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Emerging evidence has revealed the pivotal roles of C-type lectin-like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor-P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin-like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A-LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor-binding face of the C-type lectin-like domain is flat, and forms an extended β-sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte-associated C-type lectin, which binds to the CTLR member, NKp65. A LLT1-NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte-associated C-type lectin-NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Roquin is an RNA-binding protein that prevents autoimmunity and inflammation via repression of bound target mRNAs such as inducible costimulator (Icos). When Roquin is absent or mutated (Roquin(san)), Icos is overexpressed in T cells. Here we show that Roquin enhances Dicer-mediated processing of pre-miR-146a. Roquin also directly binds Argonaute2, a central component of the RNA-induced silencing complex, and miR-146a, a microRNA that targets Icos mRNA. In the absence of functional Roquin, miR-146a accumulates in T cells. Its accumulation is not due to increased transcription or processing, rather due to enhanced stability of mature miR-146a. This is associated with decreased 3' end uridylation of the miRNA. Crystallographic studies reveal that Roquin contains a unique HEPN domain and identify the structural basis of the 'san' mutation and Roquin's ability to bind multiple RNAs. Roquin emerges as a protein that can bind Ago2, miRNAs and target mRNAs, to control homeostasis of both RNA species.

Elevated cadmium (Cd) concentrations in fishery byproducts are an environmental concern, that might be reduced by enzymatic removal and adsorption of the contaminants during recycling the byproducts as animal food. We cloned the gene for Arthrobacter nicotinovorans serine protease (ANISEP), which was isolated from the hepatopancreas of the Japanese scallop (Patiopecten yessoensis) and has been found to be an effective enzyme for Cd(II) removal. The gene is 993 bp in length and encodes 330 amino acids, including the pre (1-30) and pro (31-111) sequences. The catalytic triad consists of His, Asp, and Ser. Sequence similarities indicate that ANISEP is a extracellular serine protease. X-ray crystallography revealed structural similarities between ANISEP and the trypsin-like serine protease NAALP from Nesterenkonia sp. Site-directed mutagenesis identified Ser171 as catalytic residue. The keratinolytic activity of ANISEP was 10-fold greater than that of trypsin. ANISEP digested Cd(II)-bound recombinant metallothionein MT-10a from Laternula elliptica, but did not release Cd. These results further suggest ANISEP is a trypsin-like serine protease that can release Cd from the Japanese scallop hepatopancreas because of its strong keratinolytic activity.

eIF5B and eIF1A are two translation-initiation factors that are universally conserved among all kingdoms. They show a unique interaction in eukaryotes which is important for ribosomal subunit joining. Here, the structures of two isolated forms of yeast eIF5B and of the eIF5B-eIF1A complex (eIF1A and eIF5B do not contain the respective N-terminal domains) are reported. The eIF5B-eIF1A structure shows that the C-terminal tail of eIF1A binds to eIF5B domain IV, while the core domain of eIF1A is invisible in the electron-density map. Although the individual domains in all structures of eIF5B or archaeal IF5B (aIF5B) are similar, their domain arrangements are significantly different, indicating high structural flexibility, which is advantageous for conformational change during ribosomal subunit joining. Based on these structures, models of eIF5B, eIF1A and tRNAi(Met) on the 80S ribosome were built. The models suggest that the interaction between the eIF1A C-terminal tail and eIF5B helps tRNAi(Met) to bind to eIF5B domain IV, thus preventing tRNAi(Met) dissociation, stabilizing the interface for subunit joining and providing a checkpoint for correct ribosome assembly.

Placebo-controlled randomized trials have demonstrated that prophylactic levofloxacin (LVFX) significantly reduced the incidence of febrile neutropenia (FN) in patients receiving chemotherapy for advanced solid tumors. Garenoxacin (GRNX) has been reported to be more effective than LVFX against gram-positive bacteria especially Streptococcus pneumoniae. Against this background we conducted a study to compare the efficacy and safety of GRNX with that of LVFX for the prophylaxis of FN. We retrospectively analyzed 127 patients at high risk for FN who were administered GRNX or LVFX for the prophylaxis of FN that occurred during chemotherapy for advanced solid tumors. Our primary outcome of interest was the incidence of febrile episodes. Secondary outcomes included evidence of bacterial infection and infection focus when febrile episodes were observed; adverse drug reactions and mortality were also evaluated. Febrile episodes were observed in 2 patients administered GRNX and 7 patients administered LVFX (p=0.044). Definitive pathogenic bacteria and infection focus could not be identified in any patient with febrile episodes and all cases of fever resolved simultaneously with the recovery from neutropenia. We observed 4 cases of rashes and 3 cases of liver dysfunction in the GRNX group and 2 cases of rashes and 2 cases of liver dysfunctions were observed in the LVFX group(not statistically significant in both the groups). No severe adverse effects or deaths were associated with either of these drugs. These results suggest that GRNX is useful for the prophylaxis of FN.

Background: Little is known about acute upper gastrointestinal (GI) complications associated with gemcitabine-concurrent proton radiotherapy (GPT) for inoperable pancreatic cancer. We investigated acute GI complications following GPT in patients with inoperable pancreatic cancer using small-bowel endoscopy., Methods: This prospective single center observational study was conducted at the Hyogo Ion Beam Medical Center from January 2010 to January 2012. Ninety-one patients who had clinically and medically inoperable pancreatic cancer treated by GPT were analyzed. Endoscopic examinations were performed before and after GPT to clarify the incidence rates of radiation-induced ulcers, GI hemorrhage, and GI perforation associated with GPT., Results: Post-treatment endoscopic examinations revealed that 45 (49.4 %) patients had radiation-induced ulcers in the stomach and duodenum. Of those, many ulcerative lesions were found in the lower stomach (51 %) and horizontal part of the duodenum (39 %), regardless of the primary tumor site in the pancreas. Neither GI hemorrhage, nor perforation, was found in post-treatment endoscopy examinations., Conclusion: Approximately half of the patients treated with GPT for inoperable pancreatic cancer exhibited radiation-induced ulcers in the stomach and duodenum.

Multistep catalysis of epoxide hydrolase/cyclase in the epoxide opening cascade is an intriguing issue in polyether biosynthesis. A pair of structurally homologous epoxide hydrolases was found in gene clusters of ionophore polyethers. In the epoxide opening reactions with MonBI and MonBII involved in monensin biosynthesis, we found that MonBII and catalytically inactive MonBI mutant catalyzed two-step reactions of bisepoxide substrate analogue to afford bicyclic product although MonBII alone catalyzed only the first cyclization. The X-ray crystal structure of MonBI dimers suggested the importance of the KSD motif in MonBI/MonBI interaction, which was further supported by gel filtration chromatography of wild-type MonBI and mutant MonBI. The involvement of the KSD motif in heterodimer formation was confirmed by in vitro assay. Direct evidence of MonBI/MonBII interaction was obtained by native mass spectrometry. Its dissociation constant was determined as 2.21 × 10(-5) M by surface plasmon resonance. Our results suggested the involvement of an allosteric regulation mechanism by MonBI/MonBII interaction in monensin skeletal construction.

Murine epidermal γδ T cells, known as dendritic epidermal T cells (DETCs), survey tissue stress through the invariant T-cell receptor (TCR) and non-clonotypic receptors such as NKG2D. NKG2D signaling via the DAP10-phosphatidylinositol 3-kinase (PI3K) pathway directly stimulates cytotoxicity in natural killer (NK) cells and costimulates CD8(+) T cells to augment TCR signals. In activated murine NK cells, NKG2D signals also via the DAP12-Syk/ZAP70 pathway that triggers both cytotoxicity and cytokine production. It remains controversial whether NKG2D on DETCs is a primary activating receptor or functions only as a costimulatory receptor, and signaling pathways initiated by NKG2D ligation in DETCs have not been analyzed. We show that stimulation of short-term DETC lines with recombinant NKG2D ligands triggers degranulation (exocytosis of cytotoxic granules) via the PI3K-dependent signaling pathway, but does not induce cytokine production or Syk/ZAP70 activation. Coengagement of TCR or Syk/ZAP70 signaling was not crucial for DETC-mediated killing of NKG2D ligand-expressing target cells. Thus, NKG2D can function as a coactivating stress receptor that directly triggers cytotoxicity in DETCs, at least after priming, via the PI3K-dependent, Syk/ZAP70-independent signaling pathway.

Jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors to mount adaptive immune responses. VLRs generate diversity that is comparable to immunoglobulins and T-cell receptors by a gene conversion-like mechanism, which is mediated by cytosine deaminases. Currently, three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. Crystal structures of VLRA and VLRB in complex with antigens have been reported recently, but no structural information is available for VLRC. Here, we present the first crystal structure of VLRC from the Japanese lamprey (Lethenteron japonicum). Similar to VLRA and VLRB, VLRC forms a typical horseshoe-like solenoid structure with a variable concave surface. Strikingly, its N-terminal cap has a long loop with limited sequence variability that protrudes toward the concave surface, which is the putative antigen-binding surface. Furthermore, as predicted previously, its C-terminal cap lacks a highly variable protruding loop that plays an important role in antigen recognition by lamprey VLRA and VLRB. Recent work suggests that VLRC+ lymphocytes in jawless vertebrates might be akin to γδ T cells in jawed vertebrates. Structural features of lamprey VLRC described here suggest that it may recognize antigens in a unique manner.

The avirulence gene AVR-Pia of Magnaporthe oryzae, which induces a hypersensitive reaction in rice cultivars containing the resistance gene Pia, was expressed in Escherichia coli. AVR-Pia protein was collected as inclusion bodies, denatured, and refolded. Finally, recombinant AVR-Pia (rAVR-Pia) protein was purified by column chromatography. Infiltration of rAVR-Pia triggered cell browning in the leaves of rice cultivar Aichiasahi (Pia), with accumulation of H2O2 and induction of PR1a expression in rice. On the other hand, these reactions were not observed in Shin-2 (pia) leaves after the same treatment. This observation indicated that rAVR-Pia had the function of an avirulence protein. rAVR-Pia was used for immunization of a rabbit, and anti-AVR-Pia antiserum was prepared. The specificity of this antibody was appraised by detecting native AVR-Pia in the inoculated leaf sheath extract using Western blotting in combination with immunoprecipitation. Native AVR-Pia was successfully detected, and its molecular weight was estimated to be 7.4 kDa, indicating signal peptide cleavage. Additionally, secreted native AVR-Pia was quantified as 3.7 ng/g rice sheath.

Mincle [macrophage inducible Ca(2+)-dependent (C-type) lectin; CLEC4E] and MCL (macrophage C-type lectin; CLEC4D) are receptors for the cord factor TDM (trehalose-6,6'-dimycolate), a unique glycolipid of mycobacterial cell-surface components, and activate immune cells to confer adjuvant activity. Although it is known that receptor-TDM interactions require both sugar and lipid moieties of TDM, the mechanisms of glycolipid recognition by Mincle and MCL remain unclear. We here report the crystal structures of Mincle, MCL, and the Mincle-citric acid complex. The structures revealed that these receptors are capable of interacting with sugar in a Ca(2+)-dependent manner, as observed in other C-type lectins. However, Mincle and MCL uniquely possess shallow hydrophobic regions found adjacent to their putative sugar binding sites, which reasonably locate for recognition of fatty acid moieties of glycolipids. Functional studies using mutant receptors as well as glycolipid ligands support this deduced binding mode. These results give insight into the molecular mechanism of glycolipid recognition through C-type lectin receptors, which may provide clues to rational design for effective adjuvants.

Background: Aspiration pneumonia is an emerging problem in patients receiving gastrostomy feeding. This study is designed to clarify the therapeutic effects of mosapride citrate and lansoprazole for prevention of aspiration pneumonia in patients receiving gastrostomy feeding., Methods: The study subjects were 119 patients with dysphasia who required gastrostomy feeding. They were randomly assigned to the control (without medication), lansoprazole (15 mg, 1/day), and mosapride (5 mg, 3/day) groups. The number of days with fever (≥37.8 °C), vomiting, and antibiotics administration, as well as the occurrence of pneumonia were investigated during the 6-month observation period., Results: The incidence of pneumonia during the observation period was significantly lower in the mosapride group as compared to the control (7/38 vs. 16/40, p = 0.038) and lansoprazole (vs. 20/41, p = 0.005) groups. The mosapride group also showed a significant decrease in days with fever and antibiotics administration as compared to the other groups. Multivariate analysis revealed that the presence of hiatal hernia was a significant risk factor and administration of mosapride was a significant preventive factor for pneumonia., Conclusion: Mosapride has a preventive effect on occurrence of pneumonia in patients receiving gastrostomy feeding.

Pol283-8-specific, HLA-B*51:01-restricted, cytotoxic T cells (CTLs) play a critical role in the long-term control of HIV-1 infection. However, these CTLs select for the reverse transcriptase (RT) I135X escape mutation, which may be accumulating in circulating HIV-1 sequences. We investigated the selection of the I135X mutation by CTLs specific for the same epitope but restricted by HLA-B*52:01. We found that Pol283-8-specific, HLA-B*52:01-restricted CTLs were elicited predominantly in chronically HIV-1-infected individuals. These CTLs had a strong ability to suppress the replication of wild-type HIV-1, though this ability was weaker than that of HLA-B*51:01-restricted CTLs. The crystal structure of the HLA-B*52:01-Pol283-8 peptide complex provided clear evidence that HLA-B*52:01 presents the peptide similarly to HLA-B*51:01, ensuring the cross-presentation of this epitope by both alleles. Population level analyses revealed a strong association of HLA-B*51:01 with the I135T mutant and a relatively weaker association of HLA-B*52:01 with several I135X mutants in both Japanese and predominantly Caucasian cohorts. An in vitro viral suppression assay revealed that the HLA-B*52:01-restricted CTLs failed to suppress the replication of the I135X mutant viruses, indicating the selection of these mutants by the CTLs. These results suggest that the different pattern of I135X mutant selection may have resulted from the difference between these two CTLs in the ability to suppress HIV-1 replication.

Background: Neurodegenerative diseases including Parkinson's and Alzheimer's diseases progress slowly and steadily over years or decades. They show significant between-subject variation in progress and clinical symptoms, which makes it difficult to predict the course of long-term disease progression with or without treatments. Recent technical advances in biomarkers have facilitated earlier, preclinical diagnoses of neurodegeneration by measuring or imaging molecules linked to pathogenesis. However, there is no established "biomarker model" by which one can quantitatively predict the progress of neurodegeneration. Here, we show predictability of a model with risk-based kinetics of neurodegeneration, whereby neurodegeneration proceeds as probabilistic events depending on the risk., Results: We used five experimental glaucomatous animals, known for causality between the increased intraocular pressure (IOP) and neurodegeneration of visual pathways, and repeatedly measured IOP as well as white matter integrity by diffusion tensor imaging (DTI) as a biomarker of axonal degeneration. The IOP in the glaucomatous eye was significantly increased than in normal and was varied across time and animals; thus we tested whether this measurement is useful to predict kinetics of the integrity. Among four kinds of models of neurodegeneration, constant-rate, constant-risk, variable-risk and heterogeneity models, goodness of fit of the model and F-test for model selection showed that the time course of optic nerve integrity was best explained by the variable-risk model, wherein neurodegeneration kinetics is expressed in an exponential function across cumulative risk based on measured IOP. The heterogeneity model with stretched exponential decay function also fit well to the data, but without statistical superiority to the variable-risk model. The variable-risk model also predicted the number of viable axons in the optic nerve, as assessed by immunohistochemistry, which was also confirmed to be correlated with the pre-mortem integrity of the optic nerve. In addition, the variable-risk model identified the disintegrity in the higher-order visual pathways, known to underlie the transsynaptic degeneration in this disease., Conclusions: These findings indicate that the variable-risk model, using a risk-related biomarker, could predict the spatiotemporal progression of neurodegeneration. This model, virtually equivalent to survival analysis, may allow us to estimate possible effect of neuroprotection in delaying progress of neurodegeneration.

A cell-based therapy for the replacement of dopaminergic neurons has been a long-term goal in Parkinson's disease research. Here, we show that autologous engraftment of A9 dopaminergic neuron-like cells induced from mesenchymal stem cells (MSCs) leads to long-term survival of the cells and restoration of motor function in hemiparkinsonian macaques. Differentiated MSCs expressed markers of A9 dopaminergic neurons and released dopamine after depolarization in vitro. The differentiated autologous cells were engrafted in the affected portion of the striatum. Animals that received transplants showed modest and gradual improvements in motor behaviors. Positron emission tomography (PET) using [11C]-CFT, a ligand for the dopamine transporter (DAT), revealed a dramatic increase in DAT expression, with a subsequent exponential decline over a period of 7 months. Kinetic analysis of the PET findings revealed that DAT expression remained above baseline levels for over 7 months. Immunohistochemical evaluations at 9 months consistently demonstrated the existence of cells positive for DAT and other A9 dopaminergic neuron markers in the engrafted striatum. These data suggest that transplantation of differentiated autologous MSCs may represent a safe and effective cell therapy for Parkinson's disease.

In the typical isoprenoid-biosynthesis pathway, condensation of the universal C(5)-unit precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) occurs via the common intermediates prenyl pyrophosphates (C(10)-C(20)). The diversity of isoprenoids reflects differences in chain length, cyclization and further additional modification after cyclization. In contrast, the biosynthesis of 2-methylisonorneol (2-MIB), which is responsible for taste and odour problems in drinking water, is unique in that it primes the enzymatic methylation of geranyl pyrophosphate (GPP) before cyclization, which is catalyzed by an S-adenosyl-L-methionine-dependent methyltransferase (GPPMT). The substrate of GPPMT contains a nonconjugated olefin and the reaction mechanism is expected to be similar to that of the steroid methyltransferase (SMT) family. Here, structural analysis of GPPMT in complex with its cofactor and substrate revealed the mechanisms of substrate recognition and possible enzymatic reaction. Using the structures of these complexes, methyl-group transfer and the subsequent proton-abstraction mechanism are discussed. GPPMT and SMTs contain a conserved glutamate residue that is likely to play a role as a general base. Comparison with the reaction mechanism of the mycolic acid cyclopropane synthase (MACS) family also supports this result. This enzyme represented here is the first model of the enzymatic C-methylation of a nonconjugated olefin in the isoprenoid-biosynthesis pathway. In addition, an elaborate system to avoid methylation of incorrect substrates is proposed.

Chemotherapy-induced nausea and vomiting (CINV) is one of the most distressing side effects in systemic chemotherapies. Recently, several effective agents have been developed to prevent CINV, and CINV can be prevented in 70%-80% of patients receiving chemotherapies. Conversely, 20%-30% of patients still suffer from CINV despite recommended optimal antiemetic preventions. Refractory emesis is defined as emesis occuring despite the use of antiemetic prophylaxis during the previous cycle of chemotherapy. Salvage treatments for refractory emesis are necessary, but there are few effective treatments at present. We consider medroxyprogesterone acetate to be a potentially promising agent for refractory emesis. We encountered three cases in which medroxyprogesterone acetate was extremely effective for refractory emesis induced by cisplatin-containing chemotherapy.

Synovial sarcoma is an obstinate, high-grade malignancy because of its modest responses to radiotherapy and chemotherapy; the identification of effective therapeutics for this sarcoma is therefore necessary. Inhibition of Src family kinases (SFKs) suppresses the proliferation of synovial sarcoma cells in vitro, as we have previously reported. In this study, to validate the efficacy of Src inhibition in vivo, we employed SU6656, which was originally identified as a specific SFK inhibitor. SU6656 treatment significantly impaired the growth of established, existing tumours formed by synovial sarcoma cells in mice. Tumour cell invasion into the surrounding tissues was also abolished by SU6656. It is noteworthy that SU6656 but not PP2 induced a defect in cleavage furrow formation during cytokinesis, resulting in G2/M accumulation and subsequent apoptosis. Intriguingly, SU6656 abrogated the catalytic activities of Aurora kinases and led to the down-regulation of phosphorylated histone H3 coincidently with p53 accumulation, as did the Aurora kinase inhibitor VX-680. Structural comparison indicated an extensive similarity between the catalytic domains of SFKs and Aurora kinases. The structural analysis also revealed the potential binding mode of SU6656 to the ATP-binding cleft of Aurora B via four hydrogen bonds. SU6656 prevented angiogenesis within the tumours by attenuating vascular endothelial growth factor (VEGF) production by tumour cells and the subsequent chemotaxis of endothelial cells; these effects were the result of the inhibition of SFKs but not Aurora kinases. Based on these results, we hereby report a novel property of SU6656 as a dual inhibitor of SFKs and Aurora kinases, the suppression of both of which effectively abrogates tumour development and the progression of synovial sarcoma in vivo., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: The administration of cisplatin (CDDP)-containing regimens for outpatients has been popularized with the development of supportive care such as antiemetics. Currently, the number of chemotherapies available for outpatients has increased for many kinds of cancer, and administration with a shorter duration is desirable even in CDDP-containing regimens., Methods: Between January 2008 and October 2011, we retrospectively evaluated 22 outpatients who received B 50mg/m² of cisplatin with a short hydration of within 4 hours. We instructed the patients to drink water(1, 000mL/day) on days 2 and 3 instead of receiving a drip infusion. The first course of chemotherapy was usually introduced on admission, and subsequent courses were administered in outpatient clinics., Results: Thirteen cases of lung cancer, eight with gastric cancer, and one with esophageal cancer, were retrospectively evaluated. Thirteen CDDP+S-1 regimens, four CDDP+gemcitabine regimens, and five other types of regimens were administered. The median dose of CDDP was 60mg/m² (range, 50-75mg/m²), the median amount of drip infusion was 1, 600 mL (range, 1, 350-2, 000mL), and the median duration of drip infusion was 4 hours (range 3-4 hours). The decision to use antiemetics and diuretics was made on a case-by-case basis. The average creatinine level before the initiation of a CDDP-containing regimen was 0. 778±0. 212mg/dL, and the lebel four weeks after completion of the regimen was 0. 847±0. 200mg/dL. Among these 22 patients, 20 completed cisplatin-containing regimens. However, cisplatin was reduced in one patient due to renal dysfunction, and the cisplatin regimen was interrupted in one patient due to intolerable nausea and vomiting., Conclusions: CDDP administration at doses of 50-60mg/m² for outpatients was suggested to be safe with optimal patient selection, a total duration of administration of less than four hours, and with 2, 000 mL of hydration on day 1 and without drip infusion from day 2.

Introduction: Measurement of regional cerebral blood flow (rCBF) in rodents can provide knowledge of pathophysiology of the cerebral circulation, but generally requires blood sampling for analysis during positron emission tomography (PET). We therefore tested the feasibility of using an arteriovenous (AV) shunt in rats for less invasive blood analysis., Methods: Six anesthetized rats received [15O]H2O and [15O]CO PET scans with their femoral artery and vein connected by an AV shunt, the activity within which was measured with a germanium ortho-oxysilicate scintillation detector. The [15O]H2O was intravenously injected either at a faster or slower injection rate, while animals were placed either with their head or heart centered in the gantry. The time-activity curve (TAC) from the AV shunt was compared with that from the cardiac ventricle in PET image. The rCBF values were calculated by a nonlinear least-square method using the dispersion-corrected AV-shunt TAC as an input., Results: The AV-shunt TAC had higher signal-to-noise ratio, but also had delay and dispersion compared with the image-derived TAC. The delay time between the AV-shunt TAC and image-based TAC ranged from 11 to 21 s, while the dispersion was estimated to be ∼5 s as a time constant of the dispersion model of exponential function, and both were properly corrected. In a steady-state condition of [15O]CO PET, the blood activity concentration by AV-shunt TAC was also comparable in height with the image-based TAC corrected for partial volume. Whole-brain CBF values measured by [15O]H2O were 0.37±0.04 (mean±S.D.) ml/g/min, partition coefficient was 0.73±0.04 ml/g, and the CBF varied in a linear relationship with partial pressure of carbon dioxide during each scan., Conclusions: The AV-shunt technique allows less invasive, quantitative and reproducible measurement of rCBF in [15O]H2O PET studies in rats than direct blood sampling and radioassay., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Measles virus (MV), one of the most contagious agents, infects immune cells using the signaling lymphocyte activation molecule (SLAM) on the cell surface. A complex of SLAM and the attachment protein, hemagglutinin (MVH), has remained elusive due to the intrinsic handling difficulty including glycosylation. Furthermore, crystals obtained of this complex are either nondiffracting or poorly-diffracting. To solve this problem, we designed a systematic approach using a combination of the following techniques; (1) a transient expression system in HEK293SGnTI(-) cells, (2) lysine methylation, (3) structure-guided mutagenesis directed at better crystal packing, (4) Endo H treatment, (5) single-chain formation for stable complex, and (6) floating-drop vapor diffusion. Using our approach, the receptor-binding head domain of MV-H covalently fused with SLAM was successfully crystallized and diffraction was improved from 4.5 Å to a final resolution of 3.15 Å . These combinational methods would be useful as crystallization strategies for complexes of glycoproteins and their receptors.

A water-soluble magnetic resonance imaging (MRI) contrast agent, Dextran mono-N-succinimidyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-gadolinium(3+) (Dex-DOTA-Gd(3+)), was shown to enable monitoring of the anatomical migration and the survival period of transplanted stem cells for up to 1 month. Gadolinium molecules in the cells were rapidly eliminated from the site and excreted upon cell death. Endothelial progenitor cells (EPCs) transplanted into the inguinal femoral muscle of rats migrated distally through the knee in rats after hindlimb ischemia but did not migrate in non-ischemic rats. Interestingly, the survival period of transplanted EPCs was notably prolonged in the ischemic limb, indicating that EPCs are required by the ischemic tissues and that the fate of transplanted EPCs was affected by the disease. Compared to the commonly used particle type of MRI contrast agents, the system described in this study is expected to be invaluable to help clarifying the process of stem cell transplantation therapy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Many functions have been reported for the eukaryotic ribosomal protein L30e. L30e makes several inter-subunit and intra-subunit interactions with protein or RNA components of the 80S ribosome. Yeast L30e has been shown to bind to its own transcript to autoregulate expression at both the transcriptional and the translational levels. Furthermore, it has been reported that mammalian L30e is a component of the selenocysteine-incorporation machinery by binding to the selenocysteine-insertion sequence on mRNA. As high-resolution crystal structures of mammalian L30e are not available, the purification, crystallization and X-ray structure analysis of human L30e are presented here.

Introduction: This study is intended to evaluate the feasibility of using a high-resolution pinhole SPECT system and iodine-123-N-isopropyl-4-iodoamphetamine ((123)I-IMP) for three-dimensional (3D) absolute quantitation of regional cerebral blood flow (rCBF) in mice., Methods: The pinhole SPECT system consists of a rotating stage and a pinhole collimator attached to a clinical gamma camera. The collimator's focal length is 251 mm. Phantom studies were performed to evaluate sensitivity and full-width half-maximum (FWHM) spatial resolution. The aperture-to-object distance was 15 mm. Six mice were studied. Cerebral infarctions were induced by ligating and disconnecting the distal portion of the left middle cerebral artery. Ex vivo SPECT studies were performed using harvested brains and skulls. The CBF volumetric image was computed using the standardized input function., Results: Excellent spatial resolution of 0.9-mm FWHM and uniform sensitivity throughout the 3D volume were demonstrated in the phantom experiments. The CBF images showed a defect in the infarcted areas and a reduction of CBF values in the infarcted region as compared with the control region., Conclusions: This study demonstrated the feasibility of the 3D quantitation of rCBF in mice using a high-resolution pinhole SPECT system and (123)I-IMP., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The Tom20 protein, residing on the mitochondrial surface, recognizes the N-terminal presequences of precursor proteins. We previously determined the crystal structures of the Tom20-presequence complex. The successful crystallization involved tethering the presequence to Tom20 through an intermolecular disulfide bond with an optimized linker. In this work, we assessed the tethering method. The intermolecular disulfide bond was cleaved in crystal with a reducing agent. The pose (i.e., conformation and position) of the presequence was identical to the previously determined pose. In another experiment, a longer linker than the optimized length was used for the tethering. The perturbation of the tether changed the pose slightly, but the interaction mode was preserved. These results argue against the forced interaction of the presequence by its covalent attachment to Tom20. Second, as an alternative method referred to as "molecular stiffening", we introduced a disulfide bond within the presequence peptide to restrict the freedom of the peptide in the unbound states. One presequence analogue exhibited over 100-fold higher affinity than its linear counterpart and generated cocrystals with Tom20. One of the two crystallographic snapshots revealed a known pose previously determined by the tethering method, and the other snapshot depicted a new pose. These results confirmed and extended the dynamic, multiple bound state model of the Tom20-presequence interactions and also demonstrated the validity of the molecular tethering and stiffening techniques in studies of transient protein-peptide interactions.

The biosynthetic pathway of the off-flavour terpenoid alcohol 2-methylisoborneol (2-MIB) requires geranyl pyrophosphate methyltransferase (GPPMT) to methylate GPP before the cyclization reaction. GPPMT is the first example of an S-adenosyl-L-methionine-dependent methyltransferase that acts on general intermediates such as geranyl pyrophosphate and farnesyl pyrophosphate in isoprenoid biosynthetic pathways. In this study, recombinant GPPMT was overproduced, purified and crystallized in the absence and presence of cofactor, cofactor analogue and substrate. Well diffracting crystals of apo GPPMT containing one molecule in the asymmetric unit were obtained and the structure of this form was solved by the molecular-replacement method. Two crystal forms of the tertiary complex with GPP and sinefungin were also obtained. Structure analysis of these crystals is currently under way in order to understand the enzyme reaction mechanism.

Measles virus, a major cause of childhood morbidity and mortality worldwide, predominantly infects immune cells using signaling lymphocyte activation molecule (SLAM) as a cellular receptor. Here we present crystal structures of measles virus hemagglutinin (MV-H), the receptor-binding glycoprotein, in complex with SLAM. The MV-H head domain binds to a β-sheet of the membrane-distal ectodomain of SLAM using the side of its β-propeller fold. This is distinct from attachment proteins of other paramyxoviruses that bind receptors using the top of their β-propeller. The structure provides templates for antiviral drug design, an explanation for the effectiveness of the measles virus vaccine, and a model of the homophilic SLAM-SLAM interaction involved in immune modulations. Notably, the crystal structures obtained show two forms of the MV-H-SLAM tetrameric assembly (dimer of dimers), which may have implications for the mechanism of fusion triggering.

Nonsteroidal anti-inflammatory drug (NSAID)-induced small intestinal injuries are serious clinical events and a successful therapeutic strategy is difficult. Regenerating gene (Reg) I protein functions as a regulator of cell proliferation and maintains intercellular integrity in the small intestine. The aim of this study was to evaluate the role of Reg I in NSAID-induced small intestinal injuries. First, to examine the effect of Reg I deficiency on such injuries, indomethacin, a widely used NSAID, was injected subcutaneously into 10-wk-old male Reg I-knockout (Reg I(-/-)) and wild-type (Reg I(+/+)) mice twice with an interval of 24 h, after which the mice were euthanized. Small intestinal injuries were assessed by gross findings, histopathology, and contents of IL-1beta and MPO in the experimental tissues. Next, we investigated the therapeutic potential of Reg I in indomethacin-induced small intestinal injuries. Recombinant Reg I protein (rReg I) was administered to 10-wk-old male ICR mice, then indomethacin was administered 6 h using the same protocol as noted above, after which small intestinal injuries were assessed after euthanasia. Our results showed that Reg I(-/-) mice had a greater number of severe small intestinal lesions after indomethacin administration. Histological examinations of the small intestines from those mice revealed deep ulcers with prominent inflammatory cell infiltration, whereas the mucosal content of proinflammatory agents was also significantly increased. In addition, rReg I administration inhibited indomethacin-induced small intestinal injuries in ICR mice. In conclusion, Reg I may be useful as a therapeutic agent in NSAID-induced small intestinal injuries.

Based on their differentiation ability, bone marrow stromal cells (MSCs) are a good source for cell therapy. Using a cynomolgus monkey peripheral nervous system injury model, we examined the safety and efficacy of Schwann cells induced from MSCs as a source for auto-cell transplantation therapy in nerve injury. Serial treatment of monkey MSCs with reducing agents and cytokines induced their differentiation into cells with Schwann cell properties at a very high ratio. Expression of Schwann cell markers was confirmed by both immunocytochemistry and reverse transcription-polymerase chain reaction. Induced Schwann cells were used for auto-cell transplantation into the median nerve and followed-up for 1year. No abnormalities were observed in general conditions. Ki67-immunostaining revealed no sign of massive proliferation inside the grafted tube. Furthermore, (18)F-fluorodeoxygluocose-positron emission tomography scanning demonstrated no abnormal accumulation of radioactivity except in regions with expected physiologic accumulation. Restoration of the transplanted nerve was corroborated by behavior analysis, electrophysiology and histological evaluation. Our results suggest that auto-cell transplantation therapy using MSC-derived Schwann cells is safe and effective for accelerating the regeneration of transected axons and for functional recovery of injured nerves. The practical advantages of MSCs are expected to make this system applicable for spinal cord injury and other neurotrauma or myelin disorders where the acceleration of regeneration is expected to enhance functional recovery., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

On the basis of its temporal and spatial pattern of expression during the healing of gastric ulcers, RegI is implied to be a key growth factor governing the gastric progenitor cell proliferation, which is fundamental for reconstruction of the gastric tissue; however, there is no direct in vivo evidence. The aim of this study was to use RegI-transgenic (Tg) mice to test the role of RegI protein in the healing of experimentally induced gastric ulcers. The stomachs from 48 pairs of wild-type (Wt) and Tg littermates were examined for gastric erosions after 24 h of water-immersion stress, or, 6, 12, 18 and 24 h after oral administration of HCl/ethanol. Expression levels of c-fos and c-myc proto-oncogenes were examined over time by real-time reverse transcriptase PCR to assess gastric cell proliferation. Almost all the littermate pairs tested showed superiority of Tg mice over Wt mice in the ability of decreasing ulcer index (UI) (cumulative length of erosion). The time-course study revealed that the UIs of Tg were lower in the healing phase, and not in the injury phase. The fraction of proliferating cells was higher in Tg mice than in Wt mice throughout the time course as assessed by c-fos expression levels. This is the first in vivo evidence that RegI has a role in gastric ulcer healing. We suggest that RegI exerts its effects by promoting growth and not by cytoprotection.

Alanyl-tRNA synthetase (AlaRS) catalyzes synthesis of Ala-tRNA(Ala) and hydrolysis of mis-acylated Ser- and Gly-tRNA(Ala) at 2 different catalytic sites. Here, we describe the monomer structures of C-terminal truncated archaeal AlaRS, with both activation and editing domains in the apo form, in complex with an Ala-AMP analog, and in a high-resolution lysine-methylated form. The structures show docking of the editing domain to the activation domain opposite from the predicted tRNA-binding surface. Thus, the editing site is positioned >35 A from the activation site, prompting us to model 2 different tRNA complexes: one binding tRNA at the activation site, and the other binding tRNA at the editing site. Interestingly, a gel-shift assay also implies the presence of 2 types of tRNA complex with different mobility. These results suggest that tRNA translocation via a canonical CCA flipping is unlikely to occur in AlaRS. The structure also demonstrated the binding of zinc in the editing site, in which the specific coordination of zinc would be facilitated by a conserved GGQ motif, implying that the editing mechanism may not be the same as in ThrRS. As Asn-194 in eubacterial AlaRS important for Ser misactivation is replaced by Thr-213 in archaeal AlaRS, a different Ser accommodation mechanism is proposed.

Cerebral metabolic rate of oxygen (CMRO(2)) can be assessed quantitatively using (15)O(2) and positron emission tomography. Determining the arterial input function is considered critical with regards to the separation of the metabolic product of (15)O(2) (RW) from a measured whole blood. A mathematical formula based on physiologic model has been proposed to predict RW. This study was intended to verify the adequacy of that model and a simplified procedure applying that model for wide range of species and physiologic conditions. The formula consists of four parameters, including of a production rate of RW (k) corresponding to the total body oxidative metabolism (BMRO(2)). Experiments were performed on 6 monkeys, 3 pigs, 12 rats, and 231 clinical patients, among which the monkeys were studied at varied physiologic conditions. The formula reproduced the observed RW. Greater k values were observed in smaller animals, whereas other parameters did not differ amongst species. The simulation showed CMRO(2) sensitive only to k, but not to others, suggesting that validity of determination of only k from a single blood sample. Also, k was correlated with BMRO(2), suggesting that k can be determined from BMRO(2). The present model and simplified procedure can be used to assess CMRO(2) for a wide range of conditions and species.

This study evaluated the feasibility of imaging rat brains using a human whole-body 3-T magnetic resonance imaging (MRI) scanner with specially developed transmit-and-receive radiofrequency coils. The T(1)- and T(2)-weighted images obtained showed reasonable contrast. Acquired contrast-free time-of-flight magnetic resonance angiography images clearly showed the cortical middle cerebral artery (MCA) branches, and interhemispheric differences could be observed. Dynamic susceptibility contrast MRI at 1.17 mm(3) voxel resolution, performed three times following administration of gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA, 0.1 mmol/kg), demonstrated that the arterial input function (AIF) can be obtained from the MCA region, yielding cerebral blood flow (CBF), cerebral blood volume, and mean transit time (MTT) maps. The hypothalamus (HT) to parietal cortex (Pt) CBF ratio was 45.11 +/- 2.85%, and the MTT was 1.29 +/- 0.40 s in the Pt region and 2.32 +/- 0.17 s in the HT region. A single dose of Gd-DTPA enabled the assessment of AIF within MCA territory and of quantitative CBF in rats.

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N-terminal presequences of mitochondrial-precursor proteins contain a diverse consensus motif (phi chi chi phi phi, phi is hydrophobic and chi is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20-presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR 15N relaxation analyses suggested motion on a sub-millisecond timescale at the Tom20-presequence interface. We suggest that the dynamic, multiple-mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.

In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3' end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N-terminal domain of PriA possesses a binding pocket for the 3'-terminal nucleotide residue of DNA. The interaction with the deoxyribose 3'-OH is essential for the 3'-terminal recognition. In contrast, the direct interaction with 3'-end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3' end. Thus, the N-terminal domain of PriA recognizes the 3'-end base in a base-non-selective manner, in addition to the deoxyribose and 5'-side phosphodiester group, of the 3'-terminal nucleotide to acquire both sufficient affinity and non-selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double-stranded DNA.

Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SelB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SelB-C). SelB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SelB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SelB-RNA interaction is sequence independent, possibly reflecting SelB-tRNA/-rRNA recognitions. Based on these data, the dynamic SelB-ribosome-mRNA-tRNA interactions will be discussed.

Background: Gastric emptying plays an important role in gastroesophageal reflux disease. Acid suppressants such as H2 receptor antagonists and/or proton pump inhibitors are often used in patients with gastroesophageal reflux disease. However, it remains controversial whether H2 receptor antagonists and proton pump inhibitors delay or accelerate gastric emptying. Here, the influence of acid suppressants on gastric emptying was evaluated via a cross-over study using the [13C]-labeled acetate breath test., Methods: Twenty normal male subjects without gastroesophageal reflux disease symptoms were enrolled. Gastric emptying was investigated five times in every subject by the [13C]-labeled acetate breath test with oral administration of the vehicle, domperidone, and three acid suppressants: ranitidine, famotidine and rabeprazole. Gastric emptying was estimated by the values of T(max-calc), T(1/2) and %dose/2 h calculated from the 13CO2 breath excretion curve., Results: Using the T(max-calc) values, rabeprazole, ranitidine and famotidine did not influence gastric emptying time in comparison with vehicle administration. Using the T(1/2) and %dose/2 h values, rabeprazole tended to delay gastric emptying. Domperidone produced a statistically significant acceleration of gastric emptying for all three variables (P < 0.05)., Conclusion: Oral dosage of the H2 receptor antagonists, ranitidine and famotidine, has no significant effect on gastric emptying. However, rabeprazole may delay gastric emptying more strongly than H2 receptor antagonists.

Human leukocyte Ig-like receptor B1 (LILRB1) and B2 (LILRB2) belong to "Group 1" receptors and recognize a broad range of major histocompatibility complex class I molecules (MHCIs). In contrast, "Group 2" receptors show low similarity with LILRB1/B2, and their ligands remain to be identified. To date, the structural and functional characteristics of Group 2 LILRs are poorly understood. Here we report the crystal structure of the extracellular domain of LILRA5, which is an activating Group 2 LILR expressed on monocytes and neutrophils. Unexpectedly, the structure showed large changes in structural conformation and charge distribution in the region corresponding to the MHCI binding site of LILRB1/B2, which are also distinct from killer cell Ig-like receptors and Fc alpha receptors. These changes probably confer the structural hindrance for the MHCI binding, and their key amino acid substitutions are well conserved in Group 2 LILRs. Consistently, the surface plasmon resonance and flow cytometric analyses demonstrated that LILRA5 exhibited no affinities to all tested MHCIs. These results raised the possibility that LILRA5 as well as Group 2 LILRs do not play a role in any MHCI recognition but could possibly bind to non-MHCI ligand(s) on the target cells to provide a novel immune regulation mechanism.

HLA-G is a nonclassical major histocompatibility complex class I (MHCI) molecule, which is expressed in trophoblasts and confers immunological tolerance in the maternal-fetal interface by binding to leukocyte Ig-like receptors (LILRs, also called as LIR/ILT/CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Interestingly, MHCI dimer formations have been involved in pathogenesis and T cell activation. The structure and receptor binding characteristics of MHCI dimers have never been evaluated. Here we performed binding studies showing that the HLA-G dimer exhibited higher overall affinity to LILRB1/2 than the monomer by significant avidity effects. Furthermore, the cell reporter assay demonstrated that the dimer formation remarkably enhanced the LILRB1-mediated signaling at the cellular level. We further determined the crystal structure of the wild-type dimer of HLA-G with the intermolecular Cys(42)-Cys(42) disulfide bond. This dimer structure showed the oblique configuration to expose two LILR/CD8-binding sites upward from the membrane easily accessible for receptors, providing plausible 1:2 (HLA-G dimer:receptors) complex models. These results indicated that the HLA-G dimer conferred increased avidity in a proper structural orientation to induce efficient LILR signaling, resulting in the dominant immunosuppressive effects. Moreover, structural and functional implications for other MHCI dimers observed in activated T cells and the pathogenic allele, HLA-B27, are discussed.

PriA, a DEXH-type DNA helicase, binds specifically to the 3' end of DNA through its N-terminal domain, and is a candidate sensor protein that recognizes arrested DNA replication forks in bacteria. We crystallized an N-terminal fragment of PriA in the absence and the presence of oligonucleotides to elucidate the structural basis for the specific recognition of the 3' terminus of DNA.

Most mitochondrial proteins are synthesized in the cytosol and must be imported into the mitochondria. Many mitochondrial precursor proteins have an extra leader sequence at their N-terminus called a presequence. Presequences are recognized by the Tom20 receptor protein. Based on the previously determined NMR structure of rat Tom20, a fragment corresponding to the core structure was generated. A cysteine residue was added at the C-terminus of the rat aldehyde dehydrogenase presequence to fix the presequence peptide onto the Tom20 fragment via an intermolecular disulfide bond. Two crystal forms of the complex were successfully obtained with different designs of the linker sequence which diffracted to 2.1 and 1.9 A. Crystal dehydration and subsequent annealing was essential to obtain good diffraction data for the 2.1 A crystal form.

In bacteria, the selenocysteine-specific elongation factor SelB is necessary for incorporation of selenocysteine, the 21st amino acid, into proteins by the ribosome. SelB binds to an mRNA hairpin formed by the selenocysteine-insertion sequence (SECIS) and delivers selenocysteyl-tRNA (Sec-tRNASec) at the ribosomal A site. The minimum fragment (residues 512-634) of Moorella thermoacetica SelB (SelB-M) required for mRNA binding has been overexpressed and purified. The complex of SelB-M with 23 nucleotides of the SECIS mRNA hairpin was crystallized at 293 K using the hanging-drop vapour-diffusion or oil-batch methods. The crystals diffract to 2.3 A resolution using SPring-8 BL41XU and belong to the space group P2(1)2(1)2, with unit-cell parameters a = 81.69, b = 169.58, c = 71.69 A.

In bacteria, incorporation of selenocysteine, the 21(st) amino acid, into proteins requires elongation factor SelB, which has the unusual property of binding to both transfer RNA (tRNA) and mRNA. SelB binds to an mRNA hairpin formed by the selenocysteine insertion sequence (SECIS) with extremely high specificity, the molecular basis of which has been unknown. We have determined the crystal structure of the mRNA-binding domain of SelB in complex with SECIS RNA at a resolution of 2.3 A. This is the first example of a complex between an RNA and a winged-helix (WH) domain, a motif found in many DNA-binding proteins and recently discovered in RNA-binding proteins. Notably, RNA binding does not induce a major conformational change in the WH motif. The structure reveals a new mode of RNA recognition with a geometry that allows the complex to wrap around the small ribosomal subunit.

Glycoside hydrolases have been implicated in many biological processes. To date, they have been classified into 93 glycoside hydrolase (GH) families based on amino-acid sequence similarity. alpha-Xylosidase from Escherichia coli belongs to GH family 31 and catalyzes the release of alpha-xylose from the non-reducing terminal side of alpha-xyloside. Single crystals of alpha-xylosidase have been grown by vapour diffusion at 293 K from 10%(w/v) PEG 20K, 2%(v/v) 2-propanol, 2%(v/v) glycerol and 0.1 M 2-morpholinoethanesulfonic acid pH 5.5. These crystals belong to space group P2(1)2(1)2(1) and X-ray diffraction data were collected to a resolution of 2.75 A. Crystals of selenomethionyl-substituted alpha-xylosidase were also obtained, which diffracted to at least 3.0 A. Based on the value of VM, the asymmetric unit in these crystals was assumed to contain six molecules.

PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of primosome onto PhiX174 genomic DNA during replication initiation. Here we report a 2.0-A-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli. The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold. The oligonucleotide-PriB complex model based on the oligonucleotides-SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB. Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue. The significance of these results with respect to the functional role of PriB in the assembly of primosome is discussed.

1-Aminocyclopropane-l-carboxylate deaminase (ACCD) is a pyridoxal 5/-phosphate dependent enzyme that shows deaminase activity toward ACC, a precursor of plant hormone ethylene. ACCD from some soil bacteria has been reported to be able to break the cyclopropane ring of ACC to yield a-ketobutyrate and ammonia. We reported the crystal structure of ACCD from the yeast Hansenula saturnus in the absence/presence of substrate ACC, and proposed its ingenious reaction mechanisms. In order to study the enzyme further, we overexpressed the ACCD homologue protein (phAHP) from the fully decoded hyperthermophilic archearon, Pyrococcus horikoshii OT3. However, phAHP does not show ACCD activity at high temperature as well as at room temperature, though it has significant sequence similarity. Instead of ACCD activity, the GC-MS analysis and enzymatic method show that phAHP has deaminase activity toward L and D-serine. Here, we present the crystal structures of the native and ACC-complexed phAHP. The overall topology of the phAHP structure is very similar to that of ACCD; however, critical differences were observed around the active site. Here, the differences of enzymatic activity between phAHP and ACCD are discussed based on the structural differences of these two proteins. We suggest that the catalytic disagreement between these two enzymes comes from the difference of the residues near the pyridine ring of pyridoxal 5'-phosphate (PLP), not the difference of the catalytic residues themselves. We also propose a condition necessary in the primary sequence to have ACCD activity., (Copyright 2004 Elsevier Ltd.)

Gossypol is a component present in cottonseeds and has been demonstrated to be an effective contraceptive drug in preventing spermatogenesis in mammalian species. In the present, we reported that gossypol could induce apoptosis in human promyelocytic leukemia cells (HL-60), as characterized by DNA fragmentation, poly(ADP) ribose polymerase (PARP) cleavage. The efficacious induction of apoptosis was observed at 50 microM for 6 h. Further molecular analysis showed that gossypol induced the truncation of Bid protein, the loss of mitochondrial membrane potential (DeltaPsi m), cytochrome c release from mitochondria into cytosol, and activation of caspase-3, -8, and -9. However, gossypol did not increase the level of reactive oxygen species (ROS), and antioxidants including N-acetyl cysteine (NAC) and catalase could not block gossypol-induced apoptosis in the HL-60 cells. These data suggest that gossypol induces apoptosis in HL-60 cells through ROS-independent mitochondrial dysfunction pathway.

Macrophomate synthase (MPS) is an enzyme that catalyzes an extraordinarily complex conversion reaction, including two decarboxylations, two carbon-carbon bond formations and a dehydration, to form the benzoate analogue macrophomate from a 2-pyrone derivative and oxalacetate. Of these reactions, the two carbon-carbon bond formations are especially noteworthy because previous experiments have indicated that they proceed via a Diels-Alder reaction, one of the most widely used reactions in organic synthesis. The structural evidence that MPS catalyzes an intermolecular Diels-Alder reaction has been reported recently [Ose et al. (2003), Nature (London), 422, 185-189]. Interestingly, the tertiary structure as well as the quaternary structure of MPS are similar to those of 2-dehydro-3-deoxygalactarate (DDG) aldolase, a carbon-carbon bond-forming enzyme that catalyzes the reversible reaction of aldol condensation/cleavage. Here, the structure of MPS is described in detail and compared with that of DDG aldolase. Both enzymes have a (beta/alpha)(8)-barrel fold and are classified as belonging to the enolase superfamily based on their reaction strategy. The basic principles for carbon-carbon bond formation used by both MPS and DDG aldolase are the same with regard to trapping the enolate substrate and inducing subsequent reaction. The major differences in the active sites between these two enzymes are the recognition mechanisms of the second substrates, 2-pyrone and DDG, respectively.

Health claims about food requires harmonised guidelines; the EU has started work on a regulation. The purpose of health claims should be to help consumers select foods which they can include as a natural part of their diet and which benefit their health. Importantly, the use of health claims on some foods should not undermine confidence in normal traditional foods, which is not branded or marketed with such claims. The introduction of health claims will not replace the efforts at providing general information and education aimed at consumers. Food companies have a special interest in including health claims in their marketing. However, there are as yet no studies demonstrating that such claims promote health in the general population or even healthy eating, hence the importance of a thorough evaluation of health claims.