Structural characterization of 1,3-bis-tert-butyl monocyclic benzene derivatives with agonistic activity towards retinoid X receptor alpha.

Retinoid X receptor alpha (RXRα) plays pivotal roles in multiple biological processes, but limited information is available on the structural features of chemicals that show low affinity for RXRα, but nevertheless cause significant activation, though these may represent a human health hazard. We recently discovered that several industrial chemicals having 1,3-bis-tert-butylbenzene as a common chemical structure exhibit agonistic activity towards rat RXRα. In this study, we explored the structure-activity relationship of 1,3-bis-tert-butyl monocyclic benzene derivatives for RXRα activation by means of in vitro and in silico analyses. The results indicate that a bulky substituent at the 5-position is favorable for agonistic activity towards human RXRα. Since 1,3-bis-tert-butyl monocyclic benzene derivatives with bulky hydrophobic moieties differ structurally from known RXRα ligands such as 9-cis-retinoic acid and bexarotene, our findings may be helpful for the development of structural alerts in the safety evaluation of industrial chemicals for RXRα-based toxicity to living organisms.

Pilot study on novel skin care method by augmentation with Staphylococcus epidermidis, an autologous skin microbe--A blinded randomized clinical trial.

BACKGROUND AND OBJECTIVE: Staphylococcus epidermidis is an autologous bacterium that is beneficial to skin health. Our goal was to develop a novel, personalized basic cosmetic that exploits this characteristic. METHODS: We conducted a double-blinded, randomized clinical trial on augmentation with S. epidermidis as a pilot study, in which S. epidermidis was collected from the subject, cultured for proliferation, and then continuously applied to the subject's own face before sleep twice per week for four weeks in order to increase colonization levels. RESULTS: The results showed that this treatment increased the lipid content of the skin and suppressed water evaporation, thereby markedly improving skin moisture retention. Moreover, augmentation with S. epidermidis maintained a low acidic condition on the skin surface. The low risk of undesirable effects induced by augmentation with S. epidermidis was also confirmed by measuring erythema and melanin levels. CONCLUSIONS: These results may serve as a driving force to accelerate the development of novel, personalized basic cosmetics.

Optical characterization of the antigen-antibody thin layer using the whispering gallery mode.

We immobilized an antibody (anti-ß-Galactosidase) on a polystyrene microsphere by using a covalent bond, and observed the resonance peaks in the scattered light intensity spectra related to the whispering gallery mode (WGM) excitation of the microsphere. The amount and the optical parameters, i.e., thickness and refractive index, of anti-ß-Galactosidase on the sphere surface were evaluated based on an absorbance measurement and a resonance peak shift measurement, respectively. Moreover, we measured the variation of the WGM spectra depending on the concentration of the enzyme solution (ß-Galactosidase), which allowed us to optically evaluate the thickness and the refractive index of the antigen-antibody layer from the shift of the WGM spectra peak.

Structural characteristic of folding/unfolding intermediate of pokeweed anti-viral protein revealed by time-resolved fluorescence.

The structural feature of unfolding intermediate of pokeweed anti-viral protein (PAP) was characterized using time-resolved fluorescence spectroscopic methods to elucidate protein folding/unfolding process. CD and fluorescence spectra consistently demonstrated that the unfolding of PAP completed at 4 M of guanidine hydrochloride (GuHCl). The fluorescence resonance energy transfer (FRET) and time-resolve fluorescence depolarization analysis of Trp208 and Trp237 located in the C-terminal domain of PAP suggested that peculiar unfolding intermediate populated before reaching to the unfolding state. The FRET distance of Trp237 to Tyr182 was extended to more than 28 Å with keeping the compact conformation in the unfolding intermediate state populated in the presence of 2 M GuHCl. On the other hand, Trp208 maintained the energy transfer pair with Tyr72 near the active site, although the rotational freedom was increased a little. There results suggest that the most distinguished structural feature of the unfolding intermediate of PAP is the separation of C-terminal domain from N-terminal domain. FRET and fluorescence depolarization studies also showed that C-terminal domain would be more separated to liberate the segmental motions of Trp208 and Trp237 distinctly at the unfolding state.

Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase.

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D(phys), and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (R(g)) of the guanidine unfolded state (U) is 26.9±0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the R(g) value extrapolated from kinetic R(g) data to time zero, R(g,0), is 24.3±0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in R(g)(2) was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R(g) of the minor component of D(phys) containing the non-native cis proline isomer (D(phys,cis)) to be 25.7±0.6 Å. Moreover, R(g) of the major component of D(phys) containing the native proline isomer (D(phys,tra)) was estimated as 23.9±0.2 Å based on R(g,0). Consequently, both components of the burst-phase intermediate of barnase (D(phys,tra) and D(phys,cis)) are still largely expanded. It was inferred that D(phys) possesses the N-terminal helix and the center of the ß-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.

The quenching-resolved fluorescence spectrum and its application to studies of the folding/unfolding of trypsin inhibitor from seeds of the bitter gourd.

With reference to the local conformation of a protein, it is interesting to differentiate the individual fluorescence properties of included tryptophan residues without modification. The fluorescence spectrum of bitter gourd trypsin inhibitor (BGTI) was separated into two emission bands by the quenching-resolved fluorescence method. One emission band was given as a fraction with the Stern-Volmer quenching constant, 44.9 x 10(-3) M(-1), against the fluorescence quenching by KI, and it showed an emission maximum intensity at 341 nm. The fluorescence quenching constant of the other band was 1.58 x 10(-3) M(-1), and the maximum wavelength was found at 337 nm. These separated emissions were due to the fluorescence of Trp54 and Trp9 of BGTI. The quenching resolved-fluorescence spectrum was effectively applied to the precise description of the polar circumstances surrounding the Trp residues in the unfolding intermediate state of BGTI. The results suggested that the molten globule-like state of BGTI adopted such a peculiar conformation that the helix domain including Trp9 was packed more densely while the other loop domain partially unfolded.

Heterogeneous packing in the folding/unfolding intermediate state of bitter gourd trypsin inhibitor.

The conformation and dynamics of a protein are essential in characterizing the protein folding/unfolding intermediate state. They are closely involved in the packing and site-specific interactions of peptide elements to build and stabilize the tertiary structure of the protein. In this study, it was confirmed that trypsin inhibitor obtained from seeds of bitter gourd (BGTI) adopted a peculiar but plausible conformation and dynamics in the unfolding intermediate state. The fluorescence spectrum of one of two tryptophan residues of BGTI, Trp9, shifted to the blue side in the presence of 2-3 M guanidine hydrochloride, although the other, Trp54, did not show this spectral shift. At the same time, the motional freedom of Trp9 revealed by a time-resolved fluorescence study decreased, suggesting that the segmental motion of this residue was more restricted. These results indicate that BGTI takes such a conformation state that the hydrophobic core and loop domains arranging Trp9 and Trp54 respectively are heterogeneously packed in the unfolding intermediate state.