Total synthesis of (-)-domoic acid, a potent ionotropic glutamate receptor agonist and the key compound in oceanic harmful algal blooms.

The stereo-controlled total synthesis of (-)-domoic acid is described. The critical construction of the C1'-C2' Z-configuration was accomplished by taking advantage of an unsaturated lactam structure. The side chain fragment was introduced in the final stages of synthesis through a modified Julia-Kocienski reaction, aiming for its efficient derivatization.

Diffusion tensor imaging (DTI) of human lower leg muscles: correlation between DTI parameters and muscle power with different ankle positions.

PURPOSE: To compare diffusion tensor imaging (DTI) parameters in healthy adult human lower leg muscles and to determine the correlation between DTI parameters and muscle power measurements among different types of muscle contraction. MATERIALS AND METHODS: DTI measurements of the unilateral lower leg muscles having three different types of contraction (non-contraction state, isometric contraction, and soleus shortening) were obtained from 10 healthy adults using a 3-T MRI scanner. DTI parameters (λ1, λ2, λ3, mean diffusivity, and fractional anisotropy) were calculated. The values of the DTI parameters and correlation between the DTI parameters and muscle power measurements (maximum power and maximum amount of work) obtained from a dynamometer were statistically compared among the different types of contraction. Intra- and inter-class correlation coefficients were calculated for analysis of reproducibility. RESULTS: The λ1, λ2, λ3, and mean diffusivity of the soleus muscle are significantly lower in the non-contraction state as compared with isometric contraction and soleus shortening (p < 0.05). A positive correlation of the soleus muscle in the non-contraction state was seen between the maximum power and the λ1, λ2, and mean diffusivity. There was a positive correlation between the maximum amount of work and fractional anisotropy in the non-contraction state for the soleus muscle. A negative correlation for the tibialis anterior muscle in the non-contraction state was seen between the maximum amount of work and fractional anisotropy. Overall reproducibility of the DTI parameters was excellent. CONCLUSIONS: DTI parameters were significantly changed depending on the ankle joint position and type of muscle contraction.

Calciprotein particles regulate fibroblast growth factor-23 expression in osteoblasts.

Fibroblast growth factor-23 (FGF23) is a hormone indispensable for maintaining phosphate homeostasis. In response to phosphate intake, FGF23 is secreted from osteocytes/osteoblasts and acts on the kidney to increase urinary phosphate excretion. However, the mechanism by which these cells sense phosphate intake remains elusive. Calciprotein particles are nanoparticles of calcium-phosphate precipitates bound to serum protein fetuin-A and are generated spontaneously in solution containing calcium, phosphate, and fetuin-A to be dispersed as colloids. In cultured osteoblastic cells, increase in either calcium or phosphate concentration in the medium induced FGF23 expression, which was dependent on calciprotein particle formation. When transition of calcium-phosphate precipitates from the amorphous phase to the crystalline phase was blocked by bisphosphonate, the calciprotein particle size was reduced and FGF23 expression was augmented, suggesting that small calciprotein particles containing amorphous calcium-phosphate precipitates function as a more potent FGF23 inducer than larger calciprotein particles containing crystalline calcium-phosphate precipitates. In mice, bolus phosphate administration by oral gavage transiently increased circulating calciprotein particle levels followed by a modest increase in FGF23 expression and serum FGF23 levels. However, continuous dietary phosphate load induced robust and persistent increase in circulating calciprotein particles and FGF23 levels. We confirmed by in vivo imaging that calciprotein particles injected intravenously extravasated into the bone marrow and were deposited on the inner surface of the bone, indicating that these particles have direct access to osteoblasts. Thus, we propose that osteoblasts induce FGF23 expression and secretion when they sense an increase in extracellular calciprotein particles following phosphate ingestion.

[Research on Utilization of LCD Monitors Substitute for Light Box about Hard-copy Diagnosis in an Emergency by Using Receiver Operating Characteristic Analysis].

The creation of the business continuity plan (BCP) for disaster key hospitals were mandated by the Ministry of Health, Labor and Welfare on March 31, 2017. The creation of BCP must take countermeasures assuming damage at all levels. In Japan, a light box is no longer being used by a shift to soft-copy diagnosis. However, supposing the hospital network failure occurred, we assumed the film diagnosis was necessary for radiological examination images. The purpose of this study is to investigate whether the film diagnosis using a medical monitor with high luminance (410, 800 cd/m2) instead of the light box is inferior or not to a monitor diagnosis (410 cd/m2) in detecting pulmonary nodules. Ten radiological technologists participated in the observer tests for detection of nodules, respectively. In each observer test, radiological technologists marked their confidence levels for the diagnosis of pulmonary nodules. The detection performance of radiological technologists was evaluated by receiver operating characteristic (ROC) analysis. The average area under the ROC curve (AUC) value in detecting pulmonary nodules with monitor (410 cd/m2) and film (410 cd/m2), film (800 cd/m2) were 0.770 and 0.754, 0.806 respectively. There was no statistically significant difference between monitor (410 cd/m2) and film (410, 800 cd/m2) for detection of pulmonary nodules (P=0.32, 0.09). Therefore, we believe that the film diagnosis can be used for the medical monitor instead of the light box when film operation in case of a disaster inevitable.

Crystal structure of a Ca2+-dependent regulator of flagellar motility reveals the open-closed structural transition.

Sperm chemotaxis toward a chemoattractant is very important for the success of fertilization. Calaxin, a member of the neuronal calcium sensor protein family, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis of ascidian, Ciona intestinalis. Here, we present the crystal structures of calaxin both in the open and closed states upon Ca2+ and Mg2+ binding. The crystal structures revealed that three of the four EF-hands of a calaxin molecule bound Ca2+ ions and that EF2 and EF3 played a critical role in the conformational transition between the open and closed states. The rotation of α7 and α8 helices induces a significant conformational change of a part of the α10 helix into the loop. The structural differences between the Ca2+- and Mg2+-bound forms indicates that EF3 in the closed state has a lower affinity for Mg2+, suggesting that calaxin tends to adopt the open state in Mg2+-bound form. SAXS data supports that Ca2+-binding causes the structural transition toward the closed state. The changes in the structural transition of the C-terminal domain may be required to bind outer-arm dynein. These results provide a novel mechanism for recognizing a target protein using a calcium sensor protein.

A Physicochemical and Mutational Analysis of Intersubunit Interactions of Escherichia coli Ferritin A.

Ferritin A from Escherichia coli (EcFtnA) is 24-meric protein, which forms spherical cagelike structures called nanocages. The nanocage structure is stabilized by the interface around 4-, 3-, and 2-fold symmetric axes. The subunit structure of EcFtnA comprises a four-helix bundle (helices A-D) and an additional helix E, which forms a 4-fold axis. In this study, we examined the contribution of the interface around three symmetric axes. pH-induced dissociation experiments monitored by analytical ultracentrifugation and small-angle X-ray scattering showed that the dimer related by 2-fold symmetry is the most stable unit. Mutations located near the 3-fold axis revealed that the contribution of each interaction was small. A mutant lacking helix E at the 4-fold axis formed a nanocage, suggesting that helix E is not essential for nanocage formation. Further truncation of the C-terminus of helix D abrogated the formation of the nanocage, suggesting that a few residues located at the C-terminus of helix D are critical for this process. These properties are similar to those known for mammalian ferritins and seem to be common principles for nanocage formation. The difference between EcFtnA and mammalian ferritins was that helix E-truncated EcFtnA maintained an iron-incorporating ability, whereas mammalian mutants lost it.

Identification of key neoculin residues responsible for the binding and activation of the sweet taste receptor.

Neoculin (NCL) is a heterodimeric protein isolated from the edible fruit of Curculigo latifolia. It exerts a taste-modifying activity by converting sourness to sweetness. We previously demonstrated that NCL changes its action on the human sweet receptor hT1R2-hT1R3 from antagonism to agonism as the pH changes from neutral to acidic values, and that the histidine residues of NCL molecule play critical roles in this pH-dependent functional change. Here, we comprehensively screened key amino acid residues of NCL using nuclear magnetic resonance (NMR) spectroscopy and alanine scanning mutagenesis. We found that the mutations of Arg48, Tyr65, Val72 and Phe94 of NCL basic subunit increased or decreased both the antagonist and agonist activities. The mutations had only a slight effect on the pH-dependent functional change. These residues should determine the affinity of NCL for the receptor regardless of pH. Their locations were separated from the histidine residues responsible for the pH-dependent functional change in the tertiary structure. From these results, we concluded that NCL interacts with hT1R2-hT1R3 through a pH-independent affinity interface including the four residues and a pH-dependent activation interface including the histidine residues. Thus, the receptor activation is induced by local structural changes in the pH-dependent interface.

Structural Insight into an Alzheimer's Brain-Derived Spherical Assembly of Amyloid ß by Solid-State NMR.

Accumulating evidence suggests that various neurodegenerative diseases, including Alzheimer's disease (AD), are linked to cytotoxic diffusible aggregates of amyloid proteins, which are metastable intermediate species in protein misfolding. This study presents the first site-specific structural study on an intermediate called amylospheroid (ASPD), an AD-derived neurotoxin composed of oligomeric amyloid-ß (Aß). Electron microscopy and immunological analyses using ASPD-specific "conformational" antibodies established synthetic ASPD for the 42-residue Aß(1-42) as an excellent structural/morphological analogue of native ASPD extracted from AD patients, the level of which correlates with the severity of AD. (13)C solid-state NMR analyses of approximately 20 residues and interstrand distances demonstrated that the synthetic ASPD is made of a homogeneous single conformer containing parallel ß-sheets. These results provide profound insight into the native ASPD, indicating that Aß is likely to self-assemble into the toxic intermediate with ß-sheet structures in AD brains. This approach can be applied to various intermediates relevant to amyloid diseases.

Relationship between chain collapse and secondary structure formation in a partially folded protein.

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of ß-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions.

Structural study of hNck2 SH3 domain protein in solution by circular dichroism and X-ray solution scattering.

We have done conformational study of hNck2 SH3 domain by means of far-ultraviolet (far-UV) circular dichroism (CD) and X-ray solution scattering (XSS). The results indicated that the following: (1) hNck2 SH3 domain protein exhibited concentration dependent monomer-dimer transition at neutral pH, while the secondary structure of this protein was independent of the protein concentration. (2) The hNck2 SH3 domain also exhibited pH dependent monomer-dimer transition. This monomer-dimer transition was accompanied with helix-ß transition of the secondary structural change. Moreover, the acid-induced conformation, which was previously studied by Liu and Song by CD and nuclear magnetic resonance (NMR), was found to be not compact, but the conformation of the protein at acidic pH was similar to the cold denatured state (C-state) reported by Yamada et al. for equine ß-lactoglobulin. We calculated that a structure of the equilibrium helix-rich intermediate of the hNck2 SH3 domain by DAMMIF program.

Transient helical structure during PI3K and Fyn SH3 domain folding.

A growing list of proteins, including the ß-sheet-rich SH3 domain, is known to transiently populate a compact α-helical intermediate before settling into the native structure. Examples have been discovered in cryogenic solvent as well as by pressure jumps. Earlier studies of λ repressor mutants showed that transient states with excess helix are robust in an all-α protein. Here we extend a previous study of src SH3 domain to two new SH3 sequences, phosphatidylinositol 3-kinase (PI3K) and a Fyn mutant, to see how robust such helix-rich transients are to sequence variations in this ß-sheet fold. We quantify helical structure by circular dichroism (CD), protein compactness by small-angle X-ray scattering (SAXS), and transient helical populations by cryo-stopped-flow CD. Our results show that transient compact helix-rich intermediates are easily accessible on the folding landscape of different SH3 domains. In molecular dynamics simulations, force field errors are often blamed for transient non-native structure. We suggest that experimental examples of very fast α-rich transient misfolding could become a more subtle test for further force field improvements than observation of the native state alone.

alpha-Helical burst on the folding pathway of FHA domains from Rad53 and Ki67.

We investigated refolding processes of beta-sheeted protein FHA domains (FHA1 domain of Rad53 and Ki67 FHA domain) by cryo-stopped-flow (SF) method combined with far-ultraviolet (far-UV) circular dichroism (CD, the average secondary structure content) and small angle X-ray scattering (SAXS, measuring the radius of gyration). In case of FHA1 domain of Rad53, no detectable time course was observed except the initial burst on its refolding process at 4 degrees C, suggesting that the FHA1 domain of Rad53 was already refolded to its native state within the dead time of the SF apparatus and the rate of the refolding is too fast to be observed at this temperature. In contrast, there was an observable alpha-helical burst at -15 degrees C and -20 degrees C in the presence of 45% ethylene glycol (EGOH) by CD-SF. Besides, the radius of gyration (Rg) of the burst phase intermediate at -20 degrees C shows the intermediate is already compact, and the compaction process was accompanied with the decrease of alpha-helical content at the same temperature. In case of Ki67 FHA domain, ellipticity change at 222 nm was observed on its refolding pathway at -28 degrees C in the presence of 45% EGOH and 2 mM DTT, indicating that Ki67 FHA domain also takes non-native alpha-helix-rich intermediate on its folding pathway. Time-resolved SAXS experiment was done. As the signal/noise ratio is low, we could not observe the time-dependent signal change through the time course. However, the initial Rg value was obtained as 18.2 +/- 0.5 A, which is much smaller than the unfolded Rg value (26.5 +/- 1.2 A), and is slightly larger than the native one (15.9 +/- 1.8 A). These results suggest that Ki67 FHA domain also forms compact non-native alpha-helix-rich intermediate before refolding to its native beta-structure on the refolding pathway. These results are in good agreement with other beta-proteins, such as bovine beta-lactoglobulin (BLG), src SH3 domain proteins. It seems the alpha-helical burst phases appear on the folding pathway of beta-sandwiched proteins.

Slowing down downhill folding: a three-probe study.

The mutant Tyr22Trp/Glu33Tyr/Gly46Ala/Gly48Ala of lambda repressor fragment lambda(6-85) was previously assigned as an incipient downhill folder. We slow down its folding in a cryogenic water-ethylene-glycol solvent (-18 to -28 degrees C). The refolding kinetics are probed by small-angle x-ray scattering, circular dichroism, and fluorescence to measure the radius of gyration, the average secondary structure content, and the native packing around the single tryptophan residue. The main resolved kinetic phase of the mutant is probe independent and faster than the main phase observed for the pseudo-wild-type. Excess helical structure formed early on by the mutant may reduce the formation of turns and prevent the formation of compact misfolded states, speeding up the overall folding process. Extrapolation of our main cryogenic folding phase and previous T-jump measurements to 37 degrees C yields nearly the same refolding rate as extrapolated by Oas and co-workers from NMR line-shape data. Taken together, all the data consistently indicate a folding speed limit of approximately 4.5 micros for this fast folder.

Non-native alpha-helix formation is not necessary for folding of lipocalin: comparison of burst-phase folding between tear lipocalin and beta-lactoglobulin.

Tear lipocalin and beta-lactoglobulin are members of the lipocalin superfamily. They have similar tertiary structures but unusually low overall sequence similarity. Non-native helical structures are formed during the early stage of beta-lactoglobulin folding. To address whether the non-native helix formation is found in the folding of other lipocalin superfamily proteins, the folding kinetics of a tear lipocalin variant were investigated by stopped-flow methods measuring the time-dependent changes in circular dichroism (CD) spectrum and small-angle X-ray scattering (SAXS). CD spectrum showed that extensive secondary structures are not formed during a burst-phase (within a measurement dead time). The SAXS data showed that the radius of gyration becomes much smaller than in the unfolded state during the burst-phase, indicating that the molecule is collapsed during an early stage of folding. Therefore, non-native helix formation is not general for folding of all lipocalin family members. The non-native helix content in the burst-phase folding appears to depend on helical propensities of the amino acid sequence.

Helix-rich transient and equilibrium intermediates of equine beta-lactoglobulin in alkaline buffer.

Acidic buffer conditions are known to stabilize helix-rich states of even those proteins with a predominantly beta-sheet native secondary structure. Here we investigated whether such states also exist under alkaline buffer conditions. The guanidine hydrochloride (GuHCl)-induced unfolding transition and kinetic refolding of equine beta-lactoglobulin (ELG) by GuHCl-jump were investigated at pH 8.7 by far-ultraviolet circular dichroism. We found that an equilibrium intermediate appeared in 45% ethylene glycol (EGOH) buffer with 1.5 M GuHCl. The intermediate is rich in non-native alpha-helix, which is similar to the helix-rich state of ELG at pH 4.0. A kinetic study was done on the folding rate of ELG and compared with bovine beta-lactoglobulin (BLG). Transient intermediates, which were observed as the burst phase of the refolding reaction, were also rich in alpha-helix. The activation enthalpy of ELG was calculated to be c.a. 80 kJ/mol, whereas that of BLG was c.a. 70 kJ/mol in the presence of 45% EGOH. The ellipticities of the transient intermediate of ELG show temperature dependence in the presence of 45% EGOH, whereas that of BLG did not show significant dependence. This study therefore extends the existence of helix-rich equilibrium and transient intermediates of predominantly beta-sheet proteins to alkaline buffer conditions.

A stable alpha-helix-rich intermediate is formed by a single mutation of the beta-sheet protein, src SH3, at pH 3.

Recently, we have found a transient intermediate on the folding pathway of src SH3. Intending to investigate the structure of the transient intermediate, we tested a mutant of src SH3, named A45G, using circular dichroism, fluorescence and X-ray solution scattering, and incidentally found that it forms a stable alpha-helix-rich intermediate (I(eq)) (different from the native beta-sheet-based secondary structure) at pH 3.0, but contains only beta-sheets at pH 6.0, whereas wild-type SH3 forms only beta-sheets at both pH 3.0 and pH 6.0. The intermediate I(eq) shows a circular dichroism measured at theta(222)=-10,300 deg.cm(2) dmol(-1), indicating a 31% alpha-helix proportion, as estimated by the CONTIN program. X-ray scattering gave the radius of gyration for I(eq) as 19.1 A at pH 3.0 and 15.4 A at pH 6.0, and Kratky plots showed a clear peak at pH 3.0, 4.0 and 6.0, indicating that I(eq) too is compact. In these parameters, I(eq) closely resembles the kinetically-obtained intermediate I(kin) which we found on the folding pathway of wild-type SH3 at pH 3.0 (radius of gyration 18.7 A and theta(222)=-8700 deg.cm(2)dmol(-1)), indicating a 26% alpha-helix proportion in our previous paper. Refolding experiments with A45G were done at pH 6.0 by stopped-flow apparatus monitored by circular dichroism, and compared to kinetic experiments with wild-type SH3 at pH 6.0. The result showed an alpha-helix-rich intermediate at the same dichroism amplitude, but nine times slower in formation-rate. A pH-jump experiment from pH 3.0 to pH 5.9 on A45G was also performed. This showed no bursts, and the rate of conformation-change was almost as fast as the refolding rate of A45G at pH 6.0. These kinetic experiment data would be consistent with I(eq) being nearly identical to the I(kin), which appeared on the folding pathways of both wild-type SH3 and A45G at pH 3.

An alpha-helical burst in the src SH3 folding pathway.

Src SH3 is a small all-beta-sheet protein composed of a single domain. We studied the folding behavior of src SH3 at various conditions by circular dichroism (CD), fluorescence, and X-ray solution scattering methods. On the src SH3 folding pathway, an alpha-helix-rich intermediate appeared not only at subzero temperatures but also above 0 degrees C. The fraction of alpha-helix in the kinetically observed intermediate is ca. 26% based on the kinetic CD experiment. X-ray solution scattering revealed that the intermediate was compact but not fully packed. The analysis of CD implies that the amplitude of the burst phase is proportional to the helical fraction calculated according to the helix-coil transition theory. This strongly suggests that the initial folding core is formed by the collapse of much less stably existing alpha-helices.

Solvent-tuning the collapse and helix formation time scales of lambda(6-85)*.

The lambda(6-85)(*) pseudo-wild type of lambda repressor fragment is a fast two-state folder (k(f) approximately 35 microsec(-1) at 58 degrees C). Previously, highly stable lambda(6-85)(*) mutants with k(f) > 30 microsec(-1) have been engineered to fold nearly or fully downhill. Stabilization of the native state by solvent tuning might also tune lambda(6-85)(*) away from two-state folding. We test this prediction by examining the folding thermodynamics and kinetics of lambda(6-85)(*) in a stabilizing solvent, 45% by weight aqueous ethylene glycol at -28 degrees C. Detection of kinetics by circular dichroism at 222 nm (sensitive to helix content) and small angle X-ray scattering (measuring the radius of gyration) shows that refolding from guanidine hydrochloride denatured conditions exhibits very different time scales for collapse and secondary structure formation: the two processes become decoupled. Collapse remains a low-barrier activated process, while the fastest of several secondary structure formation time scales approaches the downhill folding limit. Two-state folding of lambda(6-85)(*) is not a robust process.

Clinical usefulness of fusion of 131I SPECT and CT images in patients with differentiated thyroid carcinoma.

UNLABELLED: Precise localization of the foci of (131)I uptake for management of patients with differentiated thyroid carcinoma can be difficult because of a lack of anatomic landmarks. The objective of the present study was to demonstrate the clinical usefulness of (131)I SPECT/CT fusion images in patients with differentiated thyroid carcinoma. METHODS: CT and SPECT were performed 7 d after administration of a therapeutic dose of (131)I to 17 patients with differentiated thyroid carcinoma. External markers were placed at 3 locations on the skin of the patient to adjust the sections of CT and SPECT in the same geometric plane. Fusion images were constructed by combining the digital CT and SPECT images on a computer workstation. The data from both planar and SPECT (131)I images and CT images were first separately assessed by 2 nuclear medicine physicians. (131)I SPECT/CT fusion images were then interpreted. Fusion images were considered to improve image interpretation in comparison with CT and scintigraphy separately when they provided better localization of sites of increased radiopharmaceutical uptake. RESULTS: Both CT and (131)I SPECT showed the pathologic sites in 5 of 17 patients (29%). Fusion images were considered to be of benefit in 15 of 17 patients (88%). In 4 patients, CT showed normal-sized lymph nodes, whereas (131)I SPECT showed abnormal findings. In 3 patients with bone metastasis, fusion images confirmed the precision of the localization of abnormal (131)I uptake. Five bone metastases and 1 muscle metastasis were occult and were not seen on the CT images. Finally, (131)I scintigraphy findings were abnormal for 2 patients for whom the CT findings were initially considered normal. Fusion images confirmed the precision of the localization of physiologic (131)I uptake. CONCLUSION: For registration of anatomic and functional images in fusion imaging, the method using external markers was simple and practical. (131)I SPECT/CT fusion images using this technique may improve anatomically limited interpretation of (131)I scintigraphy alone in patients with differentiated thyroid carcinoma.