Studies on Steric and Electronic Control of 2'-3' Phosphoryl Migration in 2'-Phosphorylated Uridine Derivatives and Its Application to the Synthesis of 2'-Phosphorylated Oligouridylates.

For the synthesis of 2'-phosphorylated oligouridylates by use of new phosphoramidite building units, several masked phosphoryl groups have been examined as 2'-phosphate precursors, which should not be migrated to the 3' position when the 3' hydroxy protecting group must be removed to introduce a phosphoramidite residue into the 3'-position. As a consequence, bis(2-cyano-1,1-dimethylethoxy)thiophosphoryl (BCMETP) was found to be the most suitable 2'-phosphate precursor. This thiophosphoryl group could be introduced into the 2'-hydroxyl of 3',5'-silylated uridine derivative 7 by phosphitylation with bis(2-cyano-1,1-dimethylethoxy)(diethylamino)phosphine followed by sulfurization.

Crystal structure of 6-O-[(R)-2-hydroxypropyl]cyclomaltoheptaose and 6-O-[(S)-2-hydroxypropyl]cyclomaltoheptaose.

Crystal structures of 6-O-[(R)-2-hydroxypropyl]- and 6-O-[(S)-2-hydroxypropyl]-cyclomaltoheptaose were determined by X-ray analysis. In both structures, the 2-hydroxypropyl group is inserted into the macrocyclic cavity of the next molecule related by the two-fold screw axis, and a helically extended polymeric structure is formed by repetition of the intermolecular inclusion. The hydroxyl group of the substituent group penetrates through the macrocyclic ring from the secondary hydroxyl side and is linked to an HO-6 group by a hydrogen bond.

Self-assembling properties of synthetic peptidic lipids.

Novel peptidic lipids were synthesized by the coupling of a linear oligopeptide as the hydrophilic moiety with a glutamic acid dialkylamide as the hydrophobic moiety. Their self-assembling properties were investigated. The critical aggregate concentrations (CAC) for the peptidic lipids with a double dodecyl group were in the range of 1.

Study of blood compatible polymers. III. Copolymers of N-benzyl, N-(2-hydroxyethyl) acrylamide and 2-hydroxyethyl methacrylate.

Copolymerization of 2-hydroxyethyl methacrylate (HEMA) and N-benzyl, N-(2-hydroxyethyl) acrylamide (BENAAm) was carried out at different mole ratios of the monomers to obtain copolymers of varying composition. BENAAm content of the copolymers varies between 13 and 70%. Investigation of the interaction of rabbit platelets with these polymer surfaces showed that copolymers with higher BENAAm content inhibit the platelet deformation.

Construction and characterization of a single polypeptide chain containing two enzymatically active dihydrofolate reductase domains.

A single polypeptide chain containing two dihydrofolate reductase (DHFR) sequences from Escherichia coli was constructed to determine if a repeat sequence fusion protein could be expressed in an active form. The possibility that intersequence interactions could play a significant role for this enzyme is suggested by the results of Hall and Frieden (1989, Proc. Natl Acad.

Porous polyurethane vascular prostheses with variable compliances.

A new technique for the preparation of porous vascular prostheses was investigated. Polyurethane solution (5 to 15 wt%) was injected into a mold. After freezing at low temperature (0 degrees C-196 degrees C), solvents were dissolved out with water at 0 degrees C to form porous tubes.

X-ray structure of Glu 53 human lysozyme.

The three-dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 A resolution by X-ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild-type HL.

Expression and purification of growth hormone-releasing factor with the aid of dihydrofolate reductase handle.

Expression of a fusion protein composed of dihydrofolate reductase and a derivative of growth hormone-releasing factor resulted in the formation of inclusion bodies in Escherichia coli at 37 degrees C. Among various chemicals, such as detergents, protein denaturants, and acetic acid, tested for the ability to dissolve the inclusion bodies, acetic acid, Brij-35, deoxycholic acid sodium salts, guanidine-HCl, and urea showed a strong solubilizing effect without damaging the DHFR activity. Acetic acid was useful in terms of preparing GRF derivatives, since it could be easily removed by lyophilization, and this made it easy to perform the succeeding BrCN treatment for cutting out the GRF derivative from the fusion protein.

Activation of ATPase activity of 14S dynein from Tetrahymena cilia by microtubules.

The ATPase activity of 14S dynein was activated by the presence of microtubule-associated-protein-free microtubules. The activation was 2.5-3.

Dihydrofolate reductase from Bacillus subtilis and its artificial derivatives: expression, purification, and characterization.

The Bacillus subtilis dihydrofolate reductase (DHFR) gene was expressed in Escherichia coli. The gene product was purified to homogeneity by Butyl-Toyopearl, Toyopearl HW55, and DEAE-Toyopearl column chromatographies, and its molecular properties were compared to those of E. coli DHFR.

X-ray structural evidence for a local helix-loop transition in alpha-lactalbumin.

The three-dimensional structure of human alpha-lactalbumin for two crystal forms has been determined by x-ray analysis. One crystal (the form LT) was obtained at pH 4.2 and room temperature, while the other crystal (the form HT) was grown at pH 6.

Biosynthesis and Hydrolysis of Poly(γ-glutamic acid) from Bacillus subtilis IF03335.

Poly(γ-glutamic acid) (PGA) production in Bacillus subtilis IF03335 was studied. When citric acid as a carbon source was added to a glutamic acid medium containing L-glutamic acid and ammonium sulfate, a large amount of pure PGA was produced. On the other hand, when glucose was added to the glutamic acid medium, a by-product was produced, which seemed to be a polysaccharide.

Dihydrofolate reductase as a new "affinity handle".

Dihydrofolate reductase (DHFR) has been demonstrated to be a versatile "affinity handle" for expression of recombinant proteins. The DHFR "handle" has advantages not only in terms of efficiency of expressing the fusion protein as a soluble form but also in stabilizing unstable polypeptides and facilitating purification of the expressed protein by means of methotrexate-bound affinity chromatography and by making use of the enzyme activity. Fifteen genes encoding different lengths of polypeptides of 5 to 44 amino acids were chemically synthesized and introduced into expression vectors, pTP70-1 or its derivatives.

Dihydrofolate reductase gene as a versatile expression marker.

The Escherichia coli dihydrofolate reductase (DHFR) gene has been used as a genetic marker specifying trimethoprim resistance (TmpR). In order to use the DHFR gene as a versatile expression marker, we have constructed three types of plasmids: promoter cloning vector, terminator cloning vector, and the plasmid containing the DHFR gene cassette. In these systems, the selection of recombinant plasmids was carried out just by examining the TmpR phenotype of the transformed cells.

Study of blood compatible polymers. II. poly(N,N-disubstituted) acrylamides.

Poly(N,N-disubstituted) acrylamides with both hydrophilic and hydrophobic groups as substituents were synthesized. Different degrees of hydrophilicity were achieved by varying the bulk of the hydrophobic substituent. N-alkyl, N-(2-hydroxyethyl) acrylamides with alkyl substituents propyl (PROPAAm), octyl (OCTAAm) and benzyl (BENAAm) were synthesized.

Crystal structure of 2-O-[(S)-2-hydroxypropyl]cyclomaltoheptaose.

2-O-[(S)-2-Hydroxypropyl]cyclomaltoheptaose crystallises in the monoclinic space group P2(1) with unit-cell dimensions a = 15.072(1), b = 10.409(1), c = 20.

Fluctuation and rotation of human growth hormone-releasing factor in the presence and the absence of phospholipid bilayer analyzed by time-resolved fluorescence depolarization.

Time-resolved fluorescence depolarization measurements were carried out for human growth hormone-releasing factor analog ([Trp10]-hGRF (1-29) NH2), where the Trp10 residue was incorporated as a fluorescent probe, in the presence and the absence of 1,2-dimyristoyl-sn-glycero-3-phospho-rac- glycerol(DMPG) liposome and in aqueous 2,2,2-trifluoroethanol (TFE) solution. The fluorescence lifetimes and the rotatory correlation times of the peptide in each medium were determined. The apparent volumes of the rotatory Brownian motion unit calculated from these fluorescent parameters indicate the different mode of the fluctuation and/or the rotation of the peptide in each medium, such as: (i) In the aqueous solution, several segments of the peptide fluctuate individually.

Neoendothelial healing of modified EPTFE grafts.

Expanded polytetrafluoroethylene (EPTFE) grafts have poor neoendothelial healing characteristics and low patency rates after long-term implantation. The authors have shown that this is due to the low porosity of currently used EPTFE grafts (20-30 microns fibril length). The inner surface coated grafts made of long antithrombogenic material fibrils (40-60 microns) are desirable, especially for small diameter grafts.

Solution structure of human growth hormone-releasing factor fragment (1-29) by CD: characteristic conformational change on phospholipid membrane.

The conformations of synthetic human growth hormone-releasing factor fragment (1-29) in the presence and the absence of 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol liposome as well as in aqueous 2,2,2-trifluoroethanol solution were investigated by CD spectroscopy. The secondary structure of the peptide in each solution was analyzed by two methods. Both results show that the peptide has an unordered structure in the aqueous solution, whereas it folds into helical structure in the aqueous alcohol and in the phospholipid solution.

Hydration of clupeine in solution.

Spin-lattice relaxation times, T1, of H2(17) O at 25 degrees were measured for aqueous solutions of clupeine and its constituent amino acids, which are serine, threonine, proline and arginine. The dynamic hydration numbers, nDHN, of clupeine and amino acids were determined from a concentration dependence of T1. The coordination numbers nh, and the rotational correlation times, tau ch, of water molecules around clupeine and amino acids were estimated and compared with that of pure water.

Nucleotide specificity of the enzymatic and motile activities of dynein, kinesin, and heavy meromyosin.

The substrate specificities of dynein, kinesin, and myosin substrate turnover activity and cytoskeletal filament-driven translocation were examined using 15 ATP analogues. The dyneins were more selective in their substrate utilization than bovine brain kinesin or muscle heavy meromyosin, and even different types of dyneins, such as 14S and 22S dynein from Tetrahymena cilia and the beta-heavy chain-containing particle from the outer-arm dynein of sea urchin flagella, could be distinguished by their substrate specificities. Although bovine brain kinesin and muscle heavy meromyosin both exhibited broad substrate specificities, kinesin-induced microtubule translocation varied over a 50-fold range in speed among the various substrates, whereas heavy meromyosin-induced actin translocation varied only by fourfold.

Asymmetric inclusion by de novo designed proteins: fluorescence probe studies on amphiphilic alpha-helix bundles.

The inclusion feature and supersecondary structure of the de novo designed proteins which are constructed with several amphiphilic alpha-helices and flexible linkage parts were investigated with fluorescence probes. Five types of small proteins (or peptides) have been designed, which are composed of 2, 3, 4, 4, and 6 helices, respectively, and are linked with only linear junctions except for one of 4-helix proteins. All of these proteins have inclusion ability for hydrophobic fluorophores.

Analysis of the nucleation and crystal growth kinetics of lysozyme by a theory of self-assembly.

Concentration changes in supersaturated solutions during the nucleation and growth of the orthorhombic form of hen egg-white lysozyme crystals have been observed for 121 d at 35 degrees C and pH 4.6, and with 3% NaCl. The effect of a variation in the initial protein concentration on the rate of approach to solubility in equilibrium is analyzed, by applying a model, originally developed for the understanding of protein self-assembly.

Phosphorothioate analogs of ATP as the substrates of dynein and ciliary or flagellar movement.

The phosphorothioate analog of ATP has a sulfur atom replacing a non-bridging oxygen atom of the triphosphate moiety of ATP. Due to the tetrahedral nature of the phosphorus atom, stereoisomers are known to exist, designated as the Sp and Rp isomers. We have reported [Shimizu & Furusawa (1986) Biochemistry 25, 5787] on the hydrolytic activity of the 22S dynein from Tetrahymena cilia towards the phosphorothioate analogs of ATP.