Attempts to unravel the cell translocation mechanism of a growing number of cell-penetrating peptides (CPP) have revealed molecular determinants essential for internalization ability. The peptide sequence and the charge have been proposed to be the major factors in determining the membrane interaction mode and subsequent internalization pathway. Recent research in this field has shifted to search and design of novel CPPs with predefined vectorial properties and elucidation of the mechanism of cell entry of CPPs with high cargo delivery efficiency. Here we present a map of interaction modes with cell surface and intracellular traffic of transportan and its analogue TP10 complexed with fluorescently labeled avidin or streptavidin-gold conjugates. The protein cargo complexed with either peptide is transduced into HeLa and Bowes cells mostly in the endocytic vesicles with heterogeneous morphology and size as demonstrated by transmission electron microscopy (TEM) and confocal laser scanning fluorescence microscopy. Most of the induced vesicles are large, with 0.5-2 mum diameter, probably macropinosomes, but the complexes are present also in smaller vesicles, suggesting involvement of different pathways. Later the majority of complexes are translocated from the cell periphery into vesicles of perinuclear region and partly to lysosomes. A fraction of transportan-streptavidin complexes is present also freely in cytoplasm, both in the close vicinity of plasma membrane and more centrally, suggesting the escape from endosomal vesicles, since vesicles with discontinuous membrane were also detected by TEM. The cell-translocation process of transportan-protein complexes is temperature dependent and strongly inhibited at 8-10 degrees C and blocked at 4 degrees C when only interaction with the plasma membrane takes place.

Proteins with molecular masses ranging from 30 kDa (green fluorescent protein, GFP) to 150 kDa (monoclonal and polyclonal antibodies) were coupled to the cellular translocating peptide transportan. We studied the ability of the resulting protein–peptide constructs to penetrate into Bowes melanoma, BRL, and COS‐7 cells. After 0.5–3 h incubation with recombinant GFP coupled to transportan, most of the GFP fluorescence was found in intracellular membranes of BRL and COS‐7 cells, which suggests that transportan could internalize covalently linked proteins of about 30 kDa in a folded state. Transportan could internalize covalently coupled molecules of even larger size; that is, avidin and antibodies, (up to 150 kDa). The covalent bond between the transport peptide and its cargo is not obligatory because streptavidin was translocated into the cells within 15 min as a noncovalent complex with biotinylated transportan. Inside the cells, the delivered streptavidin was first located mainly in close proximity to the plasma membrane and was later distributed to the perinuclear region. Most of the internalized streptavidin was confined to vesicular structures, but a significant fraction of the protein was distributed in the cytoplasm. Our data suggest that transportan can deliver proteins and other hydrophilic macromolecules into intact mammalian cells, and this finding demonstrates good potential as powerful cellular delivery vector for scientific and therapeutic purposes.

The expression of the mitochondrially encoded subunit I of cytochrome-c oxidase (COX I) was investigated in cell cultures from patients with various mitochondrial diseases. Cells, grown on coverslips, were immunostained fluorescent green for the presence of COX I, while the mitochondria were co-stained fluoresent red with MitoTracker and the nuclei fluoresent blue with DAPI. Cell cultures expressing mitochondrial DNA depletion or harbouring heteroplasmic mutations in mitochondrial tRNA genes showed an intercellular mosaic staining for COX I. To analyse the results more objectively, we have developed a fully automated cytometric analysis system. The three fluorescent images of each field are captured using a low light level camera mounted on a fluorescence microscope which is fitted with a filter wheel with three excitation filters and a triple band pass emission filter. The system is used to scan 100 fields containing potentially thousands of cells. Sets of integrated images are processed to clearly define cell boundaries and to produce a binary mask image of the mitochondria. The fluorescent blue image (nuclear DNA) is utilised to define and separate individual cells. The system gave statistical credence to our earlier conclusions based on manual examination of the slides. In addition, image analysis showed a positive correlation between the intensity of COX I and MitoTracker staining (transmembrane electrical potential), and can be used to study organelle morphology. Measuring Betaine Metabolites Biochemistry Unit,Canterbury Health L.aboratories,Private Bag 151, ' 295 M . B . W . J . Dellow, M. Lever, C.M. Hayman Christchwrch, New ZeakandHomocysteine (hcy), a circulating non-protein amino acid is an independent risk factor for cardiovascular disease. Hcy is an intermediate in the metabolism of the essential amino acid methionine, a protein amino acid and precursor to S-adenosylmethionine (SAM). Methylation of hcy to methionine occurs by one of nvo pathways, a folate and B 12 (cobalamin) dependent pathway, or alternatively by methyl transfer from glycine betaine, mediated by betaine-homocysteine methyltransferase (BHMT), producing N,Ndimethylglycine. N,N-Dimethylglycine is a feedback inhibitor of BHMT and is broken down to glycine in a folate dependent pathway. The observation that elevation of N,N-dimethylglycine correlates with high hcy concentration led us to develop more sensitive and accurate methods to measure N,N-dimethylglycine and related betaine metabolites in plasma and urine samples.High performance liquid chromatography methods satisfy the requirements to measure N,N-dimethylglycine. Natural betaines are alkylated at the carboxyl terminal forming an ester using phenacyl triflates, in a base catalysed reaction. N,N-Dimethylglycine is derivatised at both the carboxyl and tertiary amine functions, producing a quaternary ammonium. Six derivatising agents have been tested with the greatest success shown by naphthacyl trifluoromethanesulphonate and 2-fluorenacyltrifluoromethanesulphonate respectively. A s...