haemolysin of Escherichia coli: Comparison of pore-forming properties between chromosome and plasmid-encoded haemolysins

Abstract Lipid bilayer experiments were performed with chromosome-encoded haemolysin of Escherichia coli . The addition of the toxin to the aqueous phase bathing lipid bilayer membranes of asolectin resulted in the formation of transient ion-permeable channels with two states at small transmembrane voltages. One is prestate (single-channel conductance 40 pS in 0.15 M KCl) of the open state, which had a single-channel conductance of 420 pS in 0.15 M KCl and a mean lifetime of 30 s. Membranes formed of pure lipids were rather inactive targets for this haemolysin. Experiments with different salts suggested that the haemolysin channel was highly cation-selective at neutral pH. The mobility sequence of the cations in the channel was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with a wide, water-filled channel with an estimated minimal diameter of about 1 nm. The pore-forming properties of chromosome-encoded haemolysin were compared with those of plasmid-encoded haemolysin. Both toxins share common features, oligomerize probably to form pores in lipid bilayer membranes. Both types of haemolysin channels have similar properties but different lifetimes.     

Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus

Summary A system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus armeniaca plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene ß-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV). The marker gene GUS was used for optical evaluation of the efficiency of the transformation system. The coat protein gene of PPV was used to introduce coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.Abbreviations GUS ß-glucuronidase - PPV Plum Pox Virus - BA 6-benzylaminopurine - NPTII neomycin phosphotransferase II - CP coat protein - CaMV Cauliflower Mosaic Virus - P35S 35S promoter - MS Murashige and Skoog - PCR polymerase chain reaction - P/C/I phenol/chloroform/isoamylalcohol - RNase ribonuclease - dNTP deoxyribonucleosidetriphosphate - DMSO dimethyl sulfoxide     

Proton and sucrose transport in isolated tonoplast vesicles from sugarcane stalk tissue

Tonoplast vesicles prepared from immature sugarcane ( Saccharum spp., hybrid cv. H65–7052) tissue and purified on a discontinuous dextran gradient take up sucrose. Uptake was stimulated by MgATP. Evidence that the mechanism is linked to proton transport is derived from "pH jump'data and from inhibition of ATP-stimulated sucrose transport by the protonophore carbonyl cyanide m -chlorophenylhydrazone (CCCP) and by the proton-channel blocker of proton-linked ATPases. N. N '-dicyclo-hexylcarbodiimide (DCCD). A saturable phase of sucrose uptake was found at low substrate concentrations, and a linear phase characterized uptake at higher concentrations. Uptake was specific for sucrose, as demonstrated by competition experiments with various sugars. Sucrose uptake by the vesicle fraction was inhibited by KNO₃, protonophores and protein modifying reagents, whereas sodium orthovanadate had no effect. Overall, the evidence suggests an ATP-hydrolysis-dependent tonoplasl antiport for sucrose transport, although a more direct influence of ATP on conformational changes in relevant tonoplast proteins cannot be ruled out.     

Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

Rapid and simple isolation of pure photosystem II core and reaction center particles from spinach

Pure and active oxygen-evolving PS II core particles containing 35 Chl per reaction center were isolated with 75% yield from spinach PS II membrane fragments by incubation with n-dodecyl--D-maltoside and a rapid one step anion-exchange separation. By Triton X-100 treatment on the column these particles could be converted with 55% yield to pure and active PS II reaction center particles, which contained 6 Chl per reaction center.Abbreviations Bis-Tris bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane - Chl chlorophyll - CP29 Chl a/b protein of 29 kDa - Cyt b ₅₅₉ cytochrome b ₅₅₉ - DCBQ 2,5-dichloro-p-benzo-quinone - LHC II light-harvesting complex II, predominant Chl a/b protein - MES 2-[N-Morpholino]ethanesulfonic acid - Pheo pheophytin - PS H photosystem II - Q_A bound plastoquinone, serving as the secondary electron acceptor in PS II (after Pheo) - SDS sodiumdodecylsulfate