Investigation of charge ratio variation in mRNA - DEAE-dextran polyplex delivery systems.

mRNA pharmaceuticals represent a new class of therapeutics, with applications, in cancer vaccination, tumour therapy and protein substitution. Formulations are required to deliver messenger RNA (mRNA) to the target sites where induction of genetic transfection following receptor mediated cell uptake & translation is required. In the current study, the cationic polysaccharide diethylaminoethylen (DEAE) - Dextran was selected as a model system carrier for the investigation of polyplex nanoparticle formation together with mRNA as a function of the molar ratio of the components. The structure of the mRNA/Dextran colloids was investigated as a function of the polymer-to-mRNA ratio and correlated with the biological activity determined by cellular transfection with luciferase coding mRNA. Dynamic light scattering (DLS), small angle x-ray scattering (SAXS), and small angle neutron scattering (SANS) with deuterium contrast variation were used to achieve structural insight into the systems. Similarly to previously investigated lipid based systems, colloidally stable particles with confined size were obtained with either excess of positive or negative charge. Highest activity was obtained with positive charge excess. From the scattering experiments information on the internal organization inside the polymer/mRNA systems was derived. Indication for the presence of structural elements in the length scale of ten to 20 nm were found in the excess of dextran, which could be due to either excess or particulate polymer. Information on the molecular organization of the mRNA nanoparticle products may provide a valuable basis for defining critical quality attributes of drug products for pharmaceutical application.

Reconstitution of bacteriorhodopsin and ATP synthase from Micrococcus luteus into liposomes of the purified main tetraether lipid from Thermoplasma acidophilum: proton conductance and light-driven ATP synthesis.

The archaebacterium Thermoplasma acidophilum is cultivated at 59 degrees C in a medium containing sulfuric acid of pH 2. The purified bipolar membrane spanning main phospholipid (MPL) of this organism can be used to produce stable liposomes of 100-500 nm in diameter either using a French pressure cell detergent dialysis or sonication. Despite a potassium diffusion potential of 186 mV very low ionic permeability of sonicated MPL liposomes was measured using the potassium binding fluorescent indicator benzofuran isophthalate PBF1, which measures net K+ uptake. The latter also remained very low, in the presence of the K(+) ionophore valinomycin and palmitic acid. Addition of valinomycin and the potent uncoupler carbonylcyanid-p-trifluormehoxyphenyl-hydrazone (FCCP), led to a stimulation in potassium uptake. The rate of proton flux can be calculated from the net K(+) uptake. Under these conditions MPL liposomes are 1-2 orders of magnitude less permeable than egg yolk lecithin vesicles. The difference in proton permeability becomes even more pronounced with increasing temperature, examined using the fluorescent pH indicator pyranine. Purified bacteriorhodopsin from Halobacterium halobium was reconstituted into MPL liposomes in order to study the light-driven proton uptake in 150 mM KCl following addition of valinomycin, gramicidin, FCCP and Triton X-100. The light-driven proton transport into the liposomes was increased 30-fold by addition of valinomycin decreased by gramicidin and FCCP, and abolished by Triton X-100. Co-reconstituted MPL proteoliposomes containing bacteriorhodopsin and ATP synthase from Micrococcus luteus were capable of light-driven ATP synthesis demonstrating the functional coupling of proton transport and nucleotide generation in liposomal MPL membranes.

ATP synthesis by purified ATP-synthase from beef heart mitochondria after coreconstitution with bacteriorhodopsin.

An ATP-synthase complex active in ATP synthesis was isolated from beef heart mitochondria by solubilization of submitochondrial particles with dodecyl-beta-D-maltoside and purified in a one-step procedure by subsequent ion-exchange chromatography. The electrophoretic analysis resulted in 14 subunits for the F0 F1 complex. ATP hydrolysis activity of the purified enzyme was 25 mumol ATP min-1 mg-1F0F1. ATP hydrolysis could be stimulated by addition of lipid vesicles. Further stimulation was observed in the presence of uncoupler. The inhibitors dicyclohexylcarbodiimide and oligomycin reduced hydrolytic activity to 70 and 40%, respectively. The preservation of ATP synthesis capability was demonstrated by reconstitution of the purified enzyme together with the light-driven proton pump bacteriorhodopsin. Upon illumination of ATP-synthase/bacteriorhodopsin proteoliposomes ATP synthesis activity was detectable for at least 7 min. At reduced temperature this time could be increased to 20 min. The maximum synthesis rate of 58 nmol ATP min-1 mg-1 F0F1 was obtained after reconstitution into liposomes made from crude soy bean lecithin by a detergent dialysis procedure using octyl-beta-D-glucopyranoside and monomeric bacteriorhodopsin. ATP synthesis was partially inhibited by oligomycin or dicyclohexylcarbodiimide and was completely abolished in the presence of uncoupler. The ability of the purified enzyme to synthesize ATP shows that the described isolation procedure results in an ATP-synthase complex which is intact in structure and function.

ERA-experiment "Space Biochemistry".

The general goal of the experiment was to study the response of anhydrobiotic (metabolically dormant) microorganisms (spores of Bacillus subtilis, cells of Deinococcus radiodurans, conidia of Aspergillus species) and cellular constituents (plasmid DNA, proteins, purple membranes, amino acids, urea) to the extremely dehydrating conditions of open space, in some cases in combination with irradiation by solar UV-light. Methods of investigation included viability tests, analysis of DNA damages (strand breaks, DNA-protein cross-links) and analysis of chemical effects by spectroscopic, electrophoretic and chromatographic methods. The decrease in viability of the microorganisms was as expected from simulation experiments in the laboratory. Accordingly, it could be correlated with the increase in DNA damages. The purple membranes, amino acids and urea were not measurably effected by the dehydrating condition of open space (in the dark). Plasmid DNA, however, suffered a significant amount of strand breaks under these conditions. The response of these biomolecules to high fluences of short wavelength solar UV-light is very complex. Only a brief survey can be given in this paper. The data on the relatively good survival of some of the microorganisms call for strict observance of COSPAR Planetary Protection Regulations during interplanetary space missions.

Purification and characterization of the inhibitory subunit (delta) of the ATP-synthase from Micrococcus luteus.

Subunit delta was isolated from the ATP-synthase from Micrococcus luteus strain (ATCC 4698). delta, in the case of M. luteus F0F1-ATPase, acts as an inhibitor of ATP hydrolysis and thus resembles subunits in E. coli and chloroplast ATP-synthase. After treatment with 1.5 M LiCl the ATP-synthase dissociated, and subsequently subunit delta (27 kDa) was purified by hydrophobic interaction chromatography. Inhibition of ATP-synthase lacking delta by addition of delta showed non-competitive kinetics with a Ki of approximately 5.9 nM. Subunit epsilon from chloroplast F1, which corresponds functionally to the M. luteus F0F1-delta, and chloroplast delta were tested for ATPase inhibitory activity by addition to the partially delta-depleted ATP-synthase from M. luteus. CF1-epsilon inhibited M. luteus ATP-synthase up to 80%, whereas CF1-delta did not show any influence.

ATP synthesis and hydrolysis of the ATP-synthase from Micrococcus luteus regulated by an inhibitor subunit and membrane energization.

After incubation for 70 min in Tris-HCl (pH 8.0), the rate of ATP hydrolysis of free and reconstituted ATP-synthase from Micrococcus luteus multiplied about three times. The apparent increase in activity is due to the reversible dissociation of the delta-subunit. Results of experiments on the temperature dependence of the ATP hydrolysis rate of substrate saturated ATP-synthase exhibited a discontinuity in the Arrhenius plot at 32 +/- 0.5 degrees C for the delta-subunit associated enzyme. Below 32 +/- 0.5 degrees C the activation energy, Ea, was 231.5 +/- 5 kJ mol-1, while above this temperature-level it decreased to 76.4 +/- 3 kJ mol-1. ATP synthesis and hydrolysis of the ATP-synthase, co-reconstituted with monomeric bacteriorhodopsin (Halobacterium halobium), showed a lag of 50 s upon the illumination with green light (505-575 nm). This retardation and the activity depended on the ATP-synthase concentration, being typical of the dissociation of an inhibitor protein. The N-terminal protein sequences of the delta- and epsilon-subunit of the ATP-synthase were identified by automated Edman degradation. Alignment of the amino acid sequence and secondary structure calculations for the delta-subunit did not reveal homology to other known ATP-synthase delta-subunits, but significant equivalence to the epsilon-subunit of E. coli. Sequence analysis of the epsilon-subunit from M. luteus showed homology to equivalent regions in delta-subunits and Oligomycin Sensitivity Conferring Protein (OSCP) of other organisms.

Purification of ATP synthase from beef heart mitochondria (F0F1) and co-reconstitution with monomeric bacteriorhodopsin into liposomes capable of light-driven ATP synthesis.

ATP synthase was isolated from beef heart mitochondria by extraction with N,N-bis-(3-D-gluconamidopropyl)deoxycholamide or by traditional cholate extraction. The enzyme was purified subsequently by ion-exchange and gel-permeation chromatographies in the presence of glycerol and the protease inhibitor diisopropylfluorophosphate. The ATP synthase consisted of 12-14 subunits and contained three tightly bound nucleotides. The co-reconstitution of crude or purified ATP synthase with monomeric bacteriorhodopsin by the method of detergent incubation of liposomes yielded proteoliposomes capable of light-driven ATP synthesis, as detected with a luciferase system for at least 30 min. The reaction was suppressed by the inhibitors oligomycin (> 90%) and dicyclohexylcarbodiimide (85%) and by the uncoupler carbonylcyanide-p-trifluormethoxyphenylhydrazone (> 95%). The purified ATP synthase was apparently free of cytochrome impurities and of adenylate kinase activity, i.e. the enzyme exhibited light-driven ATP synthesis without the dark reaction. For the first time, this is demonstrated with purified ATP synthase from beef heart mitochondria.

Structural dynamics in F1ATPase during the first reaction cycle of ATP hydrolysis.

The velocity of ATP hydrolysis, catalyzed by purified F1ATPase from Micrococcus luteus, was decelerated on decreasing the temperature. At 13 degrees C one reaction cycle is completed after 20 s. Hydrolysis was triggered upon rapid mixing of the enzyme with ATP. During the first reaction cycle, succeeding structural alterations of the F1ATPase were traced by time resolved X-ray scattering. The scattering spectra obtained from consecutive intervals of 1 s, revealed the F1ATPase to pass a conformational state exhibiting an expanded (6%) molecular shape. The expanded state was observed between 45% and 65% of the time required to complete the reaction cycle. This points out a conformational pulsation during ATP hydrolysis.

Isolation and partial characterization of a cytochrome-o complex from chromatophores of the photosynthetic bacterium Rhodospirillum rubrum FR1.

A cytochrome-o complex was isolated from chromatophores of photoheterotrophically grown Rhodospirillum rubrum FR1. The enzyme was extracted with the non-denaturating detergent taurodeoxycholate and subsequently purified by sucrose-density-gradient centrifugation and gel-permeation HPLC. The complex contains two types of cytochromes, one of them cytochrome o, and two copper atoms. It catalyzes the reduction of molecular oxygen, when N,N,N',N'-tetramethyl-p-phenylenediamine or ubiquinol 10 are offered as electron donors. The oxidase activity is inhibited by cyanide, carbon monoxide and 2-heptyl-2-hydroxyquinoline N-oxide. The molecular mass of the protein is 136 +/- 15 kDa. The subunit analysis, by SDS continuous and gradient gels, revealed four subunits with molecular mass 66 kDa (subunit I), 36 kDa (subunit II), 20 kDa (subunit III) and 11 kDa (subunit IV).

Formation of specific amino acid sequences during carbodiimide-mediated condensation of amino acids in aqueous solution, and computer-simulated sequence generation.

Carbodiimide-mediated peptide synthesis in aqueous solution has been studied with respect to self-ordering of amino acids. The copolymerisation of amino acids in the presence of glutamic acid or pyroglutamic acid leads to short pyroglutamyl peptides. Without pyroglutamic acid the formation of higher polymers is favoured. The interactions of the amino acids and the peptides, however, are very complex. Therefore, the experimental results are rather difficult to explain. Some of the experimental results, however, can be explained with the aid of computer simulation programs. Regarding only the tripeptide fraction the copolymerisation of pyroGlu, Ala and Leu, as well as the simulated copolymerisation lead to pyroGlu-Ala-Leu as the main reaction product. The amino acid composition of the insoluble peptides formed during the copolymerisation of Ser, Gly, Ala, Val, Phe, Leu and Ile corresponds in part to the computer-simulated copolymerisation data.

Neutron small angle scattering of matched proteoliposomes with incorporated F0F1 ATPase complex from Rhodospirillum rubrum FR1. An approach to the structure of membrane proteins in their natural environment.

Purified F0F1 ATPase from Rhodospirillum rubrum FR1 has been incorporated into lipid vesicles from the partially deuterated phospholipid dimyristoylglycerophosphocholine (DMPC-D54). These proteoliposomes were able to carry out energy transducing reactions. The incorporation of the membrane protein was controlled by freeze fracture electron microscopy. A method for structural research of the membrane protein in its natural environment has been developed by means of neutron small angle scattering. Using the contrast variation technique, the lipid part of the proteoliposomes was matched by adding an appropriate amount of D2O to the solvent. Thus the neutron scattering profile of F0F1 ATPase incorporated into vesicles was separated from the neutron scattering of the liposome. F0F1 ATPase incorporated in a lipid bilayer, as well as the free enzyme, yields a radius of gyration of Rg = 6.0 +/- 0.1 nm which leads to an overall diameter of 15.5 nm. This result suggests that the monomeric form of F0F1 ATPase is incorporated in DMPC-D54 membranes at 20 degrees C.