No abstract

Several shorter analogues of the cell penetrating peptide transportan have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptide into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the Nterminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity. U. S o o m e t~l *~, M. Lindgrenl, X. Gallet2, M. Hallbrinkl, A.This project relates to the field of plant molecular biology in general. The transformation of plant cells with D N A encoding an antibiotic resistance markers is clearly desirable to obtain a high level of expression of the introduced gene to enable efficient selection of the transformants. Tissue culture facilities were used to determine the lethal concentration of gentamycin and ampicillin on the tea plant (Camellia sinensis) and studied the effect of two types of media MS and B5 in the presence of auxins with or without cytokinin. It was found that gentamycin is more toxic to the tea explant in the presence of IAA without BA than in the presence of IAA and BA. There were no obvious differences between the toxicities of the MS media supplemented with 2,4-D with or without BA. The toxicity of gentamycin was detected at 200 &ml media and the lethal dose of all the samples were 400 pg/ml media in the two types of media chosen (MS & B5) with the different hormone types. B5 media which contains IAA and BA have shown some resistance to gentamycin at a concentration of 400 p g Iml media.two types of plasmids; one with Kanamycin and the other with ampicillin resistance. Tea cells were grown in either ampicillin or kanamycin depending on the marker they carry. Our preliminary results shows that the transformed tea cells have changed their metabolic pathway in relation to the synthesis of caffeine, theobromine and theophylline. Antibiotic ResistanceTransformation of tea protoplasts was carried out using 659 Cytokinin-induced epigenetic inheritance of an "extra stamens" phenotype in Brassica rapa flowers Flower development in angiosperms is affe...

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.

A 500 MHz 2D 1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities, 3JNH-alpha H coupling constants as well as 1H/2H exchange rates and delta delta/delta T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-sheet (residues 27-40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from 3JNH-alpha H coupling constants, (iii) 12 hydrogen bonds mostly deduced from 1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a beta-sheet is linked to an alpha-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.