Relative stability of poly(β-hydroxy-l-proline)

The semiempirical CNDO/2 SCF MO method using the tight-binding approximation for polymers has been applied to poly(β-hydroxy-l-proline), β-PHP, to compare the electronic structure of β-PHP with that of poly(γ-hydroxy-l-proline), γ-PHP, which we have described in a previous publication. The results obtained show the preferred orientation of the OH group at the β-position of the pyrrolidine ring. The different situation between β-PHP and γ-PHP is briefly discussed. Analysis of the calculated results shows that the energy difference between the two species is not sufficient to deny the existence of either form. This agrees well with the experimental results. The conformational stability between the trans and cis forms of the H:C:O:H group is explained by using the calculated results in connection with the previous experimental and theoretical treatments. From the analysis of the total energy, the dominant stabilizing factors are discussed.     

CNDO/2 calculations of the relative stability of poly (l-proline I) and poly (l-proline II)

The CNDO/2 method using the tight binding approximation for polymers was applied to poly(l-proline I) and poly(l-proline II). The calculations were also carried out for poly(l-alanines) and model molecules which have the same backbone geometrics as those of poly(l-prolines). The results obtained show that both forms of poly(l-proline I) and poly(l-proline II) have nearly the same energy in agreement with experimental results. From the analysis of the total energy, it was found that the intrasegment energy of poly(l-proline II) was lower than that of poly(l-proline I) while the intersegment energy of poly(l-proline I) was lower than that of poly(l-proline II). This result can be considered to correspond well with the experimental fact that poly(l-proline II) is more stable in good or polar solvents and poly(l-proline I) in poor or non-polar solvents. The analysis of the total energy of poly(l-proline) leads us to the conclusion that the α and β carbons play an important role in determining the relative stability between poly(l-proline I) and poly(l-proline II) and the γ carbon does hvae a marked effect on the electronic structures of the polymers in question. This conclusion was also confirmed by comparison of the electronic structures of poly(l-prolines) with those of poly(l-alanines) and model compounds concerned.     

Determination of Fluorescence Polarization of Double Chromophore Complexes

Polarized fluorescence of rigid double-chromophore complexes with intracomplex energy exchange between chromophores was analyzed, and the formula for the degree of polarization derived for the case of steady-state excitation: P = (3 cos²θ - 1 + 2A)/(3 + cos²θ + 4A). In this formula θ is the angle between the transition dipole moments of chromophores in complexes, and A is the parameter dependent on the spectroscopic features of chromophores and energy migration rates. The case of excitation by a δ-pulse was also analyzed, and a formula for fluorescence polarization kinetics was derived.As an example of the application of the derived formulae, the polarized fluorescence spectra and their picosecond kinetics were calculated for the β-subunits of the blue-green algae Agmenellum quadruplicatum. The results obtained were compared with experimental measurements of Mimuro et al. (1986, Biochim. Biophys. Acta848, 155-166) and found to match these data well.     

CNDO/2 calculation of the relative stability of poly(γ-hydroxy-l-proline)

The CNDO/2 method using the tight-binding approximation for polymers was applied to poly(γ-hydroxy-l-prolines) (PHP). The calculations were carried out for PHPs which have the same backbone structure as those of poly(l-proline I) (Pro-I) and poly(l-proline II)(Pro-II). The results obtained show the preferred orientation of the OH group at the γ-position, which is in agreement with the experimental results. The calculations were also carried out for the B form (PHP-B). The conformational stability between the A form (PHP-A) and PHP-B was explained by using the calculated results in connection with the previous experimental and theoretical treatments. From the analysis of the total energy, the dominant stabilizing factors for the two forms are discussed.     

Effect of cationic and non-ionic surfactants on the hydrolysis of N-glutaryl- -phenylalanine catalysed by chymotrypsin iso-enzymes

The hydrolysis of N-glutaryl- -phenylalanine p-nitroanilide catalysed by various chymotrypsin (CT) iso-enzymes (α-CT, β-CT, δ-CT, and γ-CT) has been studied in the presence of cationic and non-ionic surfactants at concentration higher than the critical micellar concentration. The enzyme activity was tested in the presence of the following surfactants: cetyltrimethylammonium bromide (CTABr), cetyldimethylethylammonium bromide (CDMEABr), cetyltripropylammonium bromide (CTPABr), Triton X100 (TX100) and polyoxyethylene 9 lauryl ether (PO9). The activity of the iso-enzymes depends on the surfactant concentration and it varies with the surfactant head group dimensions (CTPABr>CDMEABr>CTABr). For all the iso-enzymes, superactivity has been detected only in the presence of CTPABr and CDMEABr. The extent of superactivity depends on the enzyme used (δ-CT>β-CT>γ-CT>α-CT). The observed reaction rate has been compared with the prediction of a theoretical model for enzymatic activity in the presence of surfactant aggregates in aqueous media developed in a previous paper. The results can be explained by introducing an equilibrium relation between the enzyme confined in the free bulk water and in the bound water pseudo-phase, and by allowing for different catalytic behaviours of the two forms of enzyme.The theoretical model enables the initial reaction rate to be related to the substrate concentration with an overall Michaelis–Menten equation. Good agreement has been found between experimental and model predicted values of the kinetic parameters.     

Metadynamics modelling of the solvent effect on primary hydroxyl rotamer equilibria in hexopyranosides

Accurate modelling of rotamer equilibria for the primary hydroxyl groups of monosaccharides continues to be a great challenge of computational glycochemistry. The metadynamics technique was applied to study the conformational free energy surfaces of methyl α-d-glucopyranoside and methyl α-d-galactopyranoside, employing the glycam06 force field. For both molecules, seven to eight conformational free-energy minima, differing in the ω (O-5–C-5–C-6–O-6) and χ (C-3–C-4–O-4–HO-4) dihedral angles, were identified in vacuum or in a water environment. The calculated rotamer equilibrium of the primary hydroxyl group is significantly different in vacuum than in water. The major effect of a water environment is the destabilisation of a hydrogen bond between O-4–HO-4 and O-6–HO-6 groups. It was possible to calculate the free-energy differences of individual rotamers with an accuracy of better than 2 kJ/mol. The calculated gg, gt and tg rotamer populations in water are in close agreement with experimental measurements, and therefore support the theoretical background of metadynamics.     

Modified cyclodextrins as chiral selectors: molecular modelling investigations on the enantioselective binding properties of heptakis(2,3-di-O-methyl-6-O-tert.-butyldimethylsilyl)-β-cyclodextrin

Molecular modelling methods have been used to investigate the enantioselective binding properties of chiral dihydrofuranones on heptakis(2,3-di-O-methyl-6-O-tert.-butyldimethylsilyl)-β-cyclodextrin in capillary gas chromatography. A conformational analysis of the modified β-cyclodextrin was performed using annealed molecular dynamics. With the program

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the molecular interaction potential for each of the received energetically reasonable structures of the β-cyclodextrin and the dihydrofuranones was evaluated using different probe groups. The results of these computations have been used as starting points for constructing geometrically reasonable host–guest complexes between the β-cyclodextrin and the dihydrofuranones. The subsequently performed molecular dynamics simulations yielded different complex states reflecting the conformational flexibility of the diastereomeric complexes. Considering the evaluated interaction energy between the β-cyclodextrin and the dihydrofuranones as a measure of complex stability the results are in close agreement with the experimentally determined elution sequences. The methodology for the construction of the interaction model used in this study is capable of simulating the experimental data. We believe that it may serve as a basis for predictions of hitherto unknown elution sequences at modified cyclodextrins.     

The influence of branching tunnels on subterranean termites' foraging efficiency: Considerations for simulations

In our previous study, we constructed a lattice model of termite tunnel pattern to explore the relationship between tunnel geometry and foraging efficiency. The model was based on experimental data obtained from homogeneous soil substrates without food resource. In the present study, we adopted a more general rule in the model to determine branching tunnel lengths. The rule was described by two variables, the probability of tunnel branching, P_branch, and the probability for a branching tunnel to terminate, P_term. With the modified model, we explored the influence of the geometry of branching tunnel on foraging efficiency, γ, for two termite species, Coptotermes formosanus and Reticulitermes flavipes (Isoptera: Rhinotermitidae). For C. formosanus, γ map consisting of the two variables were partitioned in three regions by the level of γ value, while γ for R. flavipes was categorized as two regions: higher γ and lower γ. This result was discussed in termite foraging strategy.     

Conformational change of poly( -lysine) induced by lipid vesicles of dilauroylphosphatidic acid

The effect of negatively charged dilauroylphosphatidic acid (DLPA) vesicles on the conformation of poly( -lysine) was investigated by circular dichroism measurements. DLPA vesicles induced a confomiational change Of poly( -lysine) from the random coil to β-structure in 5 mM Tes, pH 7.0. The fraction of induced β-structure (F_β) was determined via a procedure of curve fit the observed spectra to the reference spectra. F_β increased linearly with the molar ratio, r, of DLPA to lysine residues up to r 0.7, and reached a saturation value of 1 at r > 1. Within the range 0.7 r 1, precipitation occurred. The effect of dilution of the negative charge on vesicle membranes was examined by mixing DLPA with dilauroylphosphatidylcholine (DLPC). Although the β-structure Of poly -lysine) was also induced by mixed vesicles, the saturation value of F_β decreased with decreasing DLPA content in mixed vesicles. The variation in saturation value of F_β with the composition of mixed vesicles was interpreted in terms of the change in average distance between DLPA head groups in mixed vesicles.     

A Biodegradable,Sustained-Released,Prednisolone Acetate Microfilm Drug Delivery System Effectively Prolongs Corneal Allograft Survival in the Rat Keratoplasty Model

Frequent and long-term use of topical corticosteroids after corneal transplantation is necessary to prevent graft rejection. However, it relies heavily on patient compliance, and sustained therapeutic drug levels are often not achieved with administration of topical eye drops. A biodegradable drug delivery system with a controlled and sustained drug release may circumvent these limitations. In this study, we investigated the efficacy of a prednisolone acetate (PA)-loaded poly (d,l-lactide-co-ε-caprolactone) (PLC) microfilm drug delivery system on promoting the survival of allogeneic grafts after penetrating keratoplasty (PK) using a rat model. The drug release profiles of the microfilms were characterized (group 1). Subsequently, forty-eight PK were performed in four experimental groups: syngeneic control grafts (group 2), allogeneic control grafts (group 3), allogeneic grafts with subconjunctivally-implanted PA microfilm (group 4), and allogeneic grafts with PA eye drops (group 5; n = 12 in each). PA-loaded microfilm achieved a sustained and steady release at a rate of 0.006–0.009 mg/day, with a consistent aqueous drug concentration of 207–209 ng/ml. The mean survival days was >28 days in group 2, 9.9±0.8 days in group 3, 26.8±2.7 days in group 4, and 26.4±3.4 days in group 5 (P = 0.023 and P = 0.027 compared with group 3). Statistically significant decrease in CD4+, CD163+, CD 25+, and CD54+ cell infiltration was observed in group 4 and group 5 compared with group 3 (P<0.001). There was no significant difference in the mean survival and immunohistochemical analysis between group 4 and group 5. These results showed that sustained PA-loaded microfilm effectively prolongs corneal allograft survival. It is as effective as conventional PA eye drops, providing a promising clinically applicable alternative for patients undergoing corneal transplantation.     

Capillary electrophoretic separation of nucleotide isomers via complexation with cyclodextrin and borate

The electrophoretic behaviour of monophosphorylated nucleotide isomers can be manipulated using complex-forming reactions with β-cyclodextrin (β-CD) and borate. Resolution of the 2'- and 3'-isomers of nucleotides is possible when the electrophoresis buffer contains 10 mM CD. The effect of β-CD concentration on electrophoretic mobility is used to calculate the formation constant, K, of β-CD—nucleotide complexes. The 3'-isomer of adenosine monophosphate (AMP) forms the strongest complex with β-CD probably as a result of hydrogen bonding between the phosphate group of AMP and hydroxyls of β-CD. In addition, complexation of 5'-nucleotides with borate increases the migration time window and leads to better separation. Complex-forming reactions of guanosine monophosphate and uridine monophosphate are shown to be strongly dependent on buffer pH. A mixture of 12 monophosphorylated nucleotides can be separated in less than 15 min using a buffer of 20 mM borate—10 mM β-CD.     

Gastrocnemius medialis muscle architecture and physiological cross sectional area in adult males with Duchenne muscular dystrophy

Objectives:

To describe muscle size and architecture of the gastrocnemius medialis (GM) muscle in eleven adult males with Duchenne Muscular Dystrophy (DMD, age 24.5±5.4 years), and a control group of eleven males without DMD (CTRL, age 22.1±0.9 years).

Methods:

GM anatomical cross sectional area (ACSA), volume (VOL), physiological cross sectional area (PCSA), fascicle length (Lf) and pennation angle (θ) were assessed using B-Mode Ultrasonography. GM ACSA was measured at 25, 50 and 75% of muscle length (Lm), from which VOL was calculated. At 50% of Lm, sagittal plane images were analysed to determine GM Lf and θ. GM PCSA was calculated as VOL/Lf. The ratio of Lf and Lm was also calculated.

Results:

GM ACSA at 50% Lm, VOL and PCSA were smaller in DMD males compared to CTRL males by 36, 47 and 43%, respectively (P<0.01). There were no differences in Lf and θ. GM Lm was 29% shorter in DMD compared to CTRL. Lf/Lm was 29% longer in DMD (P<0.01).

Conclusions:

Unlike previous data in children with DMD, our results show significant atrophy in adult males with DMD, and no change in Lf or θ. The shorter Lm may have implications for joint flexibility.     

Twisting of glycosidic bonds by hydrolases

Patterns of scissile bond twisting have been found in crystal structures of glycoside hydrolases (GHs) that are complexed with substrates and inhibitors. To estimate the increased potential energy in the substrates that results from this twisting, we have plotted torsion angles for the scissile bonds on hybrid Quantum Mechanics::Molecular Mechanics energy surfaces. Eight such maps were constructed, including one for α-maltose and three for different forms of methyl α-acarviosinide to provide energies for twisting of α-(1,4) glycosidic bonds. Maps were also made for β-thiocellobiose and for three β-cellobiose conformers having different glycon ring shapes to model distortions of β-(1,4) glycosidic bonds. Different GH families twist scissile glycosidic bonds differently, increasing their potential energies from 0.5 to 9.5 kcal/mol. In general, the direction of twisting of the glycosidic bond away from the conformation of lowest intramolecular energy correlates with the position (syn or anti) of the proton donor with respect to the glycon’s ring oxygen atom. This correlation suggests that glycosidic bond distortion is important for the optimal orientation of one of the glycosidic oxygen lone pairs toward the enzyme’s proton donor.     

Cloning and sequence analysis of a cDNA for the α-globin mRNA of carp, Cyprinus carpio

A cDNA for α-globin mRNA of the carp, Cyprinus carpio, was cloned by the method of Okayama and Berg (Mol. Cell. Biol. 2 (1982) 161–170) and its complete nucleotide sequence was determined. The 5′ non-coding region contained 23 nucleotides. Following this region, there was an open reading frame encoded with an α-globin polypeptide consisting of 142 amino acids. The 3′ non-coding region was 88 nucleotides in length, including two copies of the hexanucleotide AATAAA and a poly(A) site of the GC dinucleotide. There were 16 discrepancies between the reported amino acid sequence of the carp α-globin chain and the amino acid sequence predicted from the DNA sequence of the clone. The possible explanations for these differences in amino acid sequence are discussed.     

The activities of mycotoxins derived from Fusarium and related substances in a short-term transformation assay using v-Ha-ras-transfected BALB/3T3 cells (Bhas 42 cells)

Cell transformation assays using BALB/3T3 cells can mimic the two-stage process of chemical carcinogenesis in experimental animals. A short-term transformation assay using v-Ha-ras-transfected BALB/3T3 cells (Bhas 42 cells), which was developed by Ohmori et al. and modified by Asada et al., has been reported to detect both tumor initiators and promoters as transformation initiators and promoters, respectively, with their differences based on their protocols. In this new short-term assay, we examined mycotoxins derived from Fusarium and related substances for the initiation and promotion activities of the transformation. The tested substances included deoxynivalenol, nivalenol, fusarenon-X, T-2 toxin, fumonisin B₁, fumonisin B₂, zearalenone, α-zearalanol, β-zearalanol, α-zearalenol and β-zearalenol. Fumonisin B₁ and T-2 toxin were positive for promoting activity in the assay. Especially, T-2 toxin was active at concentrations as low as 0.001–0.002 μg/mL in the culture medium. From a comparison between the results of this study and published carcinogenicity assay data, it was expected that the Bhas 42 cell transformation assay had a good correlation with the two-stage carcinogenicity tests using experimental animals for estimation of the tumor-promoting activity.     

Serum fetuin--a concentrations are inversely related to cytokine concentrations in patients with chronic renal failure

Background/Aims: A close relationship exists between inflammation and vascular calcification. Although fetuin-A is known to be an inhibitor of calcification, studies correlating levels of this glycoprotein to markers of inflammation are limited. To understand these relationships, we investigated the relationship between serum fetuin-A and proinflammatory cytokine levels in patients with chronic renal failure (CRF). Methods: Thirty-two patients on haemodialysis (HD), 32 conservatively managed chronic kidney disease (CKD) patients and a control group of 25 subjects with normal renal function were enrolled in this study. Serum fetuin-A, IL-1β, IL-6 and TNF-α levels were measured by ELISA. Correlations between serum fetuin-A and IL-1β, IL-6 and TNF-α concentrations were investigated by the Spearman correlation test. Results: In 64 CRF patients (on HD and with CKD), serum fetuin-A was significantly and inversely related to IL-1β (P < 0.001), IL-6 (P = 0.025) and TNF-α levels (P = 0.007), respectively. The serum fetuin-A levels of the control subjects were not significantly correlated to levels of the inflammatory markers IL-1β, IL-6 and TNF-α (P = 0.551, 0.985 and 0.984, respectively). Conclusion: The negative correlation between serum fetuin-A and cytokine concentrations in CRF patients supports the hypothesis of inflammation-dependent down-regulation of fetuin-A expression.     

Modelling ion binding to AA platform motifs in RNA: a continuum solvent study including conformational adaptation

Binding of monovalent and divalent cations to two adenine–adenine platform structures from the Tetrahymena group I intron ribozyme has been studied using continuum solvent models based on the generalised Born and the finite-difference Poisson–Boltzmann approaches. The adenine–adenine platform RNA motif forms an experimentally characterised monovalent ion binding site important for ribozyme folding and function. Qualitative agreement between calculated and experimental ion placements and binding selectivity was obtained. The inclusion of solvation effects turned out to be important to obtain low energy structures and ion binding placements in agreement with the experiment. The calculations indicate that differences in solvation of the isolated ions contribute to the calculated ion binding preference. However, Coulomb attraction and van der Waals interactions due to ion size differences and RNA conformational adaptation also influence the calculated ion binding affinity. The calculated alkali ion binding selectivity for both platforms followed the order K⁺ > Na⁺ > Rb⁺ > Cs⁺ > Li⁺ (Eisenman series VI) in the case of allowing RNA conformational relaxation during docking. With rigid RNA an Eisenman series V was obtained (K⁺ > Rb⁺ > Na⁺ > Cs⁺ > Li⁺). Systematic energy minimisation docking simulations starting from several hundred initial placements of potassium ions on the surface of platform containing RNA fragments identified a coordination geometry in agreement with the experiment as the lowest energy binding site. The approach could be helpful to identify putative ion binding sites in nucleic acid structures determined at low resolution or with experimental methods that do not allow identification of ion binding sites.     

Myosin heavy chain isoforms expression and cyclic AMP concentrations in hypoxia-induced hypertrophied right ventricle in rats

We have previously demonstrated that the relative expression of myosin heavy chain-beta (MHC-β) in both ventricles of rats exposed to long-term hypobaric hypoxia correlated significantly with the relative ventricular mass. In the present study, we investigated whether an increased expression of MHC-β was accompanied by a reduction in cyclic AMP (cAMP) activity in hypoxia-induced hypertrophied right ventricle (RV). We used male Wistar–Kyoto rats born and raised at simulated altitudes (2200 m: H2 group or 4000 m: H4 group) compared to age-matched sea level controls (SC group). There were no significant differences between the groups in basal and forskolin-stimulated adenylyl cyclase (AC) activities. The basal and IBMX-inhibited phosphodiesterase (PDE) activities were slightly higher in both hypoxic groups (p>0.05), except that the H2 group had a higher basal PDE activity than the SC group (p<0.05). The AC/PDE activity ratios were significantly decreased in both hypoxic groups (p<0.05), suggesting that low concentrations of cellular cAMP were maintained in the RV under hypoxic conditions. However, there were no correlations between MHC-β expression and either AC activity, PDE activity, or AC/PDE activity ratio. These results provided evidence against the causal role for cAMP concentration in the expression of MHC-β associated with hypoxia-induced ventricular hypertrophy.     

Purification and properties of a low molecular weight DNA polymerase from Neurospora crassa

A third DNA polymerase ‘C’ with low molecular weight was isolated and purified 3700-fold from ground hyphae of Neurospora crassa WT 74 A, which shows similarities to β- and γ-polymerases from higher eukaryotes: preference for poly(rA)(dT) as a template/primer, inhibition by p-chloromercuribenzoate, resistance against N-ethylmaleimide up to 10 mmol/l, and molecular weight of about 40 000. This polymerase elutes as a distinct peak from DEAE-cellulose at 0.60 mol/l KCl and has an optimum for K⁺ at 2–20 mmol/l, for Mn²⁺ at 0.8 mmol/l, for Mg²⁺ at 4.0 mmol/l, the pH optimum is 8.0. Its K_m is 1.5 μmol/l using dTTP as substrate. The enzyme activity described here is free of endonuclease but contains detectable amounts of exonuclease.