Crystallization and preliminary X-ray analysis of human MTH1 with a homogeneous N-terminus.

Human MTH1 (hMTH1) is an enzyme that hydrolyses several oxidized purine nucleoside triphosphates to their corresponding nucleoside monophosphates. Crystallographic studies have shown that the accurate mode of interaction between 8-oxoguanine and hMTH1 cannot be understood without determining the positions of the H atoms, as can be observed in neutron and/or ultrahigh-resolution X-ray diffraction studies. The hMTH1 protein prepared in the original expression system from Escherichia coli did not appear to be suitable for obtaining high-quality crystals because the hMTH1 protein had heterogeneous N-termini of Met1 and Gly2 that resulted from N-terminal Met excision by methionine aminopeptidase from the E. coli host. To obtain homogeneous hMTH1, the Gly at the second position was replaced by Lys. As a result, mutant hMTH1 protein [hMTH1(G2K)] with a homogeneous N-terminus could be prepared and high-quality crystals which diffracted to near 1.1 Šresolution using synchrotron radiation were produced. The new crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.36, b = 47.58, c = 123.89 Å.

Diagnosis of depressed-type early gastric cancer using small-caliber endoscopy with flexible spectral imaging color enhancement.

BACKGROUND AND AIM: Small-caliber endoscopy has lower resolution than normal-caliber endoscopy, limiting its use in routine outpatient practice. Flexible spectral imaging color enhancement (FICE) strengthens the color contrast of depressed-type early gastric cancer without magnification. The aim of the present study was to evaluate the detection of depressed-type early gastric cancer using small-caliber endoscopy with the FICE system. METHODS: Eighty-two patients diagnosed with depressed-type early gastric cancer by standard endoscopy and biopsy were evaluated by small-caliber endoscopy. FICE images and conventional images were compared. Color differences in all 82 lesions were measured between malignant lesions and the surrounding mucosa using the Commission Internationale de L'Eclairage (CIE) 1976 color space. RESULTS: Most cancers were readily detected as reddish lesions on FICE images. Lines of demarcation between the malignant lesion and the surrounding mucosa were easily identified with FICE images, as such cancers could be clearly distinguished from the surrounding atrophic mucosa. Greater median color differences between malignant lesions and the surrounding mucosa were present in FICE images compared with conventional images, resulting in images with better contrast (27.2 vs 18.7, P<0.0001). CONCLUSIONS: Small-caliber endoscopy with the FICE system provides better color contrast of depressed-type early gastric cancers than conventional small-caliber endoscopy, and the FICE system may facilitate the diagnosis of this type of cancer as a new endoscopic modality.

Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA.

The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Down's syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 A, beta = 101.7 degrees and a V(M) value of 3.56 A(3) Da(-1). Diffraction data were collected to a resolution of 3.0 A.

Risk factors for nasal bleeding in patients undergoing transnasal gastrointestinal endoscopy.

Transnasal endoscopy is widely used in screening for upper gastrointestinal lesions because of less associated pain. Nasal bleeding is the most severe adverse effect, but specific risk factors have not been identified. The aim of this study is to identify risk factors for nasal bleeding during transnasal endoscopy. Nasal bleeding occurred in 160/3035 (5.3%) of patients undergoing transnasal endoscopy as part of health checkups. Patient data were retrospectively evaluated including anthropometric, medical, and life-style parameters with multiple logistic regression analysis. Multiple logistic regression revealed that nasal bleeding was significantly associated with age in decades [odds ratio/10 years 0.78, 95% confidence interval (CI) 0.63-0.97, p = 0.027], female gender (2.15, 95% CI 1.48-3.12, p < 0.001), a history of previous upper gastrointestinal endoscopy (0.55, 95% CI 0.36-0.82, p = 0.004), and chronic/allergic rhinitis (0.60, 95% CI 0.36-0.98, p = 0.043). Other factors including the use of antiplatelet and/or anticoagulant drugs were not significantly associated with nasal bleeding. Female and young patients are significantly associated with an increased risk of bleeding from transnasal endoscopy, but antiplatelet and/or anticoagulant medications and a history of chronic/allergic rhinitis may not be associated.

Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.

T-protein, a component of the glycine cleavage system, catalyzes the formation of ammonia and 5,10-methylenetetrahydrofolate from the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein. Several mutations in the human T-protein gene cause non-ketotic hyperglycinemia. To gain insights into the effect of disease-causing mutations and the catalytic mechanism at the molecular level, crystal structures of human T-protein in free form and that bound to 5-methyltetrahydrofolate (5-CH3-H4folate) have been determined at 2.0 A and 2.6 A resolution, respectively. The overall structure consists of three domains arranged in a cloverleaf-like structure with the central cavity, where 5-CH3-H4folate is bound in a kinked shape with the pteridine group deeply buried into the hydrophobic pocket and the glutamyl group pointed to the C-terminal side surface. Most of the disease-related residues cluster around the cavity, forming extensive hydrogen bonding networks. These hydrogen bonding networks are employed in holding not only the folate-binding space but also the positions and the orientations of alpha-helix G and the following loop in the middle region, which seems to play a pivotal role in the T-protein catalysis. Structural and mutational analyses demonstrated that Arg292 interacts through water molecules with the folate polyglutamate tail, and that the invariant Asp101, located close to the N10 group of 5-CH3-H4folate, might play a key role in the initiation of the catalysis by increasing the nucleophilic character of the N10 atom of the folate substrate for the nucleophilic attack on the aminomethyl lipoate intermediate. A clever mechanism of recruiting the aminomethyl lipoate arm to the reaction site seems to function as a way of avoiding the release of toxic formaldehyde.

Crystallization and preliminary X-ray analysis of human MTH1 complexed with two oxidized nucleotides, 8-oxo-dGMP and 2-oxo-dATP.

Human MutT homologue 1 (hMTH1) hydrolyzes a variety of oxidized purine nucleoside triphosphates, including 8-oxo-dGTP, 2-oxo-dATP, 2-oxo-ATP and 8-oxo-dATP, to their corresponding nucleoside monophosphates, while Escherichia coli MutT possesses prominent substrate specificity for 8-oxoguanine nucleotides. Three types of crystals were obtained corresponding to the following complexes: selenomethionine-labelled hMTH1 with 8-oxo-dGMP (SeMet hMTH1-8-oxo-dGMP), hMTH1 with 8-oxo-dGMP (hMTH1-8-oxo-dGMP) and hMTH1 with 2-oxo-dATP (hMTH1-2-oxo-dATP). Crystals of the SeMet hMTH1-8-oxo-dGMP complex belong to space group P4(1)2(1)2, with unit-cell parameters a = b = 45.8, c = 153.6 A, and diffracted to 2.90 A. Crystals of hMTH1-8-oxo-dGMP and hMTH1-2-oxo-dATP belong to space groups P2(1) and P2(1)2(1)2(1), with unit-cell parameters a = 34.0, b = 59.0, c = 65.9 A, beta = 90.7 degrees and a = 59.2, b = 67.3, c = 80.0 A, respectively. Their diffraction data were collected at resolutions of 1.95 and 2.22 A, respectively.

The crystal structures of semi-synthetic aequorins.

The photoprotein aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca(2+). Semi-synthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca(2+). To understand the structural basis of the Ca(2+)-sensitivity, we determined the crystal structures of four semi-synthetic aequorins (cp-, i-, br- and n-aequorins) at resolutions of 1.6-1.8 A. In general, the protein structures of these semi-synthetic aequorins are almost identical to native aequorin. Of the four EF-hand domains in the molecule, EF-hand II does not bind Ca(2+), and the loop of EF-hand IV is clearly deformed. It is most likely that the binding of Ca(2+) with EF-hands I and III triggers luminescence. Although little difference was found in the overall structures of aequorins investigated, some significant differences were found in the interactions between the substituents of coelenterazine moiety and the amino acid residues in the binding pocket. The coelenterazine moieties in i-, br-, and n-aequorins have bulky 2-substitutions, which can interfere with the conformational changes of protein structure that follow the binding of Ca(2+) to aequorin. In cp-aequorin, the cyclopentylmethyl group that substitutes for the original 8-benzyl group does not interact hydrophobically with the protein part, giving the coelenterazine moiety more conformational freedom to promote the light-emitting reaction. The differences of various semi-synthetic aequorins in Ca(2+)-sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca(2+)-binding.

Diagnosis of small flat early gastric cancer by flexible spectral imaging color enhancement.

Current conventional endoscopy often misses flat early gastric cancers (0-IIb) because they are sometimes invisible. We experienced a case of small flat early gastric cancer that had been missed by normal-caliber conventional endoscopy. By small-caliber endoscope, conventional endoscopy showed a subtle reddish change of gastric mucosa, but the image with flexible spectral imaging color enhancement clearly showed a flat reddish lesion with 10 mm diameter, distinct from the surrounding mucosa. Flat early gastric cancer was suspected even though the lesion was not clearly described by conventional endoscopy. Histological examination of biopsy specimen revealed atypical glands. Endoscopic submucosal dissection of the lesion was performed. Pathological examination of the resected specimen confirmed well-differentiated adenocarcinoma localized in the mucosal layer without any depression or protrusion compared with the surrounding mucosa, consistent with the endoscopic finding. The small flat early gastric cancer became clearly visible with the new endoscopic technology.

Crystal structure of the IL-15-IL-15Ralpha complex, a cytokine-receptor unit presented in trans.

Interleukin 15 (IL-15) and IL-2, which promote the survival of memory CD8(+) T cells and regulatory T cells, respectively, bind receptor complexes that share beta- and gamma-signaling subunits. Receptor specificity is provided by unique, nonsignaling alpha-subunits. Whereas IL-2 receptor-alpha (IL-2Ralpha) is expressed together in cis with the beta- and gamma-subunits on T cells and B cells, IL-15Ralpha is expressed in trans on antigen-presenting cells. Here we present a 1.85-A crystal structure of the human IL-15-IL-15Ralpha complex. The structure provides insight into the molecular basis of the specificity of cytokine recognition and emphasizes the importance of water in generating this very high-affinity complex. Despite very low IL-15-IL-2 sequence homology and distinct receptor architecture, the topologies of the IL-15-IL-15Ralpha and IL-2-IL-2Ralpha complexes are very similar. Our data raise the possibility that IL-2, like IL-15, might be capable of being presented in trans in the context of its unique receptor alpha-chain.

Crystal structure of mammalian alpha1,6-fucosyltransferase, FUT8.

Mammalian alpha1,6-fucosyltransferase (FUT8) catalyses the transfer of a fucose residue from a donor substrate, guanosine 5'-diphosphate-beta-L-fucose to the reducing terminal N-acetylglucosamine (GlcNAc) of the core structure of an asparagine-linked oligosaccharide. Alpha1,6-fucosylation, also referred to as core fucosylation, plays an essential role in various pathophysiological events. Our group reported that FUT8 null mice showed severe growth retardation and emphysema-like lung-destruction as a result of the dysfunction of epidermal growth factor and transforming growth factor-beta receptors. To elucidate the molecular basis of FUT8 with respect to pathophysiology, the crystal structure of human FUT8 was determined at 2.6 A resolution. The overall structure of FUT8 was found to consist of three domains: an N-terminal coiled-coil domain, a catalytic domain, and a C-terminal SH3 domain. The catalytic region appears to be similar to GT-B glycosyltransferases rather than GT-A. The C-terminal part of the catalytic domain of FUT8 includes a Rossmann fold with three regions that are conserved in alpha1,6-, alpha1,2-, and protein O-fucosyltransferases. The SH3 domain of FUT8 is similar to other SH3 domain-containing proteins, although the significance of this domain remains to be elucidated. The present findings of FUT8 suggest that the conserved residues in the three conserved regions participate in the Rossmann fold and act as the donor binding site, or in catalysis, thus playing key roles in the fucose-transferring reaction.

Crystal structure of lipoate-protein ligase A from Escherichia coli. Determination of the lipoic acid-binding site.

Lipoate-protein ligase A (LplA) catalyzes the formation of lipoyl-AMP from lipoate and ATP and then transfers the lipoyl moiety to a specific lysine residue on the acyltransferase subunit of alpha-ketoacid dehydrogenase complexes and on H-protein of the glycine cleavage system. The lypoyllysine arm plays a pivotal role in the complexes by shuttling the reaction intermediate and reducing equivalents between the active sites of the components of the complexes. We have determined the X-ray crystal structures of Escherichia coli LplA alone and in a complex with lipoic acid at 2.4 and 2.9 angstroms resolution, respectively. The structure of LplA consists of a large N-terminal domain and a small C-terminal domain. The structure identifies the substrate binding pocket at the interface between the two domains. Lipoic acid is bound in a hydrophobic cavity in the N-terminal domain through hydrophobic interactions and a weak hydrogen bond between carboxyl group of lipoic acid and the Ser-72 or Arg-140 residue of LplA. No large conformational change was observed in the main chain structure upon the binding of lipoic acid.