Reappearance of hydrolytic activities and tonoplast proteins in the regenerated vacuole of evacuolated protoplasts

Mesophyll protoplasts of tobacco (Nicotiana tabacum L. cv. Xanthi) were evacuolated by centrifugation in a density gradient. Evacuolation resulted in the quantitative loss of vacuolar hydrolytic activities. The evacuolated miniprotoplasts were cultivated under different conditions, and the regeneration of the central vacuole was investigated by light and electron microscopy as well as by the determination of activities of vacuolar marker enzymes. Vacuoles and hydrolytic activities, as well as cell wall material reappeared faster when the cells were cultivated at low osmotic strength. A newly synthesized tonoplast polypeptide could be detected using a polyspecific serum raised against tonoplast proteins of barley (Hordeum vulgare L.). Both vacuolar proton pumps, the ATPase as well as the pyrophosphatase appear to be newly synthesized during the regeneration of the vacuole.Abbreviations GAP-DH NADP-dependent glyceraldehyde 3-phosphate dehydrogenase - PEP phosphoenolpyruvate - PPi pyrophosphate - PPase pyrophosphatase We thank Dr. Ernst Wehrli, Labor für Elektronenmikroskopie I, ETH Zürich, for taking micrographs. Esther Vogt assisted in the determination of the hydrolases. Bafilomycin was kindly provided by Professor Altendorf, Osnabrück FRG. This work was supported by the Swiss National Foundation grant No. 31-25196.88.     

Emesis of rhesus monkeys induced by intragastric administration with the HEp-2 vacuolation factor (cereulide) produced by Bacillus cereus

Abstract To study the correlation between emetic toxin and HEp-2 vacuole activity produced by Bacillus cereus isolated from an outbreak of vomiting-type food poisoning, some properties and emetic activities of both purified HEp-2 factor (cereulide) and partially purified factor to rhesus monkeys were determined. The results indicate that both cereulide and partially purified factor were very stable to digestion with proteolytic enzymes, different pH, and heating. Vomiting was induced in the rhesus monkeys orally administered with both substances. From these findings, cereulide (or HEp-2 vacuole factor) is strongly suggested to be an emetic toxin itself.     

Lysosomal activation in mouse skeletal muscle induced by protamine in vitro

Summary Incubation of mouse skeletal muscle in a physiological Ringer solution containing protamine (60 g/ml) at +37° C for 1 h induced ultrastructural changes including proliferation of tubular profiles and vesicles at the I-band level close to the A-I junction, formation of numerous acid phosphatase positive lysosomes in the longitudinal sarcoplasmic reticulum and autophagic vacuolation starting at the level of the A-I junction.Biochemical determination of acid phosphatase in the incubated muscles showed that protamine caused an increase in acid phosphatase activity of about 25 % compared to enzyme activities obtained from muscles incubated without protamine at +37°C or with protamine at +4°C.The morphological findings suggest that the vesicles arising adjacent to the A-I junction originate from transverse tubules. Such vesicles, designated as endocytic, may acquire acid phosphatase activity in the longitudinal SR and be active in an autophagic process resulting in large vacuoles. A causal relationship between endocytosis and lysosomal activation is suggested.We wish to thank Miss Britt-Marie Svensson, Miss Ann-Christin Nilsson and Mrs. Lena Kvist for their able technical assistance. This work was supported by grants from the Medical Faculty, University of Lund, Sweden, and the Swedish Medical Research Council, Stockholm, Sweden (04P-4289, 14X-4286, 14X-3112) and from Muscular Dystrophy Association of America, Inc.