Craniofacial, oral, and cervical morphological characteristics in Japanese patients with Apert syndrome or Crouzon syndrome.

BACKGROUND AND OBJECTIVES: Mutations in the fibroblast growth factor receptor 2 (FGFR2) gene are responsible for both Apert syndrome (AS) and Crouzon syndrome (CS). These diseases share phenotypic characteristics, including midfacial hypoplasia and premature fusion of the calvarial suture(s). Given the extensive range of craniofacial growth and developmental abnormalities, management of these patients requires a multidisciplinary approach. This study aimed to compare craniofacial, oral, and cervical morphological characteristics in Japanese orthodontic patients with AS or CS. SUBJECTS AND METHODS: Lateral cephalograms, orthopantomograms, dental casts, medical interview records, facial photographs, and intraoral photographs of 7 AS patients and 12 CS patients on initial visits were used in this study. Cephalometric analyses were performed, and standard scores were calculated based on age- and sex-matched Japanese standard values. RESULTS: Cephalometric analysis revealed that AS patients had significantly more severe maxillary hypoplasia in two dimensions and increased clockwise mandibular rotation. Additionally, cleft of the soft palate, anterior open bite, severe crowding in the maxillary dental arch, and congenitally missing teeth occurred more frequently among AS patients. Multiple fusions between cervical vertebrae C2, C3, C5, and C6 were observed in the AS patients. LIMITATIONS: Small sample size. CONCLUSIONS/IMPLICATIONS: Our study shows that AS patients have more severe craniofacial and maxillofacial deformities than CS patients.

Structural basis of Atg8 activation by a homodimeric E1, Atg7.

E1 enzymes activate ubiquitin-like proteins and transfer them to cognate E2 enzymes. Atg7, a noncanonical E1, activates two ubiquitin-like proteins, Atg8 and Atg12, and plays a crucial role in autophagy. Here, we report crystal structures of full-length Atg7 and its C-terminal domain bound to Atg8 and MgATP, as well as a solution structure of Atg8 bound to the extreme C-terminal domain (ECTD) of Atg7. The unique N-terminal domain (NTD) of Atg7 is responsible for Atg3 (E2) binding, whereas its C-terminal domain is comprised of a homodimeric adenylation domain (AD) and ECTD. The structural and biochemical data demonstrate that Atg8 is initially recognized by the C-terminal tail of ECTD and is then transferred to an AD, where the Atg8 C terminus is attacked by the catalytic cysteine to form a thioester bond. Atg8 is then transferred via a trans mechanism to the Atg3 bound to the NTD of the opposite protomer within a dimer.

Total synthesis of the antibiotic kendomycin: a macrocyclization using the Tsuji-Trost etherification.

A highly stereocontrolled, convergent total synthesis of kendomycin [(-)-TAN2162], an ansa-macrocyclic antibiotic, is reported. The key of the strategy is an unprecedented Tsuji-Trost macrocyclic etherification, followed by a transannular Claisen rearrangement to construct the 18-membered carbocyclic framework. The oxa-six- and five-membered rings were also stereoselectively constructed respectively by a cascade oxidative cyclization at an unfunctionalized benzylic position and using a one-pot epoxidation/5-exo-tet epoxide opening.

NMR analyses of the interaction between the FYVE domain of early endosome antigen 1 (EEA1) and phosphoinositide embedded in a lipid bilayer.

Phosphoinositides (PIs) are crucial lipid components of membranes and are involved in a number of cellular processes through interactions with their effector proteins. Recently, we have established a lipid-protein nanoscale bilayer (nanodisc) containing PIs, hereafter referred to as PI-nanodisc and demonstrated that it could be used for both qualitative and quantitative evaluations of protein-membrane interactions. Here, we report further NMR analyses for obtaining structural insights at the residue-specific level between PI-binding effector protein and PI-nanodisc, using the FYVE domain of early endosome antigen 1 (EEA1), denoted as EEA1 FYVE, and PI(3)P-nanodisc as a model system. We performed a combination of the NMR analyses including chemical shift perturbation, transferred cross-saturation, and paramagnetic relaxation enhancement experiments. These enabled an identification of the interaction surface, structural change, and relative orientation of EEA1 FYVE to the PI(3)P-incorporated lipid bilayer, substantiating that NMR analyses of protein-membrane interactions using nanodisc makes it possible to show the residue-specific interactions in the lipid bilayer environment.

Solution structures of yeast Saccharomyces cerevisiae calmodulin in calcium- and target peptide-bound states reveal similarities and differences to vertebrate calmodulin.

We determined the solution structures of the calmodulin (CaM) isoform from yeast Saccharomyces cerevisiae (yCaM) in the calcium-bound form and in complex with a target peptide using NMR spectroscopy and small-angle X-ray scattering (SAXS). yCaM shows a number of unique features distinct from the vertebrate CaM isoforms: (i) it has only approximately 60% sequence identity to vertebrate CaM; (ii) its fourth Ca(2+)-binding domain is inactivated by amino acid substitution. As NMR analyses of Ca(2+)-bound full-length yCaM implied that the fourth EF-hand motif region (EF4) presents a disordered conformation, we determined the solution structure of an EF4-deletion mutant of Ca(2+)-bound yCaM. The deletion mutant showed a compact globular structure, with the target recognition sites of the N-terminal domain and the third EF-hand region bound to each other. Furthermore, we determined the solution structure of Ca(2+)-bound yCaM complexed with a calcineurin-derived peptide. Interestingly, the structure closely resembled that of the vertebrate CaM-calcineurin complex, with the EF4 region in cooperation with the peptide binding. Moreover, the results of SAXS analyses were consistent with the NMR solution structures and showed the conformational changes of yCaM in three functional stages. These unique structural characteristics of yCaM are closely related to Ca(2+)-mediated signal transduction in yeast.

Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein-protein complex structure determination.

Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein-protein and protein-ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination.

Structural basis for the transforming activity of human cancer-related signaling adaptor protein CRK.

CRKI (SH2-SH3) and CRKII (SH2-SH3-SH3) are splicing isoforms of the oncoprotein CRK that regulate transcription and cytoskeletal reorganization for cell growth and motility by linking tyrosine kinases to small G proteins. CRKI shows substantial transforming activity, whereas the activity of CRKII is low, and phosphorylated CRKII has no biological activity whatsoever. The molecular mechanisms underlying the distinct biological activities of the CRK proteins remain elusive. We determined the solution structures of CRKI, CRKII and phosphorylated CRKII by NMR and identified the molecular mechanism that gives rise to their activities. Results from mutational analysis using rodent 3Y1 fibroblasts were consistent with those from the structural studies. Together, these data suggest that the linker region modulates the binding of CRKII to its targets, thus regulating cell growth and motility.

Solution structure of the silkworm betaGRP/GNBP3 N-terminal domain reveals the mechanism for beta-1,3-glucan-specific recognition.

The beta-1,3-glucan recognition protein (betaGRP)/Gram-negative bacteria-binding protein 3 (GNBP3) is a crucial pattern-recognition receptor that specifically binds beta-1,3-glucan, a component of fungal cell walls. It evokes innate immunity against fungi through activation of the prophenoloxidase (proPO) cascade and Toll pathway in invertebrates. The betaGRP consists of an N-terminal beta-1,3-glucan-recognition domain and a C-terminal glucanase-like domain, with the former reported to be responsible for the proPO cascade activation. This report shows the solution structure of the N-terminal beta-1,3-glucan recognition domain of silkworm betaGRP. Although the N-terminal domain of betaGRP has a beta-sandwich fold, often seen in carbohydrate-binding modules, both NMR titration experiments and mutational analysis showed that betaGRP has a binding mechanism which is distinct from those observed in previously reported carbohydarate-binding domains. Our results suggest that betaGRP is a beta-1,3-glucan-recognition protein that specifically recognizes a triple-helical structure of beta-1,3-glucan.

Three-dimensional analysis of the palatal morphology in growing patients with Apert syndrome and Crouzon syndrome.

Patients with Apert syndrome or Crouzon syndrome present with severe defects in oral-maxillofacial growth and development. In this study, we conducted a quantitative three-dimensional (3D) analysis of the palatal morphology of patients with Apert syndrome and Crouzon syndrome. Four patients with Apert syndrome (average age, 11.0 ± 0.8 years) and five with Crouzon syndrome (average age, 10.1 ± 1.6 years) were investigated. The participants' maxillary dental casts were scanned and analyzed using 3D imaging. Palatal width, depth, cross-sectional area, and palatal angle (PW, PD, PCA, and PA, respectively) were measured, and standard scores were calculated based on sex- and age-matched Japanese standard values; the actual palatal surface areas (PSA) and palatal volumes (PV) were also measured. Our results show that patients with Apert syndrome and Crouzon syndrome had a very narrow PW (standard score: -3.79 and - 0.47, respectively). 3D analysis revealed that patients with Apert syndrome had a significantly shallower PD (standard score: -1.35) than those with Crouzon syndrome (standard score: 2.47), resulting in a smaller PCA (standard score: -5.13), PSA (5.49 cm2 ), and PV (1.11 cm3 ) and larger PA (standard score: -0.12) than those in patients with Crouzon syndrome. This might be due to the former having a narrower and shallower palate caused by the predominant swelling of the palatal mucosa. These findings improve our understanding of the differences in palatal morphology between Apert syndrome and Crouzon syndrome patients.

Solution structure of a novel Cdc42 binding module of Bem1 and its interaction with Ste20 and Cdc42.

Bem1 is a scaffold protein essential for the establishment of cell polarity in Saccharomyces cerevisiae. This work reports the solution structure of a Cdc42 binding module of Bem1 comprising the second SH3 domain (SH3b) and its C-terminal flanking region termed Cdc42 interacting (CI). First, the structure of Bem1 SH3b-CI was determined by NMR spectroscopy, which shows that Bem1 SH3b-CI is a structurally and functionally related domain that binds Cdc42. Next, the solution structure of Bem1 SH3b-CI in complex with the proline-rich region of p21-activated kinase Ste20 (Ste20 PRR) was determined. Finally, the interaction surface of Bem1 SH3b-CI with Cdc42 was identified based on chemical shift perturbation studies which reveals that Bem1 SH3b-CI interacts simultaneously with both Ste20 PRR and Cdc42 using the opposite surfaces. Thus, Bem1 can tether Cdc42 and Ste20 in close proximity so that Cdc42 can efficiently interact with Ste20 Cdc42 and Rac interactive binding (CRIB). Based on the present results together with the previous biochemical studies (Lamson, R. E., Winters, M. J., and Pryciak, P. M. (2002) Mol. Cell. Biol. 22, 2939-2951 and Winters, M. J., and Pryciak, P. M. (2005) Mol. Cell. Biol. 25, 2177-2190), a model was suggested that the autoinhibition of Ste20 kinase activity by CRIB is released through the Cdc42-CRIB interaction, which is mediated by Bem1, and Ste20 is subsequently activated, an initial step for the establishment of the cell polarity.

An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe.

A nuclear magnetic resonance-based ligand screening strategy utilizing a paramagnetic lanthanide probe is presented. By fixing a paramagnetic lanthanide ion to a target protein, a pseudo-contact shift (PCS) and a paramagnetic relaxation enhancement (PRE) can be observed for both the target protein and its bound ligand. Based on PRE and PCS information, the bound ligand is then screened from the compound library and the structure of the ligand-protein complex is determined. PRE is an isotropic paramagnetic effect observed within 30 Å from the lanthanide ion, and is utilized for the ligand screening in the present study. PCS is an anisotropic paramagnetic effect providing long-range (~40 Å) distance and angular information on the observed nuclei relative to the paramagnetic lanthanide ion, and utilized for the structure determination of the ligand-protein complex. Since a two-point anchored lanthanide-binding peptide tag is utilized for fixing the lanthanide ion to the target protein, this screening method can be generally applied to non-metal-binding proteins. The usefulness of this strategy was demonstrated in the case of the growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain and its low- and high-affinity ligands.

Phosphoinositide-incorporated lipid-protein nanodiscs: A tool for studying protein-lipid interactions.

Phosphatidylinositol (PtdIns) is phosphorylated at D-3, D-4, and/or D-5 of the inositol ring to produce seven distinct lipid second messengers known as phosphoinositides (PIs). The PI level is temporally and spatially controlled at the cytosolic face of the cellular membrane. Effectors containing PI-binding domains (e.g., PH, PX, FYVE, ENTH, FERM) associate with specific PIs. This process is crucial for the localization of a variety of cell-signaling proteins, thereby regulating intracellular membrane trafficking, cell growth and survival, cytoskeletal organization, and so on. However, quantitative assessments of protein-PI interactions are generally difficult due to insolubility of PIs in aqueous solution. Here we incorporated PIs into a lipid-protein nanoscale bilayer (nanodisc), which is applied for studying the protein-PI interactions using pull-down binding assay, fluorescence polarization, and nuclear magnetic resonance studies, each facilitating fast, quantitative, and residue-specific evaluation of the protein-PI interactions. Therefore, the PI-incorporated nanodisc could be used as a versatile tool for studying the protein-lipid interactions by various biochemical and biophysical techniques.

Structure determination of proteins in 2H2O solution aided by a deuterium-decoupled 3D HCA(N)CO experiment.

We developed an NMR pulse sequence, 3D HCA(N)CO, to correlate the chemical shifts of protein backbone (1)Halpha and (13)Calpha to those of (13)C' in the preceding residue. By applying (2)H decoupling, the experiment was accomplished with high sensitivity comparable to that of HCA(CO)N. When combined with HCACO, HCAN and HCA(CO)N, the HCA(N)CO sequence allows the sequential assignment using backbone (13)C' and amide (15)N chemical shifts without resort to backbone amide protons. This assignment strategy was demonstrated for (13)C/(15)N-labeled GB1 dissolved in (2)H(2)O. The quality of the GB1 structure determined in (2)H(2)O was similar to that determined in H(2)O in spite of significantly smaller number of NOE correlations. Thus this strategy enables the determination of protein structures in (2)H(2)O or H(2)O at high pH values.

Advances in high-resolution imaging and computational unfolding of the human hippocampus.

The hippocampus is often a difficult structure to visualize with magnetic resonance imaging (MRI) and functional MRI (fMRI) due to its convoluted nature and susceptibility to signal dropout. Improving our ability to pinpoint changes in neural activity using fMRI in this structure remains an important challenge. Current fMRI/MRI methods typically do not permit visualization of the hippocampus and surrounding cortex at a resolution less than 1 mm. We present here improvements to our previous methods for obtaining structural MR images of the hippocampus, which provided an in-plane resolution of 0.4 mm(2) mm and two-dimensional "flat" maps of the hippocampus with an interpolated isotropic resolution of 0.4 mm(3) (Engel, S.A., Glover, G.H., and Wandell, B.A., (1997). Retinotopic organization in human visual cortex and the spatial precision of functional MRI. Cereb. Cortex 7, 181-192.; Zeineh, M.M., Engel, S.A., and Bookheimer, S.Y., (2000). Application of cortical unfolding techniques to functional MRI of the human hippocampal region. NeuroImage 11, 668-683.). We present changes to existing structural imaging sequences that now augment the resolution of previous scans, permitting visualization of the anterior portion of CA1, parts of the dentate gyrus, and CA23. These imaging improvements are of relevance generally to the field of imaging because they permit higher overall resolution imaging of the hippocampus than previously possible (at 3 T). We also introduce a novel application of a computational interpolation method that improves our ability to capture the convoluted three-dimensional shape of the hippocampus. Furthermore, we have developed a quantitative method for obtaining group activation patterns based on producing averaged flat maps using vector field warping techniques, allowing localization of activations to specific hippocampal subregions across groups of subjects. Together, these methods provide a means to improve imaging of neural activity in the human hippocampus and surrounding cortex during cognitive tasks.

Attachment of an NMR-invisible solubility enhancement tag using a sortase-mediated protein ligation method.

Sample solubility is essential for structural studies of proteins by solution NMR. Attachment of a solubility enhancement tag, such as GB1, MBP and thioredoxin, to a target protein has been used for this purpose. However, signal overlap of the tag with the target protein often made the spectral analysis difficult. Here we report a sortase-mediated protein ligation method to eliminate NMR signals arising from the tag by preparing the isotopically labeled target protein attached with the non-labeled GB1 tag at the C-terminus.

Two-point anchoring of a lanthanide-binding peptide to a target protein enhances the paramagnetic anisotropic effect.

Paramagnetic lanthanide ions fixed in a protein frame induce several paramagnetic effects such as pseudo-contact shifts and residual dipolar couplings. These effects provide long-range distance and angular information for proteins and, therefore, are valuable in protein structural analysis. However, until recently this approach had been restricted to metal-binding proteins, but now it has become applicable to non-metalloproteins through the use of a lanthanide-binding tag. Here we report a lanthanide-binding peptide tag anchored via two points to the target proteins. Compared to conventional single-point attached tags, the two-point linked tag provides two to threefold stronger anisotropic effects. Though there is slight residual mobility of the lanthanide-binding tag, the present tag provides a higher anisotropic paramagnetic effect.

[Correlation of quantification of major bcr-abl mRNA between TMA (transcription mediated amplification) method and real-time quantitative PCR].

Imatinib mesylate has significantly improved the outcome of patients with CML. In the IRIS trial, major molecular response (MMR), which is defined as the achievement of > or =3 log reduction in bcr-abl mRNA from the standardized baseline, was observed in 40% of CML patients by 12 months. Achievement of an MMR at 18 months is associated with 100% probability of transformation-free survival at 60 months, and MMR is an important goal of therapy. The nucleic acid quantitative "DNA probe FR Amp-CML" kit based on the transcription-mediated amplification method, can measure major bcr-abl mRNA in peripheral blood leukocytes. In this study, we studied the clinical usefulness of Amp-CML for monitoring minimum residual disease by comparison with the European standard nucleic acid quantitative method and real-time quantitative PCR (RQ-PCR) with GAPDH as an internal control, using peripheral leukocytes obtained from patients receiving imatinib treatment. The results indicated that Amp-CML had a significant correlation with Fusion Quant M-BCR (R>0.971, P<0.01), a standard nucleic acid quantitative method used in Europe and RQ-PCR (R>0.974, P<0.01), especially in samples with more than 100 copies/microg RNA of major bcr-abl mRNA. These data suggest that Amp-CML is reliable for monitoring major bcr-abl mRNA in patients having achieved an MMR.

[A case of Langerhans cell histiocytosis coincident with severe external ear canal inflammation].

We report a case of Langerhans cell histiocytosis (LCH), a rare disease caused by the proliferation of abnormal Langerhans cells, coincident with severe external ear canal inflammation. A 27-year-old man with a 1-year history of external ear canal discharge was found in computed tomography (CT) to have left temporal bone erosion with tissue granulation. Chest X-ray showed pulmonary fibrosis, necessitating transbronchial lung biopsy that yielded a definitive diagnosis of multisystem, multisite LCH involving the bone, skin, lung, pituitary, thyroid, and lymph node systems. He underwent combination chemotherapy directed by the Japan LCH Study Group. LCH should therefore be considered in the case of a patient with severe external ear canal inflammation coincident with temporal bone erosion.