Application of a Hall sensor for pulsed magnetic field measurement in the FAT-CM FRC experiments.

Collisional merging experiments of a field-reversed configuration (FRC) plasma at the super-Alfvénic velocity have been conducted in the FAT (FRC Amplification via Translation)-CM (Collisional Merging) device. In the experiments, two FRCs are collided and merged in a confinement section with a quasi-static confinement magnetic field. Therefore, it is necessary to measure the high-frequency pulsed magnetic field superposed on a quasi-stationary signal. The magnetic field is generally measured by a magnetic coil probe in the pulse discharge experiments; however, in such measurements, errors arise in the low-frequency band in the conducted FRC experiments. Therefore, a Hall sensor has been applied for low-frequency magnetic field measurements in the FAT-CM experiments. Calibration of the Hall sensor involves confirming that the sensor has a sufficient response speed and linear characteristics for the magnetic field with a rising time of approximately 240 µs and that its output voltage does not saturate up to a magnetic field of 0.7 T. Combination of the Hall sensor and the magnetic coil probe ensures a comprehensive measurement of the magnetic field in the range of FAT-CM experiments. In this study, accurate magnetic measurements were performed in a collisional merging experiment in the FAT-CM device by using a combined magnetic diagnostic system.

Internal magnetic field measurements of translated and merged field-reversed configuration plasmas in the FAT-CM device.

Field-reversed configuration (FRC) Amplification via Translation-Collisional Merging (FAT-CM) experiments have recently commenced to study physics phenomena of colliding and merged FRC plasma states. Two independently formed FRCs are translated into the confinement region of the FAT-CM device, collided near the mid-plane of the device with a relative speed of up to ∼400 km/s, and a final merged FRC plasma state is achieved. To measure internal magnetic field profiles of the translated and merged FRC plasmas as well as to understand its collisional-merging process, an internal magnetic probe array, developed by TAE Technologies, has been installed in the mid-plane of the FAT-CM device. Initial magnetic field measurements indicate that both the translated and the merged FRC plasma states exhibit a clear field-reversed structure, which is qualitatively in good agreement with 2D MHD simulation. It is found and verified that a sufficient mirror field in the confinement region is required for colliding FRCs to be fully merged into a single FRC plasma state.

Super-Alfvénic translation of a field-reversed configuration into a large-bore dielectric chamber.

An experimental device to demonstrate additional heating and control methods for a field-reversed configuration (FRC) has been developed. The newly developed device, named FRC Amplification via Translation (FAT), has a field-reversed theta-pinch plasma source and a low-elongation dielectric (transparent quartz) confinement chamber with quasi-static confinement field. In the initial experiments on the FAT device, FRC translation and trapping were successfully demonstrated. Although the typical elongation of the trapped FRC in the confinement region was roughly three, no disruptive global instability, such as tilt, was observed. The FAT device increases the latitude to perform translation-related experiments, such as those concerning inductive current drive, equivalent neutral beam injection effects, and wave applications.

Characterization of compact-toroid injection during formation, translation, and field penetration.

We have developed a compact toroid (CT) injector system for particle refueling of the advanced beam-driven C-2U field-reversed configuration (FRC) plasma. The CT injector is a magnetized coaxial plasma gun (MCPG), and the produced CT must cross the perpendicular magnetic field surrounding the FRC for the refueling of C-2U. To simulate this environment, an experimental test stand has been constructed. A transverse magnetic field of ∼1 kG is established, which is comparable to the C-2U axial magnetic field in the confinement section, and CTs are fired across it. On the test stand we have been characterizing and studying CT formation, ejection/translation from the MCPG, and penetration into transverse magnetic fields.

Development of a magnetized coaxial plasma gun for compact toroid injection into the C-2 field-reversed configuration device.

A compact toroid (CT) injector was developed for the C-2 device, primarily for refueling of field-reversed configurations. The CTs are formed by a magnetized coaxial plasma gun (MCPG), which consists of coaxial cylindrical electrodes and a bias coil for creating a magnetic field. First, a plasma ring is generated by a discharge between the electrodes and is accelerated by Lorenz self-force. Then, the plasma ring is captured by an interlinkage flux (poloidal flux). Finally, the fully formed CT is ejected from the MCPG. The MCPG described herein has two gas injection ports that are arranged tangentially on the outer electrode. A tungsten-coated inner electrode has a head which can be replaced with a longer one to extend the length of the acceleration region for the CT. The developed MCPG has achieved supersonic CT velocities of ∼100 km/s. Plasma parameters for electron density, electron temperature, and the number of particles are ∼5 × 10(21) m(-3), ∼40 eV, and 0.5-1.0 × 10(19), respectively.

Pyrazinamide resistance in multidrug-resistant Mycobacterium tuberculosis isolates in Japan.

Thirty-six multidrug-resistant (MDR) Mycobacterium tuberculosis isolates collected in Japan were examined for pyrazinamide susceptibility and pyrazinamidase activity, and analysed by pncA sequencing and a hybridization-based line probe assay (LiPA), which was used to detect pncA mutations for the rapid identification of pyrazinamide-resistant isolates. Pyrazinamide resistance was found in 19 (53%) of them. All pyrazinamide-resistant isolates had no pyrazinamidase activity and at least one mutation in pncA. Among the pncA mutations, 11 had not been previously reported. The results of the LiPA were fully consistent with the DNA sequencing results. A majority of MDR M. tuberculosis isolates in Japan were resistant to pyrazinamide.

Reproducibility of temporal volume change in CT of lung cancer: comparison of computer software and manual assessment.

The purpose of this study was to investigate the reproducibility of volumetric software evaluation and manual evaluation of tumour growth. Three observers manually evaluated whether tumour volume was increasing, if it was unchanged, or if it had decreased in size in 2 serial CT examinations of 45 solid lung cancers. The tumour volumes were calculated 3 times using volumetric software and were evaluated using the same classifications as for manual evaluation. Both data sets were divided into three groups: growth or reduction with consistency among all three evaluations (group A), growth or reduction with consistency between only two evaluations (group B), and others (group C). The volume variation and relative volume variation were calculated from the median volumes measured by volumetric software. Although all 45 tumours were categorised in group A by volumetric software, only 21 tumours were categorised in group A by manual assessment. The relative volume variation of the manual assessment was 88.5 +/- 76.5%, 20.8 +/- 28.3% and 12.9 +/- 12.8% in group A, B and C, respectively. Significant differences were found between groups A and B (p<0.01) and between groups A and C (p<0.001). Inconsistency is often seen in manual assessment; in contrast, evaluation using volumetric software has good reproducibility, even when the relative change in tumour volume is small.

Regulation of the glv operon in Bacillus subtilis: YfiA (GlvR) is a positive regulator of the operon that is repressed through CcpA and cre.

Maltose metabolism and the regulation of the glv operon of Bacillus subtilis, comprising three genes, glvA (6-phospho-alpha-glucosidase), yfiA (now designated glvR), and glvC (EIICB transport protein), were investigated. Maltose dissimilation was dependent primarily upon the glv operon, and insertional inactivation of either glvA, glvR, or glvC markedly inhibited growth on the disaccharide. A second system (MalL) contributed to a minor extent to maltose metabolism. Northern blotting revealed two transcripts corresponding to a monocistronic mRNA of glvA and a polycistronic mRNA of glvA-glvR-glvC. Primer extension analysis showed that both transcripts started at the same base (G) located 26 bp upstream of the 5' end of glvA. When glvR was placed under control of the spac promoter, expression of the glv operon was dependent upon the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG). In regulatory studies, the promoter sequence of the glv operon was fused to lacZ and inserted into the amyE locus, and the resultant strain (AMGLV) was then transformed with a citrate-controlled glvR plasmid, pHYCM2VR. When cultured in Difco sporulation medium containing citrate, this transformant [AMGLV(pHYCM2VR)] expressed LacZ activity, but synthesis of LacZ was repressed by glucose. In an isogenic strain, [AMGLVCR(pHYCM2VR)], except for a mutation in the sequence of a catabolite-responsive element (cre), LacZ activity was expressed in the presence of citrate and glucose. Insertion of a citrate-controlled glvR plasmid at the amyE locus of ccpA(+) and ccpA mutant organisms yielded strains AMCMVR and AMCMVRCC, respectively. In the presence of both glucose and citrate, AMCMVR failed to express the glv operon, whereas under the same conditions high-level expression of both mRNA transcripts was found in strain AMCMVRCC. Collectively, our findings suggest that GlvR (the product of the glvR gene) is a positive regulator of the glv operon and that glucose exerts its effect via catabolite repression requiring both CcpA and cre.

Mutational analysis of catalytic sites of the cell wall lytic N-acetylmuramoyl-L-alanine amidases CwlC and CwlV.

The Bacillus subtilis CwlC and the Bacillus polymyxa var. colistinus CwlV are the cell wall lytic N-acetylmuramoyl-l-alanine amidases in the CwlB (LytC) family. Deletion in the CwlC amidase from the C terminus to residue 177 did not change the amidase activity. However, when the deletion was extended slightly toward the N terminus, the amidase activity was entirely lost. Further, the N-terminal deletion mutant without the first 19 amino acids did not have the amidase activity. These results indicate that the N-terminal half (residues 1-176) of the CwlC amidase, the region homologous to the truncated CwlV (CwlVt), is a catalytic domain. Site-directed mutagenesis was performed on 20 highly conserved amino acid residues within the catalytic domain of CwlC. The amidase activity was lost completely on single amino acid substitutions at two residues (Glu-24 and Glu-141). Similarly, the substitution of the two glutamic acid residues (E26Q and E142Q) of the truncated CwlV (CwlV1), which corresponded to Glu-24 and Glu-141 of CwlC, was critical to the amidase activity. The EDTA-treated CwlV1 did not have amidase activity. The amidase activity of the EDTA-treated CwlV1 was restored by the addition of Zn2+, Mn2+, and Co2+ but not by the addition of Mg2+ and Ca2+. These results suggest that the amidases in the CwlB family are zinc amidases containing two glutamic acids as catalytic residues.

Genetic interactions between ATM and the nonhomologous end-joining factors in genomic stability and development.

DNA ligase IV (Lig4) and the DNA-dependent protein kinase (DNA-PK) function in nonhomologous end joining (NHEJ). However, although Lig4 deficiency causes late embryonic lethality, deficiency in DNA-PK subunits (Ku70, Ku80, and DNA-PKcs) does not. Here we demonstrate that, similar to p53 deficiency, ataxia-telangiectasia-mutated (ATM) gene deficiency rescues the embryonic lethality and neuronal apoptosis, but not impaired lymphocyte development, associated with Lig4 deficiency. However, in contrast to p53 deficiency, ATM deficiency enhances deleterious effects of Lig4 deficiency on growth potential of embryonic fibroblasts (MEFs) and genomic instability in both MEFs and cultured progenitor lymphocytes, demonstrating significant differences in the interplay of p53 vs. ATM with respect to NHEJ. Finally, in dramatic contrast to effects on Lig4 deficiency, ATM deficiency causes early embryonic lethality in Ku- or DNA-PKcs-deficient mice, providing evidence for an NHEJ-independent role for the DNA-PK holoenzyme.

Increased accumulation of hybrid V(D)J joins in cells expressing truncated versus full-length RAGs.

RAG1 and RAG2 (RAGs) initiate V(D)J recombination by introducing breaks between two coding segments and flanking recombination signals (RSs). Nonhomologous end-joining (NHEJ) proteins then join the coding segments and join the RSs. In wild-type cells, both full-length and truncated ("core") RAGs lead to accumulation of "hybrid" V(D)J joins, in which an RS is appended to a different coding sequence. We now show that while hybrid joins do not accumulate in NHEJ-deficient cells that express full-length RAGs, they do accumulate in NHEJ-deficient cells that express the core RAGS; like those catalyzed by core RAGs in vitro, however, they are sealed on just one DNA strand. These results suggest a potential role for the non-core regions in repressing potentially harmful transposition events.

The CitST two-component system regulates the expression of the Mg-citrate transporter in Bacillus subtilis.

citS and citT genes encoding a new two-component system were identified in the 71 degrees region between the pel and citM loci on the Bacillus subtilis chromosome. citS- and citT-deficient strains were unable to grow on minimal plates including citrate as a sole carbon source. In addition, a strain deficient in citM, which encodes the secondary transporter of the Mg-citrate complex, exhibited the same phenotype on this medium. Northern blot analysis revealed that citM was polycistronically transcribed with the downstream yflN gene, and that CitS and CitT were necessary for transcription of the citM-yflN operon. Upon addition of 2 mM citrate to DSM, this operon was strongly induced after the middle of the exponential growth phase in the wild type, but not in the citST double null mutant. Moreover, the transcription of this operon was completely repressed in the presence of 1% glucose. We found a sequence exhibiting homology to a catabolite-responsive element (cre) in the citM promoter region. Glucose repression was lost in ccpA and citM-cre mutants. From the result of a citM-promoter deletion experiment, putative CitT target sequences were found to be located around two regions, from -62 to -74 and from -149 to -189, relative to the citM start point. Furthermore, DNase I footprinting assays revealed that these two CitT target regions extended maximally from -36 to -84 and from -168 to -194. From these findings, we concluded that the expression of citM is positively regulated by the CitST system and negatively regulated by CcpA.

Cloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli.

Aspergillus oryzae produces at least three extracellular lipolytic enzymes, L1, L2 and L3 (cutinase, mono- and diacylglycerol lipase, and triacylglycerol lipase, respectively). We cloned the triacylglycerol lipase gene (provisionally designated tglA) by screening a genomic library using a PCR product obtained with two degenerate oligonucleotide primers corresponding to amino acid sequences of L3 as probes. Nucleotide sequencing of the genomic DNA and cDNA revealed that the L3 gene (tglA) has an open reading frame comprising 954 nucleotides, which contains three introns of 47, 83 and 62 bp. The deduced amino acid sequence of the tglA gene corresponds to 254 amino acid residues including a signal sequence of 30 amino acids and, in spite of the difference in substrate specificity, it is homologous to those of cutinases from fungi. Three residues presumed to form the catalytic triad, Ser, Asp and His, are conserved. The cloned cDNA of the tglA gene was expressed in Escherichia coli, and enzyme assaying and zymography revealed that the cloned cDNA encodes a functional triacylglycerol lipase.

Overexpression, purification, and characterization of Bacillus subtilis N-acetylmuramoyl-L-alanine amidase CwlC.

N-acetylmuramoyl-L-alanine amidase CwlC of Bacillus subtilis was overproduced in Escherichia coli and purified 21-fold. The amidase hydrolyzed type A cell walls such as B. subtilis. The amidase bound slightly to the Microbacterium lacticum cell wall (type B), but did not entirely hydrolyze it. The presence of calcium or magnesium ion increased the resistance of the amidase to heat denaturation.

Resolution of a Holliday junction by vaccinia topoisomerase requires a spacer DNA segment 3' of the CCCTT/ cleavage sites.

Vaccinia virus DNA topoisomerase catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT/, that are opposed within a partially mobile four-way junction. Efficient resolution occurs on a junction with a 10 bp segment of branch mobility (5'-GCCCTTATCG) that extends 4 bp 3' of the scissile phosphate. Here we report that resolution is decreased when branch mobility is limited to an 8 bp segment extending 2 bp 3' of the cleavage site and then eliminated when branch mobility is confined to the 6 bp GCCCTT sequence 5' of the scissile phosphate. We surmise that a spacer region 3' of CCCTT is needed for simultaneous cleavage at two opposing sites at the junction. Branch mobility is not required for reaction chemistry at a junction, because topoisomerase cleaves a single CCCTT site in a non-mobile four-way junction where the scissile phosphate is at the crossover point. The junction resolvase activity of topo-isomerase may be involved in forming the hairpin telomeres of the vaccinia genome.

Accumulation of an artificial cell wall-binding lipase by Bacillus subtilis wprA and/or sigD mutants.

A recombinant lipase, CWB-LipB, localized on the Bacillus subtilis cell surface and retaining lipase activity was unstable and not accumulated in a high yield. To improve the accumulation, we examined cell wall binding protease (wprA)- and/or sigma D (sigD)-deficient mutants, and also a NprE and AprA protease-deficient mutant as host strains. The nprE aprA mutation did not lead to a significant increase in the CWB-LipB accumulation. The wprA mutant accumulated a greater amount than the wild-type only in the stationary phase, but the sigD mutant accumulated a greater amount in both the exponential and stationary phases. The double mutant exhibited great accumulation of CWB-LipB, the amount being 36% of the total proteins extracted from the cell surface.

DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway.

DNA ligase IV (LIG4) is a nonhomologous end-joining (NHEJ) protein used for V(D)J recombination and DNA repair. In mice, Lig4 deficiency causes embryonic lethality, massive neuronal apoptosis, arrested lymphogenesis, and various cellular defects. Herein, we assess potential roles in this phenotype for INK4a/ARF and p53, two proteins implicated in apoptosis and senescence. INK4a/ARF deficiency rescued proliferation/senescence defects of Lig4-deficient fibroblasts but not other phenotypic aspects. In contrast, p53 deficiency rescued embryonic lethality, neuronal apoptosis, and fibroblast proliferation/senescence defects but not lymphocyte development or radiosensitivity. Young Lig4/p53 double null mice routinely died from pro-B lymphomas. Thus, in the context of Lig4 deficiency, embryonic lethality and neuronal apoptosis likely result from a p53-dependent response to unrepaired DNA damage, and neuronal apoptosis and lymphocyte developmental defects can be mechanistically dissociated.

The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations.

We have used spectral karyotyping to assess potential roles of three different components of the nonhomologous DNA end-joining pathway in the maintenance of genomic stability in mouse embryonic fibroblasts (MEFs). MEFs homozygous for mutations that inactivate either DNA ligase IV (Lig4) or Ku70 display dramatic genomic instability, even in the absence of exogenous DNA damaging agents. These aberrant events range from chromosomal fragmentation to nonreciprocal translocations that can involve several chromosomes. DNA-dependent protein kinase catalytic subunit deficiency also promotes genome instability. Deficiency for the p53 cell cycle checkpoint protein has little effect on spontaneous levels of chromosomal instability in Lig4-deficient fibroblasts. However, in the context of ionizing radiation treatment, p53 deficiency allowed visualization of massive acute chromosomal destruction in Lig4-deficient MEFs, which in surviving cells manifested as frequent nonreciprocal translocations. We conclude that nonhomologous DNA end-joining plays a crucial role as a caretaker of the mammalian genome, and that an alternative repair pathway exists that often leads to nonreciprocal translocations.

"Face to face": a new method for the treatment of polydactyly of the thumb that maximises the use of available soft tissue.

Some patients with distal phalangeal polydactyly of the thumb have severe hypoplastic pulps that are difficult to make sufficiently large with either the fillet flap method or a symmetrical combination, the Bilhaut-Cloquet method. We have devised a new method to reconstruct the largest possible pulp by resecting only a minimum amount of soft dorsal skin tissue with one nail and not resecting any of the palmar skin of the thumbs. By contrast, other procedures require some resectioning of soft tissue of both the dorsal and the palmar skin. We have treated 13 cases with distal phalangeal polydactyly of the thumb since 1988 in this way. All the thumbs were successfully reconstructed with a sufficiently large pulp. The only problem was slight instability of the skin of the pulp after the operation, but this condition gradually improved during a five-year follow up. We think that this method facilitates the reconstruction of a sufficiently large pulp when both bifid pulps are hypoplastic.

Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development.

XRCC4 is a non-homologous end-joining protein employed in DNA double strand break repair and in V(D)J recombination. In mice, XRCC4-deficiency causes a pleiotropic phenotype, which includes embryonic lethality and massive neuronal apoptosis. When DNA damage is not repaired, activation of the cell cycle checkpoint protein p53 can lead to apoptosis. Here we show that p53-deficiency rescues several aspects of the XRCC4-deficient phenotype, including embryonic lethality, neuronal apoptosis, and impaired cellular proliferation. However, there was no significant rescue of impaired V(D)J recombination or lymphocyte development. Although p53-deficiency allowed postnatal survival of XRCC4-deficient mice, they routinely succumbed to pro-B-cell lymphomas which had chromosomal translocations linking amplified c-myc oncogene and IgH locus sequences. Moreover, even XRCC4-deficient embryonic fibroblasts exhibited marked genomic instability including chromosomal translocations. Our findings support a crucial role for the non-homologous end-joining pathway as a caretaker of the mammalian genome, a role required both for normal development and for suppression of tumours.