified cytoplasmic membrane. Imsande [2] finds that more radioactive penicillin (up to 75 %) remains associated with the protoplast, however. The amount of bound penicillin was estimated by Imsande to be 1000 molecules per cell the figure of 80 to 200 molecules per cell [Pollock and Perret, 13] may be low for technical reasons. 

It is clear that, once the induction response is well advanced, protoplasting does not stop it. On the other hand, removal of the cell wall interferes with the initial stages of induction. Induction after protoplast formation remains elusive. As Duerksen pointed out, the inability of protoplasts to bind penicillin could account for this difference between protoplasts and cells. 

Gautam, S. R, Gupta, A. K., Shrivastava, R., Awasthi, M. Protoplast formation from the thermophilic fungus Malbranchea sulfurea, using the thermostable chitinase and lamari-nasn of Paecilomyces variotii. World J Microb Biotechnol 1996, 12, 99-100. 

Kitomoto, Y, Moii, N., Yamamoto, M., Ohiwa, T, Ichiwaka, Y. A simple method of protoplast formation and protoplast regeneration fiom various fungi using an enzyme from Trichoderma harzianum. Appl Microbiol Biotechnol 1988,28,445—450. 

Mizuno, K., Kimura, O., Tachiki, T. Protoplast formation from Schizophyllum commune by a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of S. commune as a carbon source. Biosci Biotechnol Biochem 1997,61,852-857. 

Imbrie-Milligan, C.W. Hodges, T.K. (1986). Microcallus formation from maize protoplasts prepared from embryogenic callus. Planta, 168, 395-401. 

Fujiwake H, Suzuki T, Iwai K (1982) Capsaicinoid formation in the protoplast from the placenta of Capsicum fruits. Agile Biol Chem 46 2591-2592... 

During the fusion process the relative surface area decreases with increasing volume indicating a loss of membrane material (about 22% in Fig. 51). In analogy to the fusion process of protoplasts it can be assumed that the excess lipid is removed in form of small, submicroscopic vesicles (Fig. 52). The electric breakdown in the membrane contact zone leads to the formation of several pores in which lipid molecules are randomly oriented (Fig. 52 b). The molecules reorient forming submicroscopic vesicles and the new membrane of the fused vesicle (Fig. 52c). Thus, fused giant liposomes should contain small, submicroscopic vesicles. This could possibly be proven by using fluorescence-labelled lipids for liposome fusion. 

Bergounioux, C., Freyssinet, G., and Gadal, P., Callus and embryoid formation from protoplasts of Helianthus annuus, Plant Cell Rep., 7, 437-440, 1988. 

Conrad and Hepler [61] found that a subclass of voltage-gated calcium channels, L-type channels sensitive to dihydropyridines (DHP), were involved. DHP agonists induced bud initials in the absence of cytokinin while the DHP antagonist blocked bud formation even in the presence of cytokinin. Agonists did not promote complete bud development suggesting that cytokinins have other effects as well. Direct measurements of uptake in moss protoplasts demonstrated the existence of DHP-sensitive calcium transport... 

However, wheat protoplasts have no other demonstrable responses to cytokinin and, therefore, the lack of a cytokinin effect on calcium transport is not a decisive test. Zhao and Ross [69] used zeatin induced growth and chlorophyll formation in excised cucumber cotyledons as cytokinin responses to examine the effects of calcium chelators, ionophore and several anti-calmodulin drugs. None of these drugs affected the two cytokinin responses unless used at concentrations high enough to cause indirect damage. 

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