Anthony P. R. Brain

Magnetic conjugated polymer nanoparticles as bimodal imaging agents.

Hybrid nanoparticles which incorporate multiple functionalities, such as fluorescence and magnetism, can exhibit enhanced efficiency and versatility by performing several tasks in parallel. In this study, magnetic-fluorescent semiconductor polymer nanospheres (MF-SPNs) have been synthesized by encapsulation of hydrophobic conjugated polymers and iron oxide nanoparticles in phospholipid micelles. Four fluorescent conjugated polymers were used, yielding aqueous dispersions of nanoparticles which emit across the visible spectrum. The MF-SPNs were shown to be magnetically responsive and simultaneously fluorescent. In MRI studies, they were seen to have a shortening effect on the transverse T(2)* relaxation time, which demonstrates their potential as an MR contrast agent. Finally, successful uptake of the MF-SPNs by SH-SY5Y neuroblastoma cells was demonstrated, and they were seen to behave as bright and stable fluorescent markers. There was no evidence of toxicity or adverse affect on cell growth.

Induction of non-bilayer lipid phase separations in chloroplast thylakoid membranes by compatible co-solutes and its relation to therthermal stability of Photosystem II

Abstract High concentrations of compatible co-solutes such as sugars, sugar alcohols and polyols induce the phase separation of non-bilayer forming lipids in thylakoid membrane preparations. Of the co-solutes tested, sucrose, trehalose, betaine and sorbitol all led to extensive phase separation at room temperature. Glucose and glycerol were much less effective. Measurements of the temperature dependence of the fluorescence yield of chlorophyll a associated with Photosystem II (PS II), and the relative oxygen evolution efficiency of chloroplasts incubated at elevated temperatures, indicated that the presence of compatible co-solutes led to substantial increases in the threshold temperature for inhibition of PS II-mediated electron transport. The protective effect of the different co-solutes in order of efficiency, was sucrose > trehalose > sorbitol > betaine > glucose > glycerol. In general, the ability of the different co-solutes to stabilise PS II appeared to parallel their ability to induce non-bilayer lipid phase separation. Comparison of the effects of the co-solutes on isolated chloroplasts with that of heat-stress in unprotected chloroplasts suggest that there is no direct causal link between the phase-separation of non-bilayer lipids from the chloroplast membranes and the increased stability of PS II. It is concluded that the two phenomena are separate reflections of the effects of co-solutes on ordered water at lipid and protein interfaces, respectively.

Phase behaviour of the membrane lipids of the thermophilic blue-green alga Anacystis nidulans

Abstract The phase behaviour of total membrane lipid extracts of the blue-green alga Anacystis nidulans is compared with that of the individual lipid classes present in such extracts using fluorescence probe, differential scanning calorimetry, wide-angle X-ray diffraction and freeze-fracture techniques. Marked differences are observed in the properties of the isolated lipids as compared to the total lipid extracts. In particular, purified samples of monogalactosyldiacylglycerol and phosphatidylglycerol form complex high melting-point gel phases on storage which are not found in the membrane extracts. Addition of Mg 2+ ions to the extracts is also shown to lead to an extensive phase separation of monogalactosyldiacylglycerol from the extracts. The enthalpy changes associated with phase separations occurring in the lipid extracts are found to be approx. 30% higher than those for the corresponding membranes, suggesting that the presence of other components, such as membrane proteins, may influence the phase behaviour of the lipids. The significance of these observations is discussed in terms of the factors limiting the stability of membrane systems.

Stabilized Integrin-Targeting Ternary LPD (Lipopolyplex) Vectors for Gene Delivery Designed To Disassemble Within the Target Cell

Recent research in the field of nonviral gene delivery vectors has focused on preparing nanoparticles that are stabilized by the incorporation of a PEG coating and where one of the vector components is also cleavable. Here,we describe the synthesis, formulation, transfection properties, and biophysical studies of a PEG-stabilized ternary lipopolyplex vector in which, for the first time, both the lipid and peptide components are designed to be cleaved once the vector has been internalized. A series of cationic lipids, bearing short tri- or hexaethylene glycol groups, attached to the headgroup via an ester linkage, has been prepared. Trifunctional peptides have also been prepared, consisting of a Lys(16) sequence at the N-terminus (to bind and condense plasmid DNA); a spacer group (containing a sequence recognized and cleaved by endosomal enzymes) and an optional PEG4 amino acid; and an integrin-targeting cyclic peptide sequence (allowing the resulting nanoparticle to be internalized via receptor-mediated endocytosis). Differing combinations of these lipids and peptides have been formulated with DOPE and with plasmid DNA, and complex stability, transfection, and cleavage studies carried out. It was shown that optimal transfection activities in a range of cell types and complex stabilities were achieved with lipids bearing short cleavable triethylene glycol moieties, whereas the incorporation of PEG4 amino acids into the cleavable peptides had little effect. We have synthesized appropriate fluorescently labeled components and have studied the uptake of the vector, endosomal escape, peptide cleavage, and plasmid transport to the nucleus in breast cancer cells using confocal microscopy. We have also studied the morphology of these compact, stabilized vectors using cryo-EM.

The Ultrastructure and Botanical Affinity of End‐Permian Spore Tetrads

End‐Permian palynological assemblages worldwide are characterized by large numbers of unseparated tetrads of lycopsid microspores and occasional megaspore tetrads. These spores are assignable to the form genera Densoisporites, Lundbladispora, Uvaesporites, and Otynisporites. Transmission electron microscopy analysis of preserved sporoderm ultrastructure demonstrates that individual spores are juxtaposed or connected to each other by interlocking of the paraexospores at either the interradial contact areas or equatorial regions. The wall organization of Densoisporites, Lundbladispora, and Otynisporites confirms an isoetalean (Pleuromeiaceae) affinity. The Uvaesporites wall structure is far more complex than so far recognized in extant Selaginellales and extant and fossil Isoetales. Uvaesporites might be related to a distinct lycopsid lineage, with characteristics of rhizomorphic lycopsids and the Selaginalles.

Structural characteristics of thylakoid membranes of Arabidopsis mutants deficient in lipid fatty acid desaturation

The ultrastructure of thylakoid membranes from Arabidopsis thaliana wild-type, JB67 and LK3 fatty acid desaturation deficient mutants was studied by thin-section and freeze-fracture electron microscopy. There was a decrease in the amount of the appressed and non-appressed membranes in JB67 and LK3 Arbidopsis mutants when compared to the wild type, resulting in a reduction in the length of photosynthetic membrane per plastid. The results from freeze-fracture showed a decrease in size and a marked increase in packing density of membrane-associated particles on the exo- and endoplasmic fracture faces of the mutants. In addition, areas of the appressed membranes of the mutants contained particles in regular arrays under conditions where no such arrays were observed in wild-type thylakoid membranes. These observations suggest, that the decreased level of lipid fatty acid unsaturation affects the ability of the lipid matrix to mediate the assembly of chloroplast membrane components. The role of polyunsaturated membrane lipids is considered in terms of their ability to promote functional oligomeric assemblies of components of the photosynthetic apparatus.

Factors influencing PS II particle array formation in Arabidopsis thaliana chloroplasts and the relationship of such arrays to the thermostability of PS II

Chloroplasts of the thermal-stable fatty acid desaturase mutants JB67 and LK3 of Arabidopsis thaliana are characterised by the presence of regular arrays of freeze-fracture particles associated with the core and light-harvesting antennae of Photosystem II (Tsvetkova et al. (1994) Biochim. Biophys. Acta 1192, 263–271). Similar arrays were found to be induced in the membranes of chloroplasts isolated from wild-type plants by resuspending the chloroplasts in media containing Tricine and/or high concentrations of compatible co-solutes such as sorbitol. The thermal stability of their chloroplasts was also increased under such conditions. The increased tendency to form PS II particle arrays, and the enhanced thermal stability of PS II, in chloroplasts isolated from the mutants and wild-type chloroplasts suspended in different media, appear to be reflections of the increased stability of protein-protein interactions between and within PS II units, respectively. The role of lipids in determining the formation of freeze-fracture particle arrays in the mutants is discussed in terms of the observed changes in lipid composition and their possible role in the control of lipid/protein synthesis.

Effect of Mg2+ on excitation energy transfer between LHC II and LHC I in a chlorophyll-protein complex

A chlorophyll‐protein preparation has been isolated from the thylakoid membranes of Pisum sativum L. by the use of Triton X‐100 in the presence of a Tris‐HCl buffer. Analysis of this preparation by means of SDS‐PAGE and freeze‐fracture electron microscopy indicates that it contains the reaction centre complex of photosystem I (PS I) and its associated peripheral light‐harvesting complex (LHC I), together with the peripheral light‐harvesting complex of PS II (LHC II). The reaction centre complex of PS II is absent from this preparation. The polypeptide composition of the complex is similar to that of one previously isolated from barley [(1987) Eur. J. Biochem. 163, 221–2301. Fluorescence spectroscopy of the preparation at 77 K indicates that excitation of chlorophyll b at 472 nm (LHC II) gives fluorescence emission at 735 nm from LHC I, thereby indicating excitation energy transfer between the two complexes. The extent of excitation energy transfer from LHC II to LHC I is increased by the absence of Mg2+ from the medium, a phenomenon similar to effects of Mg2+ depletion promoting ‘spillover’ of excitation energy from PS II to PS I in chloroplast thylakoid membranes.

The structure of membrane lipids of the extreme halophile, Halobacterium cutirubrum, in aqueous systems studied by freeze-fracture

Abstract The structures formed by the two major membrane lipids of the extreme halophile, Halobacterium cutirubrum, namely diphytanyl ether analogues of phosphatidylglycerol phosphate and glycolipid sulphate, dispersed in either water, 1 M NaCl or 5 M NaCl were examined by freeze-fracture electron microscopy. In water, both lipids formed lamellar phases which were highly hydrated. Dispersion in 1 M NaCl caused the bilayers to stack more tightly. The presence of 5 M NaCl, mixed phases were observed at 20°C consisting of both lamellar and non-lamellar structures. Studies of binary mixtures of the two lipids in 5 M NaCl in mole ratios of 1:2, 2:1 and 3.5:1 indicated that phase separation takes place and that glycolipid sulphate tended to form bilayers at the growth temperature whereas phosphatidylglycerol phosphate preferentially formed a non-bilayer arrangement in the presence of salt. Total polar lipid extracts H. cutirubrum formed mixed phase systems that reflected the proportions of the major lipid components. Thermotropic studies performed by thermally quenching dispersions at temperatures ranging from −30°C to 70°C indicated that bilayers were formed at lower temperatures in both pure lipids and mixtures of lipids whereas there was a preference for what gave the appearance of inverted cubic phases at high temperatures. These observations are consistent with the notion that non-bilayer lipids are required to package the intrinsic membrane proteins into a lipid bilayer matrix.

Diversity of exine structure in Upper Carboniferous (Westphalian) selaginellalean megaspores.

Studies of wall structure in Mesozoic and Recent selaginellalean megaspores have been well documented. However, Palaeozoic examples have received minimal attention. The principal Palaeozoic megaspore genus of likely selaginellalean affinity is Triangulatisporites, extending from the Upper Devonian to the Upper Carboniferous. The particulate wall ultrastructure of a previously published Carboniferous (Duckmantian) megaspore assigned to this genus suggested that this form of wall construction may have been the ancestral wall structure of the group, an observation which posed difficulties in relating selaginellalean ultrastructure to that of other contemporaneous lycopsid megaspores. Subsequent investigation showed that the genus also contains more laminate exines similar to those of other extinct lycopsids and extant Selaginella species. Our new examples of Triangulatisporites ultrastructure from the Langsettian, Duckmantian and Westphalian D yield more information regarding early variation of wall structure within Carboniferous selaginellalean megaspores and suggest that a more laminate wall composition is at least as old as the particulate form. However, without further investigation of Lower Carboniferous forms, we are unable to state which is indeed ancestral. The laminate structure reported here and elsewhere is, none the less, more easily related to comparable ultrastructure in other groups of Carboniferous lycopsid megaspores and could suggest a link with such genera as Zonalesporites and early Lagenicula. This would be in keeping with current concepts regarding the most primitive ultrastructural type within lycopsid megaspore walls.