Ethylene oxide and propylene oxide block copolymers (pluronics) are widely known as agents that promote drug penetration across biological barriers. We have studied the interaction of normal and malignant blood cells with pluronics L61 and P85 that have different hydrophobicity. SP2/0 myeloma cells accumulated pluronics while normal cells adsorb most of the polymer on the surface. Interaction of pluronics with cells resulted in drastic changes of membrane microviscosity. Tumor cell membrane microviscosity decreased after pluronics adsorption, in contrast to normal cells, whose membrane microviscosity was enhanced. We suppose that sensitivity of tumor cell membrane microviscosity to the pluronics action correlates with its permeability for molecular substances.z 1999 Federation of European Biochemical Societies.

Influenza A virus matrix protein M1 is one of the most important and abundant proteins in the virus particles broadly involved in essential processes of the viral life cycle. The absence of high-resolution data on the full-length M1 makes the structural investigation of the intact protein particularly important. We employed synchrotron small-angle X-ray scattering (SAXS), analytical ultracentrifugation and atomic force microscopy (AFM) to study the structure of M1 at acidic pH. The low-resolution structural models built from the SAXS data reveal a structurally anisotropic M1 molecule consisting of a compact NM-fragment and an extended and partially flexible C-terminal domain. The M1 monomers co-exist in solution with a small fraction of large clusters that have a layered architecture similar to that observed in the authentic influenza virions. AFM analysis on a lipid-like negatively charged surface reveals that M1 forms ordered stripes correlating well with the clusters observed by SAXS. The free NM-domain is monomeric in acidic solution with the overall structure similar to that observed in previously determined crystal structures. The NM-domain does not spontaneously self assemble supporting the key role of the C-terminus of M1 in the formation of supramolecular structures. Our results suggest that the flexibility of the C-terminus is an essential feature, which may be responsible for the multi-functionality of the entire protein. In particular, this flexibility could allow M1 to structurally organise the viral membrane to maintain the integrity and the shape of the intact influenza virus.

Potato virus A (PVA) particles were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the amino acids of the coat protein was determined to assess their in situ steric accessibility. This method revealed that the N-terminal 15 amino acids of the PVA coat protein and a region comprising amino acids 27 to 50 are the most accessible at the particle surface to labeling with tritium atoms. A model of the spatial arrangement of the PVA coat protein polypeptide chain within the virus particle was derived from the experimental data obtained by tritium bombardment combined with predictions of secondary-structure elements and the principles of packing ␣-helices and ␤-structures in proteins. The model predicts three regions of tertiary structure: (i) the surface-exposed N-terminal region, comprising an unstructured N terminus of 8 amino acids and two ␤-strands, (ii) a C-terminal region including two ␣-helices, as well as three ␤-strands that form a two-layer structure called an abCd unit, and (iii) a central region comprising a bundle of four ␣-helices in a fold similar to that found in tobacco mosaic virus coat protein. This is the first model of the three-dimensional structure of a potyvirus coat protein.

Molecular mechanisms of long-term changes in brain metabolism after thiamine administration (single i.p. injection, 400 mg/kg) were investigated. Protocols for discrimination of the activities of the thiamine diphosphate (ThDP)-dependent 2-oxoglutarate and 2-oxoadipate dehydrogenases were developed to characterize specific regulation of the multienzyme complexes of the 2-oxoglutarate (OGDHC) and 2-oxoadipate (OADHC) dehydrogenases by thiamine. The thiamine-induced changes depended on the brain-region-specific expression of the ThDP-dependent dehydrogenases. In the cerebral cortex, the original levels of OGDHC and OADHC were relatively high and not increased by thiamine, whereas in the cerebellum thiamine upregulated the OGDHC and OADHC activities, whose original levels were relatively low. The effects of thiamine on each of the complexes were different and associated with metabolic rearrangements, which included (i) the brain-region-specific alterations of glutamine synthase and/or glutamate dehydrogenase and NADP+-dependent malic enzyme, (ii) the brain-region-specific changes of the amino acid profiles, and (iii) decreased levels of a number of amino acids in blood plasma. Along with the assays of enzymatic activities and average levels of amino acids in the blood and brain, the thiamine-induced metabolic rearrangements were assessed by analysis of correlations between the levels of amino acids. The set and parameters of the correlations were tissue-specific, and their responses to the thiamine treatment provided additional information on metabolic changes, compared to that gained from the average levels of amino acids. Taken together, the data suggest that thiamine decreases catabolism of amino acids by means of a complex and long-term regulation of metabolic flux through the tricarboxylic acid cycle, which includes coupled changes in activities of the ThDP-dependent dehydrogenases of 2-oxoglutarate and 2-oxoadipate and adjacent enzymes.

It has recently been found that Pluronics (block copolymers of ethylene oxide, EO, and propylene oxide, PO) favor the permeability and accumulation of anthracycline antibiotics, for example doxorubicin (Dox), in tumor cells. In an effort to understand these results, the interaction of EO(2)/PO(32)/EO(2) (Pluronic L61) with unilamellar egg yolk vesicles (80-100 nm in diameter) was examined. A partition coefficient K(p)=[Pl](membrane)/[Pl](water)=45 was determined. This corresponds to adsorption of about 20 polymer molecules to the surface of each vesicle in a 20 microM polymer solution. Despite this rather weak adsorption, Pluronic has a substantial effect upon the transmembrane permeation rate of Dox and upon the phospholipid flip-flop rate within the bilayers. Thus, the Dox permeation rate increases threefold and the flip-flop rate increases sixfold in 20 microM Pluronic. The two rates increase linearly with the amount of adsorbed polymer. The obvious ability of Pluronics to increase the mobility of membrane components may have important biomedical consequences.