Aurora A phosphorylates Ndel1 to reduce the levels of Mad1 and NuMA at spindle poles.

Dynein inactivates the spindle assembly checkpoint (SAC) by transporting checkpoint proteins away from kinetochores toward spindle poles in a process known as "stripping." We find that inhibition of Aurora A kinase, which is localized to spindle poles, enables the accumulation of the spindle checkpoint activator Mad1 at poles where it is normally absent. Aurora kinases phosphorylate the dynein activator NudE neurodevelopment protein 1 like 1 (Ndel1) on Ser285 and Mad1 accumulates at poles when Ndel1 is replaced by a nonphosphorylatable mutant in human cells. The pole focusing protein NuMA, transported to poles by dynein, also accumulates at poles in cells harboring a mutant Ndel1. Phosphorylation of Ndel1 on Ser285 is required for robust spindle checkpoint activity and regulates the poles of asters in Xenopus extracts. Our data suggest that dynein/SAC complexes that are generated at kinetochores and then transported directionally toward poles on microtubules are inhibited by Aurora A before they reach spindle poles. These data suggest that Aurora A generates a spatial signal at spindle poles that controls dynein transport and spindle function.

Crystal structure of the human N-Myc downstream-regulated gene 2 protein provides insight into its role as a tumor suppressor.

Considerable attention has recently been paid to the N-Myc downstream-regulated gene (NDRG) family because of its potential as a tumor suppressor in many human cancers. Primary amino acid sequence information suggests that the NDRG family proteins may belong to the α/ß-hydrolase (ABH) superfamily; however, their functional role has not yet been determined. Here, we present the crystal structures of the human and mouse NDRG2 proteins determined at 2.0 and 1.7 Å resolution, respectively. Both NDRG2 proteins show remarkable structural similarity to the ABH superfamily, despite limited sequence similarity. Structural analysis suggests that NDRG2 is a nonenzymatic member of the ABH superfamily, because it lacks the catalytic signature residues and has an occluded substrate-binding site. Several conserved structural features suggest NDRG may be involved in molecular interactions. Mutagenesis data based on the structural analysis support a crucial role for helix α6 in the suppression of TCF/ß-catenin signaling in the tumorigenesis of human colorectal cancer, via a molecular interaction.

Pedobacter boryungensis sp. nov., isolated from soil.

A Gram-negative, non-sporulating, non-flagellated rod, designated BR-9(T), was isolated from soil collected on the Korean peninsula. Strain BR-9(T) grew optimally at pH 6.0-7.0, at 30 °C and in the absence of NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BR-9(T) belonged to the genus Pedobacter and clustered with Pedobacter insulae DS-139(T) and Pedobacter koreensis WPCB189(T). Strain BR-9(T) exhibited 98.2 and 97.5% 16S rRNA gene sequence similarity with P. insulae DS-139(T) and P. koreensis WPCB189(T), respectively, and <96.7% sequence similarity with the type strains of other species in the genus Pedobacter. Strain BR-9(T) contained MK-7 as the predominant menaquinone and iso-C(15:0) and summed feature 3 (C(16:1)ω7c and/or iso-C(15:0) 2-OH) as the major fatty acids. The DNA G+C content of strain BR-9(T) was 38.5 mol%. DNA-DNA relatedness between strain BR-9(T) and P. insulae DS-139(T) and P. koreensis KCTC 12536(T) was 3.4-4.2%, which indicated that the isolate was genetically distinct from these type strains. Strain BR-9(T) was also distinguishable by differences in phenotypic properties. On the basis of the data presented, strain BR-9(T) is considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter boryungensis sp. nov. is proposed. The type strain is BR-9(T) (=KCTC 23344(T) =CCUG 60024(T)).

Tropicimonas aquimaris sp. nov., isolated from seawater, and emended description of the genus Tropicimonas Harwati et al. 2009.

A Gram-staining-negative, aerobic, non-motile and rod-shaped bacterial strain, designated DPG-21(T), was isolated from seawater from the South Sea in Korea, and investigated using a polyphasic taxonomic approach. Strain DPG-21(T) grew optimally at pH 7.0-8.0, at 30 °C and in the presence of 2% (w/v) NaCl. In a neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain DPG-21(T) clustered with Tropicimonas isoalkanivorans B51(T) (with a sequence similarity of 97.1%); the novel strain showed lower 16S rRNA gene sequence similarities (<95.4%) with the other species included in the tree. The mean DNA-DNA relatedness value between strain DPG-21(T) and T. isoalkanivorans DSM 19548(T) was 12%. The predominant ubiquinones of strain DPG-21(T) were Q-10 and Q-9 while C(18:1)ω7c was the strain's major fatty acid. The polar lipid profile of strain DPG-21(T) was similar to that of T. isoalkanivorans DSM 19548(T). The genomic DNA G+C content of the novel strain was 69.6 mol%. Some phenotypic properties and the phylogenetic and genetic data indicated that strain DPG-21(T) was distinct from T. isoalkanivorans and represents a novel species of the genus Tropicimonas, for which the name Tropicimonas aquimaris sp. nov. is proposed. The type strain is DPG-21(T) (=KCTC 23424(T)=CCUG 60524(T)).

Crystal structure of SmcR, a quorum-sensing master regulator of Vibrio vulnificus, provides insight into its regulation of transcription.

Quorum sensing has been implicated as an important global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens. SmcR, a homologue of Vibrio harveyi LuxR, has been proposed as a quorum-sensing master regulator of Vibrio vulnificus, an opportunistic human pathogen. Previous studies demonstrated that SmcR is essential for the survival and pathogenesis of V. vulnificus, indicating that inhibiting SmcR is an attractive approach to combat infections by the bacteria. Here, we determined the crystal structure of SmcR at 2.1 A resolution. The protein structure reveals a typical TetR superfamily fold consisting of an N-terminal DNA binding domain and a C-terminal dimerization domain. In vivo and in vitro functional analysis of the dimerization domain suggested that dimerization of SmcR is vital for its biological regulatory function. The N-terminal DNA recognition and binding residues were assigned based on the protein structure and the results of in vivo and in vitro mutagenesis experiments. Furthermore, protein-DNA interaction experiments suggested that SmcR may have a sophisticated mechanism that enables the protein to recognize each of its many target operators with different affinities.

Thalassomonas agariperforans sp. nov., an agarolytic bacterium isolated from marine sand.

A Gram-staining-negative, motile, agarolytic bacterium, designated M-M1(T), was isolated from marine sand obtained from Geoje Island, South Sea, Korea, and its taxonomic position was investigated using a polyphasic taxonomic approach. Strain M-M1(T) grew optimally at pH 7.0-8.0, at 30 °C and in the presence of 2 % (w/v) NaCl. It did not grow in the presence of >7 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain M-M1(T) fell within the clade comprising members of the genus Thalassomonas, clustering with Thalassomonas agarivorans TMA1(T), Thalassomonas loyana CBMAI 722(T) and Thalassomonas ganghwensis JC2041(T), with which it exhibited 16S rRNA gene sequence similarity values of 96.4, 96.0 and 94.9 % respectively. Strain M-M1(T) exhibited 94.7-95.2 % 16S rRNA gene sequence similarity to the other species of the genus Thalassomonas. Strain M-M1(T) contained Q-8 as the predominant ubiquinone and C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH, C(16 : 0) and C(18 : 1)ω7c as the major fatty acids. The DNA G+C content was 44.2 mol%. Strain M-M1(T) could be differentiated from phylogenetically related species of the genus Thalassomonas by differences in some phenotypic properties. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain M-M1(T) is considered to represent a novel species of the genus Thalassomonas, for which the name Thalassomonas agariperforans sp. nov. is proposed. The type strain is M-M1(T) (= KCTC 23343(T) = CCUG 60020(T)).

Oceanisphaera ostreae sp. nov., isolated from seawater of an oyster farm, and emended description of the genus Oceanisphaera Romanenko et al. 2003.

A Gram-stain-negative, motile, non-spore-forming and short rod- or rod-shaped bacterial strain, T-w6(T), was isolated from seawater of an oyster farm in the South Sea, Korea. Strain T-w6(T) grew optimally at 25 °C and in the presence of 2% (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain T-w6(T) joined the cluster comprising Oceanisphaera species with a bootstrap resampling value of 90.8%, and this cluster joined the clade comprising members of the genera Oceanimonas and Zobellella with a bootstrap resampling value of 100%. Strain T-w6(T) exhibited 16S rRNA gene sequence similarity of 95.9 and 96.6% to the type strains of Oceanisphaera litoralis and Oceanisphaera donghaensis, respectively. Strain T-w6(T) and the type strains of Oceanisphaera litoralis and Oceanisphaera donghaensis had Q-8 as the predominant ubiquinone and iso-C(15:0) 2-OH and/or C(16:1)ω7c, C(18:1)ω7c and C(16:0) as the major fatty acids. The major polar lipids were phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content of strain T-w6(T) was 56.6 mol%. Mean DNA-DNA relatedness of strain T-w6(T) with Oceanisphaera litoralis DSM 15406(T) and Oceanisphaera donghaensis KCTC 12522(T) was 13 and 10%, respectively. Phenotypic properties of strain T-w6(T) demonstrated that this strain could be distinguished from the other Oceanisphaera species. On the basis of the data presented, strain T-w6(T) is considered to represent a novel species of the genus Oceanisphaera, for which the name Oceanisphaera ostreae sp. nov. is proposed; the type strain is T-w6(T) (=KCTC 23422(T) =CCUG 60525(T)). An emended description of the genus Oceanisphaera is also presented.

ß-propeller phytase hydrolyzes insoluble Ca(2+)-phytate salts and completely abrogates the ability of phytate to chelate metal ions.

Phytate is an antinutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. To further our understanding of the chemistry of phytate's binding interactions with biologically important metal cations, we determined the stoichiometry, affinity, and thermodynamics of these interactions by isothermal titration calorimetry. The results suggest that phytate has multiple Ca(2+)-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0-9.0). We overexpressed the ß-propeller phytase from Hahella chejuensis (HcBPP) that hydrolyzes insoluble Ca(2+)-phytate salts. Structure-based sequence alignments indicated that the active site of HcBPP may contain multiple calcium-binding sites that provide a favorable electrostatic environment for the binding of Ca(2+)-phytate salts. Biochemical and kinetic studies further confirmed that HcBPP preferentially recognizes its substrate and selectively hydrolyzes insoluble Ca(2+)-phytate salts at three phosphate group sites, yielding the final product, myo-inositol 2,4,6-trisphosphate. More importantly, ITC analysis of this final product with several cations revealed that HcBPP efficiently eliminates the ability of phytate to chelate several divalent cations strongly and thereby provides free minerals and phosphate ions as nutrients for the growth of bacteria. Collectively, our results provide significant new insights into the potential application of HcBPP in enhancing the bioavailability and absorption of divalent cations.

The structure of the coiled-coil domain of Ndel1 and the basis of its interaction with Lis1, the causal protein of Miller-Dieker lissencephaly.

Ndel1 and Nde1 are homologous and evolutionarily conserved proteins, with critical roles in cell division, neuronal migration, and other physiological phenomena. These functions are dependent on their interactions with the retrograde microtubule motor dynein and with its regulator Lis1--a product of the causal gene for isolated lissencephaly sequence (ILS) and Miller-Dieker lissencephaly. The molecular basis of the interactions of Ndel1 and Nde1 with Lis1 is not known. Here, we present a crystallographic study of two fragments of the coiled-coil domain of Ndel1, one of which reveals contiguous high-quality electron density for residues 10-166, the longest such structure reported by X-ray diffraction at high resolution. Together with complementary solution studies, our structures reveal how the Ndel1 coiled coil forms a stable parallel homodimer and suggest mechanisms by which the Lis1-interacting domain can be regulated to maintain a conformation in which two supercoiled alpha helices cooperatively bind to a Lis1 homodimer.

Crystallization and preliminary crystallographic analysis of Bacillus thuringiensis AHL-lactonase.

The quorum sensing (QS) systems in Gram-negative bacteria are mostly associated with diffusible N-acyl-L-homoserine lactones (AHLs). AHL-degrading enzymes hydrolyze the AHLs into inactive molecules, thereby blocking the QS systems that are closely linked to virulence factor production and biofilm formation. Consequently, these enzymes have recently attracted intense interest for the development of anti-infection therapies for plants and animals. However, despite significant progress in the investigation of AHL-degrading enzymes, no structure is yet available. Accordingly, this study reports on the expression and purification of the AHL-lactonase from Bacillus thuringiensis subsp. kurstaki HD263, as well as the successful crystallization of the enzyme. High-quality native crystals were obtained and a complete data set collected at 2.0 A resolution. The native crystal was found to belong to the space group P2(1)2(1)2(1), with unit cell parameters a=52.7 A, b=55.9 A, and c=74.1 A and one molecule in the asymmetric unit. MAD data were also collected at 2.4 A resolution for a SeMet-substituted crystal.

Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability.

Lipase L1 from Geobacillus stearothermophilus L1 contains an unusual extra domain, making a tight intramolecular interaction with the main catalytic domain through a Zn2+-binding coordination. To elucidate the role of the Zn2+, we disrupted the Zn2+-binding site by mutating the zinc-ligand residues (H87A, D61A/H87A, and D61A/H81A/H87A/D238A). The activity vs. temperature profiles of the mutant enzymes showed that the disruption of the Zn2+-binding site resulted in a notable decrease in the optimal temperature for maximal activity from 60 to 45-50 degrees C. The mutations also abolished the Zn2+-induced thermal stabilization. The wild-type enzyme revealed a 34.6-fold increase in stabilization with the addition of Zn2+ at 60 degrees C, whereas the mutant enzymes exhibited no response to Zn2+. Additional circular dichroism spectroscopy studies also confirmed the structural stabilizing role of Zn2+ on lipase L1 at elevated temperatures.

Balloon sheaths for gastrointestinal guidance and access: a preliminary phantom study.

OBJECTIVE: We wanted to evaluate the feasibility and usefulness of a newly designed balloon sheath for gastrointestinal guidance and access by conducting a phantom study. MATERIALS AND METHODS: The newly designed balloon sheath consisted of an introducer sheath and a supporting balloon. A coil catheter was advanced over a guide wire into two gastroduodenal phantoms (one was with stricture and one was without stricture); group I was without a balloon sheath, group ll was with a deflated balloon sheath, and groups III and IV were with an inflated balloon and with the balloon in the fundus and body, respectively. Each test was performed for 2 minutes and it was repeated 10 times in each group by two researchers, and the positions reached by the catheter tip were recorded. RESULTS: Both researchers had better performances with both phantoms in order of group IV, III, II and I. In group IV, both researchers advanced the catheter tip through the fourth duodenal segment in both the phantoms. In group I, however, the catheter tip never reached the third duodenal segment in both the phantoms by both the researchers. The numeric values for the four study groups were significantly different for both the phantoms (p < 0.001). A significant difference was also found between group III and IV for both phantoms (p < 0.001). CONCLUSION: The balloon sheath seems to be feasible for clinical use, and it has good clinical potential for gastrointestinal guidance and access, particularly when the inflated balloon is placed in the gastric body.

Sequence-based approach to finding functional lipases from microbial genome databases.

A sequence-based approach was used to retrieve functional lipases from microbial genome databases. Many novel genes assigned as putative lipases were tested using the criteria of the typical lipase sequence rule, based on a consensus sequence of a catalytic triad (Ser, Asp, His) and oxyanion hole sequence (HG). To obtain the lipase genes satisfying the sequence rule, PCR cloning was performed, while the lipase activities were tested using a tributyrin/tricaprylin plate and p-nitrophenyl caproate. Among nine putative lipases from four strains, five functional lipolytic proteins were obtained from Archaeoglobus fulgidus, Deinococcus radiodurans, and Agrobacterium tumefaciens. All five lipases exhibited a relatively low sequence similarity (less than 26.7%) with known lipases and turned out to belong to different lipase families. Accordingly, the current results indicate that the proposed strategic approach based on the microbial genome is an efficient and rapid method for finding novel and functional lipases.

The N-terminal coiled-coil of Ndel1 is a regulated scaffold that recruits LIS1 to dynein.

Ndel1 has been implicated in a variety of dynein-related processes, but its specific function is unclear. Here we describe an experimental approach to evaluate a role of Ndel1 in dynein-dependent microtubule self-organization using Ran-mediated asters in meiotic Xenopus egg extracts. We demonstrate that extracts depleted of Ndel1 are unable to form asters and that this defect can be rescued by the addition of recombinant N-terminal coiled-coil domain of Ndel1. Ndel1-dependent microtubule self-organization requires an interaction between Ndel1 and dynein, which is mediated by the dimerization fragment of the coiled-coil. Full rescue by the coiled-coil domain requires LIS1 binding, and increasing LIS1 concentration partly rescues aster formation, suggesting that Ndel1 is a recruitment factor for LIS1. The interactions between Ndel1 and its binding partners are positively regulated by phosphorylation of the unstructured C terminus. Together, our results provide important insights into how Ndel1 acts as a regulated scaffold to temporally and spatially regulate dynein.

Bilateral inhibition of HAUSP deubiquitinase by a viral interferon regulatory factor protein.

Herpesvirus-associated ubiquitin-specific protease (HAUSP) regulates the stability of p53 and the p53-binding protein MDM2, implicating HAUSP as a therapeutic target for tuning p53-mediated antitumor activity. Here we report the structural analysis of HAUSP with Kaposi's sarcoma-associated herpesvirus viral interferon (IFN) regulatory factor 4 (vIRF4) and the discovery of two vIRF4-derived peptides, vif1 and vif2, as potent and selective HAUSP antagonists. This analysis reveals a bilateral belt-type interaction that results in inhibition of HAUSP. The vif1 peptide binds the HAUSP TRAF domain, competitively blocking substrate binding, whereas the vif2 peptide binds both the HAUSP TRAF and catalytic domains, robustly suppressing its deubiquitination activity. Peptide treatments comprehensively blocked HAUSP, leading to p53-dependent cell-cycle arrest and apoptosis in culture and to tumor regression in xenograft mouse model. Thus, the virus has developed a unique strategy to target the HAUSP-MDM2-p53 pathway, and these virus-derived short peptides represent biologically active HAUSP antagonists.

Ca(2+)-inositol phosphate chelation mediates the substrate specificity of beta-propeller phytase.

Inositol phosphates are recognized as having diverse and critical roles in biological systems. In this report, kinetic studies and TLC analysis indicate that beta-propeller phytase is a special class of inositol phosphatase that preferentially recognizes a bidentate (P-Ca(2+)-P) formed between Ca(2+) and two adjacent phosphate groups of its natural substrate phytate (InsP(6)). The specific recognition of a bidentate chelation enables the enzyme to sequentially hydrolyze one of the phosphate groups in a bidentate of Ca(2+)-InsP(6) to yield a myo-inositol trisphosphate (InsP(3)) and three phosphates as the final products. A comparative analysis of (1)H- and (13)C NMR spectroscopy with the aid of 2D NMR confirms that the chemical structure of the final product is myo-Ins(2,4,6)P(3). The catalytic properties of the enzyme suggest a potential model for how the enzyme specifically recognizes its substrate Ca(2+)-InsP(6) and produces myo-Ins(2,4,6)P(3) from Ca(2+)-InsP(6). These findings potentially provide evidence for a selective Ca(2+)-InsPs chelation between Ca(2+) and two adjacent phosphate groups of inositol phosphates.

Use of guiding sheaths in peroral fluoroscopic gastroduodenal stent placement.

Our purpose was to assess the safety and usefulness of guiding sheaths in peroral fluoroscopic gastroduodenal stent placement. Two types of guiding sheath were made from straight polytetrafluoroethylene tubes. Type A was 80 cm in length, 4 mm in outer diameter and 3 mm in inner diameter. Type B was 70 cm in length, 6 mm in outer diameter and 5 mm in inner diameter. The type A sheath was used in 18 patients in whom a catheter-guide wire combination failed to pass through a stricture. The type B sheath was used in 22 patients in whom a stent delivery system failed to pass through the stricture due to loop formation within the gastric lumen. The overall success rate for guiding a catheter-guide wire through a stricture after using the type A sheath was 89%. The overall success rate for passing a stent delivery system through a stricture after using the type B sheath was 100%. All procedures were tolerated by the patients without any significant complications. The guiding sheaths were safe and useful in peroral fluoroscopic gastroduodenal stent placement.

The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase.

In many Gram-negative bacteria, including a number of pathogens such as Pseudomonas aeruginosa and Erwinia carotovora, virulence factor production and biofilm formation are linked to the quorum-sensing systems that use diffusible N-acyl-L-homoserine lactones (AHLs) as intercellular messenger molecules. A number of organisms also contain genes coding for lactonases that hydrolyze AHLs into inactive products, thereby blocking the quorum-sensing systems. Consequently, these enzymes attract intense interest for the development of antiinfection therapies. However, the catalytic mechanism of AHL-lactonase is poorly understood and subject to controversy. We here report a 2.0-angstroms resolution structure of the AHL-lactonase from Bacillus thuringiensis and a 1.7-angstroms crystal structure of its complex with L-homoserine lactone. Despite limited sequence similarity, the enzyme shows remarkable structural similarities to glyoxalase II and RNase Z proteins, members of the metallo-beta-lactamase superfamily. We present experimental evidence that AHL-lactonase is a metalloenzyme containing two zinc ions involved in catalysis, and we propose a catalytic mechanism for bacterial metallo-AHL-lactonases.

Reduction of tissue hyperplasia with a rhenium-188 mercaptoacetyltriglycine-3-filled balloon: preliminary study in a canine urethral model.

PURPOSE: To evaluate the efficacy of beta-radiation therapy with rhenium-188 mercaptoacetyltriglycine-3 (MAG(3))-filled balloons to reduce tissue hyperplasia secondary to stent placement in 18 canine urethras. MATERIALS AND METHODS: Eight dogs were treated with 188-Re MAG(3)-filled balloon dilation immediately after stent placement and were killed 4 weeks later (group I, n = 4) or 8 weeks later (group II, n = 4). Five dogs were treated with 188-Re MAG(3)-filled balloon dilation 2 weeks after stent placement and were killed 4 weeks after stent placement (group III). The remaining five dogs were treated with conventional balloon dilation immediately after stent placement and were killed 4 weeks later; these animals formed the control group (group IV). Retrograde urethrography (RUG) was performed during follow-up and three histologic parameters were investigated: the number of epithelial layers, papillary projection thickness, and degree of submucosal inflammatory cell infiltration. The areas inside and outside the ends of the stents were evaluated in each case after animal sacrifice. After testing statistical significance of data for RUG and histologic findings in the four study groups, the Mann-Whitney U test was used to compare groups I and II to determine delayed effects of irradiation, groups I and III to determine benefits of delayed irradiation, groups I and IV to determine efficacy of immediate irradiation for reducing tissue hyperplasia, and groups III and IV to determine efficacy of delayed irradiation for reducing tissue hyperplasia. RESULTS: There were no significant differences in the four study groups on RUG before animal sacrifice. Between groups I and II, group II showed significantly lower mean values in five of six histologic comparisons. Between groups I and III, group III showed significantly lower mean values in only papillary projection thickness inside the stent ends. Between groups I and IV, group I showed significantly lower mean values in all three histologic parameters outside the stent ends. Between groups III and IV, group III showed significantly lower mean values in only two histologic parameters (papillary projection thickness in the in-stent area and inflammatory cell infiltration outside the stent edges). CONCLUSION: beta-Irradiation with use of a 188-Re MAG(3)-filled balloon shows the potential to reduce tissue hyperplasia secondary to stent placement in a canine urethral model. Treatment with 188-Re MAG(3)-filled balloons at the time of stent placement shows not only favorable outcomes for reducing tissue hyperplasia but also improved delayed effects until 8 weeks.