Structural insight into the active site of mushroom tyrosinase using phenylbenzoic acid derivatives

So far, many inhibitors of tyrosinase have been discovered for cosmetic and clinical agents. However, the molecular mechanisms underlying the inhibition in the active site of tyrosinase have not been well understood. To explore this problem, we examined here the inhibitory effects of 4′-hydroxylation and methoxylation of phenylbenzoic acid (PBA) isomers, which have a unique scaffold to inhibit mushroom tyrosinase. The inhibitory effect of 3-PBA, which has the most potent inhibitory activity among the isomers, was slightly decreased by 4′-hydroxylation and further decreased by 4′-methoxylation against mushroom tyrosinase. Surprisingly, 4′-hydroxylation but not methoxylation of 2-PBA appeared inhibitory activity. On the other hand, both 4′-hydroxylation and methoxylation of 4-PBA increased the inhibitory activity against mushroom tyrosinase. In silico docking analyses using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid or 4′-hydroxyl group of PBA derivatives could chelate with cupric ions in the active site of mushroom tyrosinase, and that the interactions of Asn260 and Phe264 in the active site with the adequate-angled biphenyl group are involved in the inhibitory activities of the modified PBAs, by parallel and T-shaped π-π interactions, respectively. Furthermore, Arg268 could fix the angle of the aromatic ring of Phe264, and Val248 is supposed to interact with the inhibitors as a hydrophobic manner. These results may enhance the structural insight into mushroom tyrosinase for the creation of novel tyrosinase inhibitors.     

Relationship between Alexithymia and latent trigger points in the upper Trapezius

Background

Latent trigger points (LTrPs) can be activated by future events, leading to pain. Few studies have reported LTrP risk factors. It has been suggested that alexithymia is associated with myofascial pain and diminished awareness of physical sensation. This study was designed to evaluate the relation between alexithymia and LTrPs found the upper trapezius of healthy individuals.

Methods

The correlation between LTrPs and alexithymia, and between LTrPs and depression was analyzed in 160 healthy participants (80 male, mean age: 40.5 years [20 to 66 years]). Each participant was evaluated for potential LTrPs by careful manual examination and completed the Toronto Alexithymia Scale-20 (TAS-20) and the Beck Depression Inventory (BDI) to assess potential alexithymia and depressive symptoms, respectively.

Results

LTrPs were observed in the upper trapezius of 76 participants (47.5%). TAS-20 scores were significantly higher in subjects with LTrPs than without LTrPs (p?<?0.001); in contrast, there was no significant BDI score difference between these groups (p?=?0.451). The LTrP risk for alexithymia was 2.74 (95% confidence interval [95% CI]: 2.10–3.58). There was no correlation between the TAS-20 and BDI scores (correlation coefficient: ?0.04). Significant risk factors associated with LTrPs included the TAS-20 score (odds ratio [OR]: 1.11, 95% CI: 1.07–1.15) and age (OR: 1.05, 95% CI: 1.01–1.09).

Conclusions

Alexithymia was associated with LTrPs in the upper trapezius of healthy individuals, suggesting that it may serve as a useful predictive factor.

Trial registration

UMIN000027468. Registered 23 May 2017(retrospectively registered).

     

Retrospective Proteomic Analysis of a Novel,Cancer Metastasis-Promoting RGD-Containing Peptide

Patients who undergo surgical extirpation of a primary liver carcinoma followed by radiotherapy and chemotherapy leading to complete remission are nevertheless known to develop cancerous metastases 3–10 years later. We retrospectively examined the blood sera collected over 8 years from 30 patients who developed bone metastases after the complete remission of liver cancer to identify serum proteins showing differential expression compared to patients without remission. We detected a novel RGD (Arg-Gly-Asp)-containing peptide derived from the C-terminal portion of fibrinogen in the sera of metastatic patients that appeared to control the EMT (epithelial-mesenchymal transition) of cancer cells, in a process associated with miR-199a-3p. The RGD peptide enhanced new blood vessel growth and increased vascular endothelial growth factor levels when introduced into fertilized chicken eggs. The purpose of this study was to enable early detection of metastatic cancer cells using the novel RGD peptide as a biomarker, and thereby develop new drugs for the treatment of metastatic cancer.     

Microsatellite loci in Japanese quail and cross-species amplification in chicken and guinea fowl

In line with the Gifu University''s initiative to map the Japanese quail genome, a total of 100 Japanese quail microsatellite markers isolated in our laboratory were evaluated in a population of 20 unrelated quails randomly sampled from a colony of wild quail origin. Ninety-eight markers were polymorphic with an average of 3.7 alleles per locus and a mean heterozygosity of 0.423. To determine the utility of these markers for comparative genome mapping in Phasianidae, cross-species amplification of all the markers was tested with chicken and guinea fowl DNA. Amplification products similar in size to the orthologous loci in quail were observed in 42 loci in chicken and 20 loci in guinea fowl. Of the cross-reactive markers, 57.1% in chicken and 55.0% in guinea fowl were polymorphic when tested in 20 birds from their respective populations. Five of 15 markers that could cross-amplify Japanese quail, chicken, and guinea fowl DNA were polymorphic in all three species. Amplification of orthologous loci was confirmed by sequencing 10 loci each from chicken and guinea fowl and comparing with them the corresponding quail sequence. The microsatellite markers reported would serve as a useful resource base for genetic mapping in quail and comparative mapping in Phasianidae.     

玉米根冠粘胶和铝的结合及有机酸累积

玉米根系分泌较多的植物粘胶 ,用 0 .0 1%中性红溶液使粘胶染成红色。 30 μmol/L的AlCl3 处理根系 1h后 ,不除去粘胶的根尖的铝含量大于除去粘胶的根尖的铝含量。粘胶的铝含量随着处理的铝浓度的增加而增加。用高效液相色谱仪能使粘胶 铝复合物分离成均含糖和铝的两种组分。 5 μmol/L的AlCl3 处理根系 2 4h后 ,粘胶中积累了铝诱导分泌的有机酸。粘胶中含有酸性磷酸酶 ,它的活性随着处理的铝浓度的增加而降低。这些结果证明了植物粘胶能够和铝结合、粘胶液滴能够积累有机酸及磷酸酶的假说     

Elementary steps of the cross-bridge cycle in bovine myocardium with and without regulatory proteins.

The role of regulatory proteins in the elementary steps of the cross-bridge cycle in bovine myocardium was investigated. The thin filament was selectively removed by gelsolin and the actin filament was reconstituted without tropomyosin or troponin. Further reconstitution was achieved by adding tropomyosin and troponin. The effects of MgATP and phosphate (Pi) on the rate constants of exponential processes were studied in control, actin filament-reconstituted, and thin filament-reconstituted myocardium at pCa < or = 4.66, pH 7.00, 25 degrees C. In control myocardium, the MgATP association constant was 9.1 +/- 1.3 mM(-1), and the Pi association constant 0.14 +/- 0.04 mM(-1). The equilibrium constant of the cross-bridge detachment step was 2.6 +/- 0.4, and the equilibrium constant of the force generation step was 0.59 +/- 0.04. In actin filament-reconstituted myocardium without regulatory proteins, the MgATP association constant was approximately the same, and the Pi association constant increased to 2.8x. The equilibrium constant of cross-bridge detachment decreased to 0.2x, but the equilibrium constant of the force generation step increased to 4x. These kinetic constants regained control values after reconstitution of the thin filament. These results indicate that tension/cross-bridge in the presence of regulatory proteins is approximately 1.5-1.7x of that in the absence of regulatory proteins. These results further indicate that regulatory proteins promote detachment of cross-bridges.     

The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol.

Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably alpha 2 beta 2 gamma 2. When (R)- and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R)/Km(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 A resolution. The enzyme exists as a dimer of the alpha beta gamma heterotrimer. Cobalamin is bound at the interface between the alpha and beta subunits in the so-called 'base-on' mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (beta/alpha)8 barrel that was formed by a central region of the alpha subunit. The substrate propane-1,2-diol and essential cofactor K+ are bound inside the (beta/alpha)8 barrel above the corrin ring of cobalamin. K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the alpha and beta subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.     

The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel.

OprM, the outer membrane component of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa, has been assumed to facilitate the export of antibiotics across the outer membrane of this organism. Here we purified to homogeneity the OprM protein, reconstituted it into liposome membranes, and tested its channel activity by using the liposome swelling assay. It was demonstrated that OprM is a channel-forming protein and exhibits the channel property that amino acids diffuse more efficiently than saccharides. However, antibiotics showed no significant diffusion through the OprM channel in the liposome membrane, suggesting that OprM functions as a gated channel. We reasoned that the protease treatment may cause the disturbance of the gate structure of OprM. Hence, we treated OprM reconstituted in the membranes with alpha-chymotrypsin and examined its solute permeability. The results demonstrated that the protease treatment caused the opening of an OprM channel through which antibiotics were able to diffuse. To elucidate which cleavage is intimately related to the opening, we constructed mutant OprM proteins where the amino acid at the cleavage site was replaced with another amino acid. By examining the channel activity of these mutant proteins, it was shown that the proteolysis at tyrosine 185 and tyrosine 196 of OprM caused the channel opening. Furthermore, these residues were shown to face into the periplasmic space and interact with other component(s). We considered the possible opening mechanism of the OprM channel based on the structure of TolC, a homologue of OprM.     

Mammalian HSP60 is quickly sorted into the mitochondria under conditions of dehydration.

There are few reports concerning the sorting mechanisms of mammalian HSP60 into the mitochondria from the cytoplasm. In the present study we investigated the protein import system. Based on immunoblotting and immuno-histochemistry, HSP60 was detected in both the cytoplasm and mitochondria. The purified cytoplasmic HSP60 showed chaperone activity, and the protein was imported into the mitochondria in vitro by a mitochondrial import assay. HSP60 mRNA was increased in the kidney papilla of rats that had been water restricted for three and five days, but no changes in HSP60 mRNA were detected in the cortex or the medulla of the rat kidneys. Upon immunoblotting, HSP60 was detected in both the cytoplasm and the mitochondria of normal rat kidney cortex, medulla, and papilla in almost the same quantity. HSP60 was remarkably decreased in the kidney papilla of rats that were water restricted but the protein was increased in the mitochondria of the rat kidney papilla. We also analysed binding of the protein to the signal sequence of HSP60 using signal sequence-affinity column chromatography. We identified only one protein band with a molecular mass of 70 kDa on SDS/PAGE. The protein was eluted from the affinity column by an excess of signal peptide or by 5 mm ATP. Upon immunoblotting, the 70-kDa protein cross-reacted with an antibody against HSP70. These results suggested that mammalian HSP60 is located both in the cytoplasm as a stable cytoplasmic HSP60 and also in the mitochondria under normal conditions. The cytoplasmic HSP60 is quickly imported into the mitochondria under severe conditions by cytoplasmic HSP70.