Structural studies of the interactions of normal and abnormal human plasmins with bovine basic pancreatic trypsin inhibitor

Catalytic activity of human plasmin is inhibited by bovine basic pancreatic trypsin inhibitor (BPTI, also known as aprotinin). In spite of increased interest in the function of BPTI as an inhibitor of plasmin, the 3-D structure of the plasmin-BPTI complex has not yet been determined. Therefore, in the present paper, the structure of the plasmin-BPTI complex was constructed by the homology modeling method, which provided information about the high affinity of plasmin for BPTI. Moreover, normal mode analyses of free plasmin, free BPTI and the plasmin-BPTI complex were carried out to investigate the changes in dynamics following complex formation. After study of the plasmin-BPTI interaction, we also investigated the binding of BPTI with abnormal plasmin, theoretically and experimentally. The result showing that BPTI binds to abnormal plasmin in the same way as it does to normal plasmin supports the previous finding that the difference between normal and abnormal plasmins is very small and that the abnormality is localized to the catalytic site.     

Optically active N-acetyldopamine dimer of the crude drug "Zentai," the cast-off shell of the Cicada, Cryptotympana sp

Two optically active N-acetyldopamine dimers together with four phenolic monomers were isolated from the crude drug "Zentai," a cast-off shell of the cicada of Cryptotympana sp. (Cicadidae). The former two were 2-(3',4'-dihydroxyphenyl)-1,4-benzodioxane derivatives carrying substituents at the 3 and 6 (or 7) positions, which are known to be components of sclerotized insect cuticles. Their structures including absolute configurations were determined on the basis of NMR and circular dichroism (CD) spectroscopic data.     

Synthesis, conformation, and chemical properties of new mini parallel double-stranded peptides conjugated with -Phe-Phe- and -Phe-Phe-X-sequences

To investigate the chemical conformations and functions of the -Phe-Phe-Val- or -Phe-Phe- sequences contained in the Alzheimer's disease related beta-amyloid peptide, a series of mini parallel double-stranded peptides conjugated with two peptide residues to one spacer were designed and prepared. The structure of the compounds was elucidated by circular dichroism (CD) spectrum and NMR two dimensional (2D) nuclear Overhauser enhancement and exchange spectroscopy (NOESY) measurments. The structure of 1,2-ethano-bis(L-Phe-L-Phe-L-Leu), 1,12-dodecano-bis(L-Phe-L-Phe-L-Leu), 1,12-dodecano-bis(L-Phe-L-Phe-L-Val), and 1,12-dodecano (D-Phe-D-Phe-D-Leu) conjugated with L-Leu and L-Val residues show a beta-turn-like nucleation. The dihedral angles (theta = +75 degrees, omega = +180 degrees, phi = +90 degrees, phi = -87 degrees, psi = +180 degrees) obtained from experimental coupling constant (J) data, etc. support that 1,12-dodecano-bis(L-Phe-L-Phe) adopts beta-turn mimic nucleation. The 1,12-dodecano- bis(L-Leu-L-Leu-L-Phe), 1,12-dodecano-bis(L-lle-L-Phe-L-Leu), and 1,12-dodecano-bis(L-Phe-L-Val-L-Leu), etc. adopt most probably a random structure by CD studies. It was found by titration spectrum that an inclusion complex of 1:1 ratio (association constant; azobenzene (guest, Ka=1.0 x 10(4)M-1) is formed between 1,12-dodecano-bis(L.-Phe-L-Phe-L-Leu) and [L0]=1.758 x 10(-5)M-1). Moreover, the stability of the complexes was increased in order of 1,12-dodecano-bis(L-Phe-L-Phe-L-Leu) x azobenzene> 1,12-dodecano-bis(L-Phe-L-Phe-L-Val) x azobenzene> 1,12-dodecano-bis(L-Phe-L-Val-L-Leu) azobenzene. The data show that X-Phe-L-Phe-L-spacer(S)-L-Phe-L-Phe-X (X=amino acids; S = 1,2-ethano- and 1,12-dodecano-) plays an important role as a binding site of the artificial receptor. The hydrophobic interaction of the four Phes in the two strands is a very interesting issue in the physiological action of proteins as well as the conformation of the backbone of X-L-Phe-L-Phe-spacer(S)-iL-Phe-l.-Phe-X.     

Permeation and separation characteristics for benzene/cyclohexane mixtures through tosylcellulose membranes in pervaporation

Tosylcelluloses (TosCells) with different degrees of tosylation were synthesized as membrane materials for the separation of benzene/cyclohexane (Bz/Chx) mixtures. TosCell membranes showed a high benzenepermselectivity for the Bz/Chx mixtures in pervaporation (PV). An increase in the benzene concentration in the feed mixtures increased permeation rate but decreased the benzenepermselectivity of the TosCell membranes. The increase in the permeation rate was attributed to the increase of the degree of swelling of the TosCell membranes by the feed mixtures and the decrease in the benzenepermselectivity was mainly caused by the decrease of sorption selectivity. With low benzene concentrations in the Bz/Chx mixtures, the permeation rate of a TosCell membrane with a higher degree of tosylation was greater than that with a lower degree of tosylation, but was vice versa with a high benzene concentration. The benzenepermselectivity of the former TosCell membrane was higher than that of the latter membrane. Differences of the permeation rate and benzenepermselectivity with changes in the benzene concentration in the feed mixture and degree of tosylation of the TosCell membrane were significantly influenced by the degree of swelling of the TosCell membrane and the benzene concentration sorbed into the TosCell membrane. Mechanism of separation for the Bz/Chx mixtures through the TosCell membranes is discussed by the solution–diffusion model.     

Dehydro[12]- and -[18]annulenes fused with tetrafluorobenzene: synthesis,electronic properties,packing structures,and reactivity in the solid state

Dehydro[12]- and -[18]annulenes 3 and 4 fused with tetrafluorobenzene were newly synthesized by the copper-mediated oxidative coupling of 1,2-diethynyltetrafluorobenzene. The UV-vis spectra of 3 and 4 showed the maximum absorption to be almost identical to that of the corresponding unsubstituted benzodehydro[12]- and -[18]annulenes 1 and 2, respectively, while the reduction waves in cyclic voltammetry observed at potentials of -1.48 and -1.56 V vs Fc/Fc(+) for 3 and 4 were less negative than those for 1 and 2. In agreement with these results, theoretical calculations (B3LYP/6-31G(d)) indicated that the HOMO-LUMO gap is similar for 1 and 3 and for 2 and 4 but that the LUMO levels of 3 and 4 are apparently lowered by the electronegative fluorine substituents. The X-ray crystallography of single crystals grown from 3 (crystal A), 3.C(6)H(6) (crystal B), and a mixture of 1 and 3 (crystal C) demonstrated that the molecules of 3 are stacked in a slanted manner in crystals A and B, while those of 1 and 3 form sandwichlike 1:2 complexes (3.1.3) that are stacked in a columnar arrangement in crystal C. Despite the suitable packing for topochemical polymerization, crystals A-C were quite stable against photochemical reaction. In contrast, differential scanning calorimetry showed that the thermal polymerization occurred explosively at 120-135 degrees C.     

Physicochemical properties of quaternized poly(amidoamine) dendrimers with alkyl groups and of their mixtures with sodium dodecyl sulfate

The physicochemical properties of quaternized poly(amidoamine) dendrimers (generation 4) with methyl or octyl groups and of their mixtures with sodium dodecyl sulfate (SDS) in aqueous solutions have been investigated using several techniques including surface tension, fluorescence of pyrene, and dynamic light scattering. In the single systems of the dendrimers, the dendrimer with octyl groups shows lower surface tension and lower micropolarity than the dendrimer with methyl groups. The hydrodynamic radii of two quaternized poly(amidoamine) dendrimers are considerably large, indicating the formation of aggregates. In the mixed systems of quaternized poly(amidoamine) dendrimers and SDS, the dendrimer with octyl groups-SDS mixed system shows very low surface tension and low micropolarity even in the presence of extremely low SDS concentration compared to those of the dendrimer with methyl groups-SDS mixed system. Maximum turbidity for both systems is observed at around the mixed molar ratio of dendrimer:SDS=1:1.5 where distinct changes have also been confirmed by surface tension, fluorescence of pyrene, and electrical conductivity measurements.     

Block catiomer polyplexes with regulated densities of charge and disulfide cross-linking directed to enhance gene expression

A block catiomer polyplex, showing a high stability in the extracellular medium and an efficient release of plasmid DNA (pDNA) in the intracellular compartment, was developed by controlling both the cationic charge and disulfide cross-linking densities of the backbone polycations. Poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL) was thiolated using either of two thiolation reagents, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or 2-iminothiolane (Traut's reagent), to investigate the effects of both the charge and disulfide cross-linking densities on the properties of the polyplexes. The introduction of thiol groups by SPDP proceeded through the formation of amide linkages to concomitantly decrease the cationic charge density of PLL segment, whereas Traut's reagent promoted the thiolation with the introduction of cationic imino groups to keep the charge density constant. These thiolated PEG-PLLs were complexed with pDNA to form the disulfide cross-linked block catiomer polyplexes, which had the size of approximately 100 nm. Both thiolation methods were similarly effective in introducing disulfide cross-links to prevent the polyplex from the dissociation through a counter polyanion exchange in the extracellular oxidative condition. On the other hand, the efficient release of pDNA responding to the reductive condition mimicking the intracellular environment was only achieved for the polyplex thiolated with SPDP, a system compensating for the decrease in the charge density with the disulfide cross-linking. This distinctive sensitivity toward oxidative and reductive environments was nicely correlated with the remarkable difference in the transfection efficiency between these two types of thiolated polyplexes (SPDP and Traut's reagent types): the former revealed approximately 50 times higher transfection efficiency toward 293T cells than the latter. Obviously, the balance between the densities of the cationic charge and disulfide cross-linking in the thiolated polyplex played a crucial role in the delivery and controlled release of entrapped pDNA into the microenvironment of intracellular compartment to achieve the high transfection efficiency.