Pyridoxylated Polymerized Hemoglobin Solution Processing

Glutaraldehyde hemoglobin polymerization gives too many high polymers, resulting in a too viscous solution. We describe here an alternate method leading to superior results, as compared to the classical one. This method includes a molecular fractionation step using a tangential flow ultrafiltration that secondarily lowers the unpolymerized tetramer’s content of a mildly polymerized, pyridoxylated hemoglobin solution (Pyr-Poly Hb). This leads to an adequately polymerized product with a lesser high polymer content, implying a lower viscosity. We thus obtain a pyridoxylated, polymerized molecular fractionated solution presenting suitable features as a blood substitute: A 7.5 g% hemoglobin 2 g% albumin solution had a 16% unpolymerized tetramer’s ratio, a 1.8 mPas viscosity, a P₅₀ of 2.8 kPa, a Hill coefficient of 2.1, a binding coefficient of 1.3 mL/g, a colloid osmotic pressure of 2.4 kPa, and a methemoglobin concentration of 3% Male Sprague-Dawley rats undergoing an isovolumic blood exchange with this Pyr-Poly Hb solution, down to a 2% hematocrit, present a mean survival time of 20 h.     

Continuous-wave Raman laser in H(2)

Recent developments in high-finesse cavities now make broadly tunable, continuous-wave Raman lasers possible. The design and preliminary characterization of what is to the authors' knowledge the first continuous-wave Raman laser in H(2) are presented. The threshold is currently at 2 mW of pump, making diode laser pumping possible. The maximum photon conversion efficiency observed was 35% at 7.6 mW of pump power.     

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES-X. SYNTHESES AND ANALYSES OF SULFONATED PHTHALOCYANINES

Abstract— Synthetic methods to obtain selectively sulfonated metallo phthalocyanines are compared. Both condensation and direct sulfonation procedures lead to mixtures of mono- to tetrasulfonated products which are resolved by reverse phase liquid chromatography in buffered aqueous-methanol. The proportion of sulfonated derivatives is examined as a function of the starting reagents in the case of the condensation method, and as a function of the temperature and reaction time in the case of the direct sulfonation procedure. The number of sulfonate groups per phthalocyanine molecule is determined by oxidative degradation of the phthalocyanine ring followed by quantitative chromatographic analysis of the sulfophthalamide and phthalamide fragments.     

The biologically important surfactin lipopeptide induces nanoripples in supported lipid bilayers

Under specific conditions, lipid membranes form ripple phases with intriguing nanoscale undulations. Here, we show using in situ atomic force microscopy (AFM) that the biologically important surfactin lipopeptide induces nanoripples of 30 nm periodicity in dipalmitoyl phosphatidylcholine (DPPC) bilayers at 25 degrees (i.e. well below the pretransition temperature of DPPC). Whereas most undulations formed the classical straight orientation with characteristic angle changes of 120 degrees , some of them also displayed unusual circular orientations. Strikingly, ripple structures were formed at 15% surfactin but were rarely or never observed at 5 and 30% surfactin, emphasizing the important role played by the surfactin concentration. Theoretical simulations corroborated the AFM data by revealing the formation of stable surfactin/lipid assemblies with positive curvature.     

Tilted peptides: the history

Nature has selected peptide motifs for protein functions. It is clear that specific sequence motifs can identify families of enzymes. These sequence motifs are one dimensional signatures and nature has also developed two dimension motifs which cannot be read in the one dimension of sequence language but can be detected in the three dimensional properties of a secondary structure. One of such motifs is tilted peptides. They do not correspond to any consensus of sequence but correspond to a consensus motif where hydrophobicity balance is used as a functional device. In the nineteen eighties, the first tilted peptide was deciphered from the sequence of a virus fusion protein by molecular modelling. It was described as a protein fragment hydrophobic enough to insert into a membrane but too short to span it. The fragment exhibited an asymmetric distribution of hydrophobicity along the helix long axis and hence, was unable to lie parallel or perpendicular to a membrane surface but adopted an orientation in between. Hydrophobicity motif was a very new and very challenging concept and tilted peptides were rapidly found to be involved in several mechanisms of virus fusion. They were also found to be involved in protein secretion and future studies could establish their involvement in the destabilization of 3D protein structures and in the alpha to beta transconformations, which drive the generation of amyloid deposits.     

PHOTODYNAMIC ACTIVITIES and SKIN PHOTOSENSITIVITY OF THE bis(DIMETHYLTHEXYLSILOXY)SILICON 2,3-NAPHTHALOCYANINE IN MICE

The photodynamic therapy (PDT) activity of the bis(dimethylthexylsiloxy)silicon 2,3-na-phthalocyanine (SiNc 8 ) was evaluated against the EMT-6 tumor implanted intradermally in BALB/c mice. The SiNc 8 was formulated in aqueous emulsions based on Cremophor EL or Solutol HS 15. The formulation was shown to affect plasma clearance and overall pharmacokinetics. Compared to Cremophor, Solutol promoted rapid plasma clearance and high liver retention of the dye, combined with a slight increase of dye tumor concentrations. The PDT action spectrum for tumor response of SiNc 8 in Cremophor (190 mW cm², 200 J cm², 24 h postinjection [p.i.] of 1 (jimol kg¹) showed a maximum at 780 nm, which corresponds to the absorption maximum of the monomelic dye as well as the in vivo maximum change in the “diffuse optical density” produced by the dye. The extent of tumor necrosis increased with augmented dye and light doses. Regardless of the formulation, at 1 h p.i. of 0.1 μmol kg^?! SiNc 8 , PDT efficiency (190 mW cm'², 400 J cm²) was high but accompanied by severe damage to normal tissues, at 24 h PDT resulted in complete tumor regression in 80% of the animals without adverse effects to adjacent tissues, while at 72 h p.i. PDT induced no tumor response with Cremophor and only a partial response with Solutol. At the latter time point, plasma dye clearance was nearly complete while tumor tissue levels remained high, suggesting that tumor response correlates with plasma rather than tumor dye levels. Skin sensitivity of SKhl mice to solar-simulated radiation was lower with SiNc 8 as compared to Photofrin®. Our data suggest the potential of SiNc 8 as a far-red absorbing photosensitizer in clinical PDT.     

Fusogenic tilted peptides induce nanoscale holes in supported phosphatidylcholine bilayers

Tilted peptides are known to insert in lipid bilayers with an oblique orientation, thereby destabilizing membranes and facilitating membrane fusion processes. Here, we report the first direct visualization of the interaction of tilted peptides with lipid membranes using in situ atomic force microscopy (AFM) imaging. Phase-separated supported dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers were prepared by fusion of small unilamellar vesicles and imaged in buffer solution, in the absence and in the presence of the simian immunodeficiency virus (SIV) peptide. The SIV peptide was shown to induce the rapid appearance of nanometer scale bilayer holes within the DPPC gel domains, while keeping the domain shape unaltered. We attribute this behavior to a local weakening and destabilization of the DPPC domains due to the oblique insertion of the peptide molecules. These results were directly correlated with the fusogenic activity of the peptide as determined using fluorescently labeled DOPC/DPPC liposomes. By contrast, the nontilted ApoE peptide did not promote liposome fusion and did not induce bilayer holes but caused slight erosion of the DPPC domains. In conclusion, this work provides the first direct evidence for the production of stable, well-defined nanoholes in lipid bilayer domains by the SIV peptide, a behavior that we have shown to be specifically related to the tilted character of the peptide. A molecular mechanism underlying spontaneous insertion of the SIV peptide within lipid bilayers and the subsequent removal of bilayer patches is proposed, and its relevance to membrane fusion processes is discussed.