Theoretical consideration of osmotic pressure in aqueous protein/salt systems based on extended hard core Lennard-Jones potential

A simple and analytical pair potential function was developed to represent the osmotic pressures in aqueous protein/salt systems under various conditions. Based on a hard core Lennard-Jones (HCLJ) potential model, the new potential function considers various interactions by extending the attractive Lennard-Jones potential. A temperature-dependent coefficient term was introduced to take into account the specific properties of given materials. Comparison of the new potential function with the HCLJ model in hydrocarbon and water systems showed that consideration of the temperature dependence in the potential function was effective, especially for strong polar systems such as water. To predict the osmotic pressures of aqueous lysozyme/(NH(4))(2)SO(4) solutions of various ionic strength and pH, the energy parameters of lysozyme were correlated with the experimental cloud point temperature. The proposed model agreed fairly well with the experimental osmotic pressure data with only previously obtained parameters.     

Dissociation kinetics of singly protonated leucine enkephalin investigated by time-resolved photodissociation tandem mass spectrometry

The yields of post-source decay (PSD) and time-resolved photodissociation (PD) at 193 and 266 nm were measured for singly protonated leucine enkephalin ([YGGFL + H]⁺), a benchmark in the study of peptide ion dissociation, by using tandem time-of-flight mass spectrometry. The peptide ion was generated by matrix-assisted laser desorption ionization (MALDI) using 2,5-dihydroxybenzoic acid as the matrix. The critical energy (E₀) and entropy (ΔS‡ at 1000 K) for the dissociation were determined by Rice-Ramsperger-Kassel-Marcus fit of the experimental data. MALDI was done for a mixture of YGGFL and Y₆ and the plume temperature determined by the kinetic analysis of [Y₆ + H]⁺ data were used to improve the precision of E₀ and ΔS‡ for [YGGFL + H]⁺. E₀ and ΔS‡ thus determined (E₀ = 0.67 ± 0.08 eV, ΔS‡=−24.4 ± 3.2 eu with 1 eu = 4.184 J K⁻¹mol⁻¹) were significantly different from those determined by blackbody infrared radiative dissociation (BIRD) (E₀ = 1.10 eV, ΔS‡ = −14.9 eu), and by surface-induced dissociation (SID) (E₀ = 1.13 eV, ΔS‡ = −10.3 eu). Analysis of the present experimental data with the SID kinetics (and BIRD kinetics also) led to an unrealistic situation where not only PSD and PD but also MALDI-TOF signals could not be detected. As an explanation for the discrepancy, it was suggested that transition-state switching occurs from an energy bottleneck (SID/BIRD) to an entropy bottleneck (PSD/PD) as the internal energy increases.     

Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein

Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions.     

Geometric Effects on Conductance in Single Molecule Electron Transport Junctions

We studied electron transport properties of a dithiol‐benzene molecule covalently bonded between two gold electrodes by combining ab initio calculations for the central molecule and a green function method to calculate electron transport. Due to the large computational demand, this type of calculations usually involves certain ways of simplification. The simplification commonly used is to fix the contact surface of the electrodes by ignoring the disturbance of the Au contact surface by contacting with the central molecule, i.e. without scattering region relaxation. In this study, we intended to resolve the difference between models with and without the above simplification. The large conductance found in our models without scattering region relaxation is due to the highly symmetric arrangement of the Au contact surface and those layers near the contact. The disturbance of the Au contact surface by the contact of the central molecule is important since the increase of the Au‐S bond and the distortion of the Au atom on the FCC site can lower the transmission coefficient between the two electrodes. In order to obtain better results, the model should include scattering region relaxation. However, when such relaxation is not applicable or demands too much calculation resource, the center molecule of the electronic transport junction should be at least optimized by the calculation level including electronic correlation, i.e. post‐HF methods.     

Isolation and structure elucidation of secoiridoid glucosides from <Emphasis Type="Italic">Fraxinus rhynchophylla</Emphasis> leaves

A new secoiridoid glucoside, oleuropein-4″-methyl ether, was isolated, together with two known secoiridoid glucosides, ligstroside and oleuropein, from the leaves of Fraxinus rhynchophylla Hance by repeated column chromatography over Sephadex LH-20. The structure of the new compound has been characterized on the basis of spectroscopic evidence.     

Epidemiological comparisons of codon usage patterns among HIV-1 isolates from Asia, Europe, Africa and the Americas

To investigate the genomic properties of HIV-1, we collected 3,081 sequences from the HIV Sequence Database. The sequences were categorized according to sampling region, country, year, subtype, gene name, and sequence and were saved in a database constructed for this study. The relative synonymous codon usage (RSCU) values of matrix, capsid, and gp120 and gp41 genes were calculated using correspondence analysis. The synonymous codon usage patterns based on the geographical regions of African countries showed broad distributions; when all the other regions, including Asia, Europe, and the Americas, were taken into account, the Asian countries tended to be divided into two groups. The sequences were clustered into nine non-CRF subtypes. Among these, subtype C showed the most distinct codon usage pattern. To determine why the codon usage patterns in Asian countries were divided into two groups for four target genes, the sequences of the isolates from the Asian countries were analyzed. As a result, the synonymous codon usage patterns among Asian countries were divided into two groups, the southern Asian countries and the other Asian countries, with subtype 01_AE being the most dominant subtype in southern Asia. In summary, the synonymous codon usage patterns among the individual HIV-1 subtypes reflect genetic variations, and this bioinformatics technique may be useful in conjunction with phylogenetic methods for predicting the evolutionary patterns of pandemic viruses.     

Partial-discard strategy for obtaining high purity products using simulated moving bed chromatography

A new "partial-discard" operation strategy was developed to improve the performance, especially purity, achievable in simulated moving bed (SMB) chromatography. This strategy was applied to the four-zone SMB process with two columns per zone for binary separation within the linear range. The "partial-discard" strategy significantly enhanced the purity or enrichment when the discard time and discard length were controlled. In addition, the "partial-feed with partial-discard" strategy improved remarkably the extract and raffinate purities at an intermediate feed time compared with the "partial-feed" operation. Adjustments of the discard length and discard time played key roles in achieving the desired product purity in SMB chromatographic performance.     

Antibodies with broad specificity to azaspiracids by use of synthetic haptens

The development of general, sensitive, portable, and quantitative assays for the azaspiracid (AZA) class of marine toxins is urgently needed. Use of a synthetic hapten containing rings F-I of AZA to generate antibodies that cross-react with the AZAs via their common C28-C40 domain and use of these antibodies in ELISA and immunoaffinity columns are reported. This approach has many advantages over using intact azaspiracids (AZAs) derived from environmental samples or total synthesis as haptens for antibody development. A derivative of the levorotatory C28-C40 azaspiracid domain (1) was synthesized efficiently using a one-pot Staudinger reduction/intramolecular aza-Wittig reaction-imine capture sequence to form the H-I ring spiroaminal and a double intramolecluar hetero-Michael addition to assemble the F-G ring ketal. Conjugation of the hapten 1 to cBSA and immunization in sheep generated antibodies that recognized and bound to ovalbumin-conjugated 1 in the absence of AZA1. This binding was inhibited by 1 in a concentration-dependent manner. A mixture of AZA1, AZA2, AZA3, and AZA6 caused a degree of inhibition of antibody binding consistent with its total AZA content, rather than just its content of AZA1. This result suggests that the antibodies also have a similar affinity for AZA2, AZA3, and AZA6 as they do for AZA1 and that such antibodies are suitable for analysis of AZAs in shellfish samples.     

Antibiofouling polymer-coated superparamagnetic iron oxide nanoparticles as potential magnetic resonance contrast agents for in vivo cancer imaging

We report the fabrication and characterization of antifouling polymer-coated magnetic nanoparticles as nanoprobes for magnetic resonance (MR) contrast agents. Magnetite superparamagnetic iron oxide nanoparticles (SPION) were coated with the protein- or cell-resistant polymer, poly(TMSMA-r-PEGMA), to generate stable, protein-resistant MR probes. Coated magnetic nanoparticles synthesized using two different preparation methods (in situ and stepwise, respectively) were both well dispersed in PBS buffer at a variety of pH conditions (pH 1-10). In addition, dynamic light scattering data revealed that their sizes were not altered even after 24 h of incubation in 10% serum containing cell culture medium, indicative of a lack of protein adsorption on their surfaces. When the antibiofouling polymer-coated SPION were incubated with macrophage cells, uptake was significantly lower in comparison to that of the popular contrast agent, Feridex I.V., suggesting that the polymer-coated SPION can be long-circulated in plasma by escaping from uptake by the reticular endothelial system (RES) such as macrophages. Indeed, when the coated SPION were administered to tumor xenograft mice by intravenous injection, the tumor could be detected in T2-weighted MR images within 1 h as a result of the accumulation of the nanomagnets within the tumor site. Although the poly(TMSMA-r-PEGMA)-coated SPION do not have any targeting ligands on their surface, they are potentially useful for cancer diagnosis in vivo.