Structural Basis of α-Catenin Recognition by EspB from Enterohaemorrhagic E. coli Based on Hybrid Strategy Using Low-Resolution Structural and Protein Dissection

Enterohaemorrhagic E. coli (EHEC) induces actin reorganization of host cells by injecting various effectors into host cytosol through type III secretion systems. EspB is the natively partially folded EHEC effector which binds to host α-catenin to promote the actin bundling. However, its structural basis is poorly understood. Here, we characterize the overall structural properties of EspB based on low-resolution structural data in conjunction with protein dissection strategy. EspB showed a unique thermal response involving cold denaturation in the presence of denaturant according to far-UV circular dichroism (CD). Small angle X-ray scattering revealed the formation of a highly extended structure of EspB comparable to the ideal random coil. Various disorder predictions as well as CD spectra of EspB fragments identified the presence of α-helical structures around G41 to Q70. The fragment corresponding to this region indicated the thermal response similar to EspB. Moreover, this fragment showed a high affinity to C-terminal vinculin homology domain of α-catenin. The results clarified the importance of preformed α-helix of EspB for recognition of α-catenin.     

Age at Transition from Pediatric to Adult Care Has No Relationship with Mortality for Childhood-Onset Type 1 Diabetes in Japan: Diabetes Epidemiology Research International (DERI) Mortality Study

Objective

To follow up Japanese patients with type 1 diabetes for a maximum of 40 years to examine when they transitioned from pediatric care to adult care and to explore whether the attending physician, i.e., pediatrician or internist, was associated with prognosis.

Methods

Participants consisted of 1,299 patients who had been diagnosed as having type 1 diabetes at less than 15 years old between 1965 and 1979 identified through two nationwide surveys. Patients were classified as having received either pediatric care or adult care at the age of 15 and 30, and were compared for differences in mortality associated with the attending physician.

Results

The attending physicians were confirmed for a total of 1,093 patients at the age of 15. Of these patients, 43.8% and 40.3% received pediatric care and adult care, respectively. Of the 569 patients receiving pediatric care, 74.2%, 56.6%, 53.4%, and 51.3% continued with pediatric care at 20, 30, 40, and 50 years old, respectively. The attending physicians (pediatrician or internist) at the age of 15 and 30 had no significant impact on their survival (P = 0. 892, 0.411, respectively).

Conclusions

More than half of the patients who had received pediatric care at the age of 15 continued to receive pediatric care even after the age of 30, suggesting that their transition was far from smooth, while the attending physician at the age of both 15 and 30 was not a prognostic factor for mortality. Thus, the timing for transition to adult care in these patients has no relationship with mortality in Japan.     

Quantitative and antioxidative behavior of Trolox in rats’ blood and brain by HPLC‐UV and SMFIA‐CL methods

Trolox, a water‐soluble vitamin E analogue has been used as a positive control in Trolox equivalent antioxidant capacity and oxygen radical antioxidant capacity assays due to its high antioxidative effect. In this study, the ex vivo antioxidative effects of Trolox and its concentration in blood and brain microdialysates from rat after administration were evaluated by newly established semi‐microflow injection analysis, chemiluminescence detection and HPLC‐UV. In the administration test, the antioxidative effect of Trolox in blood and brain microdialysates after a single administration of 200 mg/kg of Trolox to rats could be monitored. The antioxidative effects in blood (12.0 ± 2.1) and brain (8.4 ± 2.1, × 10³ antioxidative effect % × min) also increased. Additionally, the areas under the curve (AUC)s_0–360 (n = 3) for blood and brain calculated with quantitative data were 10.5 ± 1.2 and 9.7 ± 2.5 mg/mL × min, respectively. This result indicates that Trolox transferability through the blood–brain barrier is high. The increase in the antioxidative effects caused by Trolox in the blood and brain could be confirmed because good correlations between concentration and antioxidative effects (r ≥ 0.702) were obtained. The fact that Trolox can produce an antioxidative effect in rat brain was clarified. Copyright © 2015 John Wiley & Sons, Ltd.     

New polar constituents in the pupae of the silkworm Bombyx mori L. (II): developmental changes of the constituents

A correlation between the quantitative changes in L-methionine analogs, the ratio of D-serine/L-serine during the pupal stage, and metamorphosis was observed. The glycoside appearing at low blood sugar values during the pupal stage was isolated and characterized as D-glucosyl-L-tyrosine. (1)H-NMR indicated the appearance and increase of this glycoside, and Mirrorcle Ray CV4 equipment was used to take X-ray pictures of the pupal bodies. The results indicate that γ-cyclic di-L-glutamate and L-methionine sulfone might be concerned with ammonia assimilation in the pupae, and that D-glucosyl-L-tyrosine served as a switch for the fatty acid (pupal oil) dissimilation hybrid system.     

Effect of conformational states on protein dynamical transition

In order to examine the properties specific to the folded protein, the effect of the conformational states on protein dynamical transition was studied by incoherent elastic neutron scattering for both wild type and a deletion mutant of staphylococcal nuclease. The deletion mutant of SNase which lacks C-terminal 13 residues takes a compact denatured structure, and can be regarded as a model of intrinsic unstructured protein. Incoherent elastic neutron scattering experiments were carried out at various temperature between 10 K and 300 K on IN10 and IN13 installed at ILL. Temperature dependence of mean-square displacements was obtained by the q-dependence of elastic scattering intensity. The measurements were performed on dried and hydrated powder samples. No significant differences were observed between wild type and the mutant for the hydrated samples, while significant differences were observed for the dried samples. A dynamical transition at ∼ 140 K observed for both dried and hydrated samples. The slopes of the temperature dependence of MSD before transition and after transition are different between wild type and the mutant, indicating the folding induces hardening. The hydration water activates a further transition at ∼ 240 K. The behavior of the temperature dependence of MSD is indistinguishable for wild type and the mutant, indicating that hydration water dynamics dominate the dynamical properties.     

Inhibitiory effects of gold(III) ions on ribonuclease and deoxyribonuclease

Inhibitory effects of gold(III) ions (Au(III)) on ribonuclease A (RNase A) and deoxyribonuclease I (DNase I) were investigated at neutral pH. RNase A was completely inhibited by 3 molar equivalents of Au(III) ions. DNase I was inhibited by 10 molar equivalents of Au(III) ions. Stoichiometric analyses suggest that Au(III) ions were coordinated to RNase A molecules. The Au(III)-inhibited RNase A and DNase I were renatured to exhibit 80% and 60% of their intrinsic activity, when the bound Au(III) ions were eliminated from the nucleases by addition of thiourea, which forms a strong complex with gold ions. This suggests that RNase A and DNase I were not oxidized to lose their activity, but reversibly complexed with Au(III) ions to lose their activity. Au(III) ions were probably considered to be bound to histidine and methionine residues in the nucleases, resulting in the inhibition of their activity. CD spectra revealed that the Au(III)-induced inhibition caused a conformational change in RNase A molecules and that the addition of thiourea induced refolding of the Au(III)-inhibited RNase A.     

CD24 is expressed specifically in the nucleus pulposus of intervertebral discs

Intervertebral disc (IVD) consists of a soft gelatinous material in its center, the nucleus pulposus (NP), bounded peripherally by fibrocartilage, annulus fibrosus (AF). Despite the number of patients with IVD degeneration, gene expression analysis has not been undertaken in NP and therefore little is known about the molecular markers expressed in NP. Here, we undertook a microarray screen in NP with the other nine tissues to identify the specific cell surface markers for NP. Five membrane associating molecules out of 10,490 genes were identified as highly expressing genes in NP compared with the other tissues. Among them, we identified CD24, a glycosylphosphatidylinositol (GPI) anchor protein as a cell surface marker for NP. CD24 expression was also detected in the herniated NP and chordoma, a malignant primary tumor derived from notochordal cells, while it was absent in chondrosarcoma. Therefore, CD24 is a molecular marker for NP as well as the diseases of IVD.     

pH-dependent equilibrium between long lived near-UV intermediates of photoactive yellow protein

The long lived intermediate (signaling state) of photoactive yellow protein (PYP(M)), which is formed in the photocycle, was characterized at various pHs. PYP(M) at neutral pH was in equilibrium between two spectroscopically distinct states. Absorption maxima of the acidic form (PYP(M)(acid)) and alkaline form (PYP(M)(alkali)) were located at 367 and 356 nm, respectively. Equilibrium was represented by the Henderson-Hasselbalch equation, in which apparent pK(a) was 6.4. Content of alpha- and/or beta-structure of PYP(M)(acid) was significantly greater than PYP(M)(alkali) as demonstrated by the molar ellipticity at 222 nm. In addition, changes in amide I and II modes of beta-structure in the difference Fourier transform infrared spectra for formation of PYP(M)(acid) was smaller than that of PYP(M)(alkali). The vibrational mode at 1747 cm(-1) of protonated Glu-46 was found as a small band for PYP(M)(acid) but not for PYP(M)(alkali), suggesting that Glu-46 remains partially protonated in PYP(M)(acid), whereas it is fully deprotonated in PYP(M)(alkali). Small angle x-ray scattering measurements demonstrated that the radius of gyration of PYP(M)(acid) was 15.7 Angstroms, whereas for PYP(M)(alkali) it was 16.2 Angstroms. These results indicate that PYP(M)(acid) assumes a more ordered and compact structure than PYP(M)(alkali). Binding of citrate shifts this equilibrium toward PYP(M)(alkali). UV-visible absorption spectra and difference infrared spectra of the long lived intermediate formed from E46Q mutant was consistent with those of PYP(M)(acid), indicating that the mutation shifts this equilibrium toward PYP(M)(acid). Alterations in the nature of PYP(M) by pH, citrate, and mutation of Glu-46 are consistently explained by the shift of the equilibrium between PYP(M)(acid) and PYP(M)(alkali).     

Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis

At high resolution, we determined the crystal structures of copper-bound and metal-free tyrosinase in a complex with ORF378 designated as a "caddie" protein because it assists with transportation of two CuII ions into the tyrosinase catalytic center. These structures suggest that the caddie protein covers the hydrophobic molecular surface of tyrosinase and interferes with the binding of a substrate tyrosine to the catalytic site of tyrosinase. The caddie protein, which consists of one six-strandedbeta-sheet and one alpha-helix, has no similarity with all proteins deposited into the Protein Data Bank. Although tyrosinase and catechol oxidase are classified into the type 3 copper protein family, the latter enzyme lacks monooxygenase activity. The difference in catalytic activity is based on the structural observations that a large vacant space is present just above the active center of tyrosinase and that one of the six His ligands for the two copper ions is highly flexible. These structural characteristics of tyrosinase suggest that, in the reaction that catalyzes the ortho-hydroxylation of monophenol, one of the two Cu(II) ions is coordinated by the peroxide-originated oxygen bound to the substrate. Our crystallographic study shows evidence that the tyrosinase active center formed by dinuclear coppers is flexible during catalysis.     

Crystal structure of mitochondrial quinol-fumarate reductase from the parasitic nematode Ascaris suum

In the anaerobic respiratory chain of the parasitic nematode Ascaris suum, complex II couples the reduction of fumarate to the oxidation of rhodoquinol, a reverse reaction catalyzed by mammalian complex II. In this study, the first structure of anaerobic complex II of mitochondria was determined. The structure, composed of four subunits and five co-factors, is similar to that of aerobic complex II, except for an extra peptide found in the smallest anchor subunit of the A. suum enzyme. We discuss herein the structure-function relationship of the enzyme and the critical role of the low redox potential of rhodoquinol in the fumarate reduction of A. suum complex II.