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A method of determining the chlorine (Cl) and sulfur (S) in municipal solid waste (MSW) was studied. The quartz furnace method was improved in two ways: recovery from ash by hot extraction with dilute nitric acid, and avoid- ance of the volatilization of alkali (earth) metal chlorides by setting the sample combustion temperature at 600°C. In a comparison with the bomb method, using nine sets of kitchen garbage and waste plastics, the bomb method yielded a 15%-25% lower value than the improved quartz furnace method. Combustion in the bomb was frequently incomplete, resulting in recovery losses of Cl and S. The average kitchen garbage involved 5.2 mg Cl/g, of which at least 24.1% would be converted to HCl. Plastics contained 23 mg Cl/g generating 88.1% HCl on average. In the same way, kitchen garbage contained 3.0 mg S/g, generating 52.3% SOx, whereas plastics contained 1.1 mg S/g with 55.1% SOx formation.

X-ray crystallography is a powerful tool for the high resolution structural study of biomacromolecules. One of the most important beneficial feature of the method is the possibility of the direct observation of hydration water structure. In recent years, significant development in high-pressure macromolecular crystallography (HPMX) using a diamond anvil cell (DAC) has been performed in combination with shorter wavelength X-ray of synchrotron radiation. The number of protein structures determined by HPMX at pressure ranging from several hundred MPa to 1 GPa is gradually growing. In this chapter, we describe DAC with its usage, and then an example of HPMX study on hydration structure of 3-isopropylmalate dehydrogenase (IPMDH).

The Ile-Lys-Val-Ala-Val (IKVAV) sequence derived from laminin-1 promotes cell adhesion, neurite outgrowth, and tumor growth and metastasis. Here, we examined amyloid formation of an IKVAV-containing peptide (LAM-L: AASIKVAVSADR, mouse laminin alpha1 chain 2097-2108). The LAM-L peptide was stained with Congo red and exhibited fibrils in electron microscopy with a characteristic cross-beta X-ray diffraction pattern. Further, infrared spectra of LAM-L suggested a beta-sheet structure. These results indicate that LAM-L forms amyloid-like fibrils. We also examined amyloid-like fibril formation of LAM-L analogs. The neurite outgrowth activity of the LAM-L analogs was closely related to their amyloid-like fibril formation.

Human MRP14 (hMRP14) is a Ca(2+)-binding protein from the S100 family of proteins. This protein is co-expressed with human MRP8 (hMRP8), a homologue protein in myeloid cells, and plays an indispensable role in Ca(2+)-dependent functions during inflammation. This role includes the activation of Mac-1, the beta(2) integrin which is involved in neutrophil adhesion to endothelial cells. The crystal structure of the holo form of hMRP14 was analyzed at 2.1 A resolution. hMRP14 is distinguished from other S100 member proteins by its long C-terminal region, and its structure shows that the region is extensively flexible. In this crystal structure of hMRP14, Chaps molecules bind to the hinge region that connects two EF-hand motifs, which suggests that this region is a target-binding site of this protein. Based on a structural comparison of hMRP14 with hMRP8 and human S100A12 (hS100A12) that is another homologue protein, the character of MRP8/14 hetero-complex and the functional significance of the flexibility of the C-terminal region of hMRP14 are discussed.

The crystal structure of pyrrolidone–carboxylate peptidase (PCP) from hyperthermophilic archaea Pyrococcus horikoshii (PhoPCP) has been determined at 1.6-A resolution by X-ray crystallography. PCP belongs to the C15 family of cysteine protease, and specifically removes the amino terminal pyroglutamate residue from a wide range of N-terminal-blocking peptides. The crystal structure is very similar to that of other hyperthermophiles, Pyrococcus friosus and Thermococcus litoralis, and even that from the mesophile, Bacillus amyloliquefacience. The inter-subunit disulfide bonds, which have been proposed as one of the thermostabilizing factors of the PCP from such hyperthermophiles, was not present in PhoPCP. The result suggests that the thermostability of PhoPCP may be obtained by the accumulation of many weak factors. Abbreviations: NCS – non-crystallographic symmetry; PCP – pyrrolidone-carboxylate peptidase; rmsd – root mean square deviation

Introduction: In patients with aortic stenosis (AS) and atrial fibrillation (AF), the conventional continuity equation to evaluate aortic valve area (AVA) is cumbersome, because 5 to 10 cycles are required to ensure accuracy of results. Double-envelope (DE) is obtained by a single continuous-wave Doppler envelope with double density velocity profiles; the inner envelope represents flow across the left ventricular outflow tract and outer envelope represents flow across the aortic valve orifice. Hypothesis: The aim of this study to evaluate the usefulness of the AVA calculated from single-beat recorded inner and outer envelopes in patients with AS and AF. Methods: Eighteen AS patients (74.6±7.2 years old) with AF were examined by transthoracic echocardiography. The conventional AVA (PW/CW technique) was calculated from nonsimultaneously measured LVOT flow and AVO flow in randomly picked up 8 cardiac cycles, respectively. The AVA (DE technique) was calculated from the inner and outer envelopes simultaneously recorded. A single-beat AVA was calculated from simultaneously recorded inner and outer envelopes (by DE technique) when the preceding RR interval/pre-preceding RR interval = 1. Bland-Altman analysis was used to compare the results of two techniques and estimate the reproducibility. Results: DE profiles were successfully obtained in all patients, and mean AVA (PW/CW technique) was 1.09±0.19 cm2. AVA by Single-beat DE technique showed good correlation with that by PW/CW technique (r=0.90), and the mean bias in the AVA measurements between by PW/CW technique and by DE technique was 0.048 cm2. (Figure) Conclusions: AVA in patients with AS and AF obtained by Single-beat DE technique was feasible and in good agreement with that by PW/CW technique. We suggest that DE technique should be considered to estimate the subtle temporal change of AVA in patient with AS and AF more simply and accurately.

A new single crystal focusing monochromator for protein crystallography has been developed. In order to achieve simultaneous tuning of the beam demagnification rate and the radius of curvature for focusing over a wide wavelength range, an inclined monochromator was cylindrically bent. The monochromator has an ability to tune such two parameters simultaneously with an azimuthal rotation of the crystal. A new monochromator incorporating this idea has been developed for beamline BL6B at the Photon Factory. The monochromator was designed for use in a wavelength range 0.87 A to 1.90 A. The evaluation of the beam focusing was performed at wavelengths of 1.04 A, 1.38 A and 1.74 A.

Batch hydride generation-electrically heated quartz furnace- atomic absorption spectrometry for the analysis of arsenic, selenium and antimony is presented. Pre-reduction of arsenic was verified by the secondary injection of sodium tetrahydroborate solution. “Memory” was considered to be caused by deposition of low volatile elements or oxides onto the quartz surface, which was intensive for antimony. This problem was solved by modifying the quartz furnace structure. Humidity control was needed for selenium analysis and was achieved by employing a large hydride generator, ice cooling of the sample and a silicon gas transfer tube. The present method was applied to sea water.

We synthesized various 4-benzylamino-1-chloro-6-substituted phthalazines (15) and 4-benzylamino-1-chloro-7-substituted phthalazines (16) and evaluated their inhibitory activity toward phosphodiesterase 5 (PDE5) purified from porcine platelets. The PDE5-inhibitory activities of 15 were greater than those of the isomers (16). The preferred substituent at the 4-position of phthalazine was a (3-chloro-4-methoxybenzyl)amino group, and those at the 6-position were cyano, nitro, and trifluoromethyl groups. Compounds 15a (IC50 = 4.8 nM), 15f (3.5 nM), and 15i (5.3 nM) were more potent inhibitors than E4021 (8.6 nM). Compounds 15a and 15f also showed vasorelaxant activity in isolated porcine coronary arteries precontracted with prostaglandin F2alpha (10(-5) M). The EC50 values for vasorelaxant action of 15a, 15f, and E4021 were 150, 160, and 980 nM, respectively. These results show that novel PDE5 inhibitors possessing a potent vasorelaxant effect may exist among phthalazine derivatives.

Antimony survey in Yodo river basin, Japan was conducted in 1994 and 1995. The concentration ranged from 0.3 μg/L to 4 μg/L in average. Background level of 0.3 μg/L were detected in Kizu river and Lake Biwa. On the other hand severe anthropogenic contamination in the lower reaches of Katsura river was observed, where 3 ∼ 4 μg/L were detected in 1994. However, the contamination was presumably improved, as the concentration there decreased in 1995. Antimony load in 1995 was reduced by ca.30% compared to 1994.

Hepatocyte nuclear factor 6 (HNF-6)/OC-1, a part of liver-enriched transcription factor, controls pancreas and liver development and regulates expression of several hepatic genes. DNA-binding region of HNF-6alpha bound to a 14-mer DNA fragment has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P2 with unit cell parameters of a = 73.0 A, b = 39.0 A, c = 106.5 A, beta = 107.6 degrees. X-ray diffraction data were collected to 2.0 A resolution.

PH1061 is a hypothetical protein of 100 residues (11.4 kDa) from the hyperthermophilic archaebacterium, Pyrococcus horikoshii OT31, and is conserved among many archaeal and bacterial organisms. Although the functions in these organisms are still unknown, a Pfam2 database search revealed that PH1061 has a helix-turn-helix (HTH) motif (“HTH_5 motif” in the Pfam database), and suggested that it is a DNA-binding protein, like members of the ArsR family3. ArsR is a transcriptional repressor of the arsenic resistance operon in Escherichia coli and other members of this family involve zinc-sensing transcriptional repressors, such as CzrA4 from Staphylococcus aureus and SmtB5 from Synechococcus pcc7942. The primary sequence similarities to PH1061 are 20%, 21%, and 6.6% with ArsR, CzrA, and SmtB, respectively. There are 10 proteins with the HTH_5 motif in P. horikoshii, but none of their functions are known. For structure-based functional analysis, the three-dimensional structure of PH1061 was determined at a resolution of 2.05 Å using the multi-wavelength anomalous diffraction (MAD) method.

An analysis of the composition of waste for combustion is presented. The experimental protocol adopted for this study is the one used in Osaka city. Results over several years are given. A new format by which to describe physical, proximate and elemental composition as well as heat properties, is presented. Empirical values of the proximate composition of combustible components are given. These values are for composition estimates of some wastes. It is not clear exactly where and how much ash is generated in a municipal waste incinerator. Waste composition data enable the ash mass balance of an actual incinerator to be estimated. The methodology of elemental analysis is discussed. Chlorine and sulfur were determined by the tube combustion and bomb combustion methods. Comparisons suggest that the tube combustion method is preferable for pollution assessment.

Off-line and on-line protein data-collection systems using an imaging plate as a detector are described and their components reported. The off-line scanner IPR4080 was developed for a large-format imaging plate ;BASIII' of dimensions 400 x 400 mm and 400 x 800 mm. The characteristics of this scanner are a dynamic range of 10(5) photons pixel(-1), low background noise and high sensitivity. A means of reducing electronic noise and a method for finding the origin of the noise are discussed in detail. A dedicated screenless Weissenberg camera matching IPR4080 with synchrotron radiation was developed and installed on beamline BL6B at the Photon Factory. This camera can attach one or two sheets of 400 x 800 mm large-format imaging plate inside the film cassette by evacuation. The positional reproducibility of the imaging plate on the cassette is so good that the data can be processed by batch job. Data of 93% completeness up to 1.6 A resolution were collected on a single axis rotation and the value of R(merge) becomes 4% from a tetragonal lysozyme crystal using a set of two imaging-plate sheets. Comparing two types of imaging plates, the signal-to-noise ratio of the ST-VIP-type imaging plate is 25% better than that of the BASIII-type imaging plate for protein data collection using 1.0 and 0.7 A X-rays. A new on-line protein data-collection system with imaging plates is specially designed to use synchrotron radiation X-rays at maximum efficiency.

A new type of U-shape anti-cathode X-ray generator in which the inner surface of a cylindrical target is irradiated by an electron beam has been made by modifying a conventional rotating anti-cathode X-ray generator whose brightness in the catalog is 12 kW mm−2. A brightness of 129 kW mm−2 was thereby obtained with this new U-shape-type X-ray generator. This new X-ray generator is expected to be of keen interest for applications in academia, industry and in hospitals., A new type of U-shape anti-cathode X-ray generator in which the inner surface of a cylindrical target is irradiated by an electron beam has been made by modifying a conventional rotating anti-cathode X-ray generator whose brightness in the catalog is 12 kW mm−2. The target material (Cu), target radius (50 mm) and rotating speed (6000 r.p.m.) were not changed in this modification. A brightness of 52 kW mm−2 was obtained by this U-shape-type X-ray generator. This means that the brightness of the new type is 4.3 times greater than that of the old unmodified one. Furthermore, the new-type X-ray generator yielded a brightness of 129 kW mm−2 by adding a carbon coating on the Cu target. This means an overall increase of brightness of ten times. The original generator has the highest brightness in the generators of the same class (having a radius of 50 mm and rotation speed of 6000 r.p.m.). Observations showed that Cu Kα counts at vertical incidence of the electron beam onto the surface of the new target, which is initially optically smooth, decrease as the surface is roughened by a severe thermal stress caused by strong electron beam exposure. Further observation reveals, however, that oblique incidence of the electron beam onto the roughened surface drastically increased the X-ray output and amounts to twice as much as that from a smooth surface at vertical incidence. Thus, at the present stage, an overall increase of brightness has been realised at a level 20 times stronger than that of the original commercially offered X-ray generator that we modified.

The evolution of AR-NW12A into a multi-purpose end-station with optional high-pressure crystallography is described., The macromolecular crystallography (MX) beamline AR-NW12A is evolving from its original design of high-throughput crystallography to a multi-purpose end-station. Among the various options to be implemented, great efforts were made in making available high-pressure MX (HPMX) at the beamline. High-pressure molecular biophysics is a developing field that attracts the interest of a constantly growing scientific community. A plethora of activities can benefit from high pressure, and investigations have been performed on its applicability to study multimeric complex assemblies, compressibility of proteins and their crystals, macromolecules originating from extremophiles, or even the trapping of higher-energy conformers for molecules of biological interest. Recent studies using HPMX showed structural hydrostatic-pressure-induced changes in proteins. The conformational modifications could explain the enzymatic mechanism differences between proteins of the same family, living at different environmental pressures, as well as the initial steps in the pressure-denaturation process that have been attributed to water penetration into the protein interior. To facilitate further HPMX, while allowing access to various individualized set-ups and experiments, the AR-NW12A sample environment has been revisited. Altogether, the newly added implementations will bring a fresh breath of life to AR-NW12A and allow the MX community to experiment in a larger set of fields related to structural biology.

SCO5550 from the model actinomycete Streptomyces coelicolor A3(2) was identified as a putative transcriptional regulator, and classified into the MerR family by sequence analysis. Recombined SCO5550 was successfully produced in Rhodococcus erythropolis, which can be used to stably express recombinant protein by optimizing the temperature over a wide range (4-35 °C). Crystal structure analysis showed that the dimerization domain (C-terminal domain) of SCO5550 has a novel fold and forms a new dimer shape, whereas the DNA-binding domain (N-terminal domain) is very similar to those of MerR family members. Such the new dimer form suggests that SCO5550 may define a new subfamily as a new member of the MerR family. Binding DNA sequence analysis of SCO5550 using the genomic systematic evolution of ligands by exponential enrichment (gSELEX) and electrophoretic mobility shift assay (EMSA) indicated that SCO5550 regulates the expression of the immediately upstream gene sco5551 encoding a putative protein, probably as a transcriptional activator.

encodes an uncharacterizedprotein of 333 amino acids. The N-terminal region ofAMMECR1 (from residue 1 to 121) is rich in glycine andserine,andthesetwoaminoacidsaccountforabout50%ofthe total amino acids in the N-terminal region. TheC-terminal region of AMMECR1 (from residue 122 to 333)is well conserved

Beta-1,3-xylanase (1,3-beta-D-xylan xylanohydrolase; EC 3.2.1.32) is an enzyme capable of hydrolyzing beta-1,3-xylan. The newly cloned beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 (XYL4) exhibited a modular structure consisting of three modules: an N-terminal catalytic module belonging to glycoside hydrolase family 26 and two C-terminal xylan-binding modules belonging to carbohydrate-binding module family 31. Despite substantial crystallization screening, crystallization of the recombinant XYL4 was not accomplished. However, the deletion mutant of XYL4, composed of a catalytic module without a xylan-binding module, was crystallized. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 51.6, b = 75.8, c = 82.0 A. X-ray diffraction data were collected to 1.44 A resolution.

The flux of tributyltin(TBT) from contaminated sediment was estimated using a film transfer model. Mass transfer coefficient was around 10 −6 (m/sec) and showed diversity according to the turbulence of overlaying water. Release to the overlaying water and degradation in the sediment itself were compared in regard to contributions to removal of TBT from sediment. Releases are greater when sediment has a large porosity, whereas degradation tends to contribute more with rather smaller porosity.

A Weissenberg camera for macromolecules with imaging plate data collection system at BL6A and BL18B stations in the Photon Factory is introduced and evaluated. The special feature of these systems is considered matching for both SR‐x rays and protein crystallography. This system is user‐friendly and can collect a large amount of data to higher resolution from the crystal with large unit cell dimensions. A newly developed camera can be used as a time‐resolved Laue camera and a Weissenberg camera. A large image reader (IPR4080) that can scan 400×400 mm2 and 400×800 mm2 sizes of imaging plates has been developed and evaluated. The new data collection system combined with the new camera and IPR4080 will be installed at the BL18B station in October 1994.

With the use of extremely intense synchrotron radiation sources it has become possible to collect good quality x-ray diffraction data for protein crystals on a subsecond time-scale. This has led to the development of time-resolved protein crystallography, where the main objective is to determine the structures of short-lived intermediates during enzymatic reactions by monitoring the time dependence of the x-ray intensities. Furthermore, utilization of single-bunch mode in the 3rd generation synchrotron source enables us to shorten the time scale of the time-resolved protein crystallography to nano-or picosecond order. Possibility of nano-or picosecond protein crystallography in Accumlation Ring (KEK) and SPring-8 is disscussed.

The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3, referred to as A3) proteins are cellular cytidine deaminases that potently restrict retrovirus replication. However, HIV-1 viral infectivity factor (Vif) counteracts the antiviral activity of most A3 proteins by targeting them for proteasomal degradation. To date, the structure of an A3 protein containing a Vif-binding interface has not been solved. Here, we report a high-resolution crystal structure of APOBEC3C and identify the HIV-1 Vif-interaction interface. Extensive structure-guided mutagenesis revealed the role of a shallow cavity composed of hydrophobic or negatively charged residues between the α2 and α3 helices. This region is distant from the DPD motif (residues 128-130) of APOBEC3G that participates in HIV-1 Vif interaction. These findings provide insight into Vif-A3 interactions and could lead to the development of new pharmacologic anti-HIV-1 compounds.

バッチ式水素化物発生―加熱石英管原子吸光法によるμg/L以下の環境水中のヒ素, セレン, アンチモンの測定方法について述べた。水素化反応の際の最適な塩酸濃度と水素化ホウ素ナトリウム注入量を調べた。ヒ素, セレンについては, 試料を40mLの3M塩酸溶液に調製後, 0.5mLの4%水素化ホウ素ナトリウムを注入する。アンチモンについては, 1.5M塩酸溶液に調製後, 0.5mLの4%水素化ホウ素ナトリウムを注入する。アンチモンの予備還元には, ヨウ化カリウムとチオ尿素の双方とも効果が見られた。セレン (W) の予備還元は, 6M塩酸に調製した試料を90℃で20～30分間加熱するのがよい。この方法を環境試料に適用したところ, 河川水中のヒ素は, μg/L以下, 海水中のヒ素は数μg/Lであった。セレンは検出されなかった。アンチモンは, 0.2～4μg/Lの範囲で検出され, 1つの試料で, 要監視項目指針値である2μg/Lを越えた。

MRP14 is a protein that is specifically expressed in myeloid and epithelial cells during the stages of acute or chronic inflammatory states such as rheumatoid arthritis or sarcoidosis. MRP14 has EF-hand motifs as Ca(2+)-binding sites and belongs to the S100 family of proteins. This paper deals with the sample preparation (cloning, overexpression and purification), crystallization and preliminary crystallographic analysis of recombinant human MRP14. Crystals of MRP14 were obtained by the hanging-drop vapour-diffusion method. MRP14 crystals belong to space group P2(1), with unit-cell parameters a = 57.59, b = 178.44, c = 61.23 A, beta = 113.17 degrees, and diffract to 2.1 A resolution.

The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNA(Gln). The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNA(Glu) and Glu-tRNA(Gln). The Glu-tRNA(Gln) is then converted to Gln-tRNA(Gln) by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNA(Glu) and tRNA(Gln) with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNA(Glu)/tRNA(Gln) discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNA(Gln) complex reveals the structural determinants responsible for specific tRNA(Gln) recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine.

We synthesized various 4-benzylamino-1-chloro-6-substituted phthalazines (15) and 4-benzylamino-1-chloro-7-substituted phthalazines (16) and evaluated their inhibitory activity toward phosphodieste...

We have investigated the pharmacology of sodium (R)-2-[4-(2,2-dimethyl-1,3-dioxan-5-yl) methoxy-3,5-dimethylpyridin-2-yl]methylsulfinyl-1H-benzimidazol (E3710), a new proton pump inhibitor (PPI), and its effect on gastric acid secretion. E3710 irreversibly inhibited H(+),K(+)-ATPase activity in pig gastric vesicles with an acidic internal environment with an IC(50) of 0.28 microM. Administration of E3710 (0.1, 0.2, 0.4, and 0.8 mg/kg; n = 6) intraduodenally in a gastric fistula model in dogs inhibited histamine-stimulated gastric acid secretion at 0 to 2 and 24 to 26 h after administration with ED(50) values of 0.18 and 0.22 mg/kg, respectively. The inhibition by E3710 was 2.3 times more potent than that of another representative PPI, esomeprazole (0.2, 0.4, 0.8, and 1.6 mg/kg; n = 6) at 0 to 2 h after administration (ED(50) = 0.40 mg/kg) and 2.8 times more potent at 24 to 26 h (ED(50) = 0.71 mg/kg). In the gastric fistula dogs, the intragastric pH wasor=4 for 17% (n = 27) of a 24-h period with vehicle alone, but when E3710 was administered, at 0.2 (n = 4), 0.4 (n = 8), and 0.8 mg/kg (n = 5), the pH wasor=4 for 40, 79, and 88% of a day, respectively. The corresponding values for esomeprazole at 0.8 (n = 4) and 1.6 mg/kg (n = 8) were 55 and 59%, respectively. In a crossover study with vehicle, E3710 at 0.4 mg/kg and esomeprazole at 1.6 mg/kg (n = 6), E3710 increased the intragastric pH to4 for 82% of a day compared with 61% of a day with esomeprazole. These results show that E3710 is a long-acting inhibitor of gastric acid secretion and a promising novel therapy for acid-related diseases, such as gastroesophageal reflux disease.

CgmR (CGL2612) from Corynebacterium glutamicum is a multidrug-resistance-related transcription factor belonging to the TetR family, which is a protein family of widespread bacterial transcription factors typically involved in environmental response. Here, we report the crystal structures of CgmR homodimeric repressor in complex with two distinct inducers (1.95 and 1.4 A resolution) and with an operator (2.5 A resolution). The CgmR-operator complex showed that two CgmR dimers bound to the operator, and each half-site of the palindromic operator was asymmetrically recognized by two DNA-binding domains from different dimers on the opposite sides of the DNA. The inducer complexes demonstrated that both bound inducers act as a wedge to alter the operator-binding conformation of the repressor by steric inhibition. As steric hindrance is used, various drugs should act as inducers if they have sufficient volume for the conformation change and if their bindings sufficiently reduce free energy. The comparative structural study of CgmR free protein, in complex with operator, and with inducers, implies the other mechanism that might contribute to multidrug response of the repressor.

Polysulfide was produced and tested for use as an inexpensive chemical agent for the solidification/stabilization of lead in fly ash, generating little hydrogen sulfide gas. According to the equations S n 2 - → S + S n - 1 2 - and S n 2 - + H + → HS - + S n - 1 , this agent was expected to achieve long-term stability of disposed Pb. The leachate concentration of Pb in fly ash after sulfide treatment showed a decrease–increase–decrease trend. We also compared Pb leaching behavior from an aging/weathering experiment to thermodynamic modeling using the database from the Geochemist’s Workbench software. Thermodynamic modeling indicated the tendency that when the precipitate was formed in the order of PbO, PbCO3·PbO, and PbCO3, the total concentration of soluble Pb will be lowered. The leaching curve of Pb in fly ash without a lime slurry injection system was close to PbO, whose activity was initially near 1, but when leaching time increased, it shifted to that of PbCO3·PbO, before finally ending at that of PbCO3. However, Ca(OH)2 sprayed on fly ash interfered with the carbonation process of Pb.

To remotely control and automatically collect data in high‐throughput X‐ray data collection experiments, the Structural Biology Research Center at the Photon Factory (PF) developed and installed sample exchange robots PAM (PF Automated Mounting system) at PF macromolecular crystallography beamlines; BL‐5A, BL‐17A, AR‐NW12A and AR‐NE3A. We developed and installed software that manages the flow of the automated X‐ray experiments; sample exchanges, loop‐centering and X‐ray diffraction data collection. The fully automated data collection function has been available since February 2009. To identify sample cassettes, PAM employs a two‐dimensional bar code reader. New beamlines, BL‐1A at the Photon Factory and BL32XU at SPring‐8, are currently under construction as part of Targeted Proteins Research Program (TPRP) by the Ministry of Education, Culture, Sports, Science and Technology of Japan. However, different robots, PAM and SPACE (SPring‐8 Precise Automatic Cryo‐sample Exchanger), will be installed at BL‐1A a...

Environmental behavior and fate of butyltin compounds in a water-sediment system has been studied.Following results on butyltin behavior have been obtained experimentally :1) Sea water-sediment soil partitioning coefficient is 1,500-3,000 (l·kg-1) both for dibutyltin (DBT) and tributyltin (TBT) when the sorption is reversible ;2) Degradation of TBT in anaerobic sediment seemed to be abiotic and its half life is 460 days ;4) Flux acorss the water-sediment interface can be represented by the film model for butyltin compounds, and its transport coefficient is order of 10-6(m·s-1) and affected by turbulence of the overlaying water ; and5) TBT desorbs from sediment soil more easily than DBT : spontaneous desorption ratio are 0.16 and 0.015 (-), and first order desorption kinetic coefficient are 2.3×10-4 and 4.2×10-5 (s-1) respectively.Fate analysis of TBT in a sea water-sediment system shows ;1) TBT from paint chips has severer impact (raising concentrations and high persistence) than dissoluted TBT ; and2) Resuspension of highly contaminated sediment causes recontamination of the overlaying water : a few centimeters of erosion of TBT contaminated sediment will raise the concentration in the overlaying water up to the level of the pore water.

In ruminants, some leaf-eating animals, and some insects, defensive lysozymes have been adapted to become digestive enzymes, in order to digest bacteria in the stomach. Digestive lysozyme has been reported to be resistant to protease and to have optimal activity at acidic pH. The structural basis of the adaptation providing persistence of lytic activity under severe gastric conditions remains unclear. In this investigation, we obtained the crystallographic structure of recombinant bovine stomach lysozyme 2 (BSL2). Our denaturant and thermal unfolding experiments revealed that BSL2 has high conformational stability at acidic pH. The high stability in acidic solution could be related to pepsin resistance, which has been previously reported for BSL2. The crystal structure of BSL2 suggested that negatively charged surfaces, a shortened loop and salt bridges could provide structural stability, and thus resistance to pepsin. It is likely that BSL2 loses lytic activity at neutral pH because of adaptations to resist pepsin.

Objectives: The aim of this study is to evaluate the feasibility and efficacy of device closure of atrial septal defect (ASD) in elderly patients with permanent atrial fibrillation. Background: Little is known about the feasibility of device closure of ASD in those patients. Methods: Nine consecutive patients (mean age 68.1 years) with permanent atrial fibrillation (>1 year persistent) underwent catheter closure using the Amplatzer septal occluder. Transthoracic echocardiography and plasma B-type natriuretic peptide (BNP) level were assessed before and at 24 hours; and 1, 3, and >6 months after the closure. Before the procedure, appropriate dose of warfarin was used in all, diuretics was used in 8/9. Same amount of medications were continued after the procedure. Results: ASD could be closed in all (mean device size 27.3 mm) without hemodynamic and thromboembolic complications. New York Heart Association (NYHA) functional classification was significantly improved in all patients after device closure. No hemodynamic and thromboembolic complications were observed during the follow-up period (mean 10.6 months). Although permanent atrial fibrillation did not change in all after the procedure, resting heart rate decreased from 76.2 ± 16.0 to 68.3 ± 13.2 beats/min (P = 0.015). There was statistically significant improvement in right ventricular/left ventricular diameter ratio (1.08 ± 0.16 to 0.73 ± 0.10, P = 0.008) and plasma BNP level (183.7 ± 90.5 to 94.6 ± 47.4 pg/mL, P = 0.008) after >6 months device closure. Conclusions: Even in the patients complicated with permanent fibrillation, transcatheter closure of ASD can contribute to symptomatic improvement as well as cardiac geometric remodeling. © 2009 Wiley-Liss, Inc.

Background: Wave intensity (WI) is a new hemodynamic index that provides information about the dynamic behavior of the heart and the vascular system and their interaction. WI can be defined at any site in the circulatory system and provide a great deal of information. However, the clinical usefulness of the second peak of WI, which is related to the ability of the left ventricle to activity stop aortic blood flow, has not been fully reported. In previous study, the magnitude of W2 was related to the inertia force of late systolic aortic flow. Also, the higher augmented inertia force, which was calculated with cardiac catheterization, was associated with greater exercise capacity. Then, we hypothesize that WI is correlated to exercise capacity. In this study, we investigated whether WI and the echocardiographic parameters would be correlated with exercise capacity. Methods: A total of 24 patients of heart disease were enrolled. WI was obtained at the right carotid artery using a color Doppler system for blood velocity measurement combined with an echo-tracking method for detecting vessel diameter changes. As echocardiographic parameters, we analyzed ejection fraction (EF), the early (E) and late (A) diastolic mitral velocities, and early (Ea) and late (Aa) diastolic velocities measured using Tissue Doppler imaging. We evaluated exercise capacity (peak VO2, VE/VCO2, and AT). We used Pearson’s correlation analysis for statistics. Results: EF, the E/A ratio, and the E/Ea ratio were not correlated to exercise capacity. However, the second peak of WI was significant correlated to the peak VO2 (r=0.50, p

In 1997 the prefabricated house of the TARA Sakabe project was constructed very near to the Photon Factory ring, and many computers were installed for crystallographic data handling. A data server with high speed and large capacity was required to improve the efficiency of the protein data collection system which integrated a `high'-security computer network. The new network, based on a 100 Mbps Ethernet, consists of a DEC AlphaServer 4000 with a 115 Gbytes RAID disk, DLT as a backup device, CISCO PIX-32 as a firewall between the TARA private network and KEK, and a 100 Mbps switching hub to be linked to imaging-plate readers and workstations. Therefore, the digital output data from the imaging-plate reader are directly recorded on the server disk resulting in higher efficiency of the users' beam time. In contrast to recording on tape, there is very little problem with backup resulting in a high confidence in the data-collection system.

LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-A resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn(2+) binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed.

Background: Atrial fibrillation (AF) often occurs in patients with Brugada syndrome (BS) and BS patients with spontaneous AF often experience ventricular fibrillation (VF) attacks. We have reported that patients with BS had abnormal atrial electrophysiological vulnerability, which provides a substrate for AF, but there are no data regarding the atrial structural attribute. Our objective is to assess the atrial electrophysiological and structural characteristics according to the disease severity and their relations with gene mutations in BS. Methods: We studied 41 patients with BS: 9 patients with documented VF and spontaneous AF (Group VF+AF+), 7 patients with VF and without AF (Group VF+AF−), and 25 patients without VF and AF (Group VF−AF−). In electrophysiologic study, intra-atrial conduction time (CT) was defined as the interval from the stimulus at the high right atrium to the atrial deflection at the distal portion of the coronary sinus, and it was measured at constant pacing (CT1) and extrastimuli (CT2). In echocardiographic study, left atrial volume was calculated using the biplane modified Simpson’s method and indexed to body surface area (LA volume index). Mutations of SCN5A gene were examined in all patients. Results: CTs, especially CT2, were prolonged in both Group VF+AF+ and Group VF+AF− compared to Group VF−AF− (CT2: Group VF−AF−134.1±15.7 vs. Group VF+AF− 152.8±23.6* vs. Group VF+AF+ 173.5±12.4** ms, *p2 , respectively, *p Conclusions: High-risk patients with BS had both abnormal atrial vulnerability and remodeling even if patients did not have AF. Existence of SCN5A also induced both electrophysiological and anatomical remodeling of the atrium. To identify atrial electrophysiological and structural characteristics may be useful for the risk stratification in patients with BS.

SCO6571 protein from Streptomyces coelicolor A3(2) was overexpressed and purified using Rhodococcus erythropolis as an expressing host. Crystals of selenomethionine-substituted SCO6571 have been obtained by vapor diffusion method. SCO6571 crystals diffract to 2.3 A and were found to belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters a = 84.5, b = 171.6, c = 184.8 A. Six molecules in the asymmetric unit give a crystal volume per protein mass (V(M)) of 2.97 A (3) Da(-1) and solvent content of 58.6 %. The structure was solved by the single wavelength anomalous diffraction (SAD) method. SCO6571 is a TIM-barrel fold protein that assembles into a hexameric molecule with D(3) symmetry.

Mannosyl-3-phosphoglycerate phosphatase (MPGP) catalyzes the dephosphorylation of alpha-mannosyl-3-phosphoglycerate (MPG) to produce alpha-mannosylglycerate (MG). In the hyperthermophile Pyrococcus horikoshii, MPGP plays a role in a series of enzyme reactions that are involved in the MG-biosynthesis pathway, which is important for maintaining life under conditions of high salt concentration. Crystal structures of P. horikoshii MPGP (PhoMPGP) in the holo form and in the apo form lacking the magnesium ion were determined by the multiple-wavelength anomalous diffraction method using SeMet-substituted PhoMPGP. PhoMPGP consists of two domains: a core domain that is conserved in the haloacid dehalogenase superfamily and a cap domain that is specific to the C2B cap subclass of the superfamily. Apo-form crystals contain two PhoMPGP molecules: one in the open conformation and the other in the closed conformation. In holo-form crystals both of the two molecules are in the closed conformation with phosphate and magnesium ions. PhoMPGP has a specific hairpin loop that is bent towards the active site in the closed conformation of both the apo and holo forms. PhoMPGP has a cavity between the two domains which is considered to be the substrate-binding site as a phosphate ion is located in the cavity, mimicking the binding manner of the phosphate group of MPG. The cavity is sequestered in the closed conformation such that a conformational change is indispensable for the release of products. A salt bridge from the general acid/base Asp10 to Arg170 is observed in the holo-form PhoMPGP which is not present in the open form. The importance of the conformational change in the activity of PhoMPGP is discussed.