The Ndc80 loop region facilitates formation of kinetochore attachment to the dynamic microtubule plus end

Proper chromosome segregation in mitosis relies on correct kinetochore-microtubule (KT-MT) interactions. The KT initially interacts with the lateral surface of a single MT (lateral attachment) extending from a spindle pole and is subsequently anchored at the plus end of the MT (end-on attachment). The conversion from lateral to end-on attachment is crucial because end-on attachment is more robust and thought to be necessary to sustain KT-MT attachment when tension is applied across sister KTs upon their biorientation. The mechanism for this conversion is still elusive. The Ndc80 complex is an essential component of the KT-MT interface, and here we studied a role of the Ndc80 loop region, a distinct motif looping out from the coiled-coil shaft of the complex, in Saccharomyces cerevisiae. With deletions or mutations of the loop region, the lateral KT-MT attachment occurred normally; however, subsequent conversion to end-on attachment was defective, leading to failure in sister KT biorientation. The Ndc80 loop region was required for Ndc80-Dam1 interaction and KT loading of the Dam1 complex, which in turn supported KT tethering to the dynamic MT plus end. The Ndc80 loop region, therefore, has an important role in the conversion from lateral to end-on attachment, a crucial maturation step of KT-MT interaction.     

Relative and absolute configuration of antitumor agent SW-163D

Our interest on engineering non-ribosomal synthetase responsible for SW-163 biosynthesis prompted us to determine the relative and absolute configuration of antitumor cyclic depsipeptide SW-163s. We first isolated and identified SW-163 homologs D, F and G as known compounds UK-63598, UK-65662 and UK-63052, respectively. Both enantiomers of the unusual constitutive amino acid, N-methylnorcoromic acid, were synthesized in chiral forms starting from (R)- and (S)-1,2-propanediol. The hydrolyzate of SW-163D, a major constituent of this family, was converted with Marfey's reagent, 1-fluoro-2,4-dinitrophenyl-5-L-alanine-amide (L-FDAA), and the resulting mixture of amino acid derivatives was subjected to an LC/MS analysis. Compared with authentic samples, the analytical data unambiguously show that SW-163D consisted of L-Ala, D-Ser and (1S, 2S)-N-methylnorcoronamic acid. The remaining stereochemistry of the N-methylcysteine moieties was determined from NOE data.     

Population dynamics of phage-host system of Microlunatus phosphovorus indigenous in activated sludge

Monitoring of the phage-host system of Microlunatus phosphovorus indigenous in activated sludge was attempted. A laboratory-scale activated sludge process was operated for 5 weeks with synthetic wastewater. The phage-host system population in the process was monitored by plaque assay and FISH methods at every 3 days. During the process operation, the phage-host system populations were more or less steady, except for 1 week in the middle of the operation. In that period, initially M. phosphovorus decreased significantly and its lytic bacteriophages increased, and then M. phosphovorus increased back to its original level while its lytic bacteriophages decreased. This observation suggests that lytic bacteriophages should be considered as one of the biological factors affecting the bacterial population dynamics in activated sludge processes.     

Rapid quantification of total viral DNA in the supernatants of activated sludge samples with the fluorescent dye PicoGreen

Aims: To develop a rapid and simple method for quantifying viral DNA concentrations and determining viral quantities in activated sludge. Methods and Results: Activated sludge samples were obtained from three full‐scale and one laboratory‐scale process. They were centrifuged and the supernatant was filtered through a 0·2‐μm membrane filter. Free DNA was removed by DNase‐I treatment; any DNA within the viral capsid was liberated by heat treatment and proteinase K, and viral DNA concentrations were determined using the dye PicoGreen^®. To validate the method, we assessed the recovery of T4 phage added to filtered samples, which was 99% of those added. Viral DNA concentrations in samples from full‐scale plants ranged from 69 to 157 ng ml^?1. Monitoring of laboratory‐scale reactor samples revealed that viral DNA concentrations varied with time. Our method involves a simple sample treatment protocol and allow rapid analysis of many samples. Conclusions: A simple, rapid and sensitive method was developed and successfully used to determine the viral DNA concentrations in activated sludge. Significance and Impact of the Study: This method provides a way to investigate impact of bacteriophages on the performance of wastewater treatment processes.     

Substituted ajoenes as novel anti-cancer agents

A new synthesis of the ajoene pharmacophore core is presented involving the regioselective radical addition of a thiyl radical to a terminal alkyne as the key step. The synthesis allows structural variation of the two end groups on sulfur, and a range of novel derivatives varying the R(1) group (sulfoxide end) has been prepared and tested against CT-1 transformed fibroblast cells for anti-cancer activity. The results indicate comparable or even improved activity compared to the parent natural product ajoene isomers. This opens up the way to systematically studying the biology of the ajoene core.     

Lebetimonas natsushimae sp. nov., a novel strictly anaerobic,moderately thermophilic chemoautotroph isolated from a deep-sea hydrothermal vent polychaete nest in the Mid-Okinawa Trough

A moderately thermophilic, strictly anaerobic, chemoautotrophic bacterium, designated strain HS1857^T, was isolated from a deep-sea hydrothermal vent at the Noho site in the Mid-Okinawa Trough. Strain HS1857^T grew between 35 and 63 °C (optimum 55 °C), in the presence of 10–55 g l^?1 NaCl (optimum 25 g l^?1), and pH 5.5–7.1 (optimum 6.4). Growth occurred with molecular hydrogen as the electron donor and elemental sulfur, nitrate, or selenate as the electron acceptors. Formate could serve as an alternative electron donor with nitrate as an electron acceptor. During growth with nitrate as the electron acceptor, strain HS1857^T produced ammonium and formed a biofilm. CO₂ was utilized as the sole carbon source. The G + C content of the genomic DNA was 33.2 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain HS1857^T is a member of the order Nautiliales, showing a sequence similarity of 95.0% with Lebetimonas acidiphila Pd55^T. The fatty acid composition was similar to that of L. acidiphila, which was dominated by C_18:0 (47.0%) and C_18:1 (23.7%). Based on the genomic, chemotaxonomic, phenotypic characteristics, the name Lebetimonas natsushimae sp. nov., is proposed. The type strain is HS1857^T (= NBRC 112478^T = DSM 104102^T).     

Vitamin E inhibits PGE2 and O2- production in rat peritoneal macrophages.

In order to examine the possible role of vitamin E on the modulation of macrophages, we investigated the effect of vitamin E on O2- and PGE2 production in macrophages. The production of both PGE2 and O2- in rat peritoneal macrophages was dose-dependently stimulated by the addition of PMA and calcium ionophore A23187. However, the macrophages obtained after intraperitoneal injection of vitamin E for six successive days showed less PGE2 and O2- production when stimulated with PMA or A23187 as compared to those of control macrophages. O2- production in control macrophages stimulated with 139 nM PMA and 1 microM A23187 as 4.2 +/- 0.3 and 3.0 +/- 0.2 nmol/min per 10(6) cells, respectively. On the other hand, O2- production by the macrophages from vitamin E-treated rats was 1.5 +/- 0.4 nmol/min per 10(6) cells when stimulated with the PMA, and was not detectable when stimulated with A23187. As for the production of PGE2, control macrophages produced 2.59 +/- 0.70 ng/30 min per 10(6) cells when stimulated with PMA and 8.96 +/- 3.26 ng/30 min per 10(6) cells with the A23187, whereas PGE2 production by the macrophages from vitamin E-treated rats was reduced to 12-20% of the control. By analyzing alpha-tocopherol content and intracellular concentration of calcium ion [( Ca2+]i) in the macrophages isolated from control and vitamin E-treated rats, vitamin E treatment augmented alpha-tocopherol content (384.7 +/- 76.1 vs. 1.2 +/- 0.4 ng/10(6) cells) and decreased free [Ca2+]i when stimulated with A23187 (652 +/- 14 vs. 1201 +/- 223 nM).     

Free-radical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by alpha-tocopherol

The oxidation of rat red blood cells (RBC) by molecular oxygen was performed in an aqueous suspension with an azo compound as a free-radical initiator. The RBC were oxidized at a constant rate by a free-radical chain mechanism, resulting in hemolysis. The extent of hemolysis was proportional to the concentration of free radical. alpha-Tocopherol in RBC membranes suppressed the oxidation and hemolysis to produce an induction period. Tocopherol was constantly consumed during the induction period, and hemolysis developed when tocopherol concentrations fell below a critically low level. Among the membrane lipids, phosphatidylethanolamine, phosphatidylserine, and arachidonic acids were predominantly oxidized in the absence of tocopherol. In the presence of tocopherol, however, such lipid changes were suppressed during a 120-min incubation even when hemolysis started. Membrane proteins as well as lipids were oxidized. The formation of proteins with high molecular weight and concomitant decrease of the low-molecular-weight proteins were observed on gel electrophoresis with the onset of hemolysis. This study clearly showed the damage of RBC membranes caused by oxygen radical attack from outside of the membranes, and suggested that membrane tocopherol even below a critically low level could suppress lipid oxidation but that it could not prevent protein oxidation and hemolysis.     

Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer

Zinc-finger–FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP–scFokI). DNA cleavage assays revealed that the AZP–scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP–FokI dimer. The DNA cleavage pattern of AZP–scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP–scFokI. Furthermore, we demonstrated that AZP–scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP–scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach.