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.

Preparation and immunological characterization of ß-lactoglobulin-amylose conjugate.

ß-Lactoglobulin (BLG), a major allergen of cow's milk, was conjugated with the N-hydroxysuccinimide ester of the amylose-glycylglycine adduct (AG-ONSu) to reduce its immunogenicity, and the biochemical and immunological properties of the resulting conjugate (AG-BLG) were studied. The conjugate was prepared by modifying BLG with AG-ONSu, and was purified in a Sephadex G-100 column. The analytical data for AG-BLG indicated that 10.5 moles of AG-ONSu, with a mean molecular weight of 2,800, was covalently attached to the amino groups of the BLG molecule. Conjugation with AG-ONSu greatly decreased the reactivity of BLG with anti-BLG polyclonal antibodies owing to its shielding action for epitopes on the protein's surface. These findings suggest that AG-ONSu can be used advantageously to suppress the hypersensitivity mediated by IgG antibodies in milk allergy.

Reduction of the immunogenicity of beta-lactoglobulin from cow's milk by conjugation with a dextran derivative.

Beta-lactoglobulin (BLG) was conjugated with the N-hydroxysuccinimide ester of the dextran-glycylglycine adduct (DG-ONSu) to reduce the immunogenicity of BLG, a major allergen of cow's milk, and some immunological properties of the conjugate (DG-BLG) were studied. The conjugate was prepared by modifying BLG with DG-ONSu and purified in a Sephadex G-100 column. The analytical data for DG-BLG indicated that 5.2 moles of DG-ONSu with a mean molecular weight of 9,300 were covalently attached to the amino groups of the BLG molecule. Conjugation with DG-ONSu greatly decreased the reactivity of BLG with anti-BLG antibodies and suppressed their production in vivo due to its shielding action for epitope(s) on the protein's molecular surface. It was also found that DG-BLG was resistant to proteolytic enzymes. These findings allow us to suggest that DG-ONSu could be advantageously used to suppress the hypersensitivity mediated by IgG antibodies in milk allergy.

A novel induced-fit reaction mechanism of asymmetric hot dog thioesterase PAAI.

Hot dog fold proteins sharing the characteristic "hot dog" fold are known to involve certain coenzyme A binding enzymes with various oligomeric states. In order to elucidate the oligomerization-function relationship of the hot dog fold proteins, crystal structures of the phenylacetate degradation protein PaaI from Thermus thermophilus HB8 (TtPaaI), a tetrameric acyl-CoA thioesterase with the hot dog fold, have been determined and compared with those of other family members. In the liganded crystal forms with coenzyme A derivatives, only two of four intersubunit catalytic pockets of the TtPaaI tetramer are occupied by the ligands. A detailed structural comparison between several liganded and unliganded forms reveals that a subtle rigid-body rearrangement of subunits within 2 degrees upon binding of the first two ligand molecules can induce a strict negative cooperativity to prevent further binding at the remaining two pockets, indicating that the so-called "half-of-the-sites reactivity" of oligomeric enzymes is visualized for the first time. Considering kinetic and mutational analyses together, a possible reaction mechanism of TtPaaI is proposed; one tetramer binds only two acyl-CoA molecules with a novel asymmetric induced-fit mechanism and carries out the hydrolysis according to a base-catalyzed reaction through activation of a water molecule by Asp48. From a structural comparison with other family members, it is concluded that a subgroup of the hot dog fold protein family, referred to as "asymmetric hot dog thioesterases" including medium chain acyl-CoA thioesterase II from Escherichia coli and human thioesterase III, might share the same oligomerization mode and the asymmetric induced-fit mechanism as observed in TtPaaI.

Structure of aldolase from Thermus thermophilus HB8 showing the contribution of oligomeric state to thermostability.

2-Deoxyribose-5-phosphate aldolase catalyzes a reversible aldol condensation of two aldehydes via formation of a covalent Schiff-base intermediate at the active lysine residue. The crystal structure of 2-deoxyribose-5-phosphate aldolase from Thermus thermophilus HB8 has been determined with and without the substrate at atomic resolution. This enzyme, which has a unique homotetramer structure, has been compared with the previously reported crystal structures of two orthologues from Escherichia coli and Aeropyrum pernix. In contrast to the similar alpha/beta-barrel fold of the monomers, substantial quaternary structural differences are observed between these three enzymes. Further comparison of the subunit-subunit interface areas of these aldolases showed a clear positive correlation between the interface area and the living temperature of the source organism. From these results, it is concluded that the oligomeric state of 2-deoxyribose-5-phosphate aldolase is important for the thermostability and not for the catalytic function.

Crystallographic structure and biochemical analysis of the Thermus thermophilus osmotically inducible protein C.

The X-ray crystallographic structure of osmotically inducible Protein C from the thermophilic bacterium, Thermus thermophilus HB8, was solved to 1.6A using the multiple wavelength anomalous dispersion method and a selenomethionine incorporated protein (Se-MAD). The crystal space group was P1 with cell dimensions of a=37.58 A, b=40.95 A, c=48.14 A, alpha=76.9 degrees, beta=74.0 degrees and gamma=64.1 degrees. The two tightly interacting monomers in the asymmetric unit are related by a non-crystallographic 2-fold. The dimer structure is defined primarily by two very long anti-parallel, over-lapping alpha-helices at the core, with a further six-stranded anti-parallel beta-sheet on the outside of the structure. With respect to the beta-sheets, both A and B monomers contribute three strands each resulting in an intertwining of the structure. The active site consists of two cysteine residues from one monomer and an arginine and glutamic acid from the other. Enzymatic assays have revealed that T.thermophilus OsmC has a hydroperoxide peroxidase activity.

Expression, purification, crystallization and preliminary crystallographic analysis of osmotically inducible protein C.

Selenium-incorporated osmotically inducible protein C from the thermophilic bacterium Thermus thermophilus was overexpressed, purified and crystallized. The crystals belong to space group P1, with unit-cell parameters a = 37.58, b = 40.95, c = 48.14 A, alpha = 76.93, beta = 74.04, gamma = 64.05 degrees. Five data sets were collected from a single crystal to 1.6 A using synchrotron radiation for MAD phasing. Self-rotation functions and the Matthews coefficient are consistent with two molecules in the asymmetric unit.

Structure and implications for the thermal stability of phosphopantetheine adenylyltransferase from Thermus thermophilus.

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyzes the rate-limiting step in coenzyme A (CoA) biosynthesis by transferring an adenylyl group from ATP to 4'-phosphopantetheine (Ppant), yielding 3'-dephospho-CoA (dPCoA). The crystal structure of PPAT from Thermus thermophilus HB8 (Tt PPAT) complexed with Ppant has been determined by the molecular-replacement method at 1.5 A resolution. The overall fold of the enzyme is almost the same as that of Escherichia coli PPAT, a hexamer having point group 32. The asymmetric unit of Tt PPAT contains a monomer and the crystallographic triad and dyad coincide with the threefold and twofold axes of the hexamer, respectively. Most of the important atoms surrounding the active site in E. coli PPAT are conserved in Tt PPAT, indicating similarities in their substrate binding and enzymatic reaction. The notable difference between E. coli PPAT and Tt PPAT is the simultaneous substrate recognition by all six subunits of Tt PPAT compared with substrate recognition by only three subunits in E. coli PPAT. Comparative analysis also revealed that the higher stability of Tt PPAT arises from stabilization of each subunit by hydrophobic effects, hydrogen bonds and entropic effects.

Expression, purification, crystallization and preliminary X-ray studies of geranylgeranyl diphosphate synthase from Thermus thermophilus HB8.

Geranylgeranyl diphosphate (GGPP) synthase from Thermus thermophilus HB8 was expressed in Escherichia coli, purified to homogeneity and crystallized both as the recombinant native protein and its selenomethionine (SeMet) derivative. Well diffracting crystals of these proteins were obtained belonging to the tetragonal space group P4(1) or P4(3), with unit-cell parameters a = b = 139.88, c = 73.37 A. There were two homodimers in the asymmetric unit. A native data set was collected to 1.55 A resolution and a data set suitable for MAD phasing was collected to 2.40 A resolution on beamline BL40B2 at SPring-8.

Crystallization and preliminary X-ray crystallographic studies of NADP-dependent 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8.

3-Hydroxyisobutyrate, a central metabolite in the valine catabolic pathway, is reversibly oxidized to methylmalonate semialdehyde by a specific NADP-dependent dehydrogenase (HIBADH). HIBADH from Thermus thermophilus HB8 has been overexpressed in Escherichia coli and crystallized by the microbatch method using lithium chloride as a precipitant at 296 K. X-ray diffraction data have been collected to 1.80 A resolution at 100 K using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 85.878, b = 106.367, c = 168.639 A. A homotetramer of HIBADH is likely to be present in the asymmetric unit, giving a V(M) of 3.0 A(3) Da(-1) and a solvent content of 59.3%.

Oxyanion hole-stabilized stereospecific isomerization in ribose-5-phosphate isomerase (Rpi).

Ribose-5-phosphate isomerase (Rpi) acts as a key enzyme in the oxidative and reductive pentose-phosphate pathways for the conversion of ribose-5-phosphate (R5P) to ribulose-5-phosphate and vice versa. We have determined the crystal structures of Rpi from Thermus thermophilus HB8 in complex with the open chain form of the substrate R5P and the open chain form of the C2 epimeric inhibitor arabinose-5-phosphate as well as the apo form at high resolution. The crystal structures of both complexes revealed that these ring-opened epimers are bound in the active site in a mirror symmetry binding mode. The O1 atoms are stabilized by an oxyanion hole composed of the backbone amide nitrogens in the conserved motif. In the structure of the Rpi.R5P complex, the conversion moiety O1-C1-C2-O2 in cis-configuration interacts with the carboxyl oxygens of Glu-108 in a water-excluded environment. Furthermore, the C2 hydroxyl group is presumed to be highly polarized by short hydrogen bonding with the side chain of Lys-99. R5P bound as the ring-opened reaction intermediate clarified the high stereoselectivity of the catalysis and is consistent with an aldose-ketose conversion by Rpi that proceeds via a cis-enediolate intermediate.

Interactions of a lysozyme-monomethoxypolyethylene glycol conjugate with lipopolysaccharides and lipid bilayers and effects of conjugate on gram-negative bacteria.

We have been studying a lysozyme derivative, called mPEG-lysozyme, in which Lys 33 is bound with a monomethoxypolyethylene glycol derivative. Here, we examined the surface hydrophobicity of the derivative and also its interactions with lipopolysaccharides and lipid bilayers. These properties may affect the antimicrobial activity of mPEG-lysozyme toward Gram-negative microorganisms. The lysozyme derivative had more than 150% of the antimicrobial activity for such microorganisms with that of native lysozyme taken to be 100%. Spectroscopic analyses indicated that mPEG-lysozyme bound to lipopolysaccharides with higher affinity than lysozyme, because of the high surface hydrophobicity of the derivative. In an experiment on carboxyfluorescein-leakage, mPEG-lysozyme strongly interacted with liposomes constructed from phosphatidylcholine, releasing carboxyfluorescein from the liposomes more effectively than lysozyme did. mPEG-lysozyme may perturb the outer membrane of Gram-negative microorganisms, gaining itself access to the peptidoglycan layers of the bacterium.