Cloning and characterization of an active fragment of luciferase from a luminescent marine alga, Pyrocystis lunula

Two marine dinoflagellates, Lingulodinium polyedrum and Pyrocystis lunula, emit light in a reaction involving the enzymatic oxidation of its tetrapyrrole luciferin by molecular oxygen. The characteristic properties of P. lunula luciferase have not been clarified, whereas L. polyedrum luciferase, which has three active domains, has been characterized. A cloned partial cDNA of the P. lunula luciferase encodes an active fragment corresponding to part of domain 2 and all of domain 3 of L. polyedrum luciferase. The homology of the amino acid sequence between the two luciferases in domain 3 is about 84.3%. A recombinant His-tagged luciferase fragment containing domain 3 (Mr = 46 kDa) catalyzed the light-emitting oxidation of luciferin (lambdamax = 474 nm). This protein was purified by a single affinity-chromatography procedure. The pH-activity profile and the bioluminescence spectrum of the recombinant enzyme having a third domain are almost identical to those of an extract from P. lunula cultured in vitro. The recombinant enzyme is active at pH 8.0, although the recombinant enzyme derived from the second domain of L. polyedrum luciferase is inactive at pH 8.0. Substitution of Glu-201 by histidine in the third domain of P. lunula luciferase showed a decrease of activity above pH 7.0, suggesting that histidine residues could be responsible for pH-sensitivity in dinoflagellate luciferase.     

Molecular mobility of lyophilized poly(vinylpyrrolidone) and methylcellulose as determined by the laboratory and rotating frame spin-lattice relaxation times of 1H and 13C

Laboratory- and rotating- frame spin-lattice relaxation times (T(1) and T(1rho)) of (1)H and (13)C in lyophilized poly(vinylpyrrolidone) (PVP) and methylcellulose (MC) are determined to examine feasibility of using T(1) and T(1rho) as a measure of molecular motions on large time scales related to the storage stability of lyophilized formulations. The T(1rho) of proton and carbon was found to reflect the mobility of PVP and MC backbones, indicating that it is useful as a measure of large-time-scale molecular motions. In contrast to the T(1rho), the T(1) of proton measured in the same temperature range reflected the mobility of PVP and MC side chains. The T(1) of proton may be useful as a measure of local molecular motions on a smaller-time-scale, although the measurement is interfered by moisture under some conditions. The temperature dependence of T(1) and T(1rho) indicated that methylene in the MC molecule had much higher mobility than that in the dextran molecule, also indicated that methylene in the PVP side chain had a higher mobility than that in the MC side chain.     

Replacement of the phosphorodiester linkages of RNA with guanidinium linkages: the solid-phase synthesis of ribonucleic guanidine

[reaction: see text] Replacement of the negatively charged phosphodiester linkages of RNA with positively charged guanidinium linkages provides the polycationic ribonucleic guanidine (RNG). RNG is anticipated to bind strongly to target DNA/RNA through the specific interactions of nucleobases and the attractive electrostatic interactions of backbones. Preparation of building blocks and the solid-phase synthesis of RNG are reported. Both trimeric and pentameric uridyl RNG have been synthesized.     

Effect of Organic Ligands Used in Sol-Gel Process on the Formation of Mullite

The preparation of mullite by the sol-gel method using organic polydentate ligands and the effect of the raw materials and organic polydentate ligands on the formation of mullite were investigated. Two series of samples were prepared using tetraethoxyorthosilicate (TEOS) and aluminum nitrate nonahydrate, or dibutoxyethylacetoacetatoaluminum (Al(OBu)₂(AcAcEt)) as the silica and alumina sources, respectively, and using ethylene glycol (EG), 1,3-propanediol (PD), 1,3-butanediol (BD), 2-methyl-2, 4-pentanediol (MPD), diethlene glycol monoethyl ether (DEME) and ethoxyethanol as the ligands. When the alumina source was aluminum nitrate nonahydrate, mullite was apt to appear in the order of EG>PD>MPD. When Al(OBu)₂(AcAcEt) was the alumina source, the tendency toward the appearance of mullite crystalline phase was EG>BD>DEME>MPD. Between the two alumina sources, aluminum nitrate nonahydrate gave mullite much easier than Al(OBu)₂(AcAcEt). These relationships were discussed from the viewpoints of the coordination ability of the ligands and the miscibility between the silica and alumina.     

19F- and 1H-NMR spectra of tetrafluoroethylene–propylene copolymers

High resolution 94-MHz ¹⁹F- and 100-MHz ¹H-NMR spectra were measured on a series of tetrafluoroethylene (TFE)-propylene (P) copolymers having a range of composition (TFE/P molar ratio = 37/63–55/45) and polymerized at different temperatures (?23, 25 and 65°C). The spectra were analyzed in relation to copolymer compositions. The assignment of ¹⁹F resonance in terms of tetrads proposed previously was confirmed, and the tentative assignment of ¹H resonances was proposed in terms of triads. The spectra thus interpreted revealed the sequence distribution of the copolymers. Copolymer compositions calculated from NMR spectra and elemental analysis agreed rather well with each other. Monomer reactivity ratios were calculated from the sequence distributions and compared with those obtained from the elemental analysis. It was observed that highly alternating copolymers are obtained in this system over a wide range of monomer composition at lower temperatures and that a deviation from alternancy increases slightly with rising polymerization temperature.     

Effect of polymer excipients on the enzyme activity of lyophilized bilirubin oxidase and beta-galactosidase formulations

The effects of excipients on the protein stability during lyophilization as well as the storage stability of lyophilized bilirubin oxidase (BO) and beta-galactosidase (GA) formulations were studied using four polymer excipients: dextran, polyvinylalcohol (PVA), poly(acrylic acid) (PAA), and alpha, beta-poly(N-hydroxyethyl)-L-aspartamide (PHEA). Denaturation of BO and GA during lyophilization largely depended on the excipient used. Dextran appeared to cause severe damage to proteins, whereas PHEA protected proteins effectively from denaturation. Storage stability of BO and GA formulations also depended on the excipients, such that the formulations containing dextran and PAA were relatively unstable. Storage stability was improved by absorption of a small amount of water for all the formulations studied. Absorption of a larger amount of water, however, decreased the storage stability of the formulations containing PVA, PAA or PHEA. In contrast, the storage stability of formulations containing dextran did not decrease noticeably with increasing water. This may be because formulations containing dextran have a higher glass transition temperature than formulations containing PVA, PAA or PHEA when a large amount of water is absorbed.     

Anharmonicity and fragility in lithium borate glasses

Since the physical properties of lithium borate glasses xLi₂O-(1-x)B₂O₃ (0<x< 0.28) vary over a wide range with the composition, this binary system is particularly suitable for studying the relationship between vibrational anharmonicity and fragility. The density, the linear expansion coefficient, the longitudinal and transverse ultrasonic velocities and their respective temperature coefficients of the velocities are measured, from which the vibrational anharmonicity in lithium borate glasses is evaluated with the help of the Grüneisen parameter at the Debye cut-off frequency and the Anderson-Grüneisen parameter: these two parameters plotted vs. composition have the same characteristics with minima at x≈0.08. The fragility is evaluated from the temperature width of the glass transition; the fragility also shows a minimum at x≈0.08. The presence of minima at x≈0.08 is ascribable to the fact that the crosslinking density between six-membered rings in the glass reaches a maximum at this composition. We show that the anharmonic parameters strongly correlate with the fragility metrics. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of molecular weight in amphipathic polyethyleneglycol on prolonging the circulation time of large unilamellar liposomes

The effect in mice of the molecular weight of polyethyleneglycol on prolonging the circulation time of large unilamellar liposomes (LUVs) was examined using four different distearoyl N-(monomethoxy polyethyleneglycol succinyl) phosphatidylethanolamines (DSPE-PEGs). The molecular weights tested were 1000, 2000, 5000 and 12000. Incorporation of 6 mol% of DSPE-PEG in LUV composed of distearoylphosphatidylcholine (DSPC) / cholesterol (CH) (1:1 in molar ratio) increased the blood circulation half-life significantly more than those without DSPE-PEG derivatives. DSPE-PEGs with molecular weights of 1000 and 2000 prolonged the circulation time of liposomes more than other DSPE-PEGs with higher molecular weights, such as 5000 and 12000. Their effects are also higher than ganglioside GM1, a well described glycolipid with this effect. DSPC/CH LUV-incorporated DSPE-PEG with a molecular weight of 2000 displayed a high concentration in the blood, approximately 40% of the dose, 6 h after the injection.