Thermodynamic and kinetic analysis on oligomeric protein dissociation using high-pressure native PAGE velocity method.

High pressure is known to dissociate several oligomeric proteins, and regarded as an important tool to shift the oligomerization equilibrium. Native polyacrylamide gel electrophoresis (native PAGE) at high pressure can characterize the dissociates and clearly discriminate the aggregates. However, a band smearing of migration profiles often hinders more detailed analyses (Miwa et al., High Pressure Res. (2019) 39, 218-224). In this paper, we focused on the band smearing dependent on the migration velocity so as to extract both thermodynamic and kinetic parameters. We systematically perturbed the migration velocity by changing the gel concentration and carried out numerical analysis for a series of the migration profiles based on a simple dissociation reaction scheme with limited thermodynamic and kinetic parameters. Then, complete volumetric properties on oligomerization process can be available. We term the present analysis method as a high-pressure native PAGE velocity method. We also report the application of this method to revisit the pressure dissociation of tetrameric lactate dehydrogenase (LDH) from pig heart.

Solid-Supported Assembly Composed of n-Octyl-ß-D-glucopyranoside and 1,2-Dioleoyl-sn-glycero-3-phosphocholine in Equilibrium with Its Ambient Aqueous Solution System Including Dispersed Assembly.

In the last few decades, the preparation of solid-supported lipid bilayers by immersing a solid substrate in an aqueous solution where the lipid is dissolved with the aid of a surfactant, followed by dilution of the solution, has been reported. In this study, we attempted to interpret the evolution of supported surfactant/lipid assemblies towards the supported lipid bilayer in terms of a phase equilibrium between the supported assembly phase and its ambient solution system consisting of the dispersed surfactant/lipid assembly phase and the bulk solution phase comprising monomeric surfactant and lipid. We characterized the supported assembly formed on hydrophilized Ge or mica substrates in equilibrium with aqueous solutions containing various concentrations of the nonionic surfactant, n-octyl-ß-D-glucopyranoside (OG) and the amphoteric phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), using interaction-force-profile measurements by atomic force microscopy (AFM), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). We also investigated the ambient solution system using equilibrium dialysis to obtain the partition equilibrium profile of OG between the bulk solution and dispersed assembly phases in the micellar or vesicular states. These studies indicate that the properties of the supported assembly depend on the composition of the dispersed assembly and concentration of monomerically dissolved OG. Further, a type of micellar-bilayer state transition occurs in the supported assembly, roughly synchronized with that in the dispersed assembly.

Simple Thermodynamic Description of the Micellar-Bilayer State Transition of Assemblies Composed of n-Octyl-ß-D-glucopyranoside and 1,2-Dioleolyl-sn-glycero-3-phosphocholine Dispersed in Aqueous Media or Supported on Solid Substrates.

In the preceding paper, we investigated a mixed assembly composed of a nonionic surfactant, n-octyl-ß-D-glucopyranoside (OG), and an amphoteric lipid, 1,2-dioleolyl-sn-glycero-3-phosphocholine (DOPC), formed on hydrophilized solid substrates immersed in aqueous solutions containing OG and DOPC. The experimental data could be interpreted in terms of the phase equilibrium; thus, the partition equilibrium profile of OG between the bulk solution phase and the supported assembly phase was obtained, as well as that between the bulk solution and the dispersed assembly. The partition equilibrium profiles suggested that micellar-bilayer state transitions occur both in the supported assembly and in the dispersed one in a roughly synchronized manner, even though there are significant discrepancies between them. In this paper, we propose a simple thermodynamic model for the micellar-bilayer transition of the dispersed and supported assembly of OG and DOPC, assuming that the micellar and bilayer states are also pseudo-phases distinct from each other. Using this model, we analyzed these partition equilibrium profiles and concluded that the transition in the supported assembly should mainly be attributed to the transition in the dispersed assembly, which is partly modified by the interaction energy between the supported assembly and the substrate.

Appraisal of long-time outcomes after curative surgery in elderly patients with gastric cancer: a propensity score matching analysis.

BACKGROUND: This study was conducted to assess the long-term outcomes of elderly patients among propensity-score-matched gastric cancer patients after curative gastrectomy and to propose the proper management of elderly gastric cancer patients. METHODS: We enrolled 626 patients with gastric cancer who underwent curative gastrectomy at our institution between January 2004 and December 2015. To minimize selection bias among 2 groups, propensity score matching was performed. RESULTS: Patients were divided into an elderly group over 75 years old (EP group; n = 186) and a non-elderly group (NEP group; n = 440). After propensity score matching, patients were divided into EP group (n = 178) and NEP group (n = 175). Five-year overall survival was significantly lower in the EP group than in the NEP group, consistent with a subgroup analysis of each stage. However, the 5-year disease-specific survival among all enrolled patients and those with stage I and II disease did not differ significantly. Moreover, in the subgroup of stage III patients, 5 year disease-specific survival was significantly lower in the EP group (23.0%) than in the NEP group (59.4%; P = 0.004). Because elderly patients with stage III disease had an extremely poor prognosis, we decided to compare the two groups with stage III. The EP group contained significantly fewer patients with D2 lymphadectomy (P = 0.002) and adjuvant chemotherapy (P < 0.001) than the NEP group. C-reactive protein to albumin ratio was significantly higher in patients in the EP group than in the NEP group (P = 0.046), and the prognostic nutritional index was significantly lower in patients in the EP group than in the NEP group (P = 0.045).　Multivariate analysis revealed that the prognostic nutritional index and lymphatic invasion were independent prognostic factors. CONCLUSIONS: Elderly gastric cancer patients with stage III disease showed poorer disease-specific survival compared with non-elderly patients, which may be due to a poorer nutritional and inflammatory background, fewer D2 lymphadenectomies, and a lack of adjuvant chemotherapy. The safe induction of standard lymphadenectomy and adjuvant chemotherapy with perioperative aggressive nutritional support may improve the prognosis of elderly gastric cancer patients with stage III disease.

Negative thermal expansibility change for dissociation of lysozyme variant amyloid protofibril.

A disulfide-deficient variant of hen lysozyme, 0SS, is known to form an amyloid protofibril spontaneously, and to dissociate into monomers at high hydrostatic pressure. We carried out native PAGE at various temperatures (20-35°C) and pressures (0.1-200 MPa), to characterize the dissociation equilibrium of disulfide-deficient variant of hen lysozyme amyloid protofibril. Based on the density profiles, the partial molar volume and thermal expansibility changes for dissociation, ΔvD and ΔeD , were obtained to be -74 cm(3) /mol at 25°C and -2.3 cm(3) mol(-1) K(-1) , respectively. The dissociation of amyloid fibril destroys the cross ß-structure, and such conformational destruction in native protein fold rarely accompanies negative thermal expansibility change. We discussed the negative thermal expansibility change in terms of hydration and structural packing of the amyloid protofibril.

Modes of conformational changes of proteins adsorbed on a planar hydrophobic polymer surface reflecting their adsorption behaviors.

Infrared spectra of hen egg white lysozyme and bovine serum albumin (BSA) adsorbed on a solid poly tris(trimethylsiloxy)silylstyrene (pTSS) surface in D2O solution were measured using attenuated total reflection (ATR) Fourier transform infrared spectroscopy. From the area and shape of the amide I' band of each spectrum, the adsorption amount and the secondary structure were determined simultaneously, as a function of adsorption time. We could show that the average conformation for all the adsorbed lysozyme molecules was solely determined by the adsorption time, and independent of the bulk concentration, while the adsorption amount increased with the bulk concentration as well as the adsorption time. These results suggest that lysozyme molecules form discrete assemblies on the surface, and that the surface assemblies grow over several hours to have a definite architecture independent of the adsorption amount. As for BSA, the extent of the conformational change was solely determined by the adsorption amount, regardless of the bulk concentration and the adsorption time. These differences in the adsorption properties of lysozyme and BSA may reflect differences in their conformational stabilities.

Quantitative analysis of protein adsorption on a planar surface by Fourier transform infrared spectroscopy: lysozyme adsorbed on hydrophobic silicon-containing polymer.

The adsorption of hen egg white lysozyme onto a solid polytris(trimethylsiloxy)silylstyrene (pTSS) surface from a D(2)O solution at pD 7 containing 100 mM NaCl and 10 mM sodium deuterated phosphate was monitored at 25 degrees C by Fourier transform infrared spectroscopy using the attenuated total reflection (ATR) method. The infrared spectrum attributed to only the adsorbed lysozyme was derived from the observed spectrum, and the amount of adsorbed lysozyme was determined as a function of time and lysozyme concentration. The kinetics of adsorption could be decomposed into two components, one of which was a process with a time constant of larger than 4 h(-1) and the other was a process with one of about 0.1 h(-1). These spectra showed that the lysozyme adsorbed in the faster process had a higher beta-structure content than the dissolved lysozyme. It was also found that the slower adsorption induced some conformational change in the lysozyme adsorbed in the faster process and/or that adsorbed in the slower process. After adsorption for 24 h, the pTSS surface was rinsed out with lysozyme-free solution. The resultant spectra of the surface indicated that the lysozyme adsorbed in the faster process was bound irreversibly on the surface and was changed to a conformer with a higher beta-structure content during the slower process. The experimental procedures and the theoretical applications for such a quantitative analysis in the ATR spectroscopic method are presented in detail.